Phytosomes as Innovative Delivery Systems for Phytochemicals: A Comprehensive Review of Literature

Abstract

Nowadays, medicinal herbs and their phytochemicals have emerged as a great therapeutic option for many disorders. However, poor bioavailability and selectivity might limit their clinical application. Therefore, bioavailability is considered a notable challenge to improve bio-efficacy in transporting dietary phytochemicals. Different methods have been proposed for generating effective carrier systems to enhance the bioavailability of phytochemicals. Among them, nano-vesicles have been introduced as promising candidates for the delivery of insoluble phytochemicals. Due to the easy preparation of the bilayer vesicles and their adaptability, they have been widely used and approved by the scientific literature. The first part of the review is focused on introducing phytosome technology as well as its applications, with emphasis on principles of formulations and characterization. The second part provides a wide overview of biological activities of commercial and non-commercial phytosomes, divided by systems and related pathologies. These results confirm the greater effectiveness of phytosomes, both in terms of biological activity or reduced dosage, highlighting curcumin and silymarin as the most formulated compounds. Finally, we describe the promising clinical and experimental findings regarding the applications of phytosomes. The conclusion of this study encourages the researchers to transfer their knowledge from laboratories to market, for a further development of these products.

Full text

REVIEW
Phytosomes as Innovative Delivery Systems for
Phytochemicals: A Comprehensive Review of
Literature
Mahmood Barani
1
Enrico Sangiovanni
2
Marco Angarano
2
Mohammad Amin Rajizadeh
3
Mehrnaz Mehrabani
4
Stefano Piazza
2
Hosahalli Veerabhadrappa
Gangadharappa
5
Abbas Pardakhty
6
Mehrzad Mehrbani
7
Mario Dell’Agli
2
Mohammad Hadi
Nematollahi
8
1
Medical Mycology and Bacteriology Research
Center, Kerman University of Medical Sciences,
Kerman, 76169-13555, Iran;
2
Department of
Pharmacological and Biomolecular Sciences,
Università degli Studi di Milano, Milan, 20133,
Italy;
3
Student Research Committee, Kerman
University of Medical Sciences, Kerman, Iran;
4
Physiology Research Center, Kerman
University of Medical Sciences, Kerman, Iran;
5
Department of Pharmaceutics, JSS College of
Pharmacy, JSS Academy of Higher Education
and Research, Mysuru, India;
6
Pharmaceutics
Research Center, Institute of
Neuropharmacology, Kerman University of
Medical Sciences, Kerman, Iran;
7
Department of
Traditional Medicine, Faculty of Traditional
Medicine, Kerman University of Medical
Sciences, Kerman, Iran;
8
Herbal and Traditional
Medicines Research Center, Kerman University
of Medical Sciences, Kerman, Iran
Abstract: Nowadays, medicinal herbs and their phytochemicals have emerged as a great
therapeutic option for many disorders. However, poor bioavailability and selectivity might
limit their clinical application. Therefore, bioavailability is considered a notable challenge to
improve bio-efcacy in transporting dietary phytochemicals. Different methods have been
proposed for generating effective carrier systems to enhance the bioavailability of phyto-
chemicals. Among them, nano-vesicles have been introduced as promising candidates for the
delivery of insoluble phytochemicals. Due to the easy preparation of the bilayer vesicles and
their adaptability, they have been widely used and approved by the scientic literature. The
rst part of the review is focused on introducing phytosome technology as well as its
applications, with emphasis on principles of formulations and characterization. The second
part provides a wide overview of biological activities of commercial and non-commercial
phytosomes, divided by systems and related pathologies. These results conrm the greater
effectiveness of phytosomes, both in terms of biological activity or reduced dosage, high-
lighting curcumin and silymarin as the most formulated compounds. Finally, we describe the
promising clinical and experimental ndings regarding the applications of phytosomes. The
conclusion of this study encourages the researchers to transfer their knowledge from labora-
tories to market, for a further development of these products.
Keywords: phytochemical, nanomedicine, phytosome, delivery, vesicle, disease
Introduction
For several decades, medicinal herbs and their active constituents have been utilized
to treat different diseases.
1–5
There are some major reasons for the increased use of
herbal drugs: 1) modern medicine is unable to efciently cure all the human
pathologies, 2) there are increasing interests and attention over the assurance and
safety of synthetic drugs, and 3) many natural products are being shown to produce
better results than synthetic drugs without adverse effects.
6
However, due to poor
oral bioavailability, the clinical application of numerous active compounds of plants
is under debate.
7,8
The weak absorption rate of such constituents may be a result of
low lipid solubility, the existence of multi rings polyphenols in their structures, and
high molecular weight.
9,10
Different solutions have been suggested to face such
obstacles,
11
including preparing emulsions,
12
liposomes,
13
and nano-formulation,
14
the adjustment of molecular structure,
15
and administration of prodrugs.
16
Between
all approaches, phyto-phospholipid complexes (named phytosomes) are appeared to
be a great method to boost their bioavailability.
9
Correspondence: Mario Dell’Agli
Department of Pharmacological and
Biomolecular Sciences, Università degli
Studi di Milano, Via Balzaretti 9, Milan,
20133, Italy
Email mario.dellagli@unimi.it
Mohammad Hadi Nematollahi
Department of Clinical Biochemistry,
Kerman University of Medical Sciences,
Kerman, Iran
Email mh.nematollahi@yahoo.com
International Journal of Nanomedicine 2021:16 6983–7022 6983
© 2021 Barani et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.
php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the
work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For
permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
International Journal of Nanomedicine Dovepress
open access to scientific and medical research
Open Access Full Text Article
Received: 7 May 2021
Accepted: 13 August 2021
Published: 15 October 2021

The term “Phyto” refers to the plant, while “some” refers
to cell-like.
17
Phytosomes (or herbosomes) are the vesicular
drug delivery system enhancing the absorption and bioavail-
ability of low-soluble drugs.
9,17
Phytosomes are complex of
phospholipids and natural active phytochemicals, bound in
their structures, obtained by the reaction between phosphati-
dylcholine (or any hydrophilic polar head groups) and plant
extracts in an aprotic solvent.
10,18
These formulations exhibit
improved pharmacological and pharmacokinetic properties as
compared to prevalent preparations. The lipid-soluble phos-
phatidyl portion completely covers the hydrophilic phytocon-
stituent-choline complexes. Phytosomes have remarkable
benets such as high drug encapsulation, reveal a better stabi-
lity prole (chemical bonds are formed among the polar head
of the amphiphile molecule and phytoconstituent
19
), and have
a better bioavailability.
20
Moreover, a higher absorption rate
leads to a lower dosage of active constituents for exerting a
biological effect, also for polar phytoconstituents.
There is a variety of possible applications of phyto-
some that will be discussed in this review.
The Phytochemicals
Phytochemicals or plant chemicals are comprised of a wide
range of naturally occurring bioactive compounds produced
by plants. The term bioactive refers to the ability of these
compounds to interact with different components of living
organisms, thereby exerting their benecial effects.
Phenolics, alkaloids, carbohydrates, lipids, terpenoids, and
other nitrogen-containing compounds are the most structurally
different major categories of phytochemicals. Moreover, there
are several subcategories of phytochemicals based on differ-
ences in biogenesis or biosynthetic pathway.
Between all the phytochemicals, only those having an
active hydrogen atom (-COOH, -OH, -NH2, -NH, etc.), like
polyphenols, can be integrated into a phytosome structure.
An active hydrogen atom can form a hydrogen bond between
the herbal derivatives and the hydrophilic parts of amphiphile
molecules. Polyphenols are the major group of phytochem-
icals extensively found in plant-based foods. Potential health
effects of polyphenols were shown in different diseases
including cancer, inammation, neurodegenerative and car-
diovascular diseases, type 2 diabetes, and obesity.
21
Essentially, they are found in conjugated forms composed
of sugar residues (one or more) attached to hydroxyl groups;
however, the sugar residues may directly attach to an aro-
matic carbon.
22,23
Flavonoids and non-avonoids are two
major subgroups of polyphenols (Figure 1). The current
review updates the knowledge on the use of polyphenols
through phytosomes, paying attention to their structure, pre-
paration, and the biological activities associated with the use
of phytochemicals-loaded phytosome.
Phytosome Structure and
Preparation Methods
Bombardelli et al stated for the rst time that there is a
chemical bond between phospholipids and avonoid vegetal
Graphical Abstract
https://doi.org/10.2147/IJN.S318416
DovePress
International Journal of Nanomedicine 2021:16
6984
Barani et al Dovepress
Powered by TCPDF (www.tcpdf.org)

derivative molecules.
24
In 2016, Pu et al examined the mole-
cular docking model for the interaction of 20(S)-protopanax-
adiol (PPD) phospholipid complexes. The results indicated
that the hydrophobic section of the PPD framework was
enclosed by two hydrophobic arms of the phospholipid mole-
cule, and a hydrogen-bond with the phospholipid backbone
of the P=O section was generated by one of the hydrophilic-
OH groups. Many authors have stated that the hydrogen
interactions are the main interactions in phytosome
vesicles.
25
Phospholipids have an afnity for polyphenols and form
supramolecular adducts that have a denite stoichiometry,
which can be obtained from thermal analysis. Semalty et al
tested this parameter and found that hydrogen bond forma-
tion or hydrophobic interactions were due to the interaction
between the two molecules.
26
The phospholipid-active ingre-
dient is responsible for the creation of a hydrogen connection
between the polar head and the active ingredient’s polar
functionalities.
25,27
In summary, as presented in Figure 2,
the hydroxyl groups of polyphenols can interact effectively
with nitrate and phosphate groups of phospholipids.
Several strategies have been proposed for preparing phy-
tosome, such as the rotary evaporator method, anti-solvent
precipitation technique, freeze-drying co-solvency, and salt-
ing-out technique. The main methodologies for the prepara-
tion of the phytosome are shown in Figure 3. Popular and
commonly used techniques for producing phospholipid com-
plexes are the evaporator approach and solvent evaporation.
The solvent evaporation method for preparing evodiamine
phospholipids complex was stated by Liu et al.
28
In another
study, Yu et al prepared the berberine-loaded phytosomes by
the method of solvent evaporation and a self-assembly
approach.
29
In the process of solvent evaporation, lipid mate-
rials were dissolved in an organic solvent, which was then
removed by vacuum rotary evaporation. By the anti-solvent
precipitation technique, Singh et al reported the preparation
of lawsone-loaded phytosome.
30
In this process, dichloro-
methane was reuxed with lawsone and soya lecithin at a
temperature not exceeding 60 °C. Then, to get the precipitate
stored overnight in vacuum desiccators, n-hexane was added.
Karole et al have used the technique of anti-solvent precipi-
tation to prepare phytosomes containing Bombax ceiba
extract.
31
El-Menshawe et al described a soy thermogel
based on phytosome made by three different preparation
methods (co-solvency, solvent evaporation, and salting-
out).
32
It was observed that the optimal phytosome formula-
tion was the one prepared using the co-solvency technique,
obtaining an ideal entrapment efciency (EE) of 99.89%, a
size of 64.44 nm, and a release rate of up to 93% after 2
hours. Demir et al developed a novel liposomal formulation
in an innovative study by encapsulating both Calendula
ofcinalis extract and AuNPs.
33
Vesicle preparation was
Figure 1 Polyphenol classications. Classes of polyphenols and their relationships to each other. One structural example is presented for each class.
International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S318416
DovePress
6985
Dovepress Barani et al
Powered by TCPDF (www.tcpdf.org)

Figure 3 Thin-lm hydration as the most common method for phytosome preparation. Steps 1 to 4 are the procedures of phytosome preparation.
Figure 2 Suggested principle for phytosome formation. Hydrogen bond formation between phytochemical and polar head of phospholipid is depicted as schematic and
structural picture. Dashed lines are representative as the hydrogen bonds.
https://doi.org/10.2147/IJN.S318416
DovePress
International Journal of Nanomedicine 2021:16
6986
Barani et al Dovepress
Powered by TCPDF (www.tcpdf.org)

carried out by the conventional method of thin-lm hydration
within the extrusion. The ndings showed that this method
improved the biological activity of AuNP and calendula
extract. Other methods have been documented for the pre-
paration of phytosome complexes, such as anhydrous co-
solvent lyophilization or lyophilization.
34–36
Phytosomes are originated from the reaction of a stoichio-
metric quantity of the phospholipid (phosphatidylcholine) with
polyphenolic constituents or standardized extracts (avonoids,
tannins, terpenoids, xanthones) within a non-polar solvent.
37
Different solvents have been used in various studies as a
reaction medium to formulate phyto-phospholipid complexes.
In aprotic solvents, no hydrogen atoms exist directly connected
to an electronegative atom and have no capability at hydrogen
bonding. Traditionally, these solvents like aromatic hydrocar-
bons, methylene chloride, halogen derivatives, cyclic ethers,
and ethyl acetate have been utilized for preparing phyto-phos-
pholipid complexes. However, these are mostly substituted by
protic solvents, such as ethanol.
38,39
In protic solvents, like
methanol and ethanol, at least one hydrogen atom is directly
connected to an electronegative atom. Thanks to the higher
yield of complexes, ethanol is an effective solvent also due to
the low presence of residues. Some liposomal drug complexes
act in the existence of buffer solution or water, where the
interaction of the phytosomes with a solvent occurs with a
decreased dielectric constant.
40
Nevertheless, the use of a
single solvent is included in most preparation methods,
mixed solvent systems have been used in several studies
whereby the phospholipids are dissolved in a different solvent
from that of the drug/extract. The mixed solvent systems
include dichloromethane and methanol, water and diethyl
ether, as well as ethanol and dichloromethane.
41–43
Vesicular Systems in Phytosome
Development
Targeted delivery and sustained release rate are two rele-
vant factors for phytochemical drug carriers.
44
Several
kinds of nano-systems would be used in various disease
imaging or therapies, or as theranostics.
45
The most used
nanocarriers for phytochemicals are the vesicular drug
delivery systems,
46
in which active compounds are encap-
sulated in a spherical structure.
47
Various types of vesicular drug delivery systems such
as liposome, niosome, transfersome, and ethosome have
been developed (Table 1).
48–65
Figure 4 also depicts a
schematic representation of the different vesicle architec-
tures in phytochemical delivery.
The Liposome
Liposome originated from two Greek words “Lipos sig-
nifying fat and Soma meaning body”.
67
Liposomes are
phospholipids and cholesterol that made up the spherical
shaped vesicles with a diameter of 0.05–5.0 micrometers.
They are a very promising carrier for drug delivery in
different architectures due to their hydrophobic and lipo-
philic characters.
68–70
This drug delivery system attempts
to directly target the drug at the desired site of action.
71
Liposomes are biocompatible, biodegradable, stable, and
have a unique property that traps both hydrophilic and
lipophilic agents into their compartments and provides a
controlled-release effect.
72–74
Liposomes are used in dif-
ferent pathological conditions, such as cancer, inamma-
tion, eye and skin disease, malaria, and osteosarcomas.
75–
80
The liposomes can be designed using various
techniques.
81,82
Overall, most liposome preparatory meth-
ods are based on (1) solvation of the lipids in an organic
solvent; (2) getting lipid thin lm by evaporation; (3)
hydration of lipid layer by a hydrophilic solvent; (4) lipo-
some purication (5) and characterize the properties of the
nal liposome.
83–85
Also, other synthesis methods can
improve the encapsulation of the loaded drug.
86–88
Besides, phytoconstituent liposomes have been devel-
oped to increase the penetration, solubility, and biological
impact or to defend against degradation.
89,90
There are
many reports of the use of natural extracts via encapsula-
tion in liposomes to improve their bioactivity or to avoid
other side effects.
91,92
For example, Gautam et al
reported CD44 receptor-phyto-liposomes loaded with
stigmasterol (STS) for synergistic chemotherapy. The in
vitro anticancer activity of HA-DOX-STS-lipo was sig-
nicantly enhanced in MDA-MB-231, CD44-overexpres-
sing cells relative to MCF-7 cells demonstrating HA-
mediated targeting effect. HA-DOX-STS-lipo accumu-
lated more and increased antitumor efcacy in the
MDA-MB-231 xenograft tumor model expressing high
levels of CD44, suggesting the potential of carrier system
toward CD44-overexpressing tumors.
93
Raee et al pre-
pared nanoliposomes using a thin hydration process with
various amounts of polyphenols of pistachio green hull
extract and lecithin and characterized their particle size,
PDI, zeta potential, entrapment efciency (EE), and mor-
phology. Nanoliposomes had the highest EE (52.93%)
composed of 1% lecithin with 1000 ppm phenolic com-
pounds. The FTIR ndings show the formation of hydro-
gen bonds between both the phospholipid polar zone and
International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S318416
DovePress
6987
Dovepress Barani et al
Powered by TCPDF (www.tcpdf.org)

the phenolic compound OH groups. Also, nanoliposomes
obtained a signicant shelf life. As a result of this study,
the liposome can be used as an effective carrier for the
maintenance and enhancement of pistachio extract and
bio-functional active agents in food products.
94
In another
study, Shafaei et al evaluated the therapeutic efcacy of
sinensetin (SIN), eupatorin (EUP), rosmarinic acid (RA),
and 3-hydroxy-5,6,7,4-tetramethoxyavone (TMF) in
Orthosiphon stamineus extract (OS-E) and assessed the
formulation of OS-E-derived nanoliposomes (OS-EL) in
the plasma of Sprague-Dawley rat after oral and intrave-
nous administration. After intravenous OS-EL administra-
tion, all four compounds tended to be poorly distributed
and gradually removed from the body as opposed to OS-
E. On the other hand, in oral administration loaded for-
mulation (OS-EL), the bioavailability of all compounds
was greater than OS-E (due to higher solubility of phos-
pholipid encapsulation). These ndings indicate that OS-
EL‘s greater solubility and bioavailability may be due to
liposome encapsulation.
95
Table 1 Most Used Nanovesicle Encapsulated Herbal Formulations
Nanovesicle Phytochemicals Feature References
Liposome Aphanamixis
polystachya leaf
Great improvement in memory function, locomotive behavior, and dementia-induced
outpatient quality of mice.
[63]
Anthocyanins Increase physiological stability in vitro for 14 days and increase the activity of ROS
scavenging and skin absorption.
[62]
Curcumin Fast permeation rate endothelial cell monolayer crossing blood-brain barrier (BBB) and
good durability toward digestive enzymes.
[61]
Eleusine coracana Effective antibacterial formulations have a great nutritive value. [60]
Niosome Carum carvi Regulate release and decrease of MCF-7 cell migration, high anti-cancer behavior against
MCF-7 supported by cytometry ow (G2/M arrest).
[59]
Lawsone Entrapment efciently of 70%, a sustained release prole, and a signicant increase in
antitumor activity.
[58]
Fumaria ofcinalis Rapid degradation, stability in GI conditions simulated, and anti-diabetic and anti-
inammatory capacity.
[57]
Annona squamosa Aid with topical drug enhancers to purify the body from harmful impurities and oxidants and
can be applied directly to the skin.
[56]
Transfersome Mulberry leaves Prolonged delivery system, strong safety, and acne vulgaris care via transdermal route. [55]
Apigenin Drug entrapment of 84.24%, strong stability, enhances the permeability of apigenin in the
long-term release.
[53]
Epigallocatechin-3-
gallate (EGCG)
Increases cell viability, decreases lipid peroxidation, intracellular ROS, MMP expression in
HaCaT cells, and increases skin permeation.
[52]
Emodin High efciency and stability in encapsulation, reduces body weight, and adipocyte size
through ATGL up-regulation, down-regulation of G0S2 expression in adipose tissue, and
improved insulin sensitivity.
[51]
Ethosome Thymoquinone 99% efciency for drug trapping. Cytotoxic activity of 0.95 μg/mL against MCF-7 cell lines is
improved.
[54]
Capsaicin Ethosomal hydrogels improve performance and patient compliance with capsaicin
treatment.
[50]
Terminalia chebula Effective release comparison with extract in the gel. In vitro anti-arthritic activity
demonstrates important anti-arthritic activity as opposed to normal Diclofenac activity
[49]
Paeonol Paeonol-loaded ethosomes showed improved transdermal absorption and skin retention
(138.58 μg/cm
2
and 52.60 μg/cm
2
, respectively)
[66]
https://doi.org/10.2147/IJN.S318416
DovePress
International Journal of Nanomedicine 2021:16
6988
Barani et al Dovepress
Powered by TCPDF (www.tcpdf.org)

In a most recent study, Sinisgalli et al evaluated the
antioxidant activity of Capsicum annuum pepper extract-
loaded liposomes. The extracts exhibited no cytotoxicity
and reduced the level of ROS in the HepG2 cell line. Based
on the RT-PCR assay, the expression of endogenous antiox-
idants was increased in loaded formulations.
96
Besides the
enhanced ability in phytochemical delivery, liposomes have
also some disadvantages. Drugs encapsulated in the lipo-
somes require a high cost of development. Leakage and
fusion of encapsulated drugs may occur. Furthermore, the
phospholipid liposome may undergo hydrolysis and oxida-
tion, resulting in a shorter half-life.
The Niosome
Niosomes are nanometric lamellar vesicles that are formed by
combining non-ionic surfactant and a helper lipid-like
cholesterol.
97–99
The non-ionic surfactants create a stable
bilayer vesicle in hydrophilic systems by using energy (physi-
cal agitation and heating).
100,101
Hydrophobic parts in the
bilayer structure are guided aside from the aqueous phase,
while the hydrophilic heads stay in contact with the aqueous
side. The surfactants used in the preparation of niosomes
should be biocompatible, biodegradable, and not
immunogenic.
102,103
Niosomes act like liposomes in vivo and
in vitro, extending the circulation of the encapsulated phyto-
chemical, adjusting its organ distribution, and improving bioa-
vailability. The niosomal formulations are more leaky than
liposomes with the same cholesterol value.
99
Previous research
has been shown that cholesterol concentration is an important
inuence factor on vesicle leakage.
104
As a result, the ef-
ciency of liposomal drug trapping becomes lower than
niosomes.
105
Liposomes are expensive, and their components
are unstable for long periods and need special handling and
storage.
106
Niosomes can increase the solubility and sustain-
ability of phytochemicals, considered novel herbal delivery
systems. They are designed to target and control the release
of natural compounds.
57,107–110
Our group evaluated the nio-
some encapsulation of different antioxidant phytochemicals,
Figure 4 Possible vesicles to form phytosomes. Schematic representation of the different types of vesicles in phytochemical delivery, liposome, transfersome, niosome and
ethosome. All these possible vesicles have polar heads.
International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S318416
DovePress
6989
Dovepress Barani et al
Powered by TCPDF (www.tcpdf.org)

such as lawsone,
58
diosgenin,
111
D-limonene,
111
and Carum
spp.
111
In our last study, we designed a natural anti-cancer
niosome vesicle based on ergosterol, nonionic surfactants,
and Carum carvi extract (Carum). In vitro cytotoxicity, ow
cytometry, DNA fragmentation, and cell migration assay of
formulations were evaluated. Loaded formulations provided a
controlled release compared with free Carum extract. Based on
MTT assay and ow cytometry analysis for MCF-7 cancer cell
line, Carum encapsulated niosome (Nio/Carum) showed better
anti-cancer effects than free Carum extract. Cell cycle analysis
showed G2/M arrest in Nio/Carum formulations. Nio/Carum
remarkably decreased the migration of MCF7 cells.
111
Similarly, to improve solubility, stability, and penetration
of antioxidant avonoids (morin, quercetin, myricetin, se-
tin, rutin, and breviscapine), Lu et al loaded these phyto-
chemicals into niosome. Results revealed that quercetin
showed signicant whitening and antioxidant potential and
the loaded niosome forms a spherical shape with a size of 97
nm, 31.1 mV zeta potential, and 87.3% drug trapping
efciency.
336
Rabia et al reported an in vitro assessment of
the nanovesicles containing marigold extract and called it
phyto-niosome.
112
Their results showed marigold and its
entrapped form in a surfactant-based delivery vesicle have
a promising potential for different bio-applications as well as
food, its possible use as a component for food additives and
dermal cosmetic formulations. Niosomes greatly increased
the bioavailability and photostability of quercetin. Quercetin-
loaded niosomes had a prolonged-release, increased trans-
dermal absorption, and skin absorption 2.95 times stronger
than quercetin solution.
113
Niosomes have some additional
advantages over liposomes but also showed some disadvan-
tages. Component of niosome (non-ionic surfactants) is not
generally recognized as safe (as phospholipid in liposomes).
They are indeed more irritant than liposomes.
114
Table 1
reports some examples of phytochemical-encapsulated
niosomes.
The Transfersome
Transfersomes are a type of deformable or elastic nanocar-
riers that were rst emerged in the early 1990s.
64
The regular
liposomes do not permeate into the layers of the skin and
remain conned to the outer stratum corneum layer
(Figure 4).
115
Therefore, new types of lipid vesicles such as
transfersomes have been constructed as an improved type of
liposomes. Transfersome is an elastic and ultra-exible lipid
carrier with highly deformable membranes that enhance the
transfer of compounds to deeper skin tissues.
116
The transfer-
some consists of at least one amphipathic molecule (soy
phosphatidylcholine) and a bilayer softening agent for vesi-
cle exibility (generally a surfactant). When transfersome
components are applied to aqueous systems, they self-
assembled into a lipid bilayer that nally closes into a lipid
vesicle.
116
Studies of penetration and deformability have
shown that transfersomes give deeper penetration of the
skin. Transfersome can be used as medication carriers for
peptides, small molecules, proteins, and particularly herbal
components.
117
In a recent paper, Wu et al prepared resver-
atrol (RSV) loaded transfersomes consisting of the liposomal
system phosphatidylcholine (PC) and the non-ionic edge
stimulators (EA).
337
Results showed that a 5% ethanol and
5% PC/EA (3:1) in distilled water could make the optimum
formulation. The size of vesicles was 40 nm, and the EE%
was 60%. Based on antioxidant activity results, the transfer-
somes were nearly equivalent to the RSV (free RSV) group.
Also, the D1-20(W) formulation showed an improvement of
27% accumulation for in vitro transdermal delivery analysis.
Cell viability analysis revealed that D3-80(W) cytotoxicity
was decreased by 34.45% compared to the free RSV.
118
Because of their susceptibility to oxidative stress, transfer-
somes are not chemically stable. The purity of natural phos-
pholipids is also another factor that limits the adoption of
transfersomes as standard vehicles for the delivery of drugs.
On the other hand, transfersomes can be synthesized on a
large scale with simple and easy processes, without the use of
pharmaceutically unsuitable additives.
64
More examples of
herbal loaded transfersome are shown in Table 1.
The Ethosome
Ethosomes are non-invasive carriers that allow medicinal
products to enter deep skin layers and systemic circulation.-
119
Ethosomes are soft vesicles customized to improve the
delivery of active agents, such as drugs and natural pro-
ducts. They are primarily composed of phospholipids
(phosphatidylserine, phosphatidylcholine, and phosphatidic
acid), high ethanol concentrations, and deionized water.
120
The high concentration of ethanol makes ethosomes the
best choice for skin due to impairment of the skin lipid
bilayer. Thus, when ethanol is incorporated into the vesicle
membrane, it provides the ability to reach vesicles to the
stratum corneum. The lipid membrane in ethosomes is also
packaged less rmly than other vesicles due to the presence
of ethanol and this ability results in improved drug trafck-
ing capability in stratum corneum lipids.
121
The ethosomes
showed to be appropriate in the biotechnology, pharmaceu-
tical, cosmetic, veterinary, and nutraceutical industries for
different purposes. Therefore, these soft vesicles serve as
https://doi.org/10.2147/IJN.S318416
DovePress
International Journal of Nanomedicine 2021:16
6990
Barani et al Dovepress
Powered by TCPDF (www.tcpdf.org)

new vesicular carriers for improved skin delivery.
122
The
size of ethosomes may be modied from nanometers to
micrometers. Ethosomes have been found to be signi-
cantly superior in the quantity and depth of drugs delivered
through the skin compared to liposomes and many other
commercial transdermal and dermal delivery platforms.
123
A comparative evaluation of phytosome, liposome, nio-
some, ethosome, and transfersome in nano-delivery sys-
tems is summarized in Table 2.
Many authors have shown the advantages of ethosomes
as topical vehicles of phytochemicals. Sasindran et al
examined the cytotoxicity of combined herbal extracts
(Zingiber ofcinale, Croton tiglium, and Phyllanthus nir-
uri) and extracts loaded ethosome for transdermal delivery.
Results of the cell-line analysis indicated that ethosomes
loaded with extract inhibit testosterone and improve cell
viability similarly to the standard drug (minoxidil). Even
so, the encapsulated vesicle did not harm the rat skin layer
(based on histopathological study).
124
The drawback of
ethosome is the size variation from nanometers to micro-
meters, due to its poor consistency and evaporation of
ethanol, which leaks out from loaded compounds after a
while. To manage this deciency, alcohol can be located
with a combination of propylene glycol and trehalose.
125
Summarized examples of phospholipids and surfactants
that are used in liposome, niosome, ethosome, and trans-
ferosome preparations are presented in Figure 5. All men-
tioned vesicular systems could be used in phytosome
technology according to their applications.
Phytosome Characterization
Nanomaterial measurement approaches are a rapidly grow-
ing eld, involving effective methods for physical and
chemical characterization.
126
Phytosomes have received
tremendous attention for phytochemical delivery as a
fast-growing class of nanovesicles. Several techniques
were employed to characterize phytosomes size, elemental
composition, morphology, and a wide range of other phy-
sical characteristics. There are physical properties, which
can be investigated by more than one technique. Different
limitations and strengths affect the choice of the most
appropriate method, while a combinational methodology
for characterization is often required.
127
Also, some statis-
tical studies are needed for better application in real
world.
128,129
The main characteristics of phytosomes are
(1) size and shape; (2) surface charge; (3) chemical com-
position; (4) lamellarity and stability; (5) encapsulation
efciency and (6) release behavior. The goal of this
chapter is to provide a thorough summary and a systematic
overview of all analytical instruments used to characterize
phytosomes, including the latest papers.
Average Size and Shape
The evaluation of size and morphology is a critical phyto-
some analysis and provides valuable insight into the quality
and different forms of a sample. Different techniques such as
DLS,
130
microscopic observation
131
(TEM, SEM,
132
optical,
133
atomic force,
134
uorescence,
135
etc.), and ow
136
and size-exclusion chromatography
137
can be used for phy-
tosome size characterization. Electron microscopy is broadly
used for phytosome visualization, and Cryo-TEM and
Freeze-fracture-TEM are the most used.
138
Cryo-TEM
could show phytosomes directly in the frozen state to prevent
phytosomal disruption.
139
Freeze-fracture-TEM provides the
details on liposomal size and morphology without any struc-
tural distortion.
140
Methods of microscopy are generally of
high resolution and rapid productivity, but the sample pre-
paration is complicated and time-consuming; also, some
problems such as shrinking or shape distortion can be gen-
erated in sample preparation.
141
The measurement of phyto-
some size distribution and polydispersity gives data on their
physical stability, which can be evaluated by DLS.
142
DLS is
easy, precise, accurate, very fast and can therefore be used for
regular size distribution measurements of phytosomes.
143,144
The biggest benet of DLS is that the assessment could be
carried out in the sample’s natural environment.
145
The dis-
advantage of this approach is that the heterogeneous emula-
tions could result in false data.
146
Surface Charge
Zeta potential (complete charge generated by medium)
denes the charge of phytosomes in emulsions. Zeta
potential may be negative, positive, or neutral depending
on the composition of the phytosome.
147,148
Zeta potential
could reect the stability of phytosomes in a medium; in
fact, charged particles repel each other enough to maintain
stability. Phytosome emulsion with a zeta potential greater
than or less than 30 mV is known to be stable.
149
The
electrostatic properties of phytosomes can be measured
using Doppler velocimetry,
150
zeta sizer,
151
master size,
152
microelectrophoresis,
153
pH-sensitive uorophores,
154
high-performance capillary electrophoresis,
147
and DLS
instruments.
155
Laser Doppler velocimetry is the method
for measuring the velocity or linear or vibrational motion
of phytosome emulsions using the Doppler Effect in a
laser beam. In light-scattering methods, an electrical eld
International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S318416
DovePress
6991
Dovepress Barani et al
Powered by TCPDF (www.tcpdf.org)

Table 2 Comparative Evaluation of Phytosome, Liposomes, Niosome, Ethosome and Transfersomes in Nano-Delivery Systems
Characteristics Phytosome Liposome Niosome Ethosome Transfersome
Composition Phospholipids and polyphenolic
phytoconstituents
Phospholipids and
cholesterol
Non-ionic surfactant and
cholesterol
Phospholipid, alcohol, polyglycol and water Phospholipids and
surfactant mixture
Flexibility Rigid Rigid Rigid Elasticity Ultra-deformable
Main application Phyto-delivery Drug and gene delivery Drug delivery and cosmetics Skin delivery Skin delivery
Administration Oral, parenteral topical, transdermal Oral, parenteral topical,
transdermal
Oral, parenteral topical,
transdermal
Topical and transdermal Topical and transdermal
Key features High entrapment efciency along with a
depot formation which releases the
contents slowly
Biocompatibility, capacity
for self-assembly, ability to
carry large drug payloads
Improved dispersion of
compounds with solubility
issues, high stability, low-cost
materials
Enhance permeation of drugs across/
through the skin in an efcient manner
High deforming ability
which ensures deeper
penetration in skin
layers
Limitations Leaching of the phytoconstituents which
reduces the desired drug concentration
indicating their unstable nature
Low skin penetration, low
stability
Low skin penetration and
toxicity of surfactant
Poor yield, coalescence and fall apart on
transfer into water, Loss of product during
transfer form organic to water media
Toxic effect of surfactant
Marketed
Product
Leucoselect, Greenselect, Panax
ginseng, Sabalselect, etc.
Doxil, Abelcet, Visudyne,
DepoDur, etc.
Lancome and L’Oreal MaccabiCARE, Nanominox, Trima, etc. Daktarin
https://doi.org/10.2147/IJN.S318416
DovePress
International Journal of Nanomedicine 2021:16
6992
Barani et al Dovepress
Powered by TCPDF (www.tcpdf.org)

is applied to the cell that causes phytosome movement
within the cell. The results of the size were obtained
from these movements of particles.
Chemical Composition
Assessment of the chemical composition and interaction
between vesicle components and phytochemicals is usually
studied by NMR,
156,157
FTIR,
158
and mass spectrometry.
159
Besides, phospholipid quantication in phytosomes can be
done by reaction with an appropriate reagent, followed by a
spectrophotometric quantication.
160
Due to high signal-to-
noise, sensitivity, and selectivity, mass spectrometry is one of
the most credible techniques for determining the phytochem-
ical composition of plant extracts and phospholipids.
113
Many
authors have also applied FTIR techniques to determine the
interaction between phytochemicals and vesicle components.
For example, de Azambuja Borges et al evaluated the interac-
tion between soy isoavone genistein and asolectin-loaded
liposomes by HATR-FTIR, high-eld 31P NMR, and low-
eld 1H NMR methods. The ndings showed that isoavone
reduces the phosphate group’s degree of hydration and
mobility.
161
In another study, Mazumder et al conrmed that
DSC and FTIR can prove the formation of the sinigrin–phyto-
some complex.
35
Chen et al also prepared curcumin-lipo-
somes, and TGA and FTIR showed a successful presence of
SA and PSA in liposomal lipid bilayers and covalent bonding
between SA carboxyl group and WGA amine group.
61
Lamellarity and Stability
The word “lamellarity” represents the number of phytosomal
lipid bilayers.
162
The most used methods for the determination
of lamellarity are electron microscopy methods, 31P nuclear
Figure 5 Amphiphile molecules in phytosomes. The most amphiphile molecules used in vesicle preparation in liposome, niosome, ethosome, and transfersome. Red parts
are polar and black parts are non-polar.
International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S318416
DovePress
6993
Dovepress Barani et al
Powered by TCPDF (www.tcpdf.org)

magnetic resonance, and small-angle X-ray scattering. 31P
NMR is one of the most precise and simple methods for
determining the lamellarity. The weakness of this approach
is that it is sensitive to experimental conditions, such as the
concentration of the reagent, vesicle type, and concentration of
the buffer. Other recently applied visualization methods are
negative staining electron microscopy, freeze-fracture, and
cryo-microscopy. In order to evaluate the architecture of the
vesicle membrane, Nele et al recently merged cryogenic trans-
mission electron microscopy and small-angle neutron disper-
sion and offered insights into the impact of the formulation
method and lipid composition on the development of lipo-
somes with a dened membrane structure.
163
Phytosomal stability is another important factor in the
successful design of a successful carrier. Studies of stability
are performed to explore the phytochemical changes of phy-
tosomes during storage and general circulation. Stability can
be assessed over several months by determining mean vesicle
size, zeta potential, size distribution, and trap efciency.
Cheng et al assessed the thermal and photochemical stability
of rhamnolipids (RL) modied curcumin liposomes and
results showed improved stability of the loaded liposomes
at different pH, ionic, and heat conditions.
118
Encapsulation Efciency and Release
Behavior
Encapsulation efciency (EE percent) describes the
amount of phytochemical that is embedded in the phyto-
some. EE percent can be described as equation 1:
EE%¼IP EP
IP �100 (1)
where EE% is the efciency of encapsulation, EP is encap-
sulated phytochemical and IP is the initial content of
phytochemicals.
The process of encapsulation efciency determination
begins with the removal of free unencapsulated phytochem-
icals from the phytosome emulsion by the Sephadex gel
column, ultracentrifugation, or dialysis method (dened
cut-off) for several hours against buffer solution. Step 2 in
EE estimation is the ruination of the phytosome bilayer
(with Triton X-100, acetonitrile, methanol, and ethanol)
and the quantication of the released active agent by differ-
ent methods, such as enzymatic assays, gel electrophoresis,
uorescence spectroscopy, and eld ow fractionation
chromatographic methods, such as HPLC, UPLC, or
LC-MS.
Drug release behavior of vesicle carriers has been the
subject of extensive research over the past few years, since
the release prole obtained in vitro may provide an indi-
cator of the efciency of the carrier in vivo.
164
Membrane
diffusion strategies (dialysis, micro-dialysis, fractional dia-
lysis, and reverse dialysis), sample and separate strategy,
in situ process, and continuous ow are traditional
approaches that are most widely used to determine the
release rate of active agents.
165–170
Phytochemical release
can be spectrophotometrically determined. Table 3 shows
a summary of the experimental techniques that can be used
for the characterization of the phytosomes.
Table 3 Overview of the Analytical Methods Used for the Characterization of Phytosomes Featured in This Review
Parameter Techniques
Size, and shape DLS, SEM, TEM, Optical microscopy, Fluorescence microscopy, AFM, Field ow fractionation, Nanoparticle
tracking analysis, Scanning ion occlusion sensing, Flow Cytometry, Size-exclusion chromatography, Centrifugal
sedimentation, and DSC.
Surface charge DLS, free-ow electrophoresis, and laser Doppler velocimetry.
Chemical composition FTIR, h1 NMR, GC-MS, LC-MS, DSC, TGA, and Thin-layer chromatography.
Lamellarity and stability 31
P
nuclear magnetic resonance, Small-angle X-ray scattering, electron microscopy methods, DSC, TGA, DLS,
and UV-Vis.
Encapsulation Efciency and
release behavior
Mini-column centrifugation, HPLC, UPLC, UV-Vis, dialysis, enzymatic assays, gel electrophoresis, eld ow
fractionation, sample-and-separate approach, the in-situ method, and the continuous ow.
Optimization Design of Experiment (DoE) with Box–Behnken design
Abbreviations: DLS, dynamic light scattering; SEM, scanning electron microscope; TEM, transmission electron microscopy; AFM, atomic force microscope; DSC,
differential scanning calorimetry; FTIR, Fourier-transform infrared spectroscopy; NMR, nuclear magnetic resonance; GC-MS, gas chromatography-mass spectrometry; LC-
MS, liquid chromatography-mass spectrometry; TGA, thermal gravimetric analysis; HPLC, high-performance liquid chromatography; UPLC, ultra-performance liquid
chromatography.
https://doi.org/10.2147/IJN.S318416
DovePress
International Journal of Nanomedicine 2021:16
6994
Barani et al Dovepress
Powered by TCPDF (www.tcpdf.org)

Biological Activities of Phytosomes
The biological activities related to phytosomes are hetero-
geneous and have been evaluated in more than 100 studies.
To simplify the analysis of the results, papers were divided
based on the body district involved. Accordingly, the phy-
tosome effects on the following categories have been
investigated: cardiovascular, central and peripheral ner-
vous, gastrointestinal, genitourinary, immune, integumen-
tary, musculoskeletal, and respiratory systems. Finally, the
last paragraph was devoted to the effects of phytosomes in
metabolic syndromes. Figure 6 reports the number of
papers related to phytosome and their biological activities,
divided according to the system under study, whereas
Figure 7 collects the number of studies on phytosome
based on the main natural constituent.
Phytosomes and Cardiovascular
Protection
The isoproterenol (ISO)-induced cardiotoxicity model has
been used to evaluate the protective effects of Ginkgo
biloba phytosomes in rats. The results showed that
Ginkgo biloba phytosome (200 mg/kg) alleviated ISO-
induced myocardial necrosis considerably, as conrmed
by histopathological studies. Moreover, the myocardial
necrosis diminished and the endogenous antioxidants
were increased, thus overall making evident the cardiopro-
tective effect.
171
The same researchers explored the possi-
ble protection by cardiovascular injuries of a combined
treatment of Ginkgo biloba phytosome (100 mg/kg) and
Ocimum sanctum extract (OS) (50 and 75 mg/kg) in iso-
proterenol (ISO) (85 mg/kg)-induced myocardial necrosis
in rats. The treatment inhibited the increase of serum
marker enzymes and the lipid peroxidation marker mal-
ondialdehyde (MDA), both induced by ISO. However,
none of the combined treatments possessed better cardio-
protective or antioxidant activity than the single treatment
with Ginkgo biloba phytosome or OS.
172
Tisato et al investigated the anti-inammatory effect of
Ginkgo biloba phytosome and α-Lipoic acid on cytokines
and chemokines released by vein endothelial cells (VEC)
isolated from patients at different stages of CVD. The anti-
inammatory effects of both Ginkgo biloba derivatives
and α-Lipoic acid were conrmed by the reduction of
cell adhesion molecules ICAM-1 and VCAM-1. Ginkgo
biloba phytosome diminished the basal release of PDGF
and the TNF-α-induced PDGF, CXCL10, and RANTES
levels. Based on the data collected, α-Lipoic acid exhibited
a wider and more potent inhibitory activity on the release
of cytokines/chemokines concerning Ginkgo biloba phyto-
some. This study recognized that α-Lipoic acid markedly
Figure 6 Biological activities of phytosomes by system. The graph shows the
number of papers related to phytosomes and their biological activities, divided
according to the system under study. The gastrointestinal, nervous, genitourinary,
and musculoskeletal systems together account for almost the 75% of the published
works. Systems involved in metabolic syndrome were not considered.
Figure 7 Main natural products in phytosomes. The graph collects the number of studies on phytosomes based on the main natural constituent. The columns group
botanical or active principle with 3 or more publications, while constituents with 2 or less studies have been collected in “Others”. The data show a higher prevalence of
phytosomes loaded with pure compounds rather than natural extracts, especially curcumin. All the references corresponding to the individual studies considered are
reported along the manuscript.
International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S318416
DovePress
6995
Dovepress Barani et al
Powered by TCPDF (www.tcpdf.org)

counteracted TNF-α-induced NF-κB and p38/MAPK acti-
vation, whereas Ginkgo biloba mostly acted on Akt.
173
A commercial formulation was examined in a large
sample of CVD patients enrolled in 54 Italian centers.
The supplement contains phytosome of polyphenolic
extract from Vitis vinifera L. seeds, extract from
Melilotus ofcinalis (L.) Pall, and bromelain 100 mg. A
total of 648 patients were enrolled and received 1 tablet/
day and/or standard compression stockings for 90 days. In
all groups, it was reported a notable reduction in the
malleolus circumference, both at the left and the right
limb. A comparable pattern was observed for the severity
of the disease and symptomatology.
174
Muir et al have investigated the clinical efcacy of
Ginkgo biloba phytosome, in the treatment of Raynaud’s
phenomenon (RP). A painful condition characterized by
episodic digital ischemia. A total of 22 patients with RP
and without other associated conditions were enrolled. A
number of 11 patients were randomized to receive Ginkgo
biloba extract (120 mg three times a day for a nal amount
of 360 mg/day), while 11 patients received matching pla-
cebo. The number of RP episodes per week before treat-
ment with Ginkgo biloba phytosome (13.2 ± 16.5) was
reduced by 56%, whereas the placebo reduced the number
by only 27% (p < 0.00001). There were no signicant
dissimilarities in hemorheology among the two groups.
175
Evidence on the Role of Phytosomes in
the Nervous System
The Phytosomes in Cognitive Impairment and
Neuronal Damage
Several papers report the bioavailability of phytosome
concerning the corresponding unformulated products in
animal models, focusing on the tissue distribution of the
active ingredients. Husch et al found a greater amount of
Boswellia acids from Boswellia serrata (ie, KBA, AKBA,
βBA) following the administration of Boswellia-loaded
phytosome.
176
Another study investigated phytosome for-
mulation loaded with Annona muricata water extract
intending to ameliorate its permeability across the blood–
brain barrier (BBB), thus improving the antidepressant-
like activity due to inhibition of monoamine oxidase B
(MAO-B). Through an in vitro transwell model of BBB,
phytosome formulation registered the best performance as
a radical scavenger and MAO inhibitor, thus representing a
useful model to improve the antidepressant-like activity of
the extract.
177
La Grange et al investigated the ability of
silymarin phytosome to protect fetal rat brain by ethanol
administration. Silymarin is a complex of avonolignans
from Silybum marianum Gaertn., namely milk thistle. The
activity of antioxidant enzymes, which include gamma-
glutamyl transpeptidase, was generally higher in the
group treated with the phytosome formulation.
178
Two different studies have been carried on by Naik
et al on the biological activities of a Ginkgo biloba phyto-
some formulation in Wistar rats; in the rst study, oral
administration at 50 or 100 mg/kg, reduced pentobarbi-
tone-induced sleeping time, decreased the chlorpromazine
effects, and induced spontaneous motility in rodents.
Moreover, the formulation exhibited antidepressant effects
in the amnesia induced by scopolamine, showing general
improvement in the behavioral tests.
179
The second study
evaluated the antioxidant activity in the rat brain after
acute (7 days) or subchronic treatment (14 days). Brain
areas including the cerebellum, striatum, cerebral cortex,
and hippocampus were isolated, and the activity of the
antioxidant enzymes, GPx, SOD, CAT, and GR, was
tested, nding phytosomes-induced increased activities in
the brain areas analyzed.
180
Ullah et al studied the ability of a curcumin phytosome
to decrease glial activation in GFAPIL6 mice, an animal
model of chronic glial activation. Formulation adminis-
tered at three doses (218, 438, and 874 ppm) for four
weeks caused a decrease of neuroinammation and num-
ber of activated microglia in the hippocampus (−26.2%)
and the cerebellum (−48%).
181
Recently, our group demonstrated that Centella asia-
tica phytosome administered to adult male rats for ten
days at 20 and 100 mg/kg (calculated as triterpene equiva-
lents) induced BDNF increase in the prefrontal cortex, and
the higher dose generally counteracted cognitive impair-
ment. In the NOR test, the increase in the preference index
was accompanied by increased levels of the Bdnf expres-
sion. In addition, there were no side effects observed
during the treatment.
182
In another paper, we demonstrated
that phytosome loaded with Centella asiatica and
Curcuma longa extracts, administered chronically to rats
(50 or 250 mg/kg for ten days), affected local protein
synthesis through the modulation of BDNF-mTOR-S6
pathway. Our ndings supported the use of this prepara-
tion in subjects with memory and cognitive impairment.
101
The Phytosomes in Neurodegenerative Diseases
Neurodegenerative brain dysfunction is responsible for the
development of dementia in aged people. Bahadur S.
https://doi.org/10.2147/IJN.S318416
DovePress
International Journal of Nanomedicine 2021:16
6996
Barani et al Dovepress
Powered by TCPDF (www.tcpdf.org)

investigated the nanoparticle system to improve the drug
delivery or active compounds with poor availability to the
brain.
183
Langasco et al studied the brain delivery of the
isoavone genistein testing various nanotechnological
approaches; oxidative stress in PC12 cells (neuron cell
line) was diminished by treatment with phytosomes, and
the effect was better than the unformulated genistein.
184
Among phytochemicals, curcumin phytosome was found
to increase curcumin bioavailability in the hippocampus
and frontal lobe following repeated oral administration of
the formulation for ve days (134 mg/kg/die as curcumi-
noids equivalent) in rats. In the frontal lobe, curcumin
appeared 30 minutes after treatment, peaked at 1 hour,
and tended towards normalization after 3 hours, demon-
strating that curcumin phytosome can reach the brain in
rats.
185
Since curcumin possesses anti-amyloid and anti-
inammatory activities, which are mostly used against
neurodegenerative diseases including Alzheimer’s disease,
this nding may be useful for future studies aimed at better
design drug delivery.
186
The Phytosomes in Cerebral Ischemia
Two studies from the same group investigated the potential
positive effects of natural compounds in the middle cere-
bral artery occlusion model in rats. Rutin, a glycoside of
the avonoid quercetin, has been loaded in a phospholipid
structure and tested for its bioavailability in an animal
model of cerebral ischemia. LC-MS/MS analysis revealed
that rutin, administered at 100 mg/kg to Sprague Dawley
rats, reached the brain at concentrations ranging from 20 to
50 ng/g. Rutin-loaded formulation highly ameliorated
functional outcomes in an animal model of stroke.
187
In
the second study, a phytosomal complex containing the
ethanolic extract of Ashwagandha (Withania somnifera)
roots was administered orally (85 mg/kg) to rats 1 hour
before ischemia and six hours post-reperfusion. Treatment
provoked a strong reduction of cerebral infarction (82.7%)
and afforded better protection on all neurological decit
parameters.
188
Effect of Phytosomes in Neuropathy
Di Pajardi et al studied the clinical potential of oral treat-
ment (3 months, n=180) of, curcumin phytosome (500
mg), α-lipoic acid (300 mg), and vitamins of the B group
in subjects with carpal tunnel syndrome awaiting surgical
treatment. Patients receiving supplementation for three
months twice/day both before and after surgery showed a
decrease of night symptoms at 40 days after surgery and
were less likely to reach a positive Phalen’s test at 3
months post-surgery.
189
In neuropathic patients, a similar
formulation based on curcumin phytosome and piperine
and/or α-lipoic acid reduced pain (−66%) in all the com-
binations, after 8 weeks. The supplementation decreased
by 40% the use of conventional therapy (ie, dexibuprofen),
whereas lipoic acid alone did not show statistically sig-
nicant results.
190
The Phytosomes in Migraine
In two studies of the same research group, the efcacy of
Ginkgo biloba terpenes phytosome (60 mg), vitamin B2
(8.7 mg), and coenzyme Q10 (11 mg) as ingredients,
administered twice daily, was investigated in fty subjects
suffering from migraine with aura. Positive effects in
reducing migraine with aura, both frequency and duration,
were already clear within a four-month treatment. These
effects were probably due to the presence of ginkgolide B,
the most abundant terpene identied in the Ginkgo biloba
leaf extract.
191
Ginkgolide B was found to modulate/
reduce the glutamate neurotransmission in the CNS,
which plays a pivotal role in the onset of migraine.
192
The efcacy of the same formulation in the acute stage
of migraine with aura was tested in an open study; during
the rst symptoms of aura, patients orally consumed two
capsules of Ginkgo biloba terpenes phytosome, with no
restriction on analgesic intake during the pain phase.
About 60% of patients enrolled in the study experienced
a reduction of neurological symptoms after treatment;
moreover, the pain phase was completely abolished in
almost 20% of patients.
193
Balzano et al investigated the benecial effects of a
mixture of magnesium, vitamins (riboavin, niacin, vita-
min D), L-tryptophan, and the Boswellia serrata extract-
loaded phytosome, in patients with transient tension
migraine and migraine without aura. The authors consid-
ered pain modulation (NRS scale), monthly attack number,
and analgesic intake. Amitriptyline was used as a refer-
ence compound. The authors found an improvement in all
the outcomes, with greater compliance and no side effects
for patients who consumed the phytosome formulation.
194
The Phytosomes in Nervous System Cancer
Glioblastomas are among the most aggressive malignan-
cies affecting the central nervous system. To search for
novel strategies to cope with the disease, Mukherjee et al
studied the ability of the intranasal delivery of curcumin
phytosome (500 mg, corresponding to 96 mg
International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S318416
DovePress
6997
Dovepress Barani et al
Powered by TCPDF (www.tcpdf.org)

curcuminoids) to cause remission of glioblastoma in the
brain of GL261 (glioblastoma cells)-implanted mice.
Tumor remission was observed in 50% of mice; similar
effects were achieved also using intraperitoneal infusion.
Therefore, the authors suggest that curcumin-loaded phy-
tosome could affect the viability of glioblastoma cells and
also induce repolarization of microglia cells to the tumor-
icidal M1 state.
195
Similar results were obtained by the
same group studying the effects of curcumin phytosome in
natural killer cells and macrophages in GL261 (glioblas-
toma cells)-implanted mice. The treatment also induced
suppression of proteins STAT3 and ARG1, and IL-10
induction of STAT1; suppression of inducible nitric oxide
synthase and caspase 3 activation in the glioblastoma cells
were also observed.
196,197
The same curcumin phytosome was investigated in an
animal model (D425MED) of medulloblastoma, the most
common pediatric central nervous system cancer. The
results reveal negligible effects of formulation using either
oral or intraperitoneal administration; however, no infor-
mation on the dose used was reported.
198
Di Pierro et al studied the efcacy of a Boswellia extract
as a phytosome in cerebral edema induced by radio-che-
motherapy in patients with glioblastoma. Patients (n=20)
received temozolomide and 4500 mg/die of formulation for
a maximum of 34 weeks. The stage of the disease was
evaluated at different times ranging from 4 to 34 weeks
post-surgery, together with steroid consumption. Two sub-
jects showed a signicant decrease in brain edema, thus
leading to better surgical resection. The authors conclude
that supplementation with this type of phytosome may elicit
positive effects in reducing cerebral edema induced by radio-
chemotherapy, and the brain edema reduction may decrease
dexamethasone intake, thus minimizing steroid-induced side
effects during conventional pharmacological treatment.
199
The Phytosomes in the Gastrointestinal
System
The Phytosomes and Gut Microbiota
A recent study compared the inuence of the two different
curcumin-based products, unformulated curcuminoids, and
lecithin-curcuminoid formulation, on human colonic meta-
bolism. Both extracts were subjected to fermentation using
an in vitro fecal model mass spectrometry was used for
curcuminoid quantication and assessment of possible
curcuminoid degradation and detection of the main meta-
bolites in the human fecal fermentation. The results
showed that the fermentation of lecithin-formulated curcu-
minoids caused a more pronounced occurrence of curcu-
minoid catabolites.
200
The Phytosomes and Pancreatic Cancer
The potential synergistic effects of gemcitabine and the
curcumin phytosome in advanced pancreatic cancer were
evaluated in a prospective Phase II trial. A total of 44
patients affected by locally advanced or metastatic pan-
creatic cancer were enrolled and received 2000 mg/die
daily (4 capsules, each of 500 mg) in addition to gemcita-
bine (10 mg/m
2
/min, infusion over 100 min on days 1, 8,
15 every 28 days). The response rate was the primary
endpoint of this study; progression-free survival, overall
survival, quality of life, and tolerability were the second-
ary endpoints. The results of the study suggest that curcu-
min phytosome can be used as a complementary treatment
associated with gemcitabine in the therapy of pancreatic
cancer.
201–203
The Phytosomes Against Bowel Inammation
An open-label, observational, registry study estimated the
effects of a lecithin-based delivery form of standardized
Boswellia serrata extract in patients with minimally symp-
tomatic ulcerative colitis in the remission phase. The 43
patients freely decided to receive 1 tablet of 250 mg/day or
no supplementation for 4 weeks. Diffuse intestinal pain,
bowel movements and cramps, watery stools, blood in
stools, anemia, malaise, rectal involvement, and the num-
ber of white blood cells were attenuated in the supplemen-
tation group. The need for other drugs and medical
examinations was also reduced.
204
Two clinical studies evaluated the efcacy and safety
of Boswellia serrata extract phytosome in irritable bowel
syndrome (IBS). In the rst, 71 healthy subjects with
idiopathic IBS were assigned in three groups and received
hyoscine butyl bromide, papaverine hydrochloride +
Atropa belladonna extract, both administered when
needed, or 1 tablet of phytosome (250 mg/day) for 4
weeks. IBS symptoms showed improvements in all
groups, but only in the phytosome consumption group a
substantial decrease in the need for medical attention and a
lower occurrence of side effects, mainly stypsis, was
detected.
205
The second perspective, a controlled, rando-
mized study evaluated the long-term efcacy and the
safety of phytosome for the prevention of symptoms in
healthy subjects with mild IBS. The same management
strategies of the previous study were applied to 71
https://doi.org/10.2147/IJN.S318416
DovePress
International Journal of Nanomedicine 2021:16
6998
Barani et al Dovepress
Powered by TCPDF (www.tcpdf.org)

subjects. At the follow-up (6 months), compared to the
groups receiving the standard treatment, the phytosome
group exhibited a lower mean score value for nearly all
self-assessed IBS symptoms and a considerably lower
need for medicines and consultations or medical evalua-
tion/admissions.
206
Efcacy of the Phytosomes in Bowel Cancer
The benecial effects of oral silibinin and silybin-phyto-
some against human colorectal HT29 xenograft growth
were compared in vivo in athymic nude mice. A dosage
of 100 mg/kg of the silybin-phytosome exhibited an ef-
cacy close to silibinin 200 mg/kg in reducing tumor weight
and volume.
207
Efcacy of the combination of oxaliplatin and curcu-
min phytosome was investigated in vitro, in oxaliplatin-
resistant cells, and in vivo, in colorectal tumor-bearing
mice. This combination, compared with oxaliplatin alone
and control, improved the antiproliferative capacity of
oxaliplatin in vitro. A positive effect was observed also
in the HCT116 nude mouse xenograft model, with a
decrease of tumor volume, a decrease of pharmacody-
namic markers Ki-67 and Notch-1, and an increase of
cleaved caspase-3.
208
Another study investigated the ef-
cacy of phytosomal curcumin or in association with 5-
uorouracil (5-FU) in vitro or in vivo model of colon
tumor with the presence of colitis. In CT26 cells, curcumin
inhibited cell growth in a concentration-dependent fashion
(0–1000 µg/mL) and notably improved the expression of
E-cadherin. A combination of curcumin (25 mg/kg/day) +
5-FU (35 mg/kg/weekly) diminished the tumor-number
and tumor-size in mice for curcumin or 5-FU alone.
209
The same combination of phytosomal curcumin and 5-FU
was used in a xenograft mouse model of colorectal cancer.
The study showed tumor growth reduction, an increase in
the antitumor effect of 5-FU, and anti-angiogenic effects
across modulation of VEGF and VEGFR2.
210
Hepatoprotective Effects of the Phytosomes
La Grange et al investigated the efcacy of silymarin-
phytosome in the protection of the fetus from maternal
ethanol ingestion in rats. It was compared to the activity of
oral with subcutaneously injected phytosome, with doses
ranging from 400 to 800 mg/kg. All doses suppressed
gamma-glutamyl transpeptidase (GGTP) activity induced
by ethanol in brain and liver tissue, in both the fetuses and
the dams. The highest dose of phytosome administered
orally appeared optimal in reducing maternal brain and
fetal GGTP activity. According to the authors, there may
be a protective activity of the formulation of ethanol
toxicity, as well as direct inhibition of GGTP without
protective activity.
178
The hepatoprotective effects of Ginkgo biloba phyto-
some on carbon tetrachloride (CCl
4
)-induced hepatotoxi-
city were investigated in rats. Ginkgo biloba phytosome
was administered for 10 days in two doses, 25 mg/kg and
50 mg/kg i.p., and silymarin (200 mg/kg P.O.) was used as
the standard reference. Phytosome decreased enzyme
levels of glutamic oxaloacetic transaminase (GOT), gluta-
mic-pyruvic transaminase (GPT), and alkaline phosphatase
(ALP) in serum; levels of SOD, CAT, GPx, GR, albumin,
and total proteins were signicantly increased, and the
GSH levels were found close to control levels. On some
parameters, the effect of the higher dose of Ginkgo biloba
phytosome was comparable with silymarin.
211,212
The
same group investigated the hepatoprotective effects of
phytosome on rimpfacin-induced hepatotoxicity in rats.
Also in this study, Ginkgo biloba phytosome was adminis-
tered at 25 mg/kg and 50 mg/kg, showing hepatoprotective
effects by reducing the levels of serum marker enzymes
and lipid peroxidation; treatment increased the levels of
SOD, GSH, GPx, GR, CAT, albumin, and total protein in a
dose-dependent manner.
213
A Phase III, double-blind, placebo-controlled, rando-
mized clinical trial evaluated the benecial activities of
silybin combined with vitamin E and phosphatidylcholine
on liver function in patients with non-alcoholic fatty liver
disease (NAFLD). Several 180 patients with NAFLD (36
with HCV chronic infection) were enrolled to receive
orally active treatment (n=91) (silybin 94 mg, phosphati-
dylcholine 194 mg, vitamin E acetate 50% 89.28 mg,
twice daily) or placebo (n=88) for 12 months. In patients
receiving the active treatment, improvements in insulin
resistance, transaminases and γ-glutamyltransferase (γGT)
levels and several aspects of liver histology were
observed. In patients HCV-positive, the active treatment
improved markers of brogenesis.
214
Ali et al examined the effects of silybin phytosome
(400 mg/kg), curcumin (400 mg/kg), or α-R-lipoic acid
(200 mg/kg), all given orally, in a model of thioacetamide-
induced liver cirrhosis in rats. All supplements signi-
cantly decreased serum levels of GPT, GOT, LDH, and
γGT; only serum ALP levels were not decreased by silybin
phytosome. Collagen deposition, matrix metalloproteinase
(MMP)-2 activity (MMP-2), TGF-b1 level, and HSP-47
gene expressions were also reduced. Moreover, all
International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S318416
DovePress
6999
Dovepress Barani et al
Powered by TCPDF (www.tcpdf.org)

supplements improved the oxidative stress status through
the increase of liver GSH and the reduction of MDA
levels.
215
Another study compared hepatoprotective activities of
silymarin phytosomes and milk thistle extract (both given
orally as 200 mg/kg/day silybin equivalent for 10 days) in
CCl
4
-induced hepatotoxicity in rats. Silymarin phytosome
increased SOD and decreased GPT levels more efciently
than milk thistle extract (p < 0.05). No signicant differ-
ence between the two treatments regarding other biochem-
ical parameters was observed.
216
The bioavailability of a standardized pomegranate extract
(30% w/w punicalagin – SPE) and soy phospholipids was
compared with unformulated SPE in rats treated with CCl
4
.
Pharmacokinetic studies showed that the formulation of
pomegranate extract and soy phospholipids (500 mg/kg
equivalent SPE) led to the serum concentration of punicala-
gin higher than SPE (C
max
466.3 ng/mL and 192.5 ng/mL
respectively). Antioxidant activity was evaluated at two
doses (100 and 200 mg/kg) as well. Compared with SPE,
pomegranate extract and soy phospholipid combination sig-
nicantly preserved the concentrations of the liver enzymes
SOD, glutathione system, CAT.
217
In vivo Boswellia serrata extract phytosome signi-
cantly decreased the serum levels of the pro-inammatory
cytokines TNF-α and IL-6 and increased the levels of the
anti-inammatory cytokine IL-10 in lipopolysaccharide-
induced systemic inammation in mice. Phytosome
showed antioxidant capacities through a signicant
attenuation of lipid peroxides and increased levels of
GSH, glutathione disulde, and total glutathione concen-
trations. Moreover, treatment was able to restore CYP
transformation and consequently re-establish the biotrans-
formation capacity in the liver.
218
The hepatoprotective activity of a phytosome formu-
lated with the combination of dry ethanolic extracts from
Piper longum fruits and Abutilon indicum leaves was
compared with dry ethanolic extracts from each plant
alone and with LIV 52, an Ayurvedic formulation indi-
cated for liver disorders. Phytosome (100 mg/kg), dry
ethanolic extracts (100, 200, 400 mg/kg), and LIV 52 (1
mL/kg) were administrated orally to rats with liver damage
induced by CCl
4
. Phytosome reduced liver damage mar-
kers (GPT, GOT, ALP, and bilirubin) to a greater extent
than dry ethanolic extracts and in a similar manner to LIV
52.
219
Hepatoprotective effects of curcumin phytosome were
investigated in a model of aluminum chloride (AlCl
3
)
induced hepatotoxicity in rats. Compared with the
untreated AlCl
3
group, treatment with phytosome (200
mg/kg/day for 21 days) notably normalized the hepatic
markers increased by AlCl
3
(GOT, GPT, ALP, LDH, and
bilirubin).
220
The chemopreventive effect of curcumin phytosome
was evaluated and compared with unformulated curcumin
on hepatitis B virus related-hepatocellular carcinoma by
using a transgenic mouse model. Phytosome showed
greater efcacy in reducing hepatocellular carcinoma
growth, reduction of total tumor volume, and anti-inam-
matory activity than unformulated curcumin.
221
The Phytosomes Effect in the
Genitourinary System
This section describes the biological activities, which
affect the genitourinary tract, including the breast, as a
gland linked to the reproductive system.
The Phytosomes and Breast Cancer
In the rst study, twelve early breast cancer patients were
treated for 4 weeks with a commercial lecithin formulation
containing catechins from green tea, at a daily dose of 300
mg (corresponding to 44.9 mg of epigallocatechin-3-gal-
late or EGCG) before surgery. The research showed the
ability of the active principles to reach human breast
tissue; concentrations up to 8 ng/g of EGCG were found
in all the tumor tissues tested. The evaluation of Ki-67, as
a biomarker of proliferation, demonstrated a signicant
inverse correlation with EGCG plasma levels for each
patient.
222
The same research group evaluated the activity of a
complex of silybin-phosphatidylcholine, in another group
of 12 breast cancer patients, 2.8 g per day for 28 days. The
concentration of silybin reached up to 177 ng/g in breast
tumor tissues, but non-changes in Ki-67 was noted, as well
for insulin-like growth factor 1 (IGF-I) and nitric oxide
blood levels.
223
In vitro, silybin-phosphatidylcholine treat-
ments obtained a concentration- and time-dependent
decrease in viability of SKBR3, a cell line of human breast
adenocarcinoma, conrming a superior membrane trans-
mission (more than 1.5 times) and inhibitory effect on
growth (more than 2 times) compared to pure silybin.
Both silybin and silybin-phosphatidylcholine downregu-
lated Human Epidermal Growth Factor Receptor 2
(HER2) expression, but the complex gave better results
in longer treatment times (72 h).
224
A study prepared and
evaluated the effect of phytosomes containing luteolin in
https://doi.org/10.2147/IJN.S318416
DovePress
International Journal of Nanomedicine 2021:16
7000
Barani et al Dovepress
Powered by TCPDF (www.tcpdf.org)

MDA-MB 231 breast tumor cells. Phytosomes increased
the activity of doxorubicin in inhibiting the growth of
cancer cells, compared to the pure compound. Besides,
the related phytosomes were more active than luteolin in
inhibiting the gene expression of nuclear factor erythroid
2-related factor 2 (Nrf2), lowering the antioxidant defense
of cancer cells.
225
Similarly, quercetin phytosomes also increased the ef-
ciency of doxorubicin on the growth of MCF-7 human
breast cancer cells. Despite no signicant effects were
observed on Nrf2 gene expression, quercetin phytosomes
decreased the expression of two Nrf2-activated genes,
NAD(P)H dehydrogenase (quinone) 1 (35%) and multi-
drug resistance-associated protein 1 (43%), more ef-
ciently than pure quercetin.
226,227
MCF-7 cells treated with a commercial phytosomal-cur-
cumin, showed a dose–response inhibition of proliferation
and invasion, linked to higher levels of E-cadherin and
MMP-9. Moreover, phytosomal-curcumin enhanced the bio-
logical activity of uorouracil in inhibiting tumor growth of a
xenograft mouse model (female BALB/c), by positively
regulating MDA levels, catalase, total thiol concentration,
and SOD in breast cancer tissue. However, the phytosome
alone, without uorouracil, reduced growth to a lesser extent,
without modulating the individual parameters.
228,229
Phytosomal-curcumin was tested in female BALB/c mice
with metastatic breast tumor (4T1). Mice were fed with the
phytosomal-curcumin for 14 days with 10 mg once every 3
days. While the treatment alone had minor effects on the
primary tumors, it signicantly decreased the number of
metastases in the lung at a dose of 10 mg/mouse. Although
this study lacks comparative data on pure curcumin, the
animals treated with cryoablation and phytosome did not
improve their survival rate with respect to the animals with
saline, or just cryoablation or phytosomal-curcumin alone.
230
Finally, two studies by the same research group evaluated
phytosome bilayer-enveloped casein micelles or phosphati-
dylcholine-casein micelles, containing Monascus yellow pig-
ments (Monascus purpureus) and resveratrol, by comparing
also folate conjugated and PEGylated phytosome modica-
tions. All forms of phytosomes induced higher toxicity in
MCF-7 cells comparing to the cotreatment of free resveratrol
and Monascus yellow pigments. Tumor-bearing BALB/c
mice received through injection the pure compounds/mix-
tures or phytosomes, corresponding to 5 mg/kg per day of
resveratrol, for consecutive 21 days. At 250 μg/mL, the
percentage of hemolysis induced by phytosomes was lower
than 5%. Phytosomes were superior in tumor regression
concerning coadministration of free resveratrol and
Monascus yellow pigments. Treatments with phytosomes
better-reduced aromatase, NF-κB, VEGF, and CD1 levels,
and increased caspase-3 level and necrosis.
231,232
The Phytosomes Role in Prostate Diseases
Three studies evaluated the effects of silibinin-loaded phyto-
some, in the eld of prostate cancer. In the rst in vivo study,
TRAMP male mice, characterized by a palpable prostate
tumor, were exposed to 0.5% or 1% w/w of a phytosome
diet. After 11 weeks, the diet dose-dependently decreased the
weight of the prostate together with tumors (up to 60%),
suppressing metastasis formation by reducing broblast
growth factor (bFGF), VEGF, MMP-2, and MMP-3.
Silibinin led to higher levels of E-cadherin in parallel with
a reduction of vimentin and also snail-1 in tumors.
233
Two
clinical studies evaluated the effects on humans. The rst
pharmacokinetic Phase I study involved 13 subjects with
prostate cancer. Phytosome was increased from a 2.5 g to a
20 g orally daily dose, but a persistent grade 2 hyperbilir-
ubinemia was registered at 15 and 20 g. Silibinin, rapidly
conjugated, was released into the urine, pointing out a short
plasma half-life, in a range of 1.79–4.99 h. None of the
patients under study achieved a 50% reduction in PSA, but
several patients experienced a prolonged stable disease.
234
The second study from the same research group admi-
nistered 13 g of silibinin phytosome daily to 6 prostate
cancer patients, for 14–31 days before radical prostatect-
omy. The plasma silibinin levels were relatively low (1.2
μM) at the end of the treatment. Only three patients out of
six showed silibinin values reaching from 14.9 to 496.6
pmol/g in prostatic tissues. IGF-I, IGFBP-3, or PSA levels
were not signicantly changed.
235
The latest research investigated the application of cur-
cumin phytosome formulation in patients with benign pro-
static hyperplasia. The phytosome was administered as
two tablets per day (2 × 500 mg per day, equivalent to
200 mg of daily curcumin) to 33 subjects (range: 55–65
years) in association with the best standard management.
All symptoms including urination frequency, intermit-
tency, urgency, straining, and nocturia improved with cur-
cumin administration compared to standard management,
except for stream weakness.
236
The Phytosomes in Female Reproductive System
Conditions
A clinical study evaluated the effect of curcumin phyto-
some in 6 patients with endometrial cancer. Patients
International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S318416
DovePress
7001
Dovepress Barani et al
Powered by TCPDF (www.tcpdf.org)

received the supplement for 2 weeks with 2 g (4 × 500 mg
per day) without simultaneous oncological treatments.
Supplementation lowered MHC expression on leukocytes,
the number of monocytes, and ICOS protein levels on
CD8 +T cells. No other signicant changes were observed
in inammatory markers, such as number of different
immune cell types, activation of T cell, and the protein
levels of cyclooxygenase-2 (COX-2).
237–239
A second study evaluated the effect of a quercetin phyto-
some (10 or 50 mg/kg, per os) in 48 rats subjected to ovar-
iectomy. Treatment with phytosome induced a signicant
increase of calcium, inorganic phosphorus, and glutathione
in serum, compared to the corresponding doses of free quer-
cetin. Compared to quercetin, the phytosome signicantly
lowered serum alkaline phosphatase, TNF-α, acid phospha-
tase, MDA, and glucose level and also positively modied
the lipid prole.
131
Furthermore, a study evaluated an icariin-
containing phytosome in OVCAR-3 ovarian cancer cells.
The phytosomes showed higher cytotoxicity versus ovarian
cancer cells compared to pure icariin (6.31 vs 13.1 µM) and
in particular, the number of cells in the G2-M phase, the
caspase-3 content, and intracellular ROS were enhanced
following incubation with phytosomes.
240
The Phytosomes in Urinary Tract Dysfunctions
Two clinical trials evaluated the biological effects of phy-
tosomes in the urinary tract. In the rst research, cranberry
was studied in 13 healthy volunteers to evaluate the
Candida albicans antiadhesive properties of urine after
cranberry extract phytosome or the corresponding standar-
dized extract consumption. The subjects consumed 2 cap-
sules of cranberry phytosome or cranberry extract per day,
for a week and urines were analyzed at different times.
The fractions retrieved after 12 h of extract or phytosomal
form treatment signicantly and similarly inhibited the
adhesion of C. albicans, but phytosome contained only
33% of the cranberry extract (phytosome: 12 mg proantho-
cyanidins/capsule; extract: 36 mg proanthocyanidins/
capsule).
241
The second study explored the effect of curcumin in
asymptomatic patients suffering from temporary kidney
dysfunction. Patients consumed 3 capsules/day for 4
weeks, containing curcumin phytosome (300 mg of curcu-
min). The subjects treated with curcumin phytosomes had
signicantly higher improvement in micro- and macro-
albuminuria and oxidative stress levels than those on stan-
dard management. The number of patients experiencing
fatigue was signicantly reduced by curcumin phytosome,
and compliance and tolerability were good.
242
The Phytosomes as Modulators of the
Immune System
A couple of studies evaluated the effects of phytosome on
parameters related to immune function. Silymarin loaded
on liposome/phytosome (lecithin: cholesterol ratio 6:1)
showed improved prevention of ROS release compared
to unformulated silymarin in RAW 264.7, a murine macro-
phage cell line. In vivo study performed for seven days in
Wistar rats (50 mg/kg) exerted protection against liver
toxicity and inammation induced by paracetamol.
243
Another study evaluated the immunomodulatory effects
of the phytosomal formulation of grape seed extract, that
is particularly rich in epigallocatechin 3-O-gallate. One-
month administration of grape seed phytosome (300 mg/
die) to elderly patients inuenced the immune response, as
shown by serum cytokine assessment. In particular, the
treatment increased both IL-2 and INFγ production, thus
suggesting a possible role in the Th1/Th2 rebalance in
atopic frail elderly or the enhancement of antiviral
response.
244
The Phytosomes Effect in Integumentary
System
The formulations evaluated at the skin level are more
disparate and can be collected in three main areas: skin
inammatory conditions, wound healing, and skin cancer.
The Phytosomes in Skin Inammatory Conditions
Two clinical studies showed the effect of phytosomes in the
eld of skin inammation. A rst blind trial with 30 volunteers
investigated the topical effect of a quercetin phytosome, in
comparison to a formulation containing 1% dexchlorphenir-
amine, in different types of skin insults. Quercetin phospholi-
pids 1% and dexchlorpheniramine 1% obtained similar results
by signicantly reducing UV-induced erythema (−10.05% vs
−14.05%, respectively) and in histamine prick test (−13.25%
vs −12.23%, respectively). When erythema was induced by
sodium lauryl sulfate (SLS) or glycolic acid (GA) only quer-
cetin phospholipids 1% induced a signicant increase in
hydration, but both the formulations reduced erythema.
245
In
a Phase III randomized, single-dose, and double-blind placebo
clinical trial, 49 patients with chronic psoriasis were treated
orally for 12 weeks with phytosome (2 g per day) or placebo,
while topically applying once daily methylprednisolone ace-
ponate 0.1% ointment on psoriasis plaques. Curcumin
https://doi.org/10.2147/IJN.S318416
DovePress
International Journal of Nanomedicine 2021:16
7002
Barani et al Dovepress
Powered by TCPDF (www.tcpdf.org)

phytosome obtained a better effect on PASI compared to
placebo. No signicant reduction of IL-17 serum levels was
observed between the groups, but IL-22 serum levels were
lower in the curcumin treated group (−11.8 pg/mL).
246
Another study evaluated the effects of curcumin phytosomes
in carrageenan-treated mice. Indomethacin, curcumin, or
nano-phytosome of curcumin at 15 mg/kg were administered
orally to animals for one week. The nano-phytosome treatment
was more antioxidant than curcumin (P < 0.05) in the case of
SOD, CAT, GPx, and GR and had a higher latency time
compared to curcumin in hot plate and tail-pinch tests.
247
Three studies evaluated the topical effects of three
different phytosomes in carrageenan-induced edema in
Wistar rats. The Lawsone-containing phytosome complex
(Lawsonia inermis L.) had a higher anti-inammatory
effect than plant extract gel at 4 h (P < 0.001).
30
Escin
β-sitosterol (ES) phytosome 5% hydrogel showed signi-
cantly improved efcacy in antihyperalgesic activity com-
pared to escin and ibuprofen 5% gel.
248
A resveratrol
phospholipid complex, topically applied with patches,
reduced the swelling to 6.1% after 24 h, a value signi-
cantly lower than control (38.4%) and diclofenac sodium
gel groups (23.2%) (P < 0.05). Resveratrol phospholipid
complex containing patches resulted in non-irritant effects
in albino rabbits, with a skin irritation score (erythema and
edema) of less than 1.
249
Silymarin in nanostructured lipid
carriers (NLC) complex was topically applied in rats sub-
jected to UVB irradiation (0.115–0.23 J/cm
2
). Silymarin-
NLC gel application decreased the epidermal thickness
and wrinkle score in UV exposed animals.
250
The Phytosomes Effect in Wound Healing
A combination of Ginkgo biloba, α-lipoic acid, and grape
seed phytosome associated with advanced medications,
was benecial in the treatment of chronic diabetic ulcers
in subjects with diabetic foot ulcers.
251
Phytosomes con-
taining Moringa oleifera aqueous leaf extract were found
to be non-toxic in NHDF cells till 3.0 mg/mL. The for-
mulation at 1 mg/mL provided the shortest gap closure
time (94.8% at 24 h) compared to the extract at the same
concentration. Conversely, higher doses (1.25 and 1.50
mg/mL) did not reach statistically signicant results, as
well for lower doses.
252
A second in vitro study of NHDF
cells evaluated a combination of gold nanoparticle (AuNP)
and Calendula ofcinalis in phytosomal systems. The for-
mulations reduced the interruptions of cell monolayer by
about 42.2% for Calendula phytosomes and 58.7% for
AuNP–Calendula phytosomes (p < 0.01). The
combination did not show toxic effects up to 400 μg/
mL.
33
A complex of sinigrin-phytosome displayed bene-
cial effects on wound healing with respect to sinigrin
alone, in HaCaT cells. After 42 h, the phytosome at 0.14
mg/mL completely solved the wound, whereas pure sini-
grin reached only 71%, with negligible cytotoxicity
towards cells.
35
Evidence of the Phytosomes Efcacy in Skin Cancer
Only two studies evaluated the potential effectiveness of
phytosomes in ghting skin cancers. The rst study
showed a cytotoxic effect of the aforementioned sinigrin-
phytosome complex in A-375 melanoma cells. At 0.14
mg/mL, the complex inhibited by almost 74% the cell
viability, more than 46% displayed by free sinigrin, but
only minimal toxicity was observed in non-tumoral
HaCaT cells.
35
The second study considered the effect of
silymarin in nanostructured lipid carriers (NLC) in vitro.
Silymarin-NLC showed a higher inhibition (IC
50
: 21 μg/
mL) in cell viability of the human melanoma cell line (SK-
MEL-2) in comparison to a non-specied phytosome com-
mercial formulation (IC
50
: 26 μg/mL).
250
The Phytosomes Effect in Musculoskeletal
System
Pharmacological treatment of musculoskeletal dysfunc-
tions is mostly based on non-steroidal anti-inammatory
drugs (NSAIDs) or analgesics; unfortunately, the therapy
is often accompanied by several side effects. Among 16
studies regarding natural product-loaded phytosomes for
treatment of musculoskeletal disorders, 62.5% were
related to turmeric (Curcuma longa) extracts or curcumin,
and 31.2% related to Indian Frankincense (Boswellia ser-
rata) extracts.
One pilot study investigated the treatment of patients
with osteopenia. Subjects with low bone density and no
symptoms were treated for 24 weeks with the curcumin-
based supplementation curcumin phytosome. The bone
density of the heel, small nger, and upper jaw was assessed
at 4, 12, and 24 weeks. A general improvement in bone
density was observed in the group treated with 1 tablet/day
containing 1000 mg of curcumin phytosome, whereas no
signicant changes were observed in the control group.
253
The same formulation, tested at the same dose either or not
combined with other nutritional supplements and exercise,
showed positive results in elderly subjects (>65 years)
characterized by loss of strength, contributing to improving
strength and physical performance.
254
International Journal of Nanomedicine 2021:16 https://doi.org/10.2147/IJN.S318416