A guide to the use of bioassays in exploration of natural resources

Abstract

Bioassays are the main tool to decipher bioactivities from natural resources thus their selection and quality are critical for optimal bioprospecting. They are used both in the early stages of compounds isolation/purification/identification, and in later stages to evaluate their safety and efficacy. In this review, we provide a comprehensive overview of the most common bioassays used in the discovery and development of new bioactive compounds with a focus on marine bioresources. We present a comprehensive list of practical considerations for selecting appropriate bioassays and discuss in detail the bioassays typically used to explore antimicrobial, antibiofilm, cytotoxic, antiviral, antioxidant, and anti-ageing potential. The concept of quality control and bioassay validation are introduced, followed by safety considerations, which are critical to advancing bioactive compounds to a higher stage of development. We conclude by providing an application-oriented view focused on the development of pharmaceuticals, food supplements, and cosmetics, the industrial pipelines where currently known marine natural products hold most potential. We highlight the importance of gaining reliable bioassay results, as these serve as a starting point for application-based development and further testing, as well as for consideration by regulatory authorities.

Full text

Biotechnology Advances 71 (2024) 108307
Available online 5 January 2024
0734-9750/© 2024 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-
nc/4.0/).
Research review paper
A guide to the use of bioassays in exploration of natural resources
Jerica Sabotiˇ
c
a
,
*
, Engin Bayram
b
, David Ezra
c
, Susana P. Gaudˆ
encio
d
,
e
,
Berat Z. Haznedaro˘
glu
b
, Nika Janeˇ
z
a
, Leila Ktari
f
, Anna Luganini
g
, Manolis Mandalakis
h
,
Ivo Safarik
i
,
j
, Dina Simes
k
,
l
, Evita Strode
m
, Anna Toru´
nska-Sitarz
n
,
Despoina Varamogianni-Mamatsi
h
, Giovanna Cristina Varese
g
, Marlen I. Vasquez
o
a
Department of Biotechnology, Joˇ
zef Stefan Institute, 1000 Ljubljana, Slovenia
b
Institute of Environmental Sciences, Bogazici University, Bebek, Istanbul 34342, Turkey
c
Department of Plant Pathology and Weed Research, ARO, The Volcani Institute, P.O.Box 15159, Rishon LeZion 7528809, Israel
d
Associate Laboratory i4HB – Institute for Health and Bioeconomy, NOVA School of Science and Technology, NOVA University Lisbon, 2819-516 Caparica, Portugal
e
UCIBIO – Applied Biomolecular Sciences Unit, Department of Chemistry, Blue Biotechnology & Biomedicine Lab, NOVA School of Science and Technology, NOVA
University of Lisbon, 2819-516 Caparica, Portugal
f
B3Aqua Laboratory, National Institute of Marine Sciences and Technologies, Carthage University, Tunis, Tunisia
g
Department of Life Sciences and Systems Biology, University of Turin, 10123 Turin, Italy
h
Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, 71500 Heraklion, Greece
i
Department of Nanobiotechnology, Biology Centre, ISBB, CAS, Na Sadkach 7, 370 05 Ceske Budejovice, Czech Republic
j
Regional Centre of Advanced Technologies and Materials, Czech Advanced Technology and Research Institute, Palacky University, Slechtitelu 27, 783 71 Olomouc,
Czech Republic
k
Centre of Marine Sciences (CCMAR), Universidade do Algarve, 8005-139 Faro, Portugal
l
2GenoGla Diagnostics, Centre of Marine Sciences (CCMAR), Universidade do Algarve, Faro, Portugal
m
Latvian Institute of Aquatic Ecology, Agency of Daugavpils University, Riga LV-1007, Latvia
n
Department of Marine Biology and Biotechnology, Faculty of Oceanography and Geography, University of Gda´
nsk, 81-378 Gdynia, Poland
o
Department of Chemical Engineering, Cyprus University of Technology, 3036 Limassol, Cyprus
ARTICLE INFO
Keywords:
Bioassay selection
Bioactivity
Natural products
Drug discovery
Blue biotechnology
Bioactivity-guided purication
Validation
Preclinical trials
Biodiscovery
ABSTRACT
Bioassays are the main tool to decipher bioactivities from natural resources thus their selection and quality are
critical for optimal bioprospecting. They are used both in the early stages of compounds isolation/purication/
identication, and in later stages to evaluate their safety and efcacy. In this review, we provide a compre-
hensive overview of the most common bioassays used in the discovery and development of new bioactive
compounds with a focus on marine bioresources. We present a comprehensive list of practical considerations for
selecting appropriate bioassays and discuss in detail the bioassays typically used to explore antimicrobial,
antibiolm, cytotoxic, antiviral, antioxidant, and anti-ageing potential. The concept of quality control and
bioassay validation are introduced, followed by safety considerations, which are critical to advancing bioactive
compounds to a higher stage of development. We conclude by providing an application-oriented view focused on
Abbreviations: ABTS/TEAC, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)/Trolox®-Equivalent Antioxidant Capacity; ADMET, absorption, distribution,
metabolism, excretion, toxicity; AFST, antifungal susceptibility testing; BSL, biosafety level; CADD, computer-aided drug design; CC
50
, 50 % cytotoxicity concen-
tration; CLSI, Clinical and Laboratory Standards Institute; COST, European Cooperation in Science and Technology; CFU, colony forming unit; CPE, cytopathic effect;
CTA, cell transformation assays; CUPRAC, CUPric Reducing Antioxidant Capacity; DPPH, 2,2-Diphenyl-1-picrylhydrazyl; EC
50
, 50% effective concentration; EFSA,
European Food Safety Authority; EGCG, epigallocathechin-3-gallate; EMA, European Medicines Agency; ET, electron transfer; EUCAST, European Committee on
Antimicrobial Susceptibility Testing; FDA, United States Food and Drug Administration; FFA, focus-forming assay; FMCA, uorometric microculture cytotoxicity
assay; GI
50
, 50 % growth inhibition; GLP, good laboratory practice; HA, hemagglutinin; HAT, hydrogen atom transfer; HCS, high content screening; HIA, hemag-
glutination inhibition assay; HTS, high-throughput screening; LLPS, liquid-liquid phase separation; LOD, limit of detection; LOQ, limit of quantitation; MALDI-TOF,
matrix-assisted laser desorption ionization–time-of-ight mass spectrometry; MIC, minimum inhibitory concentration; MOI, multiplicity of infection; MBC, minimum
bactericidal concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MS, mass spectrometry; MSPE, magnetic solid phase extraction;
NAM, new approach methodology; OECD, Organisation for Economic Co-operation and Development; ORAC, oxygen radical absorbance capacity; PBPK, physiology-
based pharmacokinetic; PRA, plaque reduction assay; qPCR, quantitative real-time polymerase chain reaction; RBC, red blood cells; SI, selectivity index; SPE, solid
phase extraction; VOC, volatile organic compound; VRA, virus yield reduction assay.
* Corresponding author.
E-mail address: Jerica.Sabotic@ijs.si (J. Sabotiˇ
c).
Contents lists available at ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
https://doi.org/10.1016/j.biotechadv.2024.108307
Received 24 July 2023; Received in revised form 5 December 2023; Accepted 1 January 2024

Biotechnology Advances 71 (2024) 108307
2
the development of pharmaceuticals, food supplements, and cosmetics, the industrial pipelines where currently
known marine natural products hold most potential. We highlight the importance of gaining reliable bioassay
results, as these serve as a starting point for application-based development and further testing, as well as for
consideration by regulatory authorities.
1. Introduction
The most common approach to discovering new bioactive com-
pounds is an extensive screening of crude natural extracts using
bioassay-guided protocols to determine their activity, followed by
isolation and characterization of the active compounds, which are then
used in a variety of biotechnological applications, including food, feed,
agriculture, cosmetics, and veterinary and human medicine. The dis-
covery of new marine natural products in the last ve years has been
driven primarily by marine fungi, but also by sponges, tunicates (as-
cidians), molluscs and cyanobacteria, which are the source of most of
the approved drugs in the marine pharmacology pipeline (see e.g., www.
marinepharmacology.org). Bacteria associated or symbiotic with marine
invertebrates are recognized as an important source of marine natural
products (El-Seedi et al., 2023; Jim´
enez, 2018; McCauley et al., 2020;
Newman and Cragg, 2020; Rotter et al., 2021a). In addition, marine
archaea, green algae, thraustochytrids, and dinoagellates, have long
been studied as sources of natural bioactive products. To increase the
chemical space and diversity of activities detected in bioassays, modi-
cations of culture conditions or co-cultivation are used in the search for
natural products from culturable microorganisms (e.g., (Lauritano et al.,
2016; Marmann et al., 2014; Oh et al., 2005; Romano et al., 2018). Other
sources of marine natural products include actinomycetes, brown, and
red algae, cnidarians, bryozoans, echinoderms, crustaceans, and sh
(Barreca et al., 2020; Carroll et al., 2021; Jimenez et al., 2020; Rotter
et al., 2021a). The ecological diversity of the marine environment and
(micro)organisms in this habitat, combined with the large genetic di-
versity, represents a unique and rich source of compounds that can be
exploited by the pharmaceutical industry and potentially provide solu-
tions to the increasing number of drug-resistant infectious and non-
infectious diseases (Bettio et al., 2023; Hughes and Fenical, 2010;
Liang et al., 2019; Liu et al., 2019).
The authors of this review are members of COST Action CA18238
Ocean4Biotech, a network of >150 blue biotechnology scientists and
practitioners from 37 countries (Rotter et al., 2020, 2021b). Our goal is
to provide a guide for decision making in the selection and use of bio-
assays to improve the efciency of bioprospecting and discovery of
bioactive marine compounds. A comprehensive overview of bioassays
currently used in the marine bioprospecting community is provided,
along with their strengths and weaknesses, followed by considerations
for bioassay-guided identication and isolation. We also consider the
importance of incorporating in vitro, ex vivo, and 3D human cell- or
tissue-based bioassay protocols as important tools in the preclinical
process to avoid drug failure in clinical trials, most often due to lack of
clinical efcacy and/or unacceptable toxicity. We then present quality
control procedures, including validation, that are required for further
safety and efcacy testing, which will then pave the way for eventual
regulatory approval for commercialization. The procedures and work-
ows described are general in nature and can be applied to a wide range
of potential applications of bioactive compounds, from industrial en-
zymes to pharmaceuticals for human consumption. Therefore, we use
the term bioactive compounds to refer to all structural variants of nat-
ural molecules, from small molecules to large polymers, including, for
example, proteins and polysaccharides. Finally, we provide an
application-oriented overview of the industrial pipelines most
commonly supplied with marine-derived natural products, including
those focused on the development of pharmaceuticals, dietary supple-
ments, and cosmetics. By providing insight into the assays used to
evaluate bioactivity and best practices in bioassays, this review aims to
guide the natural products and blue biotechnology community in deci-
sion making for natural product discovery and development.
2. Bioassay types and their use in bioactive compound discovery
The biological relevance of natural extracts and pure compounds,
whether natural or synthetic, is determined by the bioactivity assays or
bioassays used (Weller, 2012). The term “bioactive” is dened as
“having or causing an effect on living tissue” (Str¨
omstedt et al., 2014).
Different characteristics of bioassays such as throughput, complexity,
speed, and cost are relevant to different stages of the biodiscovery
process (Fig. 1). In the pre-screening and screening phase, the goal is to
detect and potentially quantify bioactivity potential. Therefore, bio-
assays should be performed in a high-throughput screening format
(HTS) that allows rapid and cost-effective testing of large number of
samples or large libraries of extracts, extract fractions, or pure com-
pounds. In the monitoring phase, bioassays are used to guide purica-
tion or fractionation processes to isolate and identify single pure
bioactive compounds (bioactivity-guided approach), so they must be
designed to have a high throughput capacity, be fast and easy to
perform, and be cost-effective. Interestingly, innovative in silico ap-
proaches have recently been developed that do not require extract
fractionation and are known as compound activity mapping (CAM) and
are freely available (www.npanalyst.org) (Gaudˆ
encio et al., 2023; Kurita
et al., 2015; Lee et al., 2022; O’Rourke et al., 2020). Finally, in the
secondary phase, bioassays are used to identify and characterize the
biological mode of action of the bioactive compound, which typically
requires a series of bioassays that must be highly specic and accurate
and are usually time-consuming and expensive (Claeson and Bohlin,
1997; Str¨
omstedt et al., 2014; Suffness and Pezzuto, 1991).
Bioassays can be performed in silico, in vitro, ex vivo, or in vivo at
any of the levels described, and usually a combination of these methods
is used to characterize a new compound or the bioactivity potential of a
natural resource. When screening an extract for medicinal activity, in
silico and in vitro assays are typically used to identify the bioactive
compound and its mode of action, while ex vivo and in vivo assays (e.g.,
animal studies) provide information on pharmacological activity and
toxicity (Mbah et al., 2012; Str¨
omstedt et al., 2014).
3. Practical considerations in choosing bioassays to detect
target bioactivity
The following paragraphs provide a list of questions and consider-
ations, the answers to which provide information on what to consider
when selecting or designing a bioassay (Table 1, Fig. 2).
At what stage of the discovery process and for what purpose will the
bioassay be performed? Considering the target bioactivity of interest,
appropriate bioassays can be selected and used to screen crude or frac-
tionated extracts, to guide subsequent purication, or to explain un-
derlying mechanisms of action, as described in the previous section. First
and foremost, the target bioactivity should be selected. An overview of
the most commonly used bioassays can be found in Table 2.
Is there an interest in a specic or general activity? In general, bio-
assays can be divided into two distinct categories: “single-target bio-
assays” and “functional multi-target bioassays”. Single-target bioassays
are generally designed to detect the effect of the tested compounds on a
particular target with a high degree of specicity and based on a distinct
mechanism of action (Claeson and Bohlin, 1997). Examples include the
analysis of specic enzymatic activities, such as the degradation of
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Biotechnology Advances 71 (2024) 108307
3
proteins or breakdown of plastics, or the inhibition of enzymatic activ-
ities, such as the inhibition of proteases and the blocking of target re-
ceptors. Another variation of single-target bioassays is “chemical-
genetic proling” in yeast. A panel of yeast strains with selective mu-
tations that highlight sensitivity to specic drugs is used to screen
known compounds with unknown modes of action or mixtures of com-
pounds such as natural product extracts (Harvey, 2008). The second
category, “functional multi-target bioassays”, includes bioassays that
use whole animals, organs or cells. These bioassays are non-specic in
their outcome and measure phenotype change or a general biological
effect, such as an antimicrobial or cytotoxic effect. The response to the
bioactive compound tested cannot necessarily be attributed to a specic
mode of action. These are often referred to as the “phenotype-based
approach” (Claeson and Bohlin, 1997; Swinney, 2013).
Which are the most common bioassays for determining target ac-
tivity? The target bioactivity can be assessed using a variety of bioassays,
but the scientic community may prefer certain assays for which trou-
bleshooting, appropriate controls, and interpretation support are avail-
able (Table 2).
Are resources available to perform bioassays (in terms of ease of
execution or technical complexity)? Specialized equipment and/or
trained personnel are required to perform certain bioassays. In terms of
safety, it is also important to consider whether the bioassay uses haz-
ardous chemicals or organisms that must be handled in safety chambers
and comply with local regulations (e.g., consider the biosafety level
(BSL) of the target organisms, the use of genetically modied organisms
(GMOs), and waste management).
What are the associated costs for personnel, equipment, and mate-
rials? Will the bioassay be used as a routine method? A bioassay may be
simple (e.g., an enzymatic reaction detected by a colour change) and
performed by a technician, whereas some types of bioassays (e.g., bio-
assays using cell culture) require extensive training. Similarly, bioassays
may be more or less labour-intensive and require specialized equipment
or expensive consumables.
Is high throughput and full automation of the analytical process
required? Bioassays often use a 96- or 384-well plate format, whereas a
higher density layout of 1536-wells is also available but less popular.
Performing a manual 384-well plate assay is challenging, especially for
assays where precise time intervals between stages are critical. Never-
theless, it is feasible for selected bioassays. A common plate-related
phenomenon is the so-called “edge effect”, in which the response in
peripheral wells differs from the response observed in the inner wells of
Fig. 1. Characteristics of bioassays used at different stages of biodiscovery. The biodiscovery process consists of several stages (centre), which place different de-
mands on bioassays’ characteristics (bottom) in order to achieve the progressive goals of biodiscovery (top).
Table 1
What to consider when selecting a bioassay to search for a selected bioactivity.
Purpose
Is it aimed at general or specic bioactivity?
How selective should it be?
Are quantitative or qualitative results needed?
How sensitive should it be (what is the requirement for the minimal amount of
compound)?
Cost
Time requirement
Labour intensiveness
Cost of material
Requirement of special equipment (different modes of detection)
Effect of the extraction procedure on bioactivity
Selection of source material (amount available, possibility to reacquire)
Availability of source material (seasonal, geographic, legal)
Organic solvent or water-based
Temperature of extraction
Length of extraction
Homogenization steps
Cultivation steps
Stability of bioactive compound
Interference with materials used for extraction (e.g., plastic, solvent components)
Feasibility
Errors caused by the colour or viscosity of extracts
Reproducibility
High-throughput capacity or automation possibility
Ease of results interpretation
Other
Availability of standards
Bioactivity threshold
Capability of dereplication
Regulatory requirements (e.g., use of BSL2 or GMO organisms)
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Biotechnology Advances 71 (2024) 108307
4
a microplate. There are several approaches to avoid this problem, such
as using only the inner wells, randomization in plate design, or repli-
cation (White et al., 2019). Recently, some manufacturers offer plates
with a built-in moat surrounding the outer wells (or even both inner and
outer wells), that is lled with water, and serving as an evaporation
buffer during prolonged incubation. Depending on the desired
throughput, robotic liquid handling systems can be used to fully auto-
mate almost any bioassay workow, but the initial cost of such systems
can be prohibitive for small laboratories.
Are standardized forms of bioassay available? Although standardi-
zation of bioassays facilitates interpretation and comparison of data
between laboratories and allows better monitoring of bioassay perfor-
mance, standardized bioassay protocols are available for only a limited
number of bioassays. Inter-laboratory reproducibility or precision under
the same operating conditions becomes more and more valuable in
stages of higher levels of technology readiness (TRL).
What is required to interpret the results of the bioassay? What are the
appropriate controls to distinguish true results from false positives or false
negatives? Before beginning to interpret the results, it is assumed that the
test performance was appropriate. This can be veried by including an
external positive or negative control (or sometimes an internal standard)
in the assays, such as organisms with a known phenotype, to ensure that
the bioassay performance was optimal. The measurements obtained can
be compared to positive and/or negative controls, as well as to blank
measurements, to evaluate the effects of medium/buffer/background.
Although method validation at the discovery level is not essential, eval-
uation of precision, i.e., the degree of scatter between a series of replicate
measurements obtained from multiple samplings of the same homoge-
neous sample under the same conditions – expressed as coefcient of
variation (CV) - makes the data more robust and reliable.
How are the results to be interpreted in a meaningful way? Is the
extract/compound bioactive? Benchmarks and thresholds for bioac-
tivity must be considered, as there are common thresholds below which
an extract is considered very active or moderately active, while above
these thresholds it is considered of little interest for further develop-
ment. Meaningful evaluation of the results in combination with chemi-
cal dereplication strategies (i.e., evaluating the presence of known
compounds in the crude extracts) (Gaudˆ
encio and Pereira, 2015;
Gaudˆ
encio et al., 2023) plays a very important role in prioritizing
samples for further development and deciding which samples are
worthwhile for further development investment.
What is the expected content of bioactive compounds in the extract?
How complex is the crude extract and what is the level of background
substances that would interfere with the measurement of bioactivity?
Advanced dereplication methods are used for natural product proling/
ngerprinting of complex extracts (Gaudˆ
encio and Pereira, 2015;
Gaudˆ
encio et al., 2023). An estimate of the expected content of bioactive
target compounds helps in the selection of the bioassay to avoid false
positives in terms of required sensitivity (high sensitivity for low-content
compounds), selectivity (the extent to which the bioassay can differen-
tiate and detect a target analyte without interference from concurrently
present irrelevant compounds), and specicity, which is a measure of
high selectivity (the ability to unambiguously detect the target analyte
in the presence of other substances, including those with similar
chemical structures). It also helps in the selection of appropriate controls
and thus in the interpretation of data. For some compounds, spiking
samples with a reference standard can be a solution for detection and
quantitation, but a suitable standard must be available.
What is the desired level of quantitative response (qualitative, semi-
quantitative, quantitative results)? Does the potency need to be accu-
rately assessed? Measurements can be binary (activity present or ab-
sent), or quantitative information can be obtained by comparison with
appropriate controls. Although only quantitative bioassays are suitable
for unambiguous determination of potency, the need for such accurate
information may be more important at later stages of discovery, puri-
cation, safety, and efcacy testing. Potency is usually expressed as a
percentage of the extract volume or as a unit of mass in the screening
stages if the bioactive compound is not known; later, molar concentra-
tions are used for pure compounds with known molecular and functional
properties. Quantitative assays often use standard compounds (spiking,
calibration curves), and it is worthwhile to check the availability of
appropriate standards. In the context of interpretation of results,
determination of the limit of detection (LOD) and limit of quantication
(LOQ) provides better reliability of data. In addition, selection of bio-
assays with lower limits of detection and quantitation usually results in a
higher degree of condence in the nal data.
Fig. 2. Characteristics of a good bioassay. For each bioassay, different characteristics must be balanced (indicated by scales) in order to arrive at a complete
description (indicated by a puzzle) of a good bioassay. The blue biotechnology theme of the review is indicated by the wave symbol, but these characteristics apply to
every bioassay.
J. Sabotiˇ
c et al.

Biotechnology Advances 71 (2024) 108307
5
Table 2
Principles and characteristics of popular bioassays used in pre-screening and screening of bioactivities.
Bioassay designation Principle and general characteristics Advantages Limitations References
Antimicrobial bioassays
Disc diffusion =Agar disc
diffusion method =Kirby-
Bauer test =Disc-diffusion
antibiotic susceptibility test
In vitro detection of effects on
microbial growth or survival on solid
media. A microbial inoculum
suspension (e.g., 1–2 10
8
CFU/mL for
bacteria) is spread on agar plates and
the test extract/compound is applied
on impregnated paper discs. After
12–24 h incubation (bacteria) or
24–48 h incubation (fungi) in suitable
growth conditions for the tested
microbial strain inhibition zone
diameters are read at the point where
no growth is observed. Variations are
available for yeasts and molds.
- Simple
- Standardized protocols available for
bacteria and yeast (CLSI, EUCAST)
- Versatile (suitable for majority of
bacterial pathogens)
- Controls for bioassay performance
available in form of antibiotics and
characterized type strains with
known phenotype and antibiogram
- No special equipment, only basic
microbiological utilities required
- Easily used in routine
- Reproducible and accurate if
standard protocols are followed
- Inexpensive
- Easy to interpret
- Adequate for primary screening
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Not appropriate for all bacterial
pathogens
- Diffusion of the extract/
compound can be non-
homogeneous and affect accuracy
- Not appropriate for large
molecules, amphiphilic molecules
- Importance of the inoculum size
and preparation
- Importance of growth medium
used
- Not quantitative – cannot
determine MIC value
- Qualitative categorization into
susceptible, intermediate or
resistant is possible based on
standardized MIC breakpoints
- Cannot distinguish between
bactericidal and bacteriostatic
effect
- Few interpretative criteria are
available
- Not adapted for lamentous
fungi as breakpoints for standard
antibiotics are not dened
(Alastruey-Izquierdo
et al., 2015; Balouiri
et al., 2016;
Matuschek et al.,
2014; Str¨
omstedt
et al., 2014)
Antimicrobial gradient method
=Epsilometer testing
(commercial version Etest®)
In vitro detection of effects on
microbial growth or survival on solid
media. Variant of agar diffusion
method that combines the principle of
dilution and diffusion methods to
determine MIC. Exponential gradient
of substance applied on a plastic or
nitrocellulose strip (marked with
concentration scale) and placed on a
previously inoculated agar surface.
After 12–24 h incubation (bacteria) or
24–48 h incubation (fungi) in suitable
conditions ellipse-shaped zone of
inhibition indicates the MIC that can
be read off the strip.
- Simple
- Used for antibiotics, also
antimycobacterials
- High sensitivity (can detect trace
amount of beta-lactamase (ESBL)
- Quantitative (provides MIC value)
- Can be used to test interaction of
two antimicrobials
- Cost-effective
- Useful also for yeast and
lamentous fungi
- No special equipment, only basic
microbiological utilities required
- Easy to interpret
- Commercial kits available that can
be used as controls
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Not appropriate for all bacterial
pathogens
- Subjective interpretation
- Diffusion of the extract/
compound can be non-
homogeneous and affect accuracy
- Not appropriate for large
molecules, amphiphilic molecules
- Cannot distinguish between
bactericidal and bacteriostatic
effect
- Not used for marine natural
products (MNPs) (problematic
preparation of gradient strip)
(Idelevich et al., 2018)
Agar plate assay =Poisoned food
method for lamentous fungi
In vitro evaluation of antifungal effect
against lamentous fungi. The
substance or extract is incorporated
homogeneously into the molten agar
and mycelia disc are inoculated at the
center of plate. After incubation under
suitable growth conditions the
diameters of growth inhibition are
read and compared with the
unexposed control.
- Simple
- Standardized protocols available
(CLSI, EUCAST)
- Easy to interpret
- Relatively sensitive
- Low cost
- Adequate for primary screening
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- There are some commercial kits
that combine identication-
susceptibility testing assay for
Candida and Aspergillus spp.
- Resources for work with fungi
- Not quantitative
- Possible interference with growth
medium components
- Not appropriate for heat labile
compounds
- Requires large amounts of
compounds
- Time consuming
(Chadwick et al.,
2013)
Broth (micro)dilution for
determination of MIC
(Minimum Inhibitory
Concentration)
In vitro detection of microbial growth
inhibition in liquid culture containing
a known concentration of drug. Two-
fold dilutions of antimicrobial agent or
extract are mixed with the inoculum in
liquid medium and after suitable
growth time period of incubation
(12–24 h), MIC value is determined by
detecting the lowest concentration
that inhibited visible microbial
growth. Usually performed in 96-well
plates (microdilution). Detection of
- Standard protocols are available
(CLSI, EUCAST)
- Gold standard in clinical
microbiology
- High-capacity bioassay
- Versatile
- Accurate and reproducible
- Applicable to both yeasts and molds
- Economic if plates are produced in
the laboratory
- Can be used for any new discovered
antimicrobials
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Solubility of organic extract in
broth medium can be challenging
- Not suitable for large
polycationic, amphiphilic
molecules
- Plastic interference of 96 well
plates for peptide antimicrobial
assessment
- Importance of the inoculum size
(Arendrup et al.,
2008; Balouiri et al.,
2016; Rodriguez-
Tudela et al., 2008;
Str¨
omstedt et al.,
2014)
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Biotechnology Advances 71 (2024) 108307
6
Table 2 (continued )
Bioassay designation Principle and general characteristics Advantages Limitations References
growth is by naked eye or colorimetric
assays using tetrazolium salts,
resazurin, or ATP can be used to detect
metabolically active cells.
Different procedures are adapted for
yeasts and molds including longer
incubation time (24–72 h).
- Low sample volume required
- Cost-effective
- Adequate for primary screening
- Appropriate for high-throughput
screening
and preparation
- Importance of growth medium
used
- Subjective interpretation by CLSI
methodology alleviated using
EUCAST protocol
- Labor-intensive
- Technical training requirement
high
- Risk of error with dilution
preparation
- Edge effect
MBC (Minimum bactericidal
concentration), or MFC
(minimum fungicidal
concentration), or MLC
(minimum lethal
concentration)
Common estimation of bactericidal or
fungicidal activity determined after
broth dilution by subculturing
samples from wells with incubation
time from 24 h to 72 h. It is the lowest
concentration of antimicrobial agent
needed to kill 99.9 % of the nal
inoculum after 24 h incubation in
standardized conditions.
- Simple
- Quantitative
- Cost-effective
- Adequate for primary screening
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Labor intensive
- Importance of growth medium
used
- Only culturable cells are detected
(Balouiri et al., 2016)
Time-kill assay =Time-kill
curve =Growth curve analysis
In vitro test to measure the kinetics of
dynamic interaction between the
compound and the microbial strain to
reveal a time-dependent or a
concentration dependent
antimicrobial effect. The log CFU/mL
of microbial/antimicrobial solution is
determined on time scale depending
on the bacteria strain and the media
used. Alternatively, growth is
followed in a microplate reader
measuring optical density at 600 nm.
Typically used in secondary testing.
- Existing standard guidelines CLSI
and ASTM
- Growth curve analysis offers many
variables that may indicate mode of
action: growth rate, growth
dynamics
- Can be used to study synergy/
antagonism between substances
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Special software needed for
growth curve analysis
- Labor intensive
- Specialized equipment needed
- Inoculum size, growth phase,
growth medium affect outcome
- Possible interference with growth
vessels, medium components and
method of growth detection
(Balouiri et al., 2016)
Bioautography In vitro direct detection of
antibacterial compounds on TLC (Thin
Layer Chromatography) plate based
on incubation (12–24 h) and
visualization of microbial growth
using vital stains or metabolic stains or
dehydrogenase-activity-detecting
reagent to reveal zones of inhibition. A
variation is possible using
bioluminescent bacteria as reporters.
Particularly adequate for monitoring.
- Simple
- Rapid
- Results easily visualized
- Inexpensive
- Applicable to both bacteria and
fungi
- Can be utilized for spore-producing
fungi
- Little amount of extract/compound
required
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Volume of agar or broth has to be
well dened otherwise resulting in
poorly dened inhibition zones or
irregular bacterial growth
- Not quantitative
- Difcult to standardize
(Balouiri et al., 2016;
Choma and Grzelak,
2011; Dewanjee et al.,
2015; Kl¨
oppel et al.,
2008; Patil et al.,
2017)
Volatile antibiotics bioassays All versions of these bioassays use the
same principle to detect volatile
organic compound (VOC) activity. The
source of the volatile (a living
organism or chemical) is placed on
one side of a chamber without direct
contact with the target organism,
while the target is grown or located on
another side or compartment of the
chamber. The effect of the volatile on
the growth (inhibition) or survival of
the target organism is compared to a
control using the same container and
conditions without the volatiles.
- Easy to perform and interpret
- Low cost
- Sensitive
- Adequate for primary screening
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Not quantitative
- Special equipment or material
required (sealed chambers)
(Ezra, 2004; Liarzi
et al., 2016;
Tomsheck et al.,
2010)
Antibiolm bioassays
Crystal violet Gold standard for biolm
quantication in microtiter plates.
Inoculum in liquid medium incubated
for 24–72 h at selected temperature
under static conditions. Washing steps
and short incubation times in crystal
violet, are followed by the
colorimetric detection of the stained
biomass.
- Adapted protocols available for
different bacterial species
- Different surfaces can be assayed
using coupons
- Versatile: both for G+and G-
- Qualitative or quantitative, but
characterized control strains need to
be incorporated for interpretation
- Low cost
- Can be used to monitor biolm
growth and biolm eradication
- High-throughput (96-well plates)
- Adequate for primary screening
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Non-specic binding to anionic
proteins and other negatively
charged molecules, like capsules,
lipopolysaccharides, and DNA/
nucleic acids, leading to an
inability to distinguish between
live and dead bacterial
populations and/or
exopolysaccharides
- Large variability between
samples leading to possibly
(Haney et al., 2021;
O’Toole, 2011)
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Biotechnology Advances 71 (2024) 108307
7
Table 2 (continued )
Bioassay designation Principle and general characteristics Advantages Limitations References
complicated interpretation
- Medium composition important
- Culture conditions important
- Strain to strain variability is high,
need to know primary biolm
phenotype
- Interference of the stain with
experimental setup possible
CFU (Colony Forming Units) Biolm is sonicated to dislodge
adhered biomass and serial dilutions
of homogenized bacterial suspension
is plated onto agar plates, incubated
24–48 h to count the colony forming
units (CFUs).
- Simple
- Low cost
- Adequate for primary screening
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Need for specialized equipment
- Sonication parameters important
(can reduce viability of recovered
CFUs),
- Sonication parameters are
different for different bacterial
species
- Aggregation of bacteria can affect
CFU count
- Labor intensive
- Only culturable cells are detected
(Haney et al., 2021)
The BioFilm Ring Test Mobility measurement of magnetic
microbeads mixed with bacterial
suspension in a polystyrene
microplate. Without biolm growth
beads gather together in a visible
central spot under magnetic action,
while no spot indicates bead
immobilization by biolm formation.
- Simple
- Rapid
- No dyes or stains
- No washing steps
- Low sample volume required
- High-throughput (96- well plates)
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Need for specialized equipment
- Interpretation may be
challenging
- Qualitative
(Olivares et al., 2016)
The Calgary Biolm device Two-part reaction vessel containing a
lid with 96 pegs that sit in channels of
the reaction vessel that allows ow of
medium across pegs to create
consistent shear force.
- Standardized protocols available
- High-throughput (96-well plates)
- Quantitative
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Need for specialized equipment
- Use of multiple sterile
microplates for treatment and
washing steps
- Relies on viable cell counting for
experimental validation
(Haney et al., 2021;
Kırmusao˘
glu, 2019)
MBEC (Minimum biolm
eradication concentration)
Assay®
High-throughput screening of
antibiolm activity. Plastic lid with 96
pegs on which biolms establish under
batch conditions and the lid with pegs
is transferred to a new 96 well for
testing, biolm is dislodged by
sonication and CFUs are determined.
- Standardized method for
Pseudomonas aeruginosa (ASTM
E2799-17)
- BSL2 and BSL3 level
microorganisms require work in
suitable facility
- Aggregation of bacteria can affect
CFU count
- Labor intensive
- Only culturable cells are detected
(ASTM, 2022; Parker
et al., 2014)
SIMBA – simultaneous detection
of antimicrobial and
antibiolm activity
The SIMultaneous detection of
antiMicrobial and anti-Biolm
Activity (SIMBA) method combines
the testing of antimicrobial and
antibiolm activity against bacteria
with the evaluation of the 20-h growth
curve of the Salmonella Infantis ˇ
ZM9
strain determined with absorbance
measurements at 600 nm in a 96-well
plate.
- Simple
- Rapid
- No dyes or stains
- Cost-effective
- Information on both antimicrobial
and antibiolm activity in one assay
- Low sample volume required
- High-throughput (96-well plates)
- Possibility of automation
- Optimized for one Salmonella
strain
- Not suitable for dark colored
samples
- Need for specialized equipment
(spectrophotometer with
temperature control and shaking
capabilities)
(Sterniˇ
sa et al., 2022,
2023)
Cytotoxicity bioassays
MTT (also MTS, XTT, WST) In vitro colorimetric assay usually
performed in 96-well plates to
evaluate cellular metabolic activity -
glycolytic production of NADH. Based
on tetrazolium salts (MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; XTT,
2,3-bis-(2-methoxy-4-nitro-5-sulfo-
phenyl)-2H-tetrazolium-5-carboxani-
lide; MTS, 3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium; WST,
water-soluble tetrazolium salts) –
difference between them is the
tetrazolium salt used and the
solubility and/or absorption spectrum
of the formazan product.
- Commercial kits with standardized
protocols available
- Cost-effective
- Relatively simple
- Assay for whole cells
- Linearity between absorbance and
cell count
- Versatile: suitable for both adherent
and suspended cell cultures
- One-step procedure variants using
water soluble tetrazolium salts
include XTT, MTS, WST
- Possibility of automation
- Appropriate for high-throughput
screening
- BSL2 and BSL3 level cell lines
require appropriate facility
- Lengthy two-step procedure
- Highly variable results depending
on: the number of cells per well,
and the high pH of the culture
medium
- Requires optimization of cell
density (untreated cells have
absorbance values that fall within
the linear portion of the growth
curve (conditions not too close to
saturation)
- Requires optimized incubation
time
- Not suitable for reducing
compounds
(Balbaied and Moore,
2020; Jo et al., 2015;
Mccauley et al., 2013;
Riss et al., 2019)
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Biotechnology Advances 71 (2024) 108307
8
Table 2 (continued )
Bioassay designation Principle and general characteristics Advantages Limitations References
Eukaryotic cells are treated for 24 - 48
h with different concentrations of
compounds to determine the
concentration of the tested
compounds, which produces 50% of
cytotoxicity (CC
50
).
Tetrazolium salt (e.g., MTT) is then
added to the cells for 2 h at 37 ◦C. MTT
is reduced by a cellular mitochondrial
enzyme (succinate dehydrogenase) to
violet formazan precipitates, which
are subsequently solubilized by
organic solvents before absorbance is
read. Alternatively, water-soluble
tetrazolium salts can be used, omitting
the nal solubilization step.
- Not for metabolically poor cells,
i.e. thymocytes and splenocytes
- Linearity between absorbance
and cell count is lost when cells are
conuent and cellular metabolism
slows down
- The result can be variable
because metabolic activity
depends not only on the number of
cells per well but also on several
other factors
Sulforhodamine B (SRB) assay Used for cell density determination,
based on the measurement of cellular
protein content. Toxicity screening of
compounds to adherent cells in a 96-
well format. After an incubation
period, cell monolayers are xed with
10% (wt/vol) trichloroacetic acid and
stained for 30 min, after which the
excess dye is removed by washing
repeatedly with 1% (vol/vol) acetic
acid. The protein-bound dye is
dissolved in 10 mM Tris base solution
for OD determination at 510 nm using
a microplate reader.
- Simple
- Cost-effective
- Results linear over a 20-fold range
of cell numbers
- Sensitivity comparable to those of
uorometric methods
- Appropriate for high-throughput
screening
- Requires microplate reader
(absorbance)
(Vichai and Kirtikara,
2006)
ATP-based test Gold standard luminescence test. See
MTT for the procedure. Quantication
of released intracellular ATP by
enzymatic reaction between the
enzyme luciferase and its substrate,
luciferin, to produce luminescence.
There is a linear relationship between
the intensity of the light signal and the
ATP concentration or cell number.
It is one of the most sensitive
endpoints for measuring cell viability.
- One-step procedure
- Faster than MTT and MTS
- Reduction of artifacts
- Sensitive measure of intracellular
ATP rather a specic biological effect
- More sensitive than conventional
biochemical methods
- Sensitive compared to other
cytotoxicity tests
- Interferences minimal
- Commercial kits available
- Possibility of being automated
- BSL2 and BSL3 level cell lines
require suitable facility
- More expensive than MTT and
MTS and uorescent methods
- The ATP assay sensitivity is
usually limited by reproducibility
of pipetting
- Replicate samples rather than a
result of the assay chemistry
- Need for specialized equipment
(luminescence detection)
(Aslantürk, 2018;
Herzog et al., 2007;
Ponti et al., 2006)
Automated uorometric
microculture cytotoxicity
assay (FMCA)
Based on the measurement of
uorescence generated from cellular
hydrolysis of uorescein diacetate
(FDA) to uorescein by viable cells
with intact plasma membranes after a
48–72 h culture period in microtiter
plates. See MTT for procedure.
- Highly standardized and
reproducible one-step procedure
- Possibility of being automated
- BSL2 and BSL3 level cell lines
require suitable facility
- Need for specialized equipment
(uorescence detection)
(Burman et al., 2011;
Lindhagen et al.,
2008)
Dye exclusion method The membrane integrity of cell is
determined by its permeability to
several dyes (eosin, Trypan blue,
erythrosine B, Congo red assays).
Trypan blue has been used the most
extensively to assess the percentage of
viable cells in suspension culture.
- Simple
- Rapid
- Small numbers of cells needed
- Can be applied in non dividing cell
populations
- BSL2 and BSL3 level cell lines
require suitable facility
- Can be challenging to process a
large number of samples
simultaneously, particularly when
the exact timing of progressive
cytotoxic effects is taken into
consideration
- Careful interpretation needed for
living cells with metabolic activity
loss (trypan blue)
- Its toxic side effect of some dyes
on mammalian cells (trypan blue)
- Not suitable for adherent
monolayer cell cultures
- Labor intensive
(Aslantürk, 2018)
LDH (lactate dehydrogenase)
cytotoxicity assay
LDH is a cytosolic enzyme present in
many different cell types that is
released upon damage to the plasma
membrane. The assay quantitatively
measures the activity of stable,
cytosolic LDH released from damaged
cells. It is a colorimetric assay.
- Suitable for both adherent and
suspended cell cultures
- Commercial kits available
- Detects low level damage to cell
membranes which cannot be
detected using other methods
- BSL2 and BSL3 level cell lines
require suitable facility
- LDH assay is limited to serum-
free or low-serum culture
conditions to avoid high
background readings.
- Interference with serum
components
(Kocherova et al.,
2020)
Clonogenic cell survival assay Determines the ability of a cell to
proliferate indenitely, retaining its
reproductive ability to form a colony
- Simple
- Cost-effective
- Gold standard
- BSL2 and BSL3 level cell lines
require suitable facility
- Suitable only for adherent cells
(Munshi et al., 2005)
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Biotechnology Advances 71 (2024) 108307
9
Table 2 (continued )
Bioassay designation Principle and general characteristics Advantages Limitations References
or a clone. These cells are considered
clonogenic. Cells are seeded at low
density and growth of colonies/clones
is analysed after a week by staining
and counting. The gold standard for
measuring cellular reproductivity.
- Not suitable for all adherent cell
lines (not all cells are able to form
colonies in vitro – cell-to-cell
contacts and self-produced growth
factors are limited at low cell
density)
DNA synthesis assay
3H-labeled thymidine (3HT)
The process of DNA synthesis is
relatively specic for cell division and
can therefore be considered a marker
of cell proliferation activity.
Nucleoside analogue incorporation
assays are based on the introduction of
chemically or radio-labeled
nucleosides that are subsequently
incorporated into DNA strands
synthesised during S phase. A
scintillation beta counter is used to
measure radioactivity in DNA
recovered from cells to determine the
extent of cell division that has
occurred in response to a test agent.
The nucleoside analogue 5-bromo-2’-
deoxyuridine (BrdU) is used to avoid
the use of radioisotopes and is
detected with monoclonal antibodies.
Alternatively, thymidine analogues
are available that do not require
antibody detection.
- This assay is commonly regarded as
reliable and accurate.
- Suitable for immunohistochemistry
or immunocytochemistry, in-cell
ELISA, ow cytometry
- It can be performed in experiments
in vitro and ex vivo, but not in vivo
- Not suitable for screening, used for
mechanistic studies
- Commercial kits available
- Allows quantitative assessment of
proliferation levels
- Direct measures of proliferation
- Appropriate for high-throughput
studies
- BSL2 and BSL3 level cell lines
require suitable facility
- Potential use of radioisotopes
- It is an endpoint assay because of
the DNA extraction step, and so no
further studies can be performed
with the treated cells.- synthetic
analogues such as 5-bromo-2
′
-
deoxyuridine (BrdU) or 5-ethynyl-
2’-deoxyuridine (EdU), are usually
preferred (can be used not only in
vitro or ex vivo but also in vivo)
- Cannot identify cells that have
undergone numerous divisions
- Need for specialized equipment
(Romar et al., 2016)
Antiviral bioassays
Flow cytometry cell count assay
(FACS)
Cytotoxicity-based antiviral assay
based on the detection of intact and
damaged cells using a ow cytometer
and dyes to stain the cells (e.g.,
propidium iodide, carboxyuorescein
diacetate).
- Three populations discriminated
(dead, viable, injured)
- Reproducible
- Rapid (2-6 h to results)
- BSL2 and BSL3 level cell lines
and/or viruses require suitable
facility
- Need for specialized equipment:
ow cytometry equipment
- Need for trained personnel
- Not easy to interpret
- Specic cell lines known to be
susceptible to and allowing viral
infection with the virus of interest
(Zamora and Aguilar,
2018)
Cytopathic effect assay (CPE) Suitable for primary in vitro antiviral
screening. In this assay, cells
permissive for a virus are infected with
the same virus at serial dilutions. Cells
are observed daily until a cytopathic
effect is detected. The virus
concentration is expressed as
infectious tissue culture dose
(TCID
50
), which is the multiple of
dilutions that result in CPE in 50% of
wells. Direct method.
- Commercial kit available allowing
standardization and automated
procedures
- For all types of viruses that do or do
not form viral plaques
- Cell xation and staining not
required
- Cost-effective
- Operator independent
- Technically simple in respect to
plaque reduction assay (PRA) or
virus reduction yield assay (VRA)
- Labor intensive and time consuming
- Reduced reading time
- Appropriate for high-throughput
screening
- Infectious virus detection
- BSL2 and BSL3 level cell lines
and/or viruses require suitable
facility
- Method applicable only to viruses
that cause morphological changes
in infected cells (CPE inducing
viruses)
- Lengthy: the time required for the
cytopathic effect to become
apparent
- Relatively subjective reading
- Works only with specic cell lines
known to be susceptible and
permissible to viral infection with
the virus of interest.
- Equipment required to work with
viruses and specialized virology
trained personnel
(El Sayed, 2000;
Suchman and Blair,
2007)
Plaque reduction assay (PRA) Primary in vitro antiviral screening for
the detection of infectious viral
particles.
A viral inoculum of approximately 50-
70 viral plaques/well is adsorbed onto
permissive cells in the presence of the
test substance. After viral adsorption,
the unbound virus is removed and the
culture is covered with a semi-solid
medium (agar, Avicel,
methylcellulose). After an incubation
period equal to the duration of the
replication cycle of the virus, the cells
are xed and stained to count the viral
plaques microscopically. Titers are
expressed as the number of plaque-
- Validation with a positive control,
such as a commercial compound with
known antiviral activity
- Commonly used
- No special equipment is required in
addition to a cell culture laboratory
- Results are easily visualized under a
microscope or with the naked eye
- Cost-effective
- Sensitive
- Protocols vary from laboratory to
laboratory and depend on the type of
cells used
- Appropriate for high-throughput
screening
- Infectious virus detection
- BSL2 and BSL3 level cell lines
and/or viruses require suitable
facility
- Only for viruses that form
plaques
- Labor intensive
- Sometimes lengthy
- Results not reproducible:
depends on cell density, CPE and
plaque size
- Counting of plaques can be
subjective
- Specic cell lines known to be
susceptible and permissible for
viral infection with the virus of
interest
(El Sayed, 2000)
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Biotechnology Advances 71 (2024) 108307
10
Table 2 (continued )
Bioassay designation Principle and general characteristics Advantages Limitations References
forming units (PFU) per milliliter
(PFU/mL). Direct method.
- Protocol must be adapted for
each host-virus combination
Virus reduction yield assay
(VRA)
Primary in vitro antiviral screening to
detect infectious viral particles.
Permissive cell cultures are infected
with a specic amount of virus, and
after virus adsorption (usually 2 h at
37 ◦C or 33 ◦C for temperature-
sensitive viruses), the unbound virus is
removed, and different concentrations
of the same compound are added.
After an incubation period that allows
virus replication, the total viral yield is
titrated and determined. Direct
method.
- Less operator-dependent than the
PRA
- Cost-effective
- Sensitive
- Infectious virus detection
- BSL2 and BSL3 level cell lines
and/or viruses require suitable
facility
- Time/material-intensive
- Not-automatable
- Not reproducible: results depend
on harvesting time
- Specic cell lines known to be
susceptible and permissible to
viral infection of the specic virus
in focus
(Collins and Bauer,
1977; Hu and Hsiung,
1989)
Focus Forming assay (FFA) Primary in vitro antiviral screening for
viruses that do not induce CPE.
Procedure identical to PRA. FFA doses
are expressed as concentration units
per milliliter (FFU/mL). Direct
method.
- Faster than PRA or TCID
50
- Reading time varies depending on
the replication cycle of the virus
- Sensitive
- BSL2 and BSL3 level cell lines
and/or viruses require suitable
facility
- Expensive
- Specic reagents and equipment
required
- Specic cell lines that are known
to be susceptible and permissible
to infection with the virus of
interest
- Reading time of foci depends on
the size of the area the operator is
counting. A larger area will take
longer, but may provide a more
accurate representation of the
sample.
- Based on the antibody used, no
discrimination between viable
viruses and non-infective ones
(Flint et al., 2009)
Hemagglutination inhibition
assay (HIA)
Primary in vitro antiviral screening to
detect infectious and noninfectious
viral particles for viruses that do not
form plaques or cause CPE.
For HIA, viral samples are rst mixed
with dilutions of compounds that take
time to bind the virus. Then red blood
cells (RBCs) are added to the mixture.
Antiviral activity: means that there are
no free virus particles and the RBCs
fall to the bottom of the well by
gravity, creating a distinct red spot in
a U or V bottom plates.
No antiviral activity: the erythrocytes
clump together, resulting in a lattice-
like structure.
Indirect method
- Simple
- Does not require special equipment
- Fast evaluation of virus particles
- Standardized protocols available
- Validation of a modied HAI: more
sensitive, easy to analyse, required
only a single source of erythrocytes
and allowed utilisation of virus
strains which are difcult to handle
by the standard HAI (e.g., H3N2,
H5N1 and H1N1pdm09)
- Infectious virus detection
- BSL2 and BSL3 level cell lines
and/or viruses require suitable
facility
- Less sensitive than other methods
- Only for hemagglutinating
viruses
- The red blood cells used depend
on the type of inuenza virus in
the test
- Required source of suitable red
blood cells (horse, rabbit, chicken,
guinea pig)
- Optimization of the type and
concentration of red blood cells
used is necessary to obtain reliable
results.
- Requires skilled personnel
- Manual evaluation may lead to
misinterpretation of results
- Non-specic inhibition of
hemagglutination possible
- Low sensitivity
- Semiquantitative data
(Joklik, 1988;
Morokutti et al., 2013)
Quantitative polymerase chain
reaction (qPCR)
qPCR involves amplifying short
stretches of longer genomic molecules
in a thermocycler, a device that
exposes the reaction to a series of
different temperatures for a specied
time (1 amplication cycle). With
each PCR cycle, the amount of target
sequence (amplicon) in the reaction
theoretically doubles. In quantitative
polymerase chain reaction, the
amplication rate is monitored in real
time during PCR using nonspecic
intercalating uorescent dyes or
uorescently labeled sequence-
specic DNA probes. Direct method.
- Rapid (1-4 h response)
- Sensitive
- High specicity
- Possible to validate
- Quantitative or semi-quantitative
- Protocol needs to be adapted for
each virus, but the general guidelines
are the same
- Cell lines and/or viruses of BSL2
and BSL3 levels require a suitable
facility
- More complex compared to PRA
- Need for specialized equipment:
ow cytometry equipment
- Need for trained personnel
- Positive detection does not
equate to viable (or infectious)
virus, therefore not recommended
for initial screening
- Expensive
(Engstrom-Melnyk
et al., 2015; Kralik
and Ricchi, 2017)
Antioxidant assays
(continued on next page)
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Biotechnology Advances 71 (2024) 108307
11
Table 2 (continued )
Bioassay designation Principle and general characteristics Advantages Limitations References
DPPH (2,2
′
-diphenyl-1-
picrylhydrazyl radical) assay
Based on the reaction of the tested
antioxidant with the stable synthetic
radical 2,2-diphenyl-1-picrylhydrazyl
(DPPH•), accompanied by a colour
shift of the latter. Aliquots of the
extracts are mixed with a methanolic
solution containing DPPH radicals,
and the mixture is incubated in the
dark for 30 min. Absorbance is
measured with a spectrophotometer at
517 nm. Usually, quercetin is used as a
reference standard, and DPPH results
are expressed as quercetin equivalents
(QE) in
μ
mol per 100 mL.
- Commercial kits available
- Simple
- Cost-effective
- Good repeatability
- Quantitative
- Adequate for primary screening
- Appropriate for high-throughput
screening
- Applicable only for compounds
soluble in organic solvents
- Radical strongly affected by light,
oxygen, pH and type of solvent
- Steric hindrance effects for bulky
antioxidants
- Narrow linear range
- Limited relevance to biological
systems
- Need for specialized equipment
(spectrophotometer, microplate
reader)
(Apak et al., 2006;
Awika et al., 2003;
Molyneux, 2004)
ABTS/TEAC
(2,2’-azino-bis(3-
ethylbenzothiazoline-6-
sulfonic acid)/Trolox
equivalent antioxidant
capacity
With the help of an oxidizing agent,
the colorless ABTS salt is converted
into its radical cation with
characteristic blue-green colour,
which is then reduced back to its
original colorless ABTS form by
reaction with the tested antioxidant.
Antioxidant activity is dened as the
amount of ABTS• + quenched after a
given time (usually 5 min) and is
expressed in Trolox (6-hydroxy-
2,5,7,8-tetramethylochroman-2-
carboxylic acid) equivalents as TEAC
(Trolox Equivalent Antioxidant
Capacity).
- Rapid
- Simple
- Sensitive
- Reproducible
- More sensitive than DPPH assay,
high response to antioxidants
- Can be performed in a 96-well
microplate.
- Diverse, exible usage in multiple
media (pH, solvents)
- Applicable to both lipophilic and
hydrophilic anti-oxidants
- Commercial kits available
- Quantitative
- Adequate for primary screening
- Limited relevance to biological
systems
- Difculties in the formation of
the colored radical and limited
stability
- Steric hindrance effects for bulky
antioxidants
- Specialized equipment required
(spectrophotometer, microplate
reader)
(Apak et al., 2007;
Awika et al., 2003;
Erel, 2004; Lee et al.,
2015; Re et al., 1999)
Cupric ion (Cu
2+
) reducing
assay (CUPRAC)
In vitro assay for measurement of the
absorbance of the colored Cu(I)-
neocuproine (Nc) chelate formed as a
result of the redox reaction between
the chromogenic oxidizing CUPRAC
reagent (i.e., Cu(II)-Nc) and the chain-
breaking antioxidant under study.
Trolox is used as the standard.
- Applicable to both lipophilic and
hydrophilic antioxidants
- Selective detection of antioxidants
- Simulates antioxidant action under
nearly physiological conditions
- Favorable redox potential
- High stability of reagents
- No steric hindrance effects
- Commercial kits available
- Quantitative
- Adequate for primary screening
- Appropriate for high-throughput
screening
- Unable to react with compounds
having isolated hydrocarbon
double bonds or alternating
double and single bonds (e.g.,
ferulic acid, β-carotene)
- An incubation at elevated
temperature may be required for
slow-reacting compounds (e.g.,
naringin and naringenin)
- Need for specialized equipment
(spectrophotometer, microplate
reader)
(Apak et al., 2006,
2007; Gulcin, 2020; ¨
O
zyürek et al., 2011)
Folin-Ciocalteu The Folin-Ciocalteu phenolic reagent
is used to obtain a rough estimate of
the total amount of phenolic
compounds present in an extract.
Specically, the phenolic compounds
undergo a complex redox reaction
with the phosphotungstic and
phosphomolybdic acids present in the
reaction mixture, yielding a blue
colour proportional to the amount of
phenols. The assay can be performed
in a 96-well microplate. The
absorbance is read at 760 nm and
quantication is based on a calibration
curve generated using gallic acid
standards (GA).
- Adequate for primary screening
- Simple
- Reproducible
- Excellent correlation between
measured “antioxidant capacity” and
“total phenolic content”
- Quantitative
- Commercial kits available
- Adequate for primary screening
- Non-specic to phenolics (it
reacts with many non-phenolic
compounds)
- not applicable to lipophilic
components
- Need for specialized equipment
(spectrophotometer, microplate
reader)
(Apak et al., 2007;
Bravo et al., 2016;
Singleton et al., 1999)
Oxygen radical absorbance
capacity (ORAC)
This method is based on the ability of
antioxidants to protect uorescein, a
highly uorescent protein, from
oxidative damage caused by peroxyl
radicals. The experimental procedure
of ORAC involves the addition of the
extract under study and a free radical,
usually AAPH (2,2’-azobis(2-
amidinopropane) dihydrochloride),
which forms a moiety together with
uorescein, followed by heating in a
phosphate buffer. Thermal
decomposition produces free radicals
that react with antioxidant
compounds, resulting in loss of
uorescence due to decrease in radical
- Easily automated and largely
standardized
- Adaptable for numerous sample
matrices
- High biological relevance
- Quantitative
- Commercial kits available
- Appropriate for high-throughput
screening
- It is based on uorescence
detection and it requires more
expensive instrumentation
- Need for specialized equipment
(uorescence detection,
microplate reader)
(Awika et al., 2003;
Bravo et al., 2016; Ou
et al., 2001)
(continued on next page)
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Biotechnology Advances 71 (2024) 108307
12
Table 2 (continued )
Bioassay designation Principle and general characteristics Advantages Limitations References
concentration. The test can be
performed in a 96-well microplate.
Anti-ageing enzyme-based
assays
Anti-elastase This in vitro assay is performed in Tris-
HCl buffer and at room temperature
using porcine pancreatic elastase
(PPE; E.C.3.4.21.36) and N-succinyl-
Ala-Ala-Ala-p-nitroanilide (Suc-Ala3-
pNA) as substrate. Inhibition of PPE by
natural extracts is determined
spectrophotometrically by monitoring
the release of p-nitroaniline from Suc-
Ala3-pNA at 410 nm. Can be
performed in a 96-well microplate.
Epigallocathechin-3-gallate (EGCG) is
commonly used as a positive control.
- Rapid
- Simple
- Provide effective approaches to
evaluate inhibitory effects of
unknown samples against skin-
ageing enzymes
- Quantitative
- Commercial kits available
- Appropriate for high-throughput
screening
- High cost and limited lifetime of
enzymes used
- Considerable consumption of
tested compounds/samples
- Do not closely mimic cellular
processes and in vivo conditions
- Need for specialized equipment
(absorbance detection with
spectrophotometer or microplate
reader)
(Pastorino et al.,
2017; Thring et al.,
2009)
Anti-collagenase The ability of the extracts to inhibit
collagenase activity is evaluated by a
spectrophotometric method based on
hydrolysis of the synthetic substrate
N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-
Ala (FALGPA) using collagenase from
Clostridium histolyticum (ChC –
EC.3.4.23.3). Can be performed in a
96-well microplate. EGCG is usually
used as positive control.
- Rapid
- Simple
- Provide effective approaches to
evaluate inhibitory effects of
unknown samples against skin-
ageing enzymes
- Quantitative
- Commercial kits available
- Appropriate for high-throughput
screening
- High cost and limited lifetime of
enzymes used
- Considerable consumption of
tested compounds/samples
- Do not closely mimic cellular
processes and in vivo conditions
- Need for specialized equipment
(absorbance detection with
spectrophotometer or microplate
reader)
(Thring et al., 2009;
Van Wart and
Steinbrink, 1981)
Anti-hyaluronidase In vitro assay that determines activity
indirectly by measuring the amount of
undegraded hyaluronic acid (HA)
substrate remaining after the enzyme
is allowed to react with the HA for 30
min at 37 ◦C.
- Rapid
- Simple
- Provide effective approaches to
evaluate inhibitory effects of
unknown samples against skin-
ageing enzymes
- Standardized protocol
- Commercial kits available
- Quantitative
- High cost and limited lifetime of
enzymes used
- Considerable consumption of
tested compounds/samples
- Do not closely mimic cellular
processes and in vivo conditions
- Need for specialized equipment
(turbidimeter)
(Bailey and Levine,
1993; Kim et al.,
1995)
Anti-tyrosinase The ability of the extracts to inhibit
the catalytic action of tyrosinase in the
oxidation of L- DOPA, a precursor of
melanin biosynthesis, is usually
determined by an enzymatic
procedure using the substrate L- DOPA
and fungal tyrosinase followed by
incubation in a phosphate buffer. The
absorbance of the nal solutions is
measured at 492 nm using a
microplate reader. Kojic acid (500
mM) is usually used as a reference
inhibitor.
- Rapid
- Simple
- Provide effective approaches to
evaluate inhibitory effects of
unknown samples against skin-
ageing enzymes
- Quantitative
- Commercial kits available
- Appropriate for high-throughput
screening
- High cost and limited lifetime of
enzymes used
- Considerable consumption of
tested compounds/samples
- Do not closely mimic cellular
processes and in vivo conditions
- Need for specialized equipment
(absorbance detection with
spectrophotometer or microplate
reader)
(Momtaz et al., 2008)
Anti-ageing broblast-based
assays
Cytotoxicity/cytoprotection Cultured human broblast cell lines
are pretreated with the samples and
subjected to UV irradiation. Cell
viability is measured by the
colorimetric 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. The amount of formazan
is measured by recording the
absorbance changes at 570 nm with a
spectrophotometer.
- Rapid
- Precise
- Avoids manipulation of radioactive
isotopes
- Constitutes a vital cellular setting
and a real-life model for simulating
oxidative damages and assessing the
protective role of natural extracts/
compounds
- Handling and preservation of
human broblast cell lines can be
cumbersome
- Results should be interpreted
with caution as the biological
effect is evaluated against a
specic type of cells (the
interaction of the tested substance
with other cell types are not taken
into account)
- Need for specialized equipment
(cell culture, absorbance
detection)
(Mosmann, 1983;
Ramata-Stunda et al.,
2013; Ratz-Lyko et al.,
2012; Riss et al.,
2004, 2019)
Regenerative potential This assay involves exposure of seeded
human broblast cells to extracts
followed by washing with chemical
reagents and measurement of
procollagen type I or hyaluronic acid
content in cell-free supernatants by
enzyme-linked immunosorbent assay
(ELISA).
- Constitutes a vital cellular setting
and a real-life model for simulating
oxidative damages and assessing the
protective role of natural extracts/
compounds
- Expensive
- Results should be interpreted
with caution as the biological
effect is evaluated against a
specic type of cells (the
interaction of the tested substance
with other cell types are not taken
into account)
- Need for specialized equipment
(Koudan et al., 2022)
(continued on next page)
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Biotechnology Advances 71 (2024) 108307
13
It is useful to know what may affect the precision or repeatability of
bioassays. Some metabolites show synergistic effects and bioactivity is
lost after fractionation, or metabolites may act antagonistically and
activity is detected only after fractionation. Fractionation may also lead
to an apparent loss of compounds due to their dilution or binding to
discarded material (e.g., with pelleted debris in clarication steps). In
addition, physical parameters of the extract (viscosity, pH, colour, etc.)
can lead to false-positive and false-negative results. Potential in-
terferences can arise from the material of the sample containers (usually
polypropylene and polystyrene, treated or untreated, or glass), and these
should be carefully selected based on the charge and polarity of the
molecules to be tested, if known (Str¨
omstedt et al., 2014).
What is the solubility and stability of the compound of interest? Is it a
small molecule or a complex molecule? The solvent used for extraction
must not be toxic or should not be used at a concentration that is toxic to
the microorganisms, cells, tissues, organs, or organisms. When aqueous
solutions are not used for extraction, extractions are usually performed
with dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF),
methanol, or ethanol, which can be tolerated in microbial or cell-based
assays only at low concentrations (e.g., up to 1 % DMSO) and whose
presence may affect nal results (Dyrda et al., 2019; Hipsher et al., 2021;
Rekha et al., 2006). Compounds extracted with organic solvents can be
vacuum dried to mitigate this issue. Nevertheless, the effect of extraction
solvents can be evaluated by performing the bioassay with the solvent as
a control. In addition, poor water solubility can lead to misleading re-
sults. Bioassay optimization strategies are recommended to improve
bioassay performance for poorly soluble compounds (Di and Kerns,
2006). As mentioned earlier, the effect of extraction medium is evalu-
ated by performing the bioassay with the extraction solution alone. If
necessary, this control is performed each time the bioassay is conducted.
Characteristics of the extraction medium such as thermostability, vola-
tility, and complexity (sedimentation properties and migration) can also
affect the design of the bioassay, while characteristics of the target
substance such as thermostability, susceptibility to proteolytic degra-
dation, and complexity that affect the temperature and timing of
extraction can also affect the desired bioactivity. For example, enzymes
are typically isolated at low temperatures because they can be sensitive
to proteolytic degradation or thermal denaturation, which can lead to
loss of bioactivity. In addition, natural products should be handled at
temperatures below 40 ◦C to avoid degradation and loss of bioactivity.
In general, it is preferable to work with compounds that are stable under
various conditions, especially with regard to further development and
for practical reasons with regard to the application and marketing of the
nal products.
Do seasonal and geographic differences or legal aspects of sampling
affect samples used for bioactivity screening and thus affect bio-
discovery? For many types of natural samples, re-sampling is limited due
to large seasonal or geographic variations. In addition, issues of safety
Table 2 (continued )
Bioassay designation Principle and general characteristics Advantages Limitations References
Pesticidal bioassays
Feeding bioassay =poisoned
food assay
Compound is incorporated into food
(mixing in an articial diet or
producing a genetically modied
organism) or spread/sprayed over
food. Different parameters can be
followed after exposure depending on
the pest – e.g., survival, weight gain,
size gain, offspring count, food
consumption or a specic trait
- Simple
- Easy interpretation
- Qualitative or quantitative –
depending on the set up
- Live animals (e.g., arthropods,
gastropods) are used so a rearing
facility is required
- Dependent on test animal
availability – laboratory cultures
or seasonal collection
- Time-consuming
- Development of articial diet or
GM food can be challenging
(Burgess et al., 2020;
Phan et al., 2020;
Portilla, 2020;
Razinger et al., 2014;
Sanan´
e et al., 2021; ˇ
S
mid et al., 2015)
Volatile organic compounds
(VOCs) Anti-insect activity test
The bioactivity of metabolites can be
based on different mechanisms, two of
which that are most often studied are
to repel or to kill the insect.
- Simple
- Easy interpretation
- Qualitative or quantitative –
depending on the set up
- Live animals (e.g., arthropods,
gastropods) are used so a rearing
facility is required
- Dependent on test insect
availability – laboratory cultures
or seasonal collection
- Time-consuming
- Need for specialized equipment
(sealed chambers)
(Daisy et al., 2002;
Sternberg et al., 2014)
Other
Enzymatic activity or inhibition
of enzymatic activity
To determine enzymatic activity, the
sample is incubated with the substrate
in an appropriate buffer and at an
appropriate temperature, and the
reaction is followed by measuring
absorbance or uorescence change
(depending on the substrate used).
For inhibition of enzymatic activity,
the sample is added to an enzyme in a
suitable buffer, and after pre-
incubation period of 10 to 60 min the
substrate is added and the reaction is
followed with a spectrophotometer or
uorimeter kinetically or at a selected
endpoint (incubation time).
- For some enzymes SOPs (Standard
Operating Procedures) available
- Simple
- Versatile
- Quantitative or qualitative
- Mechanism of action can be
determined
- Commercial kits available for
selected enzymes
- High-throughput
- High cost and limited lifetime of
enzymes used
- Can be time-consuming
- Optimization of conditions
(buffer, pH, temperature,
cofactors, incubation time) needed
for each enzyme
- Prone to false positive and false
negative results
- Enzyme inhibitors in the extracts
may affect activity
- Specic for each enzyme-
substrate pair
(Brooks et al., 2012;
Mohan et al., 2018;
Pohanka, 2019;
Sabotiˇ
c et al., 2009;
Sepˇ
ci´
c et al., 2019)
In-gel detection of enzymatic
activity
Sample is resolved in polycrylamide
gel under nondenaturing conditions
and gel is then incubated in a series of
solutions until colored or uorescent
bands appear. The enzyme substrate
can be incorporated into the gel or
applied during staining process.
- Additional info on size of enzyme
- Can be simple one-step but also
multiple step staining
- Qualitative, can be
semiquantitative
- Not all enzymes withstand the
conditions of in-gel separation
- Optimization of each enzymatic
reaction required with many
variables
- Can take variable time for signal
development (e.g., from minutes
to days)
(Covian et al., 2012;
Rivoal et al., 2002;
Sabotiˇ
c et al., 2007;
Sepˇ
ci´
c et al., 2019;
Sims, 1965; ˇ
Zun et al.,
2017)
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Biotechnology Advances 71 (2024) 108307
14
and sustainability should be considered. Legal issues can also limit
transnational access to (marine) biological resources, but this obstacle
can be effectively addressed under the Nagoya Protocol, and the con-
ventions CITES (Convention on International Trade in Endangered
Species of Wild Fauna and Flora) and CBD (Convention on Biological
Diversity), and CMS (Convention on the Conservation of Migratory
Species of Wild Animals) by following well-regulated procedures
(Kuunal et al., 2020; Schneider et al., 2022, 2023).
Is there a need and possibility to validate the bioassay? Validation of
bioassays in the discovery phase is useful for evaluating efcacy of
candidate bioactivities with high precision and accuracy. This is also
important for planning safety and efcacy testing and clinical trials,
establishing the basis for discussions with regulatory authorities during
planning. At later stages, at the quality control level, bioassays should
also reliably assess the quality across different product batches.
What are the relevant target organisms? In bioassays involving living
organisms, e.g., microorganisms, cell lines, or animals, it is important to
select appropriate target organisms with respect to their relevance and
the particular requirements for handling these organisms. An important
aspect to consider is the growth conditions, as different growth condi-
tions may affect the outcome of the bioassay.
Do we have a clear idea of the intended application? If there is a clear
idea of an application/use, the local regulatory authority should be
approached early in biodiscovery, as it is benecial to use those bio-
assays that are congruent with product development, as this can be very
useful to expedite the process.
3.1. Specics of marine samples
When working with marine extracts or marine microorganisms in
bioassays, special considerations should be made and methods adapted
to account for the unique challenges posed by the presence of salt,
poorly hydrophilic, often highly colored or autouorescent, and chem-
ically complex materials. These features characteristic of the marine
environment require customized protocols for working with samples
that may exhibit increased background interference, altered solubility
properties, and greater chemical diversity. Moreover, when working
with higher organisms as a source of bioactivity, it should be veried
whether the bioactivity originates from the macroorganism or from the
associated microbiota (Beutler, 2009; De La Calle, 2017; Macedo et al.,
2021). Geographic or seasonal variations in the production of bioactive
metabolites, which have been demonstrated for different marine
organisms (El-Wahidi et al., 2011; Heavisides et al., 2018; Hellio et al.,
2004; Henrikson and Pawlik, 1998), are another important issue.
4. Prevalent bioassays in marine biodiscovery
Using a keyword search of the PubMed database, we analysed
research efforts on marine natural product discovery between 2000 and
2022 (Fig. 3). There is a panoply of bioassays that can be used to screen
natural resources for their bioactive properties. We have compiled the
most common of these in Table 2 and provided a critical overview of
their advantages and disadvantages. Here, we provide an overview of
antimicrobial, antifungal, antiviral, and cytotoxicity bioassays, as well
as those that investigate the antioxidant and anti-ageing potential of
marine samples. These include both phenotype-based and single-target
bioassays to varying degrees, e.g., antimicrobial assays are mostly
phenotype-based, whereas both phenotype-based and single-target
bioassays can be used to assess cytotoxicity.
4.1. Antimicrobial bioassays
The most research efforts in the eld of bioactivity of natural marine
sources have been dedicated to the detection of antimicrobial activities
using phenotypic assays (Fig. 3). The increased efforts are mainly due to
the worldwide decline in the development of antibiotics, while the
increasing emergence of microorganisms resistant to antimicrobials is
becoming a global health threat (Dadgostar, 2019). The problem is of
particular concern for the Gram-positive and Gram-negative bacterial
pathogens that belong to the ESKAPE group (Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa, and Enterobacter spp.), and some fungal path-
ogens (Candida auris, Candida glabrata, Aspergillus fumigatus, Crypto-
coccus neoformans), for which an increasing number of multidrug-
resistant strains have been identied worldwide (Arendrup and Patter-
son, 2017; Liu et al., 2019; Minarini et al., 2020). The term antimicrobial
activity is used in studies investigating compounds that kill or inhibit the
growth of bacteria and fungi, and therefore includes both antibacterial
and antifungal activities. However, the term antimicrobial activity is
also often used in studies that focus solely on bacteria, which should lead
us to use this term with caution. In addition, there are studies that focus
on one group of organisms and investigate either antibacterial or anti-
fungal bioactivity.
The most commonly used bioassay to investigate the antimicrobial
activity of marine natural products is the determination of minimum
inhibitory concentration (MIC) in the form of broth microdilution,
macrodilution, and agar dilution, followed by the disc diffusion/Kir-
by–Bauer method (Fig. 4, Table 2). These bioassays determine the lowest
concentration of an antimicrobial agent that prevents visible or
measurable growth of a microorganism.
The main advantages of dilution methods are cost-effectiveness,
practicability, accuracy, reproducibility, versatility, availability of
standard protocols, low sample volume requirements, and the ability to
obtain quantitative MIC values (minimum concentration that inhibits
microbial growth) and MBC values (minimum bactericidal concentra-
tion, lowest concentration at which 99.9% of bacteria are killed). Pub-
lished MIC values for marine extracts vary from
μ
g/mL to even mg/mL
and are generally below 100
μ
g/mL for pure compounds (Choudhary
et al., 2017). There are common thresholds at which the extract is
considered very active (<10
μ
g/mL), moderately active (10-250
μ
g/
mL), and with little or no activity (>250
μ
g/mL) (Fajarningsih et al.,
2018; Nweze et al., 2020; Pech-Puch et al., 2020). The optimal MIC and
IC
50
(concentration at which 50 % of growth inhibition is achieved) for a
pure substance should be below 1
μ
g/mL, while concentrations above
10
μ
g/mL are considered of little interest for further research (Cushnie
et al., 2020). Following a detailed structural characterization of the
bioactive compound, potency can be dened in molar units, which may
require consideration of the characteristics of the active site (e.g., the
Fig. 3. Distribution of research efforts to assess the bioactivity of marine nat-
ural products from 2000 to 2022 based on the PubMed database. For each
bioactivity, a keyword search (together with keyword marine compound) was
performed for all publications and only for reviews in the two specied time
periods (2000 to 2020 and 2021 to 2022). The number of publications found for
each keyword, excluding reviews, is shown here. The last two years are high-
lighted with the number of publications (excluding reviews) shown next to the
columns. The greatest increase in research efforts has been in antioxidant, anti-
inammatory, antiviral, and neurodegenerative bioactivities, with >25% of
publications in the last two-year period compared to the entire 2020-
2022 period.
J. Sabotiˇ
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Biotechnology Advances 71 (2024) 108307
15
oligomeric state required for bioactivity). In drug discovery, compounds
are often considered highly bioactive if they are active at micromolar
(
μ
M) or nanomolar (nM) concentrations. In the diffusion-based method,
there is no quantitative result or only a limited one. However, both types
of bioassays can be useful to analyse the difference in antimicrobial
activity of individual natural products observed in different strains of a
given species (e.g., resistant and non-resistant mutants). In vitro assays
are characterized by simplicity of design and performance. They are
traditionally time-consuming but can be automated. However, the re-
sults are usually not available within a day and do not provide infor-
mation on the mechanism of action. To ensure the quality of the bioassay
performed, a positive control of a standard antibiotic should be tested
against authenticated microbial strains, preferably from a type culture
collection such as national type cultures collections (e.g., National
Collection of Type Cultures (NCTC) in the United Kingdom; German
Collection of Microorganisms and Cell Cultures DSMZ; American Type
Culture Collection - ATCC). A biosafety level 3 (BSL-3) laboratory is
required for antimicrobial screening against certain pathogens (e.g.,
Mycobacterium tuberculosis, Brucella sp.). Reagent sterility controls and
negative controls (e.g., inuence of solvents) should also be included in
each bioassay. When working with complex samples such as natural
extracts, the presence of other metabolites in the extract can potentially
serve as a carbon source for the microorganism used, which can mask
the effect. Both technical and biological replicates should be performed
to increase measurement accuracy.
Gram-positive bacteria are more sensitive to the effects of many
known agents than Gram-negative ones, which increases the likelihood
of hits in screening studies (Cos et al., 2006). For this reason, microor-
ganisms from different groups should be included in the screening
process. For each microorganism tested, the optimal growth medium
and inoculum size should be determined to avoid underestimation or
masking of antimicrobial activity (Wiegand et al., 2008). In most cases,
rich complex media (e.g., Mueller-Hinton broth - MHB, tryptic soy broth
- TSB, nutrient broth - NB) are used without supplements for non-
fastidious organisms and with supplements (e.g., salts, dyes, vitamins,
minerals) for fastidious organisms. Many published studies have used
Lysogeny Broth (LB) media for antibacterial testing, but their use should
be avoided due to the imbalanced composition of carbohydrates, low
availability of divalent cations, and occasional contamination with bile
salts (Nikaido, 2009; Sezonov et al., 2007).
When choosing methods for antimicrobial bioassays, the type of
solvent used to prepare the extracts should be taken into account. For
example, lipophilic compounds do not diffuse well into solid culture
media, whereas strongly charged molecules may undergo ion exchange
processes in agar. Therefore, the agar diffusion method is more suitable
for the analysis of single metabolites with known polarity and not for
complex extracts.
Two organizations develop standardized reference methods for
antimicrobial susceptibility testing: the Clinical & Laboratory Standards
Institute (CLSI) (https://clsi.org/) and the European Committee on
Antimicrobial Susceptibility Testing (https://www.eucast.org/).
Although some guidelines from standardized protocols should also apply
to bioassays performed on marine samples, noncompliance with these
guidelines is relatively common. Items whose standardization has a
critical impact on the repeatability and reliability of results include the
selection of microbial species and strains, the size and age of the inoc-
ulum, the type of culture medium, and the duration of incubation.
To further investigate the antimicrobial activity of natural mole-
cules, time-kill assays and ow cytometry methods can be used to pro-
vide information on the nature of the inhibitory effect and the cellular
damage inicted on the test microorganism (Balouiri et al., 2016). This
bioassay is used in a second phase of testing to determine the dynamics
of microbial inhibition kinetics (Dinarvand et al., 2020). Most antimi-
crobial bioassays are performed in vitro, but secondary screening for
highly potent compounds may also include in vivo assays, (e.g., in
murine models), to gain better insight into their preclinical potential
(Martín et al., 2013). In vivo bioassays are generally not performed with
extracts because of the difculty of interpreting effects based on an
unknown mixture of compounds. However, in some examples, in vivo
testing is recommended early in the development timeline because po-
tential systemic side effects may be antagonistic or synergistic (Sabotiˇ
c
et al., 2020).
4.1.1. Antibiolm assays
In recent years, the control of microbial biolms has gained signi-
cant attention as it is increasingly recognized that biolms are respon-
sible for microbial persistence. Antibiolm agents are therefore
considered as an alternative to ght microbial resistance to antibiotics,
since microorganisms do not need to develop resistance to adapt, as their
population is not decimated, but merely prevented from persisting in the
selected environment. However, the tested compound may have anti-
microbial activity, which then also has an effect on biolm development
by inhibiting growth, but not on the biolm properties themselves.
Therefore, determination of both antibiolm (i.e., inhibition of biolm
formation or promotion of biolm dispersion) and antimicrobial (i.e.,
inhibition of growth and/or survival) activity is important to understand
Fig. 4. Distribution of research methods and target microorganisms for the antimicrobial bioactivity of marine natural products from 2000 to 2022 based on the
PubMed database. For each category, a keyword search (together with the keyword marine compound) was performed for all publications and only for reviews in the
two specied time periods (2000 to 2020 and 2021 to 2022). The number of publications found for each keyword, excluding reviews, is shown for each method or
microorganism. The last two years are highlighted, with the number of publications (excluding reviews) shown next to the columns. (A) Research effort by bioassay
method. The greatest increase in research effort was in the use of time-kill and in vitro pharmacokinetic methods, with >35% of publications in the last two-year
period compared to the entire 2020-2022 period, while the number of publications for all these methods increased by >25 % in the same period; (B) Research efforts
by microbial species. The greatest increase in research efforts was for Klebsiella pneumoniae, Listeria sp., Acinetobacter baumanii, Staphylococcus aureus and
Campylobacter sp., with >25% of publications in the last two-year period compared to the entire 2020-2022 period. MIC, minimum inhibitory concentration assay
determines the lowest concentration of a substance that inhibits the visible growth of a microorganism.
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Biotechnology Advances 71 (2024) 108307
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whether the compounds tested affect biolm formation directly or
indirectly. Antibiolm strategies for combating microorganisms focus
on the one hand on preventing biolm formation by inhibiting adhesion
or bacterial cell to cell communication (quorum sensing) and on the
other hand on eliminating biolms by dispersion.
Biolms can be grown using various conditions and formats, but
commonly they are grown in a microplate format that can be adapted for
high-throughput screening evaluation of antibiolm efcacy under
laboratory conditions. Alternative methods have been developed that
provide a better approximation of real biolm conditions but require
specialized equipment, such as delicate microuidic systems (Goeres
et al., 2005; Millar et al., 2001; Tremblay et al., 2015), the Calgary
Biolm Device (Ceri et al., 1999) or the BioFilm Ring Test (Olivares
et al., 2016). Biolm formation is usually monitored by crystal violet
staining, which is used to stain the biomass of the biolm. Other
commonly used methods include measuring the metabolic activities of
biolm cells with tetrazolium salts, culturing biolm cells after
sonication to determine the number of CFUs (colony forming units) in
the biolm, or microscopy, which can be either scanning electron
microscopy or confocal laser scanning microscopy (Bridier et al.,
2010; Haney et al., 2021; Kırmusao˘
glu, 2019; Klanˇ
cnik et al., 2017;
Peeters et al., 2008). Quorum sensing reporter strains are typically
used to detect interference in quorum sensing. However, this approach
has some limitations, including negative effects on the growth of
reporter strain, so appropriate control experiments are essential to
obtain reliable results (Defoirdt, 2018; Defoirdt et al., 2013; Taga and
Xavier, 2011; Zhao et al., 2020). Simultaneous detection of
antimicrobial and antibiolm activity against important pathogenic
bacteria is also possible by studying their growth kinetics with a
microplate reader and using a growth curve analysis (Sterniˇ
sa et al.,
2022). Antibiolm activity is often expressed as minimum biolm
inhibitory concentration (MBIC) or CFU log reduction. In antibiolm
assays, typically screening of individual compounds at concentrations
of up to 100
μ
M is used and identifying active hits as those that
inhibit biolm formation by ≥80% while simultaneously inhibiting
bacterial growth by ≤40% (Kwasny and Opperman, 2010). Inhibiting
biolm formation without affecting bacterial growth is preferable
because there is less pressure on survival and consequently on the
development of resistance (Sterniˇ
sa et al., 2022).
To date, there is only one standardized assay for antibiolm activity,
namely the single-tube method (ASTM E2871), which is supported by a
standard practice for biolm growth in a CDC biolm reactor (ASTM
E3161) optimized for biolms of Pseudomonas aeruginosa and Staphylo-
coccus aureus (ASTM E2871-21, 2021; ASTM E3161-21, 2022; Lozano
et al., 2020).
4.1.2. Special consideration for antifungal bioassays
The prevalence of fungal infections (both invasive and opportunistic
fungal infections) is rising due to the increase in the ageing population
and immunocompromised patients (Webb et al., 2018). In addition,
acquired resistance has emerged in clinically relevant fungi such as
Candida spp. and Aspergillus spp. Therefore, antifungal susceptibility
testing (AFST) is of increasing importance in clinical microbiology lab-
oratories, both for selection of appropriate therapy and to provide in-
formation on resistance rates at local and global levels in
epidemiological studies. The same assays used for antibacterial activity
are also used for the screening of natural products and guiding the dis-
covery of new antifungal agents. Many factors can inuence the
outcome of in vitro AFST tests, including the denition of the endpoint,
the inoculum size of the studied fungus, the incubation period, the
temperature, and the culture media used for the test (Berkow et al.,
2020). For this reason, AFST is not recommended for every fungal
pathogen detected in a sample and is performed in clinical microbiology
laboratories primarily for yeasts.
The nature of lamentous fungal growth requires the use of adapted
antimicrobial bioassays described above to test the antifungal activities
of metabolites and molecules. Broth microdilution bioassays are
routinely used for fungi, and there are two standard methods for broth
microdilution testing of yeasts in clinical laboratories (Clinical and
Laboratory Standards Institute, 2017a; Rodriguez-Tudela et al., 2008)
and two others for molds (Arendrup et al., 2008; Clinical and Laboratory
Standards Institute, 2017b): those established by the Clinical and Lab-
oratory Standards Institute (CLSI) and those established by the European
Committee on Antimicrobial Susceptibility Testing (EUCAST). The four
standards use the same criteria to dene the test endpoint and use
similar criteria to develop clinical breakpoints and thus interpret anti-
fungal resistance and/or susceptibility. However, they differ in several
aspects regarding media composition, test microorganism preparation
(including inoculum size), measurement methods, and positive controls.
Standardized protocols based on disk diffusion are available for both
yeasts (Clinical and Laboratory Standards Institute, 2009) and la-
mentous fungi (Clinical and Laboratory Standards Institute, 2010).
Although the qualitative results of the disk diffusion method are suitable
for routine use in the clinical laboratory, the quantitative MIC data are
more relevant for the treatment of invasive infections. Agar-based
antifungal screening or “poisoned food assays”, in which fungal
growth on a standard agar containing antifungal agents is evaluated.
Alternative methods for determining antifungal activity using
specialized equipment have also been developed. These techniques
include ow cytometry, in which changes in uorescence are inter-
preted as changes in cell viability and damage (Chaturvedi et al., 2004).
With MALDI-TOF, changes in the proteome compared to a drug-free
control are interpreted as indicators of antifungal activity (Sanguinetti
and Posteraro, 2016). Isothermal microcalorimetry is used to determine
changes in metabolic heat ow of cultured fungi in response to an
antifungal agent and indirectly assess its activity (Furustrand Tan et al.,
2013).
Fig. 5. Distribution of research methods used between 2000 and 2022 to assess
the cytotoxic activity of marine natural products (based on the PubMed data-
base). For each category, a keyword search (together with the keyword marine
compound) was performed for all publications and only for reviews in the two
specied time periods (2000 to 2020 and 2021 to 2022). The total number of
publications found for each keyword, excluding reviews, is shown for each
method used, with the last two years highlighted in light blue and the number
next to each column. The greatest increase in research effort was seen in the use
of NRU, ATP, and alamar blue methods, with >25% of publications in the last
two-year period compared to the entire 2020-2022 period. NRU, neutral red
uptake cytotoxicity assay; alamar blue is a metabolic dye used to quantify
proliferation; calcein assay measures cell viability by following conversion of
calcein-AM to uorescent calcein in living cells; LDH measures the activity of
lactate dehydrogenase released from damaged cells; SRB, sulforhodamine B is a
uorescent dye used to quantify cellular proteins; PI, propidium iodide is a
uorescent dye that can pass freely through the cell membranes of dead cells
and is excluded from viable cells; ATP, adenosine triphosphate assay measures
cell viability based on the presence of ATP; Annexin-V is a protein that binds to
phosphatidylserine on the plasma membrane and is used to detect apoptosis;
MTT, MTS, XTT are tetrazolium salts that are reduced to formazan in living
cells, with MTS and XTT yielding a water-soluble formazan dye that is detected
spectrophotometrically.
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Biotechnology Advances 71 (2024) 108307
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4.2. Cytotoxicity bioassays
Cytotoxic activity is the second most studied bioactivity for marine
natural products in the last twenty years (Figs. 3, 5, 9). Cytotoxicity is
often studied in terms of possible anticancer activity. There are several
types of bioassays to analyse the cytotoxic properties of natural prod-
ucts, which include phenotypic and single-target bioassays. They are
based either on the selective penetration of dyes into dead and living
cells or on the detection of markers leaking from the cytoplasm of dead
cells. Cytotoxicity bioassays based on selective dye penetration can be
divided according to the nature of their endpoints into colorimetric as-
says (e.g., tetrazolium salts such as MTT, MTS, XTT, or WST, trypan
blue, sulforhodamine B (SRB), neutral red uptake (NRU), crystal violet),
uorometric assays (Alamar Blue (AB), 5-carboxyuorescein diacetate,
acetoxymethyl ester (CFDA-AM), carboxyuorescein succinimidyl ester
(CFSE), propidium iodide (PI), Hoechst-33,342, protease viability using
glycylphenylalanyl-aminouorocoumarin (GF-AFC) as substrate), and
luminometric assays (ATP-based and real-time viability) as reviewed
elsewhere (Aslantürk, 2018; Riss et al., 2019). The most commonly used
bioassays based on markers leaking from dead cells measure the activity
of lactate dehydrogenase (LDH), adenylate kinase (AK), glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) or aminopeptidase. Similarly, the
commonly used Annexin-V detects phosphatidylserine, which is nor-
mally located in the inner membrane but is exposed during apoptosis.
Another option is to preload cells with a measurable marker such as
calcein-AM or radioactive
51
Cr, which is typically used for mixed cell
assays in immunology (Aslantürk, 2018; Riss et al., 2019). Assays are
usually performed either in microplate format or ow cytometrically.
Regarding the evaluation criteria for cytotoxic activity, it was suggested
that crude extracts showing 50 % growth inhibition (GI
50
) at concen-
trations below 100
μ
g/mL should be considered cytotoxic, while those
holding promise for further investigation should have a GI
50
below 30
μ
g/mL (Suffness and Pezzuto, 1991). For pure compounds, GI
50
values
in the nanomolar (nM) or low micromolar (below 10
μ
M) range are
considered potentially effective. The accuracy of cytotoxic bioassays is
strongly inuenced by cell type, seeding density, and medium compo-
sition. Therefore, it is important to include appropriate controls such as
background control (no cells), negative control (untreated cells), and
positive control (all cells dead) and to test different cell types (Aslantürk,
2018; Carlsen et al., 2020; Cox et al., 2021; Riss and Moravec, 2004). In
addition to cancer cell lines, non-malignant cells, preferably rst derived
from the same tissue and then also using more normal cell types, should
be used to evaluate the selectivity of anticancer bioactivity. Based on the
cytotoxic activity against cancer cells compared to normal cells, the
selectivity index (SI) is calculated (SI =GI
50
in normal cells/GI
50
in
cancer cells). A higher SI value (at least above 2) reects better cytotoxic
selectivity (Lopez-Lazaro, 2015; Nguyen and Ho-Huynh, 2016).
Testing different cell types is essential, especially in the context of
cancer research, as each cell type may respond differently to treatment
(Niepel et al., 2017). The screening of 60 human tumour cell lines for
anticancer drugs (NCI60) by the US National Cancer Institute (NCI) was
developed in the late 1980s as a tool for in vitro drug discovery and then
expanded into a service screening to support cancer research. In 2018,
the NCI established a Program for Natural Product Development
(NPNPD) to develop a publicly accessible HTS-amenable library of
>1,000,000 fractions from 125,000 marine, microbial, and plant ex-
tracts gathered from around the world to advance HTS efforts and
accelerate drug development. By 2019, 384-well plates containing over
326,000 fractions were made available for free screening against any
disease target (Gaudˆ
encio et al., 2023; Thornburg et al., 2018).
Although cytotoxicity screening aims to identify compounds with
growth inhibitory or toxic effects on specic tumour types (disease-
oriented approach), the patterns of relative drug sensitivity and resis-
tance generated with standard anticancer drugs can also help to deter-
mine the mechanisms of action of the compounds tested. The
information-rich nature of the screening data thus provides additional
insight into cytotoxic effects (Shoemaker, 2006). The pattern recogni-
tion algorithm COMPARE assigns a biological response pattern to the
60-cell line dose-response data for a compound and evaluates whether
the response is unique or resembles a known or prototypical compound
to assign a putative mechanism of action to a tested compound. As more
data are collected on the characterization of different cellular molecular
targets of the compounds tested, the compounds most likely to interact
with a particular molecular target can be selected (Park et al., 2010;
Zaharevitz et al., 2002).
An important aspect to consider when selecting an appropriate
bioassay is understanding the mechanism of cell death and the resulting
kinetics. In this context, apoptosis-specic (e.g., Annexin-V binding or
addition of a caspase inhibitor) or necrosis-specic assays (e.g., detec-
tion of the released High mobility group box 1 (HMGB1) protein or
addition of specic inhibitors) can be used (Raucci et al., 2007; Riss and
Moravec, 2004; Shounan et al., 1998). Preferably, cytotoxicity assays
should be performed to cover multiple endpoints and determine multi-
ple parameters from the same cell sample that can reveal the actual
cause of cell death (Aslantürk, 2018; Santacroce et al., 2015). Another
aspect to consider is whether the effect is cytotoxic or cytostatic (Anttila
et al., 2019; Mervin et al., 2016). Understanding the mode of action and
molecular mechanisms targeted by cytotoxic compounds is important
for rational decision making about their use in specic cancer types, and
for assessing the risk of potential cross-reactivity with other treatments,
and side effects.
4.3. Antiviral bioassays
Viral infections are a major cause of disease in the world because of
their complexity, diversity, and rapid spread, which is often accelerated
by urbanization, increased migration, and globalization (Drexler, 2011).
The 21
st
century is characterized by major viral epidemics and pan-
demics, such as inuenza A (H1N1) pdm/09, Ebola, Zika, severe acute
respiratory syndrome (SARS), Middle Eastern respiratory syndrome
(MERS) and SARS-CoV-2 (Ong et al., 2020). In light of these emerging
viruses, as well as endemic viruses and the emergence of viral resistance,
attention has focused on natural products as sources of new antiviral
drugs, including those from the marine environment (Bhadury et al.,
2006; da Silva et al., 2006; Dias et al., 2018; Linnakoski et al., 2018;
Tziveleka et al., 2003). The very rst step before an antiviral assay is to
determine the potential toxicity of the compounds or extracts to host
cells (Fig. 6) followed by a selected antiviral assay. Several different
assays can be used to determine antiviral activity, which can be divided
into direct and indirect methods. Direct methods detect the presence of
the virus itself, while indirect methods observe the effects of the virus on
cell lines used in vitro (Table 2)(De Clercq et al., 1980; Louten, 2016;
Luganini et al., 2008; Sauer et al., 1984; Sidwell, 1986; WHO Scientic
Group, 1987). In general, all the assays described below allow the
detection of infectious viruses, with the exception of some that will be
highlighted later, which allow to determine the presence of the virus but
not to distinguish whether the virus is viable or non-infectious.
Prior to the antiviral assay, it is essential to rule out the possibility
that the antiviral properties observed in vitro are not due to cytotoxicity.
For cytotoxicity screening, any of the methods described in the previous
section can be used. Although the MTT assay has been widely used in the
past, the ATP-based assay has proven to be the gold standard for
measuring cell viability to date. It is more sensitive than conventional
biochemical methods because it detects cell death by a general rather
than a specic biological mechanism (Herzog et al., 2007; Ponti et al.,
2006). However, assays based on cell metabolism are not suitable for
metabolically inactive cells, for which the uorometric microculture
cytotoxicity assay (FMCA) is becoming increasingly popular. The FMCA
assay is based on the hydrolysis of the uorescein diacetate (FDA) probe
by the cytosolic esterases of intact cells (Burman et al., 2011; Lindhagen
et al., 2008; Str¨
omstedt et al., 2014), and cell survival is reported as an
index of survival after treatment. Usually, the concentration of the
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Biotechnology Advances 71 (2024) 108307
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compounds to be tested is between 400
μ
M and 1.5
μ
M. According to ISO
10993-5, a cell viability of >80 % indicates no cytotoxicity, 80-60 %
indicates weak cytotoxicity, 60-40 % indicates moderate cytotoxicity
and <40 % indicates strong cytotoxicity (ISO 10993-5:2009, 2022), so
that compounds with a viability between 74 % and 100 % are used for
the subsequent antiviral tests. If the results of the cytotoxicity assays
indicate no effect on cell line tness, the compounds can then be tested
with primary antiviral assays (Table 2, Fig. 6)(Gomes et al., 2016).
In cytotoxicity evaluation, the value of the 50% cytotoxicity con-
centration (CC
50
), dened as the concentration of a compound that
produces a 50% cytotoxic effect (Hu and Hsiung, 1989), is determined
and used together with the value of the 50% effective concentration
(EC
50
, i.e., the concentration of a compound that produces a 50% inhi-
bition of viral replication) to evaluate the efcacy of an antiviral
candidate. This relative efcacy of a compound in inhibiting viral
replication with respect to inducing cell death is dened as the thera-
peutic or selectivity index (SI) and calculated as SI =CC
50
/EC
50
.
Theoretically, a high SI ratio corresponds to a safer and more effective
compound that is cytotoxic only at very high concentrations and exhibits
antiviral activity at very low concentrations (Naesens et al., 2006;
Reymen et al., 1995). The antiviral activity is considered effective/
useful when the CC
50
value is 20 times higher than the EC
50
value (Cao
et al., 2015). Since the CC
50
and EC
50
values for a given compound
depend on the assays used, the SI value varies from laboratory to labo-
ratory. Nevertheless, the SI value is a widely accepted parameter of a
compound that expresses its in vitro efcacy in inhibiting viral repli-
cation (Naesens et al., 2006; Reymen et al., 1995).
At this point, it is necessary to determine the cell system(s) best
suited for virus replication on which to test new antiviral agents.
Depending on the cell type used, the replication capacity of the virus and
its actual effect on cells varies considerably (i.e., some viruses may cause
a cytopathic effect (CPE), while others may form plaques or induce
specic functions such as hemagglutination (e.g., orthomixyxovirus and
paramixovirus) or hemadsorption. Biosafety issues must be considered
when working with viruses and other microorganisms. Therefore, when
performing antiviral bioassays, specialized equipment and trained
personnel should be considered with regard to biosafety level (BSL)
requirements. These requirements depend on various factors, such as the
pathogenicity of the virus strain under investigation, its biological sta-
bility, its transmission potential, the nature of the procedures and ma-
nipulations with the pathogen, and the availability of effective vaccines
or therapeutic interventions (CDC and NIH, 2020). In general, bioassays
and relevant research activities with viral strains that are unlikely to
cause disease in humans should be conducted under BSL-1 (e.g., canine
adenoviruses). Strains that can cause disease but for which immuniza-
tion or antiviral/antibiotic treatment is available should be handled in
BSL-2 (e.g., hepatitis A and E), while in the case of severe or potentially
fatal disease due to inhalation of pathogens, BSL-3 facilities should be
used (e.g., highly pathogenic avian inuenza). In addition, viral path-
ogens that pose a high individual risk of aerosol-borne laboratory in-
fections and life-threatening diseases should be handled in BSL-4
facilities (e.g., Ebola and Marburg virus).
A cytopathic effect (CPE) test is based on the observation of
morphological changes that occur in a conuent monolayer of host cells
as a result of viral infection and replication, and therefore requires
experienced personnel. The CPE-based assay was the rst assay devel-
oped to evaluate whether a compound is antivirally effective, and it can
also be scaled up for high-throughput screening (Maddox et al., 2008;
Severson et al., 2007). Because viral replication leads to cell death,
cell viability assays can be considered a substitute for CPE assessment
as they are more accurate, automatable, and objective compared to
visual assessment by an operator. Although the CPE assay was one of
the rst antiviral assays developed, commercial kits (e.g., Viral ToxGlo
Assay) that measure cellular ATP as an indicator of host cell survival
have enabled standardization of the procedure in many laboratories,
and ATP depletion can be correlated with viral load. Since CPE is an
Fig. 6. Screening for antiviral activity begins with determining the potential
toxicity of the compounds or extracts to cell lines that allow viral replication
using bioassays such as tetrazolium salts or ATP-based assays or uorometric
microculture cytotoxicity assays (FMCA). It must then be determined which cell
system(s) is best suited for virus replication to test for antiviral activity. The
ability of the cell line to support viral replication varies and can be measured by
cytopathic effect (CPE), focus-forming assay (FFA), plaque quantication (PRA,
VRA), or hemagglutination inhibition (HI). Once specic antiviral activity has
been established, it needs to be veried in more complex systems and using in
vivo models.
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Biotechnology Advances 71 (2024) 108307
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indirect measure of viral load, the result regarding the protective effect
of drugs against a virus may also vary and be lower than other tests that
measure viral load directly (PRA, VRA, see below) (Gorshkov et al.,
2021).
Plaque reduction assay (PRA) is widely used direct viral detection
method for viruses that produce plaques on target cell lines. It is based
on counting plaques formed by lysis of infected cells in a monolayer. The
plaques are visible to the naked eye or under a light microscope after
staining with neutral red or crystal violet. The plaque assay is the
preferred method of viral titration because it is economical and tech-
nically simple, but it can be tedious because visible viral plaques can
take from 24 h to several weeks to form (El Sayed, 2000). Conicting
results may be obtained due to various limitations (see Table 2).
Therefore, in addition to PRA, the virus yield reduction assay (VRA) is
recommended to determine the EC
50
value by assessing viral progeny
production in a growth experiment performed on a conuent monolayer
of cells permissive to infection. The assay conditions must be optimized,
especially the multiplicity of infection (MOI, i.e., the ratio of virus to cell
number), because this single parameter can signicantly affect the
evaluation of antiviral activity and a high MOI can reduce the sensitivity
of the virus to an antiviral agent (Collins and Bauer, 1977; Sauer et al.,
1984). Therefore, it is advisable to perform VRA at both low MOI
(multicycle viral replication, e.g., MOI of 0.0001 to 0.1) and at high MOI
(single-cycle replication, e.g., MOI of 1 to 5), to compare the resulting
EC
50
values, and to evaluate the range of action of the antiviral molecule
as accurately as possible (Yang et al., 1989). Since many factors inu-
ence how easily viruses can infect their target cells, the MOI range to be
used varies by several orders of magnitude, depending on the applica-
tion, target cells, type of virus to be used and especially its replication
kinetics (e.g., slow rate for cytomegalovirus, fast rate for herpes simplex)
(Abedon and Bartom, 2013; Fields et al., 2007).
For viruses that do not cause cytopathic effects, the focus-forming
assay (FFA), a direct method for virus measurement, can be used. This
is a variant of the plaque assay that relies on immunohistochemical
techniques, as it uses chemically or uorescently labeled antibodies
specic for a viral antigen to detect infected cells (Flint et al., 2009). If
the antibody used recognises a viral antigen that is expressed early in the
replication cycle, this assay may not detect non-infectious viruses as
there may be an arrest of the replication cycle that prevents the for-
mation of complete infectious virions. For example, quantication of
infectious viral particles for
α
- (hCoV229-E) and β- (hCoV-OC43) coro-
naviruses relies on an enzymatic antigen detection method that uses
horseradish peroxidase (HRP) to label antigen-antibody complexes
(Lambert et al., 2008).
For the viruses expressing hemagglutinin (HA), an envelope glyco-
protein (e.g., inuenza virus, respiratory syncytial virus), the hemag-
glutination inhibition assay (HIA) can be used. This indirect method is
based on measuring the ability of virions to adsorb to and agglutinate
red blood cells (RBCs) by binding to glycans (e.g., sialic acid) on the
surface of red blood cells (usually from rabbits, horses, chickens or
guinea pigs). In practice, the hemagglutination assay is used to deter-
mine the viral concentration that agglutinates an exact (standard)
number of erythrocytes, making it extremely accurate, although it is
only applicable to certain viruses (Joklik, 1988). Standardization of the
HIA assay has been described (Kaufmann et al., 2017). In particular,
before performing the assay, the following should be considered: (i)
although HIA assays provide consistent results across multiple plates,
the same amount of virus particles must be used in each plate; (ii) ac-
cording to WHO, the standard amount of HA used in the HIA assay is 4
units per 25
μ
L [HA unit is the amount of virus required to agglutinate an
equal volume of standardized RBC suspension]; (iii) the RBCs used
depend on the type of inuenza virus in the assay; and (iv) for different
types of 96-well microtiter plates (V- or U-bottom), the incubation time
and the occurrence of nonagglutinated cells are different (Kaufmann
et al., 2017).
An example of a direct detection method is the use of recombinant
viruses, especially, uorescent protein-expressing viruses or viruses
expressing reporters fused to viral proteins, as they are rapidly detect-
able and even quantiable, making these recombinant viruses suitable
for high-throughput applications, e.g., large-scale screening of antiviral
drugs (Falzarano et al., 2014). Indeed, some in vivo applications of GFP/
Cherry/reporter viruses have also been developed, such as monitoring
the efcacy of antiviral therapies and more detailed pathogenesis
studies. Unfortunately, a foreign gene or an alteration of existing viral
genes can change the biological properties of “modied” viruses, which
may, for example, result in reduced virulence of these viruses. In addi-
tion, such alterations can put pressure on the virus to eliminate the
genetic information encoding the reporter protein, resulting in attenu-
ation/loss of expression of the reporter gene.
Modern assays such as ow cytometry, tunable resistive pulse
sensing (TRPS), and quantitative real-time PCR (qPCR) are also
increasingly being developed to determine antiviral activity. In partic-
ular, qPCR was widely used as direct method to detect SARS-CoV-2 virus
during the SARS-CoV-2 pandemic because it allowed testing of antiviral
activity of many molecules against this pathogen in a short time. How-
ever, it is important to emphasize that during viral replication, the ratio
of whole virions to nucleic acid copies is rarely 1:1 and that the viral
assembly process can produce complete virions, empty capsids, and/or
an excess of free viral genomes. Therefore, positive qPCR results may
also be due to the presence of residual viral nucleic acid (i.e., nonin-
fectious virus) rather than infectious virus (Tandon and Mocarski,
2012). For this reason, many molecules with true antiviral activity might
be rejected a priori simply because they are unable to reduce viral
genome copy number in a solution, even if the viruses present are no
longer active or infectious. Therefore, it is better to use qPCR-based
methods for routine laboratory testing and to conrm the results ob-
tained with the classical methods described above when necessary.
After a certain type of antiviral activity is detected, it is necessary to
further investigate this activity using several specialized secondary
bioassays for screening and/or monitoring purposes. These in vitro or in
vivo assays are time-consuming, more expensive, and more challenging
than the primary screening bioassays and require the expertise of bio-
chemists or pharmacologists. Therefore, they can only be performed by a
Fig. 7. Distribution of research methods for antioxidant and anti-ageing ac-
tivities of marine natural products from 2000 to 2022 based on the PubMed
database. For each category, a keyword search (together with the keyword
marine compound) was performed for all publications and only for reviews in
the two specied time periods (2000 to 2020 and 2021 to 2022). The number of
publications, excluding reviews, is shown for each method. The last two years
are highlighted, with the number of publications (excluding reviews) shown
next to the columns. The greatest increase in research effort was seen in the use
of ABTS, ORAC and CUPRAC methods, with >35% of publications in the last
two-year period compared to the entire 2020-2022 period, while the number of
publications for all these methods increased by >25 % in the same period.
CUPRAC, CUPric Reducing Antioxidant Capacity; ORAC, oxygen radical
absorbance capacity; ABTS, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid)/Trolox®-equivalent Antioxidant Capacity; DPPH, 2,2-diphenyl-1-
picrylhydrazyl.
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multidisciplinary team. Secondary testing of compounds that interact
with the target, for example, examines whether this interaction occurs in
biological systems and attempts to determine the structure-activity
relationship between the compounds and the target. Secondary tests
also include in vitro enzyme activity tests with mechanistic relevance or
resonance energy transfer (FRET), as well as pharmacokinetic and
pharmacodynamic experiments performed in vitro or in vivo in an an-
imal model. Human viruses adapted to infect animal models (Ruiz et al.,
2013) or humanized animal models (Crawford et al., 2015; Lai and
Chen, 2018) can be used at this stage. Such secondary assays are
necessary/mandatory to select potential candidates to be tested in
human clinical trials (Gomes et al., 2016; ¨
Oberg and Vrang, 1990).
4.4. Bioassays for cosmetics and cosmeceuticals with a focus on
antioxidant and anti-ageing effects
A variety of specialized bioassays have been developed and routinely
used to evaluate the overall cosmetic activity of a marine extract (Fig. 7).
The majority of these bioassays are single-target bioassays, but pheno-
typic bioassays are also available. In the primary screening and sec-
ondary testing phases for potential cosmetics and cosmeceuticals,
bioassays are mostly based on in vitro assays for cytotoxicity, antioxi-
dant and anti-inammatory activities, using either biochemical cell-free
assays or immortalized cell lines (e.g., THP-1 and HaCaT cells). These
bioassays are leading in terms of their simplicity, speed, throughput, and
cost-effectiveness, even though they may not adequately reect the
actual biological processes in skin cells. Therefore, in later stages, the
active extracts or compounds are tested for safety, activity, and mode of
action in preclinical assays using primary cells (e.g., keratinocytes) and/
or ex vivo skin tissue models (Brancaccio et al., 2022), with the option to
perform nal testing in clinical trials.
In vitro bioassays are used to investigate the antioxidant capacity of
extracts by mimicking the damage caused by radicals in the skin and by
assessing the efcacy of natural extracts in combating this damage
(Thring et al., 2009). Depending on the mechanism by which radicals
are scavenged, antioxidant capacity assays are broadly divided into two
categories: electron transfer (ET) and hydrogen atom transfer (HAT)
based assays (Apak et al., 2007). Compared to HAT-based assays, the ET
reaction is relatively slow, and its actual rate depends greatly on labo-
ratory conditions, such as solvent and pH (Apak et al., 2007; Huang
et al., 2005). ET assays widely used in cosmetics include the DPPH (2,2-
Diphenyl-1-picrylhydrazyl), ABTS/TEAC (2,2’-azino-bis(3-ethyl-
benzothiazoline-6-sulfonic acid)/Trolox®-Equivalent Antioxidant Ca-
pacity), CUPRAC (CUPric Reducing Antioxidant Capacity), and Folin-
Ciocalteu methods, each of which uses a different chromogenic re-
agent with different redox potential (Ratz-Lyko et al., 2012), as shown in
Table 2. Although the actual reducing capacity of an extract or com-
pound is not directly related to its ability to scavenge radicals, these
biochemical assays are useful for initial screening procedures (Amorati
and Valgimigli, 2015; Apak et al., 2007). Most HAT−based assays are
kinetic and rely on a competitive reaction scheme in which the antiox-
idants of a natural extract and an oxidizable probe compete for peroxyl
radicals, the latter being thermally generated in a solution by the
decomposition of azo compounds (Apak et al., 2007; Huang et al.,
2005). This is the case with the oxygen radical absorbance capacity
(ORAC) assay, which is widely used to measure the antioxidant capacity
of natural products with anti-ageing and cosmetic potential (Baldisser-
otto et al., 2012; D´
avalos et al., 2004; Dudonn´
e et al., 2011; Ky and
Teissedre, 2015; Le Lann et al., 2016). However, it must be emphasized
that most HAT and ET assays are sensitive to either hydrophilic or hy-
drophobic antioxidants and therefore may underestimate the total ac-
tivity of an extract (Fraga et al., 2014; Ratz-Lyko et al., 2012). Thus, a
combination of these biochemical methods may be required to obtain
reliable results (Ratz-Lyko et al., 2012).
The anti-ageing effect of the extracts is usually investigated in the
screening of cosmetics and cosmeceuticals, including antioxidant
(described above) and anti-inammatory activities (Brancaccio et al.,
2022). The anti-inammatory activity of extracts or pure compounds
can be assessed by TNF-
α
or IL-1β production measured in LPS-
stimulated THP-1 activated human macrophage cells (Lauritano et al.,
2016). The anti-ageing activity may also be related to the specic ability
to block enzymes involved in the breakdown of skin rmness (Thring
et al., 2009). These include matrix metalloproteinases (e.g., collage-
nase), serine proteases (e.g., elastase), and endoglycosidases (e.g.,
mucopolysaccharide hyaluronidase), which degrade the major compo-
nents of the extracellular matrix (ECM) of the skin: collagen, elastin, and
hyaluronic acid (Li et al., 2019; Rittie and Fisher, 2002). Maintaining
high levels of these components is critical for skin elasticity, rmness,
and hydration, and thus inhibitors of these hydrolytic enzymes are being
sought (Madan and Nanda, 2018). In addition, there is particular in-
terest in the regulation of melanin levels in the skin (i.e., changes in skin
pigmentation), the overproduction of which leads to aesthetic problems
such as pigmentation spots (Lall and Kishore, 2014; Saghaie et al., 2013)
as well as other skin conditions such as discoloration, freckles, and skin
cancer (An et al., 2005). Specic assays are available to study the
inhibitory properties of extracts on the activity of the enzyme tyrosinase,
which catalyses the rst rate-limiting steps of the melanin biosynthetic
pathway in melanocytes (Parvez et al., 2006). Typically, L-DOPA (an
intermediate in melanogenesis) is used as a substrate and its enzymatic
oxidation to the red-colored dopachrome is monitored spectrophoto-
metrically to assess inhibition of tyrosinase. Despite the widespread use
of (bio)chemical antioxidant assays, they are usually performed under
non-physiological conditions without taking into account the cellular
uptake of compounds and their mode of action at the subcellular level,
which inherently limits their ability to predict the true antioxidant effect
in living systems.
To investigate the regenerative properties of extracts on specic skin
cell lines (e.g., broblasts), in vitro phenotypic assays based on the
monitoring of stimulatory effects on the production of ECM components
are used (Adil et al., 2010; Boonpisuttinant et al., 2014; Pastorino et al.,
2017; Roh et al., 2013; Yodkeeree et al., 2018), as well as their photo-
protective effects in terms of cell viability (Moon et al., 2008). The
protective role of extracts against photooxidative skin damage can also
be evaluated by ex vivo approaches. Specically, a cosmetic formulation
is applied to the skin of human volunteers and after a short period of
time, strips of the outermost skin layers are removed, exposed to UV
radiation, and lipid peroxidation is assessed by measuring the losses of
unsaturated fatty acids and the amounts of primary, secondary, or end
products of the reaction (Alonso et al., 2009). Cell line-based bioassays
are also used to estimate safety parameters by assessing skin irritation by
evaluating direct cytotoxicity or other types of damage to the epithelial
barrier of the skin by measuring the permeability of uorescein through
epithelial cell monolayers (OECD test no. 460). In addition, mutage-
nicity and carcinogenicity (OECD test no. 451) are assessed using cell
cultures, e.g., the in vitro micronucleus test (OECD test no. 487) to
detect chromosomal aberrations and the bacterial reverse mutation test
(OECD test no. 471) to detect gene mutations. An alternative to animal
models for carcinogenicity testing is cell transformation assays (CTA),
which are used in combination with other approaches to evaluate
carcinogenic potential (Creton et al., 2012; Mascolo et al., 2018; Orga-
nisation for Economic Co-operation and Development - OECD, 2022;
Scientic Committee on Consumer Safety - SCCS, 2021).
Ex vivo bioassays using skin tissues have been developed for toxi-
cological studies, such as the reconstructed human epidermis (RhE) test
methods (OECD test no. 439, 431), using four validated commercial
human skin models, viz. i.e., EpiSkin™, EpiDerm™, SkinEthic™, and
EpiCS®, which use reconstructed human epidermis equivalents to
evaluate cell viability and are used to assess skin corrosion or irritation
potential. Bioassays for the assessment of ocular damage include orga-
notypic assay methods using tissues from slaughterhouses, such as
bovine corneas (OECD test no. 437) or chicken eyes (OECD test no. 438),
or in vitro assays using corneal epithelial cell lines to assess irritation by
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Biotechnology Advances 71 (2024) 108307
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measuring direct cytotoxicity on rabbit corneal cell lines (OECD test no.
491) or human cornea-like epithelium (OECD test no. 492) (e.g., Epi-
Ocular™). For assessment of genotoxicity or reproductive toxicity, new
alternative approach methodologies to animal testing are being imple-
mented worldwide, including in vitro methods using the whole embryo
culture test (WEC) to evaluate developmental toxicity in rodent embryos
maintained in culture during the early stages of organ formation, the
MicroMass Test (MM), which uses embryonic limb mesenchyme or
central nervous system cells from chickens, mice, or rats to evaluate
effects on cell differentiation into chondrocytes and neurons as an
indication of potential teratogenicity, and the embryonic stem cell assay
(EST), which is based on permanent cell lines to predict embryotoxicity
by evaluating effects on cell differentiation (Organisation for Economic
Co-operation and Development - OECD, 2022; Scientic Committee on
Consumer Safety - SCCS, 2021; Seiler and Spielmann, 2011).
5. Quality control and bioassay validation
5.1. The concept of validation
The concept of validation can be dened as a systematic approach to
collecting and analysing a sufcient amount of data under specied
conditions and based on documented evidence (validation report) and
scientic judgment, to provide reasonable assurance that the process of
interest will reliably and consistently reproduce results within pre-
determined specications when operated within specied parameters
(Haider, 2006).
The main objective of the validation process is to produce reliable
and consistent data (quality data). In addition, four critical components
of data quality are identied, including analytical instrument quali-
cation, analytical method validation, system stability testing, and
quality control sampling (United States Pharmacopeial Convention,
2018), with each of these components contributing to overall quality:
- Analytical instrument qualication (AIQ) is the collection of docu-
mented evidence that an instrument is t for its intended purpose
and that its use provides condence in the validity of the data pro-
duced. It includes (i) design qualication (DQ), which is performed
by the manufacturer prior to purchase to ensure the technical char-
acteristics required by the user; (ii) installation qualication (IQ),
which is performed prior to and at the time of installation; (iii)
operational qualication (OQ), which is performed after installation
and major repairs; and (iv) performance qualication (PQ), which is
performed periodically to ensure continued satisfactory performance
during routine operation and includes preventive maintenance,
Table 3
The summary of selected validation guidelines and corresponding organizations.
Organisation Abbreviation Sample Guideline(s) Area of Interest Remarks and References
European Medicines Agency EMA Guideline on bioanalytical method
validation (EMEA/CHMP/EWP/192217/
2009)
Bioanalytical assays for drug
development studies (with all
clinical trials)
Biological matrices such as blood,
urine, tissues etc. (European
Medicines Agency, 2011)
European Network of Forensic
Science Institutes
ENFSI Guidelines for the single laboratory
Validation of Instrumental and Human
Based Methods in Forensic Science
Forensic Biological matrices such as blood,
urine, tissues etc. (De Baere et al.,
2014)
International Council for
Harmonisation
ICH Validation Of Analytical Procedures: Text
And Methodology Q2(R1)
Pharmaceutical QC analyses Pharmaceutical samples such as;
Active Pharmaceutical Ingredient
(API), nished drug samples (ICH
Expert Working Group, 2005)
Bioanalytical method validation and study
sample analysis (M10)
Bioanalytical assays for drug
development studies
Biological matrices such as blood,
urine, tissues etc.,
Draft document (European Medicines
Agency, 2019)
United States Food and Drug
Administration
USFDA Bioanalytical Method Validation-
Guidance for Industry
Bioanalytical assays for drug
development studies (with all
clinical trials) and for veterinary
drug development as well
Biological matrices such as blood,
urine, tissues etc.(USFDA, 2018)
Association of Analytical
Communities
AOAC Guidelines for Single Laboratory
Validation of Chemical Methods for
Dietary Supplements and Botanicals
Food & Feed Quality Food and feed stuffs (Harnly et al.,
2012)
International Union of Pure &
Applied Chemistry
IUPAC Harmonized Guidelines for Single
laboratory Validation of Methods of
Analysis
General terminology on analytical
method characteristics
Sample matrices are not specied (
Thompson et al., 2002)
European Directorate for the
Quality of Medicines &
HealthCare-The Directorate-
General for Health and Food
safety
EDQM/DG-
SANTE
Analytical Quality Control and Method
Validation; Procedures for Pesticide
Residues Analysis in Food and Feed
(SANTE/12682/2019)
Food & Feed Quality Specied on the pesticide analysis in
food and feed samples (Philstr¨
om
et al., 2019)
EURACHEM n/a The Fitness for Purpose of
Analytical Methods-
A Laboratory Guide to Method Validation
and Related Topics
General terminology on analytical
method performance characteristics
Sample matrices are not specied (
Barwick et al., 2014)
European Commission
Joint Research Centre
Institute for Health and
Consumer Protection
ECJRC-IHCP Guidelines for performance criteria and
validation procedures of analytical
methods used in controls of food contact
materials (EUR 24105 EN - 1st edition/
2009)
Food Quality Migration analysis (from the food
contacting part of the packing
materials) (Bratinova et al., 2009)
United States Pharmacopeia USP General Chapter <1225>Validation of
Compendial Procedures
Pharmaceutical QC analyses Pharmaceutical samples such as
Active Pharmaceutical Ingredient
(API) and nished drug samples (USP
40, 2017)
United States Environmental
Protection Agency
USEPA Guidance for Methods Development and
Methods Validation for the RCRA Program
Environmental analysis Test Methods for Evaluating Solid
Waste (SW-846) Methods (EPA Ofce
of Solid Waste, 1992)
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Biotechnology Advances 71 (2024) 108307
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recalibration, and performance testing (Bansal et al., 2004; Kaminski
et al., 2010; Valigra, 2010)
- Analytical method validation is the collection of documented evi-
dence that demonstrates that an analytical method is t for its
intended purpose and provides assurance that its use with qualied
analytical instruments will generate accurate data of acceptable
quality (Haider, 2006).
- System suitability tests (SSTs) are used to verify that the system
meets predened criteria. They are performed in conjunction with
sample analyses to ensure that the system is functioning properly at
the time of testing.
- Quality control (QC) samples help to ensure the quality of analytical
results by being included immediately prior to or during sample
analysis.
5.2. Validation of the analytical method
The concept of bioassay validation is often associated with com-
pounds that are classied as drugs by regulatory authorities, because the
development, production and testing of these products are strictly
regulated. Consequently, bioassay validation is an integral part of the
quality control system. This may not be the case for cosmetic prepara-
tions or dietary supplements, where product characteristics and claims
dictate testing or trial requirements, however, in practice many cosmetic
preparations claiming bioactivity are also subject to rigorous testing. For
biodiscovery and research, it is not usually necessary to meet quality
control requirements, but it is good to keep the concepts of validation in
mind and apply them wherever possible. This can facilitate the transi-
tion from research to industrial development, as well as communication
with regulatory agencies, regardless of the type of application.
It is important that the operator performing the validation of the
analytical procedure has the scientic and technical understanding,
process knowledge, and/or risk assessment capability to adequately
perform the quality functions of analytical method validation (Chan,
2011). The parameters to be evaluated for validation depend on the type
of method, and the measures used to describe the performance of the
analytical method are typically: accuracy (trueness), precision (repeat-
ability), limit of detection (LOD), limit of quantitation (LOQ), linearity
(calibration curve), range, selectivity, specicity, and robustness. All of
these parameters must be determined for validation of a quantitative
analytical method, whereas specicity and limit of detection may be
sufcient for a qualitative method. There are numerous guidelines
(>30) published by regulatory organizations; some of them are sum-
marized in Table 3. These guidelines can be used as a frame of reference
for the validation process. Unlike instrument qualication, the type of
analytical method (e.g., sample matrix, analytical equipment) de-
termines the parameters to be evaluated, so it is important to select an
appropriate guidance document as a frame of reference. It is important
to note that the terminology used in different guidelines varies. For
example, selectivity, specicity, or diagnostic specicity are dened
differently in different guidelines (Borman and Elder, 2017; Chan, 2011;
Kadian et al., 2016).
Validation of analytical methods is a progressive, dynamic, and time-
consuming process, so it is recommended that a validation schedule (or
protocol) be established (EURL, 2022; Shabir, 2003). In addition, there
are fundamental differences in validation parameters between different
types of assays (e.g., chromatography-based or ligand-binding assays),
and this issue is addressed differently by different regulatory agencies,
either by providing separate validation guidelines (e.g., ICH, EMA) or by
specifying certain aspects in a guideline (e.g., FDA) (Borman and Elder,
2017; EMA Committee for human Medicinal Products, 2011; USFDA,
2018).
5.3. Data integrity and documentation
The term data integrity refers to the degree of a data-generating
system in which the acquisition and storage of data is undivided,
coherent, reliable, and accurate. This does not depend on whether the
data are in paper or electronic form (Wingate, 2004). The critical issue in
ensuring the quality of analytical procedures and data integrity is the
documentation of all steps. Good documentation practices (GDocP) is a
term used in the pharmaceutical industry to describe the guidelines,
standards, and regulations for creating, maintaining, and archiving
documents. These apply to all parties involved in a process and to all
activities. GDocP-based records have the following characteristics: they
are complete, truthful, clear, permanent, accurate, consistent, legible,
and concise (Davani, 2017).
5.4. Good laboratory practice (GLP)
It is recommended that the principles of good laboratory practice
(GLP) are followed at all times when performing bioassays. GLP is a
quality assurance system that addresses the organizational process and
conditions under which nonclinical health and environmental safety
studies are planned, performed, monitored, recorded, archived, and
reported (OECD Series on Principles of Good Laboratory Practice (GLP)
and Compliance Monitoring, https://www.oecd.org/chemicalsafety/t
esting/oecdseriesonprinciplesofgoodlaboratorypracticeglpandcomplia
ncemonitoring.htm, accessed 4 May 2022).
6. Bioactivity-guided fractionation and/or purication
With the desired bioactivity in mind, a series of fractionation and
analytical steps can be applied to natural resources to isolate and/or
purify specic compounds that exhibit the bioactivity of interest. The
path from a natural extract exhibiting a specic bioactivity to a der-
eplicated, puried, identied, and characterized compound exhibiting
that bioactivity is often quite long and labour intensive.
A signicant portion of the labour and operating costs in a
biochemical and analytical laboratory is devoted to the preparation
(extraction) of samples for subsequent analytical separation. During the
extraction process, the target compound is pre-concentrated and con-
verted into a form suitable for subsequent instrumental analysis and
chromatographic or electrophoretic separations, and the complexity of
the matrix is reduced. Depending on the solvents and procedures used
for extraction, we expect to isolate either small molecules such as pol-
yketides, alkaloids, and terpenoids or complex polymers such as proteins
and polysaccharides, and the purication steps are then designed
accordingly (Fig. 8). The solvents used for the extraction of small mol-
ecules usually consist of either a single solvent (e.g., methanol, ethanol,
ethyl acetate, acetone or water) or a mixture of solvents with a wide
range of polarity (e.g., mixtures of ethanol and acetone or ethanol and
water) (Varijakzhan et al., 2021). Complex biopolymers are usually
extracted using water or buffer solutions (Kazir et al., 2019). The
biomass remaining after the primary extraction step can be subjected to
further extraction with different solvent(s) to extract components with
different properties (Izanlou et al., 2023).
Extraction and subsequent removal of solid particles is the rst
important step in the screening process, and the selection of extraction
method and solvent(s) is critical for successful downstream processing.
For example, bioactive compounds may be present in both a highly
polar/aqueous extract and a moderately nonpolar/organic extract. In
addition, the physicochemical properties of the starting material
determine the steps in the extraction process. For example, microalgae
have a rigid cell wall that acts as a natural barrier to prevent solvent
molecules from diffusing into the cells and must be broken by me-
chanical and/or physical techniques such as high-pressure homogeni-
zation, shear mixing (high-speed homogenization), ultrasound-assisted
extraction (UAE), or microwave-assisted extraction (MAE) prior to or
simultaneously with chemical extraction (Benbelkhir and Medjekal,
2022; Tian et al., 2022). It is important to consider all available alter-
natives of the extraction procedure, including sequential extraction
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Biotechnology Advances 71 (2024) 108307
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using various solvents (Zhang et al., 2018) to optimize the extraction
process and avoid possible structural or conformational changes of the
extracted molecules that can alter their bioactivity. Such changes are
more likely to occur in large molecules (e.g., polysaccharides, oligo-
saccharides). Switching from slow extraction methods (e.g., hydro-
thermal extraction), which require longer processing time, to faster
technologies such as UAE, MAE, or UMAE (ultrasound and microwave
assisted extraction) can help shorten the extraction process and increase
the likelihood that the molecule will remain intact (Guo et al., 2022; Qiu
et al., 2022). However, chemical reactions can also occur when micro-
waves and/or ultrasound are used for extraction.
Since a natural extract contains a mixture of molecules, the concept
of bioactivity-guided purication is based on the sequential application
of different types of fractionations that separate molecules from a
mixture and the concurrent application of the selected bioassay to
identify fractions containing the bioactive compounds until a satisfac-
tory level of purity is achieved. In each purication step, the individual
fractions are tested with the bioassay to select the fractions with the
highest bioactivity for further purication. Since numerous fractions
usually need to be tested, it is optimal to use a rapid and inexpensive
bioassay with low volume requirements. A qualitative bioassay is suf-
cient to guide the purication.
Purication is usually performed by either liquid-liquid phase sep-
aration (LLPS) or the currently predominant solid phase extraction
(SPE). SPE has become a standard analytical procedure for the enrich-
ment of target analytes by partitioning and/or adsorption onto a solid
stationary phase. SPE is currently the most widely used method for the
extraction, concentration, purication, and fractionation of organic
compounds from a variety of samples, as well as for solvent exchange; in
addition, SPE is also used efciently for the desalting of proteins and
glycan samples. SPE offers several advantages over liquid-liquid
extraction, including higher recoveries, avoidance of emulsion forma-
tion, lower organic solvent consumption, simpler operation and auto-
mation capability, improved selectivity and reproducibility, and shorter
sample preparation time. The standard SPE procedure begins with the
application of an analysed solution to a solid phase (sorbent), usually in
a cartridge, in which the target analytes are eluted with a suitable sol-
vent and collected (Andrade-Eiroa et al., 2016; Faraji et al., 2019).
There are numerous adsorbents for the extraction of different types
of molecules. Various SPE mechanisms can be applied to separate target
molecules using specic sorbent materials, such as adsorption (e.g.,
using silica gel, alumina, orisil, or graphitic carbon-based packing),
normal separation (e.g., cyanogen-, diol-, or amino-based silica),
reversed phase separation (e.g., octadecyl-, octyl-, butyl-, or phenyl-
bonded silica), ion exchange (various cation or anion exchangers), size
exclusion (e.g., macroporous silica or organic gels), afnity separation
(carriers with immobilized afnity ligands), and immunoafnity sepa-
ration (carriers with immobilized specic antibodies); often two sepa-
ration mechanisms can be used simultaneously (e.g., ion exchange and
reverse phase separation) (Andrade-Eiroa et al., 2016).
Efcient SPE can also be performed with magnetically responsive
adsorbents. Magnetic SPE (MSPE) is becoming increasingly popular due
to its ease of use, high extraction efciency, and straightforward auto-
mation (Jiang et al., 2019; Pena-Pereira et al., 2021; ˇ
Safaˇ
ríkov´
a and
ˇ
Safaˇ
rík, 1999; Vasconcelos and Fernandes, 2017). MSPE uses various
types of magnetically responsive adsorbents based on ferrimagnetic iron
oxides (magnetite, maghemite) or ferrites to which specic afnity li-
gands are immobilized. A popular variation of MSPE is immuno-
magnetic separation (IMS), which uses magnetic nano/microbeads with
immobilized specic antibodies (monoclonal, polyclonal, or
Fig. 8. The approach for the discovery of new bioactive compounds from marine extracts, with the methodology indicated separately for small (left) and large (right)
biomolecules. After extraction, bioassays are performed to determine the potential bioactivities of the extract, and several purication steps are performed to
fractionate the extract for analysis and prioritise the puried compounds according to their novelty, for which the dereplication step is crucial. Several purication
and analysis runs are required to narrow down the selection of bioactive compounds. Finally, a purication procedure is applied to obtain larger amounts of bioactive
compounds that can be further used for compound identication and structure elucidation. The general approach for the discovery of new bioactive compounds is the
same for each type of molecule, but the analysis and separation methodology differs depending on the properties.
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Biotechnology Advances 71 (2024) 108307
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engineered) to capture target analytes or cells via antigen-antibody in-
teractions (De Meyer et al., 2014; He et al., 2018; Safarik et al., 2012;
ˇ
Safaˇ
rık and ˇ
Safaˇ
rıkov´
a, 1999). Magnetically responsive materials can
also be used to separate and purify various biologically active com-
pounds on a larger scale (Franzreb et al., 2006; Safarik and Safarikova,
2004, 2014). Stir-bar sorptive extraction (SBSE) is based on the use of a
magnetic stir bar covered with a suitable sorbent (usually poly-
dimethylsiloxane or ethylene glycol-modied silicone material) into
which the analytes are extracted. The technique has been successfully
used for the analysis of samples of varying complexity and for the
detection, concentration or removal of marine toxins in crude extracts
(Chen et al., 2019; Gonz´
alez-Jartín et al., 2020; Pena-Pereira et al.,
2021; Wang et al., 2017b).
Various SPE mechanisms are used to separate compounds from the
extracts, which can be performed in a column chromatography format.
These are used to fractionate either by size (e.g., size exclusion chro-
matography), charge (e.g., ion exchange chromatography), hydropho-
bicity (e.g., hydrophobic interaction chromatography), polarity (e.g.,
reversed-phase vs. normal phase chromatography), or other specic
binding interactions (e.g., afnity chromatography). These chromato-
graphic stationary phases can be used in a variety of platforms/equip-
ment, such as fast protein liquid chromatography (FPLC), generally used
for proteins or nucleic acids, or high-performance liquid chromatog-
raphy (HPLC) or ultra-performance liquid chromatography (UPLC),
used for both proteins and small molecules. In addition to column mode,
other SPE formats can be used such as extraction disks and membranes,
which are usually composed of glass bers forming a matrix on which
particles of pure or modied silica gel are anchored (Andrade-Eiroa
et al., 2016). Supercritical uid adsorption (SFA) or supercritical uid
chromatography (SFC) are another option, especially for nonpolar vol-
atile compounds. SFA can also be used for polar compounds that are
poorly soluble in supercritical CO
2
by using a suitable co-solvent such as
ethanol (Dinarvand et al., 2020). Various types of chromatography used
for isolation, purication, and characterization of natural products have
been reviewed (e.g., (Bucar et al., 2013; Nehete et al., 2013; Saini et al.,
2021; Sarker and Nahar, 2012; Yang et al., 2020). Alternatively, variants
of preparative polyacrylamide gel electrophoresis (PAGE) (e.g., native
PAGE, isoelectric focusing, 2D PAGE) can be used to separate mixtures
of compounds from extracts. Miniaturized analytical techniques can also
be used for sample processing. Pipette tip or in-syringe SPE is a minia-
turized version of standard SPE in which the absorbent material is
packed in plastic micropipette tips or in the needle of syringes; analytes
are extracted by repeated aspiration and desorption of the sample. Solid
phase microextraction (SPME) can also be used for in vivo analyses, such
as sh tissue sampling, due to its low invasiveness. Headspace SPME
allows selective extraction of volatile and semi-volatile compounds from
samples. Thin lm microextraction (TFME) increases the volume of the
extraction phase and the surface-to-volume ratio, allowing higher
extraction efciency and rapid analysis (Faraji et al., 2019; Pena-Pereira
et al., 2021).
An important step in the isolation process is dereplication
(Gaudˆ
encio and Pereira, 2015; Gaudˆ
encio et al., 2023), which is usually
performed using tandem mass spectrometry (MS/MS), which de-
termines the presence of known compounds. The bioactive extracts
containing unknown compounds are usually selected for further frac-
tionation. Alternatively, known compounds can be tested for new types
of bioactivities using other types of bioassays, a process known as
repurposing (Dinarvand et al., 2020; Houssen and Jaspars, 2012;
Nothias et al., 2018; Pereira et al., 2020; Pushpakom et al., 2019;
Veerapandian et al., 2020).
Information about the properties of the bioactive compound can be
derived from the purication process, and separation into specic
fractions provides information about their characteristics. The number
of purication steps required to purify compounds varies from case to
case and usually ranges from two to eight. Finally, the structures of
compounds are elucidated using 1D and 2D nuclear magnetic resonance
(NMR), high-resolution mass spectrometry (HR-MS), X-ray diffraction
(for crystalline compounds), and other techniques to determine the
absolute conguration (for non-crystalline compounds) (Gaudˆ
encio
et al., 2023). It is important to note that the use of low-resolution tan-
dem mass spectrometers (e.g., triple quadrupole mass spectrometers)
may be sufcient for targeted analysis of known compounds, but for
untargeted analysis of unknown compounds, the use of a high-resolution
mass spectrometer (HR-MS) in tandem mode (e.g., quadrupole time-of-
ight, Orbitrap) is essential for accurate measurement of both molecular
and fragment ions (Berlinck et al., 2022; Guo et al., 2022).
7. Application-oriented development
Given the enormous richness of the marine environment in terms of
global biodiversity, almost unlimited resources of bioactive compounds
are available for various applications (Atanasov et al., 2021; Newman
and Cragg, 2020; Rotter et al., 2021a). Over 38,000 compounds of
marine origin are listed in the Dictionary of Marine Natural Products
(https://dmnp.chemnetbase.com), the MarinLit database (http://pubs.
rsc.org/marinlit/), and the Comprehensive Marine Natural Products
Database CMNPD (https://www.cmnpd.org/) (Lyu et al., 2021).
Currently, around 1500 new marine compounds are reported annually
(Carroll et al., 2021), a substantial increase from the annual average of
1200 compounds reported nearly a decade ago (Kiuru et al., 2014).
However, marine natural product discovery faces several challenges.
Despite support from research funding organizations in the EU and
worldwide, access to the marine environment and sampling of aquatic
organisms remain very challenging, while several technical issues,
including supply of active compounds and sustainable production, can
hinder the biodiscovery process (Schneider et al., 2022, 2023).
Furthermore, extracts derived from marine organisms are very complex,
and the potentially bioactive components are usually present at low
concentrations or are characterized by high structural novelty/
complexity, making their identication and isolation in sufcient
quantities for extensive biological testing difcult.
By overcoming the above-mentioned challenges, a limited number of
promising bioactive compounds are eventually isolated in quantities
large enough to enable bioactivity studies and to support the different
stages of natural product development. There are no universal sets of
bioassays that should be used for specic research applications, while
different types of bioassays are important for different phases of bio-
discovery and product development. Much practical information on
selecting a bioassay has been discussed in Section 3, but it is prudent to
keep in mind the potential uses and regulatory requirements associated
with the various intended applications from early discovery on. To
illustrate this point, we consider the development pipeline of a general
natural source value chain and focus on marine products intended for
specic target markets, namely the pharmaceutical industry (medi-
cines), the cosmetics industry, and the food industry (dietary supple-
ments and/or ingredients for food or feed).
7.1. Pharmaceutical drug discovery
The entire process to approval of a new drug can take 12–15 years for
the pharmaceutical industry and costs up to $2.8 billion (Wouters et al.,
2020). In particular, drug discovery based on natural products has
proven to be an extraordinary laborious, costly, and time-consuming
process. Nevertheless, this is the most effective approach to new drug
development, and the number of natural-product-inspired drugs is much
higher than synthetic drugs, as over 69% of modern drugs are based on
natural products or their derivatives. Many pharmaceutical companies
have turned to combinatorial chemistry for drug structure discovery and
optimization; however, only three new chemical drugs have been
approved based on this methodology (Jimenez et al., 2020; Newman
and Cragg, 2020). To date, 15 approved marine drugs are in clinical use,
including 10 anticancer drugs, and 43 marine natural products are in
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Biotechnology Advances 71 (2024) 108307
25
clinical trials (20 in Phase I, 18 in Phase II, and 5 in Phase III). The vast
majority of the latter (i.e., 37 of 43) are being tested as anticancer drugs,
whereas others are being investigated for viral diseases, Alzheimer’s
disease, chronic pain, relapsed or refractory systemic amyloidosis, and
hypertriglyceridemia (https://www.marinepharmacology.org, accessed
03 December 2023). The drug development process (Fig. 9.) involves
ve major steps: (i) discovery and development; (ii) preclinical research;
(iii) clinical development; (iv) review by a health authority (e.g., FDA or
EMA); and (v) postmarketing surveillance, including numerous phases
and stages within each of these steps. Bioassays are primarily used
during the rst two steps of (i) discovery, including screening and
bioactivity-guided purication, and during (ii) preclinical research,
which serves as the decision-making basis for the next step of clinical
trials. For pharmaceutical and nutraceutical products, both of which
promise health benets and are subject to the same regulatory re-
quirements, preclinical testing is followed by (iii) the clinical develop-
ment phase, which includes a sequence of clinical trial phases. Phase I
clinical trials focus on testing safety, dose and side effects in a small
group of healthy volunteers. Phase II then enrols a medium-sized group
of patients with the target disease or condition and treats them for
several months to two years, comparing them to a placebo control group
or an approved standard drug to obtain efcacy and additional safety
data. Phase III studies are larger and of longer duration (1-4 years) and
include approximately 300-3000 patients who are treated and compared
to a control group. Data collected in phase III provide information on
long-term and rare side effects compared to the last two phases. After the
drug has been approved (iv) by the regulatory authorities, i.e., the Eu-
ropean Medicines Agency (EMA) and the European Food Safety Au-
thority (EFSA) in Europe and the Food and Drug Administration (FDA)
in the U.S.A., (iv) post-marketing surveillance (Phase IV) is conducted to
obtain additional information on the benets and risks of using a
particular drug.
The screening phase relies on in silico and in vitro biochemical assays
to identify bioactive extracts, fractions, or lead compounds, with high-
throughput screening playing a central role. However, in recent de-
cades, interest from the pharmaceutical industry in conducting HTS
programmes, particularly for natural products, has tended to decline
(Harvey et al., 2015). This is primarily due to a number of bottlenecks
associated with the complexity of biological extracts that can affect the
accuracy of targeted molecular screening (e.g., the effects of active
compounds can be masked by other components in the crude extract),
associated costly efforts to reduce matrix complexity, and the limited
success of large HTS campaigns previously conducted by companies.
Nonetheless, interest in HTS natural products for drug discovery remains
a hot research topic in academia. Laboratory-scale studies have reported
the application of HTS techniques to a repertoire of natural products to
identify potential therapeutic agents for tumour metastasis (Gallardo
et al., 2015), cancer and necroptosis (Li et al., 2016), cell stress and
cytotoxicity (Judson et al., 2016), metabolic and age-related disorders
(Wang et al., 2017a), and, more recently, COVID -19 (Chen et al., 2021;
Coelho et al., 2020; Gaudˆ
encio et al., 2023). Other studies have inves-
tigated natural product-like small molecules for their antimalarial ac-
tivity (Kato et al., 2016) and their suitability for genome engineering
technologies (e.g., inhibition of CRISPR-Cas9 (Maji et al., 2019).
Over the past decade, High Content Screening (HCS) has made sig-
nicant technological advances and evolved into a robust cell-based
approach that is gaining increasing interest in biological testing and
drug discovery. HCS enables automated confocal uorescence imaging
of living cells and is increasingly used to determine whether a natural
product or drug candidate elicits a specic bioactivity by monitoring the
changes induced in specic cellular pathways (Artusa et al., 2022;
Romerio et al., 2023). This is a phenotypic screening approach that
considers the nal effect on the phenotype of the cells without exam-
ining specic molecular targets. By applying a multiparametric HCS
approach, the phenotypic function of metabolites from Jaspis splendens
sponges against Parkinson’s disease was recently investigated (Wang
et al., 2016). In a similar study, natural products puried from soft corals
were screened using an HCS assay to identify potent inhibitors of the
ubiquitin-proteasome system (Ling et al., 2018). Currently available
HCS platforms can provide rich descriptive quantitative phenotypic data
for various cellular markers and parameters (e.g., cell viability, specic
protein expression, cell size, etc.), which can be used to detect different
types of bioactivities. By considering the entire cellular mechanisms,
including compensatory mechanisms, HCS enables the assessment of the
biological effect of a molecule as a whole and not just on a specic
target. This is particularly valuable for the discovery of bioactivities
against complex and multifactorial diseases such as neurodegenerative
diseases or cancer.
In addition to experimental efforts, complementary dry-lab ap-
proaches (e.g., virtual screening) have emerged under increasing pres-
sure to reduce costs and improve the speed and simplicity of the
biodiscovery process (David et al., 2015). These efforts primarily
Fig. 9. Overview of the different stages of drug discovery in the early discovery and preclinical phases of natural product development. Examples are shown of
various bioactivity and safety assays that can be used specically at each stage.
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Biotechnology Advances 71 (2024) 108307
26
involve the use of structure-assisted drug design in conjunction with
virtual HTS. With respect to natural products, this approach has been
applied in a substantial number of studies to accelerate the discovery of
antiviral agents against coronaviruses (Jin et al., 2020; Naik et al.,
2020), while others have focused on identifying molecular entities with
inhibitory activity against typical disease-related enzymes (e.g., cancer,
diabetes, and neurodegenerative disorders) (Jhong et al., 2015; Khan
et al., 2019; Mohammad et al., 2019).
Natural products that have been evaluated for pharmacological or
biological activity and have the potential to be therapeutically useful
can be considered drug hits. However, in the early stages of drug
development, a hit-to-lead (H2L) process is used that includes
mechanism-of-action studies to identify the pharmacological targets of
potent hits and a limited optimization of their chemical structure to
reduce potential side effects, increase afnity and selectivity, improve
efcacy, potency, metabolic stability (half-life) and oral bioavailability.
A lead-optimization (LO) process is then performed to synthesize,
evaluate, and modify the bioactive compounds using medicinal chem-
istry approaches to form new chemical entities (NCEs) that improve
efcacy and reduce side effects. Lead optimization also involves exper-
imental in vitro and in vivo testing in a variety of efcacy studies,
pharmacokinetic studies, and toxicological assessments, as well as
ADMET (absorption, distribution, metabolism, excretion, toxicity) as-
sessments through the use of in silico models and animal testing to
develop therapeutically effective drugs. For this reason, the preclinical
phase is typically more time-consuming, more expensive, and requires
less testing capacity than the preceding screening phases, and may
require more qualied personnel working according to the principles of
good laboratory practice (Andrade et al., 2016; Claeson and Bohlin,
1997; Collins et al., 2020).
Before the bioactive compound (lead structure) enters a new phase of
development for a specic application, its toxicity to humans, animals,
and the environment must be determined. The conclusions drawn from
the safety and toxicity tests are highly dependent on the results of the
bioassays used. Bioactivity must be quantied at this stage to determine
dose (exposure) and derive potency. Different types of bioassays may be
required for these steps, but often only validated versions of the quan-
titative bioassays already used in the discovery phase are used. The pure
compounds (lead compounds) are tested in vitro on primary cell lines or
ex vivo tissue models, or combinations thereof, specically designed for
the application of interest. The lack of adequate human disease models
has been described as a major limitation in preclinical drug development
(Khanna, 2012). Recently, however, several preclinical human disease
models have been developed for several common chronic inammatory
diseases (e.g., osteoarthritis, cardiovascular disease, chronic lung dis-
ease, psoriasis, atopic dermatitis) and various cancer types, using two-
dimensional (2D) cell culture methods, ex vivo and co-culture models
and three-dimensional (3D) organoid structures. These disease models
serve as immediate in vivo testing platforms to evaluate the efcacy and
safety of drug candidates prior to entering clinical phases (Araújo et al.,
2020; Ho et al., 2018; Jessica E Neil et al., 2022; Muenzebrock et al.,
2022; Veldhuizen et al., 2019). Results from disease models form the
basis for designing and planning potential clinical trials or conducting
other safety and efcacy testing required by regulatory authorities for a
particular application (e.g., pharmaceutical, nutraceutical or cosmetic).
It should be emphasized that the safety evaluation of pharmaceutical,
food, and cosmetic ingredients is more stringent than that of well-
characterized non-food substances, such as industrial chemicals or
pesticides (´
Sliwka et al., 2016). Moreover, cosmetics and dietary sup-
plements are not required to be approved for sale by the FDA or EMA.
Nevertheless, the cosmetics industry has recently become interested in
incorporating marine bioactive compounds into cosmetic products (e.g.,
creams and lotions) that have medicinal or drug-like effects. In this
context, the term “cosmeceuticals” has been coined to describe the
combination of cosmetics and pharmaceuticals, but it does not yet have
any legal meaning under current regulations.
The potential toxicity of compounds is determined based on their
chemical structure and mechanism of action to characterize
concentration-dependent effects, long-term effects, and effects of expo-
sure at low concentrations. Animal testing can provide valuable infor-
mation on toxicity and pharmacological activity, including
pharmacokinetics (ADME) and pharmacodynamics (interaction with the
organism), but interspecies differences in drug toxicity and efcacy can
become an important issue. Despite the recognized limitations and
benets, there are ongoing efforts to reduce the use of animals for
testing. Indeed, in vivo testing in animals and humans is subject to strict
ethical constraints, is costly, and therefore is generally performed only
in the nal stages of development (Ferdowsian and Beck, 2011). Current
regulatory approaches to toxicity testing and evaluation continue to rely
primarily on a checklist of in vivo tests that follow standardized test
guidelines or protocols. The Interagency Coordinating Committee on the
Validation of Alternative Methods ICCVAM, along with other organi-
zations, is promoting the development of non-animal alternatives to
current in vivo acute systemic toxicity tests (Clippinger et al., 2018;
Hamm et al., 2017; Kleinstreuer et al., 2018). There is a trend toward
increased use of new technologies such as high-throughput screening
(HTS), tissue chips, and computational modelling to better predict
human, animal, and environmental responses to a wide range of sub-
stances relevant to new product development. The International Coop-
eration on Alternative Test Methods (ICATM) partnership was created to
establish international cooperation in validation studies and the devel-
opment of harmonized recommendations to ensure global acceptance of
alternative methods and strategies (https://ntp.niehs.nih.gov/whatwes
tudy/niceatm/iccvam/international-partnerships/icatm/index.html).
Signicant efforts are being made to develop in vitro tests that cover
endpoints and target organs/tissues that are most relevant to humans
(Bal-Price et al., 2015). However, in some cases, animal models may still
be needed to address specic developmental toxicity questions (Clip-
pinger et al., 2018; Leist et al., 2013; Wambaugh et al., 2018). In this
context, zebrash-based bioassays offer an interesting combination of an
in vivo model and the possibility of high-throughput screening with low
compound consumption. For example, zebrash embryos have been
established as an in vivo model for the analysis of angiogenesis and
vascular development and can be further developed for other specic
high-throughput screening (Crawford et al., 2011). Another alternative
to these assays is the use of the whole-animal Caenorhabditis elegans (e.g.,
(Durai et al., 2013; Palacios-Gorba et al., 2020). In addition, phenotype-
based bioassays are also used to retarget known compounds to unknown
and novel targets (Pushpakom et al., 2019).
In recent years, computer-assisted methods have been used to predict
or model the ADMET properties of lead compounds, enabling drug
design and identication of potentially problematic structures in the
early stages of drug discovery to avoid late-stage failures (Ortega et al.,
2012). Computer-aided drug design (CADD) is increasingly being used
in drug discovery. Existing tools for predicting and visualising ADME/
toxicity data include: i) predictors of ADME parameters, ii) predictors of
metabolic fate, iii) predictors of metabolic stability, iv) predictors of
cytochrome P450 substrates, and v) software for physiology-based
pharmacokinetic (PBPK) modelling (Romano and Tatonetti, 2019;
Wishart, 2007, 2009). These enable pharmacophore modelling (PM),
molecular docking (MD), inverse docking, chemical similarity search
(CS), development of quantitative structure-activity relationships
(QSAR) (Pereira et al., 2014, 2015), virtual screening (VS) (Cruz et al.,
2018; Dias et al., 2018; Gaudˆ
encio and Pereira, 2020) and molecular
dynamics simulations (MDS), which effectively predict the therapeutic
outcome of lead structures and drug candidates and accelerate the dis-
covery process. The importance of predictive models for clinical phar-
macology is recognized by regulatory agencies, and this approach is
being used for various applications. These models combine different
types of data and parameters to estimate pharmacological activities and
are commonly referred to as physiologically based pharmacokinetic
(PBPK) models. By linking the properties of individual lead molecules to
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Biotechnology Advances 71 (2024) 108307
27
physiological properties, PBPK models also provide a rational approach
to predicting drug similarity (Benjamin et al., 2010; Deepika and Kumar,
2023; Karnati et al., 2023; Mbah et al., 2012; Str¨
omstedt et al., 2014).
By exploring structural and other data about the target (enzyme/
receptor) and ligands, CADD approaches have identied compounds
that can treat disease. Examples of approved drugs that have been
supported by CADD include dorzolamide, saquinavir, ritonavir, indin-
avir, captopril, and tiroban (Dar et al., 2019). Given the success of this
approach, the development of ”go/no-go” selection criteria and opti-
mization strategies for drug candidate development should include the
use of advanced CADD for drug metabolism and pharmacokinetics
(DMPK) proling in the development of safe and effective drugs.
7.2. Cosmetics
Cosmetic products are intended to be applied to the external parts of
the human body, including the teeth and oral mucous membranes, to
cleanse, protect, change their appearance, improve their odour or keep
them in good condition. Their use is regulated in the EU by the EU
Cosmetics Directive (Directive 1223/2009) and in the US by the Federal
Food, Drug, and Cosmetic Act (FD&C Act) and the Fair Packaging and
Labelling Act (FPLA). In the EU, all cosmetic products are registered
with the EU Cosmetic Products Notication Portal (CPNP) and must
undergo a safety assessment, have a product information le, and report
serious undesirable effects. Manufacturing must be in accordance with
good manufacturing practice (GMP), must not involve animal testing,
and labelling is subject to strict rules (Regulation EC 1233/2009). In the
U.S., registration under the FDA’s Voluntary Cosmetic Registration
Program (VCRP) is not required but it is encouraged, the use of animals
for testing is not prohibited, and truthful labelling is also regulated. It is
also important to distinguish between pharmaceuticals and cosmetics,
as pharmaceuticals require FDA approval and include products that
claim, for example, hair restoration, pain relief, anti-ageing effects, re-
lief of eczema, dandruff or acne, sun protection, etc. Therefore, the path
of regulation may vary depending on the product’s intended use. Simi-
larly, if a product corrects or alters physiological functions by exerting a
pharmacological, immunological or metabolic effect, it should be clas-
sied as a medicinal product in the EU (Regulation EC 1233/2009, FDA
Cosmetics Laws & Regulations https://www.fda.gov/cosmetics/cosme
tics-guidance-regulation/cosmetics-laws-regulations, accessed May 6,
2023).
The ingredients of cosmetic products must not be harmful or toxic
and must comply with the lists of prohibited and restricted substances.
Only approved colorants, preservatives, and UV lters may be included
in cosmetic products. The International Nomenclature Committee (INC)
manages internationally recognized systematic names for cosmetic in-
gredients such as plant extracts, oils and chemicals with the abbrevia-
tion INCI (International Nomenclature Cosmetic Ingredient), which are
used in the European Commission’s database for information on
cosmetic substances and ingredients CosIng (https://ec.europa.eu/gro
wth/tools-databases/cosing/index.cfm, accessed May 6, 2023), but in-
clusion in the database does not imply approval for use. INCI names are
primarily used for cosmetic product labelling to avoid confusion, as an
ingredient may have different chemical names (e.g., common names,
CAS or IUPAC names) in different countries.
Typical safety assessment procedures for cosmetic ingredients
include the following elements: (i) hazard identication to identify the
intrinsic toxicological properties of the substance using New Approach
Methodology; (ii) exposure assessment calculated based on the declared
functions and uses of a substance as a cosmetic ingredient, the amount
present in each cosmetic product category, and the frequency of its use;
(iii) dose-response assessment; and (iv) risk characterization, which
usually focuses on systemic effects. The ban on animal testing and the
requirement to use only validated replacement alternative methods in
Europe ensure that the New Approach Methodology (NAM) is followed,
which includes in vitro, ex vivo, in chemico, and in silico approaches,
read-across, and combinations thereof, to support regulatory decision-
making by providing information for hazard and risk assessment (Sci-
entic Committee on Consumer Safety - SCCS, 2021).
Marine resources offer an interesting repertoire of bioactive in-
gredients with cosmetic potential. Extracts from seaweed, algae, soft
corals, or other marine life are rich in proteins, amino acids, exopoly-
saccharides, carbohydrates, vitamins (A, B and C), fatty acids, and trace
elements that contribute to hydration, rming, slimming, shine, and
protection of human skin, as well as bioactive compounds with, for
example, antioxidant and anti-inammatory properties that protect the
skin from ageing and photooxidation (Guillerme et al., 2017). Therefore,
beauty products with marine ingredients are becoming increasingly
widespread.
7.3. Food and feed supplements
Food supplements are foods whose purpose is to supplement the
normal diet and consist of concentrated sources of nutrients (e.g., vita-
mins, amino acids, and minerals) or other substances with nutritional or
physiological effects. Their use is regulated by the establishment of
substance lists that are positively evaluated by a food safety authority,
such as the European Food Safety Authority (EFSA) or United States
Food and Drug Administration (FDA) for safety of ingestion and
bioavailability (i.e., the effectiveness with which the substance is
released into the body). These agencies also provide guidance on the
type and extent of information that should be submitted to demonstrate
bioavailability and toxicological data. Special regulations apply to foods
for infants and young children and to foods for special medical purposes
(Younes et al., 2021)(https://www.fda.gov/food/guidance-regulation-
food-and-dietary-supplements, accessed May 6, 2023).
Safety testing evaluates safety based on biological, physical, and
chemical parameters. Physical tests check for the presence of foreign
objects. Biological safety tests ensure the absence of pathogens and
toxins, and chemical tests detect trace elements or contaminants such as
food additives, avourings, contaminants such as heavy metals, nitrates,
disinfectants, pesticides, dioxins, residues of veterinary drugs including
antibiotics, and components of food contact materials (EU Food safety
2022, https://ec.europa.eu/food/safety_en, accessed May 6, 2023).
There is a growing interest in functional food ingredients and dietary
supplements for which the marine environment is an important
resource. Numerous compounds such as enzymes, proteins, peptides,
polysaccharides, polyunsaturated
ω
-3 fatty acids (PUFA), phenols, pig-
ments, and other secondary metabolites have already found use in the
food industry (Boziaris, 2014; ˇ
Simat et al., 2020). In addition to routine
identication of known toxins or contaminants using analytical chem-
istry methods, bioassays for detection of potentially unknown or unex-
pected toxic components are important for food and feed safety. Apart
from animal testing, bioassays are the only way to identify novel risks in
food or feed ingredients, especially when new and alternative resources
are introduced. This will become especially important with the advent of
the circular economy and green waste plans, which will increase the
input of waste streams into the food chain (Gerssen et al., 2019).
8. Conclusions
Many new and repurposed biologically active natural products from
microorganisms and macroorganisms from the marine environment
have been detected and characterized using in vivo, in vitro, and in silico
bioassays. The choice of bioassays used in biodiscovery is critical to the
successful path from extract to marketed product. Therefore, it is
important to realise that each extract contains many bioactivities and
that when pursuing a bioactive compound using a series of bioassays to
isolate and purify the targeted bioactive compound, the other compo-
nents of the extract should not be discarded as inactive. Additional
valuable bioactivities may be revealed by other bioassays. Conversely, a
bioactive compound targeted for a particular application can be
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Biotechnology Advances 71 (2024) 108307
28
reassessed for other types of bioactivities as part of the repurposing
process. Only when all these aspects are taken into account is it possible
to optimize the potential and make the best use of the various natural
resources and, in particular, the marine environment, which is now
being increasingly explored.
A careful inspection of the literature reveals many questions
regarding the performance of bioassays used for screening and identi-
cation of bioactivity. Some of these issues relate to possible artifacts in
assay results, variations in activity within different methods, differences
in solubility, synergy of compounds in the tested extract, proper use of
controls, storage conditions of extracts, etc. For many bioassays
routinely used in research laboratories, there are no standardized assay
procedures, so it is often very difcult to compare results reported by
different laboratories. To improve the potential for standardization of
bioassays, fundamental properties such as robustness, reproducibility,
relevance, sensitivity, cost-effectiveness, automation, accuracy, and
selectivity should be considered in the development and selection of
bioassays to be used. A practical aspect is the use of validated protocols,
appropriate controls, and biologically relevant concentrations in bio-
assays. In this way, it can be assessed at an early stage of biodiscovery
whether the selected bioactivity has realistic potential, for example, for
pharmacological or cosmetic applications, or whether it is merely an
interesting but descriptive discovery.
It is important to note that computational approaches should be
widely incorporated into biodiscovery screenings for two reasons: (i)
these approaches are data-driven, so their inclusion in screening pro-
tocols will provide large amounts of data that can be examined for
valuable patterns for further discovery; and (ii) large amounts of data
are already available for analysis, so systematic analysis of data should
become routine, including genome sequences, gene expression, chemi-
cal structures analytical data, genotype or proteome data, human
microbiome, or electronic health records. These analyses, performed
using computational tools, can save time through dereplication, pre-
diction of new targets for already known compounds, and information
on modes of action. Understanding the molecular mode of action of
bioactive compounds is particularly important because this knowledge
helps in the development of new ways to elicit the same effect when the
original bioactive compound proves toxic or immunogenic, cannot be
synthesised, and/or is not available in sufcient quantity or is lost from
natural resources.
Finally, scientic research must be supported by innovation. The
search for products for human and environmental health and well-being,
including the development of new bioassays, must consider the princi-
ples of ethics, responsible research and innovation (RRI) (Schneider
et al., 2022), good laboratory practices, and respect for natural ecosys-
tems and habitats.
Declaration of generative AI and AI-assisted technologies in the
writing process
During the preparation of this work the authors used InstaText in
order to improve readability, grammar and language style. After using
this tool, the authors reviewed and edited the content as needed and take
full responsibility for the content of the publication.
Acknowledgments & funding
This publication is based upon work from COST Action CA18238
(Ocean4Biotech), supported by COST (European Cooperation in Science
and Technology) program.
Research of Jerica Sabotiˇ
c and Nika Janeˇ
z was supported by Slove-
nian Research Agency (J4- 2543, J4-4555, P4-0127, P4-0432).
Research of Evita Strode was supported by ERDF post-doctoral
research grant 1.1.1.2/16/I/001 (application No 1.1.1.2/VIAA/3/19/
465).
Susana P. Gaudˆ
encio: This work is nanced by national funds from
FCT - Fundaç˜
ao para a Ciˆ
encia e a Tecnologia, I.P., in the scope of the
project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit
on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/
2020 of the Associate Laboratory Institute for Health and Bioeconomy -
i4HB.
Research of Anna Luganini and Giovanna Cristina Varese was
nanced by the University of Torino (Ricerca Locale) and the European
Commission – NextGenerationEU, Project “Strengthening the MIRRI
Italian Research Infrastructure for Sustainable Bioscience and Bio-
economy”, code n. IR0000005.
Research of David Ezra was supported by The Chief Scientist of the
Israeli Ministry of Agriculture and Rural Development (MOARD), grant
number 20-02-0122, and Copia Agro Israel.
Research of Dina Simes was funded by the Portuguese National
Funds from FCT—Foundation for Science and Technology, through
projects UIDB/04326/2020, UIDP/04326/2020 and LA/P/0101/2020
and AAC n◦41/ALG/2020 - Project n◦072583 – NUTRISAFE.
Declaration of competing interest
None.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biotechadv.2024.108307.
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