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Molecules 2021, 26, 802. https://doi.org/10.3390/molecules26040802 www.mdpi.com/journal/molecules
Article
Positive Effect of Cannabis sativa L. Herb Extracts on Skin
Cells and Assessment of Cannabinoid-Based
Hydrogels Properties
Martyna Zagórska-Dziok *, Tomasz Bujak, Aleksandra Ziemlewska and Zofia Nizioł-Łukaszewska
Department of Technology of Cosmetic and Pharmaceutical Products, Medical College,
University of Information Technology and Management in Rzeszow, Kielnarowa 386a, 36-020 Tyczyn, Poland;
tbujak@wsiz.rzeszow.pl (T.B.); aziemlewska@wsiz.rzeszow.pl (A.Z.); zniziol@wsiz.rzeszow.pl (Z.N.-Ł.)
* Correspondence: mzagorska@wsiz.rzeszow.pl
Abstract: The skin is an organ that is constantly exposed to many external factors that can affect its
structure and function. Due to the presence of different cannabinoid receptors on many types of
skin cells, cannabinoids can interact directly with them. Therefore, as part of this work, the impact
of two types of Cannabis sativa L. herb extracts on keratinocytes and fibroblasts was assessed. The
content of biologically active compounds such as phenols, flavonoids, chlorophylls and
cannabinoids was evaluated. The antioxidant capacity of prepared extracts using the DPPH radical,
H
2
DCFDA probe and measurement of superoxide dismutase activity was also assessed. The
cytotoxicity of hemp extracts was determined using the Alamar Blue, Neutral Red and LDH assays.
The ability of the extracts to inhibit the activity of matrix metalloproteinases, collagenase and
elastase, was assessed. Preparations of model hydrogels were also prepared and their effect on
transepidermal water loss and skin hydration was measured. The obtained results indicate that
hemp extracts can be a valuable source o f biologically activ e substances that red uce oxidative stress ,
inhibit skin aging processes and positively affect the viability of skin cells. The analysis also showed
that hydrogels based on cannabis extracts have a positive effect on skin hydration.
Keywords: Cannabis sativa L.; antioxidants; metalloproteinase inhibitors; cytotoxicity; skin cells;
hydrogel
1. Introduction
Plant cells produce numerous chemicals that are secreted as physiological
components or as by-products of metabolism. They show anti-inflammatory, cytotoxic,
but also antibacterial or antifungal properties [1–3]. A very large group of them have
strong antioxidant properties [4]. To a large extent, their properties can be associated with
the regulatory effect of plant substances on the oxidative-reduction processes occurring
in cells, including maintaining redox balance [5,6]. Many factors can disturb the oxidative
balance, leading to oxidative stress, as a result of which the amount of substances capable
of neutralizing free radicals is much lower than the amount of free radicals. Therefore, it
is very important to support the body’s endogenous protective system by exogenous
antioxidants, which may have a significant impact on the function of various types of
important proteins, cell signaling process or a number of enzyme systems [6–8].
The variety of chemical compounds found in plant material has been inspiring
scientists for years and contributes to the fact that active compounds contained in plant
materials are used in many industries [9]. Plant extracts are increasingly used in
pharmaceutical and cosmetic research, which aims to create new drugs, supplements and
cosmetic products [10]. Modern research methods allow standardization of extracts
composition and full quality control of products. Their widespread use in many industries
Citation: Zagórska-Dziok, M.;
Bujak, T.; Ziemlewska, A.;
Nizioł-Łukaszewska, Z. Positive
Effect of Cannabis sativa L. Herb
Extracts on Skin Cells and
Assessment of Cannabinoid-Based
Hydrogels Properties. Molecules
2021, 26, 802. https://doi.org/
10.3390/molecules26040802
Academic Editors: Giuseppe Caruso,
Nicolò Musso and Claudia Giusep-
pina Fresta
Received: 30 December 2020
Accepted: 31 January 2021
Published: 4 February 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(http://creativecommons.org/licenses
/by/4.0/).
Molecules 2021, 26, 802 2 of 23
results from their multifunctionality. In addition to antioxidant activity, plant extracts
contain a number of active substances that have protective, regenerative and anti-
inflammatory properties [7,11,12]. Particularly noteworthy is the group of compounds
called cannabinoids occurring naturally in plants as phytocannabinoids and in animal
organisms as endocannabinoids. Cannabinoids are lipophilic compounds interacting with
cannabinoid receptors present in mammalian cells. Hemp is one of the main sources of
phytocannabinoids [13,14] and belongs to the group of phenolic terpenoids and are
synthesized primarily in the secretory gland of female flowers. Currently, more than 100
different phytocannabinoids are known [15] and the most important include delta-9-
tetrahydrocannabinol (THC), cannabidiol (CBD), cannabidiol acid (CBDA), cannabinol
(CBN) and cannabigerol (CBG). One of the most common cannabinoids found in different
varieties of cannabis plants is THC. The content of THC in plants ranges from less than
0.2% (in fibrous varieties) up to even 30% in female flowers in other varieties of cannabis
[15–17]. The phytocannabinoids also include CBD, which, unlike THC, does not activate
G-protein-related endocannabinoid receptors, causing no psychostimulatory effect of this
compound. In addition, CBD may enhance the beneficial effects of THC by increasing its
therapeutic range. It has also been shown that the polyphenolic nature of CBD makes it a
powerful antioxidant [18]. It should be noted that in fresh plant material, 95% of CBD is
in the form of cannabidiolic acid (CBDA), which has a strong anti-inflammatory effect.
Cannabinol (CBN) and cannabigerol (CBG) also exhibit this effect [19–21].
In addition, hemp is a rich source of many active substances. A large group of
compounds are terpenes and sesquiterpene which are responsible for the characteristic
smell of cannabis. In addition, terpene components of hemp may have synergistic
properties with phytocannabinoids and enhance their health-promoting effects [22,23].
Polyphenolic compounds, which include flavonoids (flavones and flavonols), stilbenes
and lignans, are largely responsible for the antioxidant activity of the plant material.
Thanks to the lignin content, hemp raw materials also provide effective protection against
UV radiation [19,20].
The aim of this work was to present the hemp extract as a multifunctional ingredient
in cosmetic and pharmaceutical preparations intended for skin care. For this purpose, the
antioxidant and cytotoxic properties of water-ethanol extracts from Cannabis sativa L. were
assessed. Due to the fact that oxidative stress significantly affects the condition of the skin,
this work includes an assessment of the ability of hemp to scavenge exogenous free
radicals, affect the intracellular level of reactive oxygen species and increase the activity
of antioxidant enzyme-superoxide dismutase. The work also contains the determination
of the content of biologically active compounds such as phenols, flavonoids and
cannabinoids. Various cytotoxicity assays have also been carried out, such as Alamar Blue,
Neutral Red and LDH to assess the cytotoxicity of the obtained extracts for skin cells-
keratinocytes and fibroblasts. In addition, to determine potential anti-aging properties,
the ability to inhibit collagenase and elastase activity as well as the effect of hydrogels
based on cannabis extracts on transdermal water loss and skin hydration were
determined.
2. Results and Discussion
2.1. Determination of Biologically Active Compounds
Polyphenols and flavonoids are the basic active ingredients of plant extracts. They
are responsible for their antioxidant activity, by neutralizing free radicals that may
generate oxidative stress. Oxidative stress is one of factors inducing skin aging processes
and inhibiting its regeneration ability [7]. Long-term oxidative stress in skin cells
(occurring e.g., after prolonged skin exposure to the sun, as well as due to many external
factors, e.g., smog) may leads to DNA, protein and lipid damage, disrupting many natural
processes, including degradation and synthesis of collagen and elastin-basic proteins that
play the most important role in a skin aging process, regeneration or wound healing [24–
Molecules 2021, 26, 802 3 of 23
26]. Chlorophyll may also enhance the antioxidant effect [6,24]. The research showed that
the extraction method of the hemp herb has significant impact on the concentration of
flavonoids and polyphenols, as well as chlorophyll in the obtained extract [15,25]. The
results (calculated per 1 g of dry extract and 1 g of dry hemp herb) are presented in Table
1. The extract obtained by ultrasonic extraction is characterized by a higher content of
polyphenols, flavonoids and chlorophyll compared to the extract obtained using the
traditional method. For the ultrasound (UAE) extract, 20% higher concentration of
polyphenols, about 30% higher of flavonoid and twice higher chlorophyll concentration
were obtained (Table 1).
Table 1. Total content of polyphenols (TPC), flavonoids (TFC) and chlorophyll in ultrasound
assisted (UAE) and magnetic stirrer assisted (MAE) hemp extracts (DWE—dry weight of extract,
DWH—dry weight of herb). The content of phenolic compounds was determined using gallic acid
(GAE) and flavonoids using quercetin (QE).
TPC
[mg GAE/g DWE]
TFC
[mg QE/g DWE]
Chlorophyll a
[mg/g DWE]
Chlorophyll b
[mg/g DWE]
Chlorophyll a +
b [mg/g DWE]
MAE 42.524 ± 0.005 a 8.091 ± 0.010 a 1.923 ± 0.04 a 0.241 ± 0.015 a 2.642 ± 0.023 a
UAE 51.322 ± 0.012 b 10.374 ± 0.009 b 4.372 ± 0.022 b 0.821 ± 0.010 b 5.404 ± 0.042 b
TPC
[mg GAE/g DWH]
TFC
[mg QE/g
DWH]
Chlorophyll a
[mg/g DWH]
Chlorophyll b
[mg/g DWH]
Chlorophyll a +
b [mg/g DWH]
MAE 2.511 ± 0.011 a 0.483 ± 0.011 a 0.113 ± 0.005 a 0.014 ± 0.016 a 0.156 ± 0.019 a
UAE 3.184 ± 0.008 b 0.642 ± 0.005 b 0.271 ± 0.009 b 0.051 ± 0.015 b 0.335 ± 0.022 b
a,b Different letters in the table indicate significant differences between groups (p < 0.05).
Concentration of active ingredients in hemp extract depends on the extraction
method, temperature, a type of solvent, as well as the part and variety of the plant from
which they are obtained. As shown in previous studies [27–32], the total content of
phenols per 1 g of dried plant or dry extract is about 0.09–0.56 mg in hemp leaves, 4.7–8.1
mg in flowers, 0.77–51.6 mg in seeds and 10.51–52.58 mg in inflorescences. In the research
of Maqsood et al. [26] the influence of different solvents on the concentration of
antioxidants in cannabis leaf extracts was compared. It was shown that the highest content
of phenols was characterized in aqueous and methanolic extracts, while extracts obtained
using organic solvents did not show the content of phenols. The highest content of
flavonoids (about 55–60 mg/g of the extract) was found in methanolic and ethanolic
extracts. Lower concentrations of these substances were found in chloroform and acetone
extracts (about 20 mg/g of the extract). Flavonoids were not found in root extract while
inflorescences were characterized by a content of flavonoids about three times lower than
the leaves [29]. The lower concentration of flavonoids in extracts analyzed in this work is
due to the fact that herb of hemp was used, containing leaves, inflorescences and stems of
hemp.
As part of this work, a chromatographic assessment of the amount of individual
cannabinoids in the prepared extracts was also carried out. The analysis confirmed that
the selection of ultrasonic extraction results in a higher concentration of cannabinoids in
Cannabis sativa L. extracts compared to the extractions using a traditional method using a
magnetic stirrer. The results of the tests showed that the cannabinoids that occur in the
largest amount in the obtained extracts are Cannabidiol Acid (CBD-A) and Cannabidiol
(CBD) (Table 2).
Table 2. The content of individual cannabinoids in hemp UAE and MAE extracts (DW—dry
weight of extract).
Chemical Compound MAE [mg/g DW] UAE [mg/g DW]
Cannabidiol (CBD) 12.00 ± 1.43 31.00 ± 2.86
Cannabidiol acid (CBD-A) 130.00 ± 1.92 150.00 ± 16.84
Molecules 2021, 26, 802 4 of 23
Cannabigerol (CBG) Not detected Not detected
Cannabigerolic acid (CBG-A) 4.20 ± 0.38 6.30 ± 0.52
Delta-9-tetrahydrocannabinol (THC) 2.60 ± 0,19 4.00 ± 0.34
Tetrahydrocannabinolic acid (THC-A) 4.10 ± 0,46 6.50 ± 0.52
Cannabinol (CBN) Not detected Not detected
2.2. Assessment of Antioxidant Activity
The antioxidant properties of plants are extremely important in the context of
protecting cells against the adverse effects of various external factors. Therefore, the next
stage of the study was to assess the prepared UAE and MAE extracts from hemp in terms
of their ability to scavenge the DPPH free radical, changes the activity of superoxide
dismutase (SOD) and the impact on the amount of reactive oxygen species produced
inside the tested cells-fibroblasts and keratinocytes.
Analysis aimed at assessing the antioxidant properties of the analyzed extracts by
assessing the ability to scavenge the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical clearly
indicated that the extracts of Cannabis sativa L. have the ability to neutralize the DPPH
radical in a dose-dependent manner. UAE extracts, in which analysis showed a greater
amount of biologically active compounds compared to MAE (Tables 1 and 2), showed
better antioxidant properties compared to MAE extracts at the concentration of 500 and
1000 μg/mL. After using the UAE extract, more than 40% free radical scavenging was
observed, while in the case of the extract obtained on a magnetic stirrer, this inhibition
reached up to 30% for the highest concentration used (1000 μg/mL). In the analysis, it was
observed that as the concentration of both extracts increased (in the range of analyzed
concentrations), the antioxidant capacity of the extracts was also higher (Figure 1A,B).
This correlates with the results obtained by other authors who pointed to the antioxidant
properties of chemical compounds whose presence in the analyzed hemp extracts was
confirmed in this work [33,34].
Figure 1. Kinetics of the absorbance changes in DPPH• solutions in the presence of various concentrations (1–1000 μg/mL)
of UAE (A) and MAE (B) extracts of Cannabis sativa L. herb. Values are mean of three replicate determinations (n = 3).
Before starting the analysis using 2′,7′-dichlorodihydrofluorescein diacetate
(H
2
DCFDA), it was checked whether the cannabis extracts alone (without the cells tested)
affect H
2
DCFDA fluorescence. After excluding these interactions, the plant extracts were
tested on cell lines. The research showed that the hemp extract effect varies depending on
the concentration used and the cell type. In the case of fibroblasts (BJ), after using UAE
extract from Cannabis sativa L. in the concentration range of 1–250 μg/mL, the level of
intracellular reactive oxygen species (ROS) was below the value obtained for the control
(cells not treated with the extract). The cells also treated with 1 mM hydrogen peroxide
(H
2
O
2
) were used as a positive control. Compared to the control, doses above 250 μg/mL
resulted in a statistically significant increase in intracellular ROS production. (Figure 2A).
Similar results were obtained for keratinocyte cells (HaCaT). The two highest
concentrations of hemp extract caused a statistically significant increase in ROS
Molecules 2021, 26, 802 5 of 23
production, while the values of normalized fluorescence for concentrations of 250 μg/mL
and lower are below the control value which indicates a reduced amount of reactive
oxygen species (Figure 2B). In the case of fibroblasts, a statistically significant decrease in
ROS production was observed at the concentrations of 1 and 100 μg/mL, while in the case
of keratinocytes at the concentrations from 1–250 μg/mL. The obtained results show that
lower concentrations of the tested extracts show a protective effect on the cells tested,
thereby reducing oxidative stress inside the cells.
Molecules 2021, 26, 802 6 of 23
Figure 2. The effect of UAE Cannabis sativa L. extract on the 2′,7′-dichlorofluorescein (DCF) fluorescence in HaCaT (A) and
fibroblasts cells (B). The data are expressed as the mean ± SD of 3 independent experiments, each of which consisted of 3
replicates per treatment group.
In the next step, the ability of the hemp extracts to change the activity of superoxide
dismutase (SOD), which is an enzyme that acts as the first stage of antioxidant defense
and protects cells from damage by ROS, was assessed [35]. The activity of SOD was
measured in cell-free in vitro assay. The conducted analysis indicated that both analyzed
extracts show similar ability to increase SOD activity. This effect is dependent on the
concentration used and correlates with other analyses performed as part of this work
assessing antioxidant capacity (using DPPH and H2DCFDA) indicating that as the
concentration of the extract increases, the antioxidant properties are stronger (Figure 3).
The obtained results indicate that all analyzed concentrations (both UAE and MAE
extract) cause statistically significant differences in SOD activity. Superoxide dismutase
activity was highest at a concentration of 1000 μg/mL and reached up to 178% and 165%
for UAE and MAE extract, respectively. The ability of chemical compounds contained in
cannabis extracts to increase SOD activity has also been confirmed by other authors,
including in vivo studies [36,37]. Antioxidant abilities of plant extracts, including Cannabis
sativa L. extract, are extremely important because they allow cells to be protected against
oxidative stress, lipid peroxidation or DNA damage. Protection against free radicals is
extremely important in the context of skin cells, because these radicals cause damage to
the skin’s structure and significantly affect its aging processes [37]. Hence, natural
compounds that are capable of scavenging ROS are intensively sought.
Figure 3. Effect of UAE and MAE extracts from Cannabis sativa L. (100, 250, 1000 μg/mL) on
superoxide dismutase activity. Data are the mean ± SD of three independent experiments, in
which each concentration was tested in duplicate. *** p < 0.001, ** p < 0.01 versus the control
(100%).
2.3. Cytotoxicity Assessment
In the next stage of our research, the cytotoxicity of Cannabis sativa L. extracts against
skin cells (fibroblasts and keratinocytes) was evaluated in vitro using three types of assays
(Neutral Red uptake assay, Alamar Blue test and lactate dehydrogenase (LDH)
cytotoxicity test). The Neutral Red uptake assay is one of the most used cytotoxicity tests
with many biomedical applications [38]. It is based on the ability of viable cells to
incorporate and bind the supravital dye neutral red. This weakly cationic dye penetrates
cell membranes by nonionic passive diffusion and concentrates in the lysosomes, where
it binds by electrostatic hydrophobic bonds to anionic and/or phosphate groups of the
lysosomal matrix [39]. Our results obtained with the Neutral Red test have found that
Cannabis sativa L. extract at all tested concentrations (1–1000 μg/mL) showed no cytotoxic
effect on keratinocytes and fibroblasts, and thus does not affect the integrity of cell
membranes. Due to the fact that keratinocytes are cells that are directly exposed to various
Molecules 2021, 26, 802 7 of 23
external factors as well as cosmetic preparations applied directly to the skin, it is
reasonable to perform cytotoxicity tests on these cells. The highest increase in
keratinocytes proliferation, and thus the highest number of active metabolic cells, was
observed for extracts at a concentration of 100 μg/mL and this increase reaches even 120%
(Figure 4A). What is more, it can be seen that the level of proliferation decreases as the
concentration of extracts increases. Due to the possibility of penetration of the ingredients
of cosmetic and pharmaceutical preparations through individual skin layers during their
topical application, the work examined the cytotoxic effect of obtained hemp extracts on
cells that are found in the deeper layers of the skin-fibroblasts. The highest cellular
proliferation of fibroblasts was observed at a concentration of 500 μg/mL and this increase
reached 188%. The extract obtained by ultrasound-assisted extraction increased the
viability of both types of skin cells to a greater extent. The analysis shows that fibroblasts
are more sensitive to the effects of C. sativa L. extracts compared to keratinocytes (Figure
4B).
Figure 4. The effect of increasing concentrations of Cannabis sativa L. extract (1–1000 μg/mL) on Neutral Red Dye uptake
in cultured (A) keratinocytes (HaCaT) and (B) fibroblasts (BJ) after 24 h of exposure. Data are the mean ± SD of three
independent experiments, each of which consists of four replicates per treatment group. For BJ **** p < 0.0001, ** p = 0.04,
* p < 0.03 versus the control (100%). For HaCaT *** p = 0.004, ** p < 0.01 versus the control (100%).
The next test used to assess cytotoxicity of Cannabis sativa L. extracts was Alamar Blue
assay which is a fluorometric method for the detection of metabolic activity of cells. This
method is based on the reduction of resazurin (oxidized form 7-hydroxy-3H-phenoxazin-
3-1-10-oxide) to resorufin (reduced form), by mitochondrial enzymes that carry
diaphorase activity, like NADPH dehydrogenase. Optically, the blue and poorly
fluorescent resazurin is gradually transformed by cells into the red, highly fluorescent,
resorufin [40,41]. Our results obtained with the Alamar Blue test have also found that
analyzed extracts at all tested concentrations (1–1000 μg/mL) showed no cytotoxic effect
on two types of cells and thus does not slow down metabolic processes. The cytotoxicity
tests performed on keratinocytes showed that the extract at a concentration of 250 μg/mL
showed the most favorable effect, where the increase in metabolic activity of the tested
cells reaches 115% (Figure 5A). The analysis carried out using the fibroblast cell line
demonstrated slight changes in proliferation depending on the concentration of the
extracts. The level of cell proliferation of fibroblasts for all concentrations is similar and
this only increase reaches 101% (Figure 5B).
Molecules 2021, 26, 802 8 of 23
Figure 5. The reduction of resazurin after 24 h exposure to the Cannabis sativa L. extract (1–1000 μg/mL) in cultured (A)
keratinocytes (HaCaT) and (B) fibroblasts (BJ). Data are the mean ± SD of three independent experiments, each of which
consists of three replicates per treatment group. *** p < 0.0005, ** p < 0.01, * p = 0.0356 versus the control (100%).
The cytotoxic effect of the obtained extracts was also assessed using LDH cytotoxicity
assay which is a simple, reliable colorimetric method of quantitatively assaying cellular
cytotoxicity. The assay can be used with different cell types for assaying cell mediated
cytotoxicity as well as cytotoxicity mediated by chemicals and other test compounds. The
assay quantitatively measures a stable cytosolic enzyme LDH, which is released upon cell
lysis. The released LDH is measured with a coupled enzymatic reaction that results in the
conversion of a tetrazolium salt (INT) into a red color formazan. The LDH activity is
determined as NADH oxidation or INT reduction over a defined time period [42,43]. At
each concentration, there was no significant membrane damage (LDH release). The cell
viability results indicate that C. sativa L. extracts are nontoxic to keratinocytes and
fibroblasts. There were also no changes in extracellular LDH levels after exposure to C.
sativa L. extract. These values are given as the percentage of the negative control
(untreated with tested extracts). A correlation between extract concentration and LDH
release was also observed in both analyzed skin cells. With decreasing concentration of
extract, membrane damage was less noticeable. In summary, it should be noted that all
cytotoxicity tests performed indicate that the extract of Cannabis sativa L. does not show
toxic effects on the cell lines tested (Figure 6A,B).
Molecules 2021, 26, 802 9 of 23
Figure 6. The release of LDH after 24 h exposure to the Cannabis sativa L. extract (1–1000 μg/mL) in cultured (A)
keratinocytes (HaCaT) and (B) fibroblasts (BJ). Data are the mean ± SD of three independent experiments, each of which
consists of three replicates per treatment group. **** p < 0.0001 versus the control (0%).
The results obtained in these studies indicate the lack of cytotoxicity of Cannabis sativa
L. extracts to skin cells, especially fibroblasts, which may suggest their potential use as
biologically active compounds in the pharmacological, dermatological and cosmetic
industries. As mentioned earlier, C. sativa L. extracts can be seen as extremely valuable
ingredients not only in food products, but also in cosmetic preparations or dietary
supplements due to their good protective effect on our body. Studies on keratinocytes and
fibroblasts have confirmed that the cannabinoids present in this plant exert anti-
inflammatory and protective effects [44]. The skin cells like keratinocytes and fibroblasts
are involved in wound healing with the other skin cells [45]. Keratinocytes function are
regulated by a variety of cytokines, growth factors and chemokines and in turn, these cells
release a few of proinflammatory mediators including interleukin-1 beta (IL-1β), IL-6, IL-
8, tumor necrosis factor alpha (TNFα), and transforming growth factor alpha and beta, as
well as vascular endothelial growth factor (VEGF), a potent mitogen for endothelial cells,
playing a pivotal role in angiogenesis and psoriasis [46,47]. Many authors have shown
that cannabinoids present in Cannabis sativa L. can have protective effects on the skin. Δ9-
THC, CBN and CBD were shown to inhibit keratinocyte proliferation in the low
micromolar range and in a cannabinoid receptor independent manner [48]. Sangiovanni
et al. have shown that C. sativa. L. extract is able to inhibit the release of mediators of
inflammation involved in wound healing and inflammatory processes occurring in the
skin. What is more, UV radiation, especially UVB, also decreased endocannabinoids:
anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and significantly increased
palmitoylethanolamide (PEA) levels. These changes were significantly greater in
keratinocytes from psoriatic patients compared to healthy individuals. CBD counteracted
both the reduction of AEA in keratinocytes from healthy individuals, as well as the
increase in the level of PEA in psoriatic keratinocytes (both with and without UV
irradiation) [49,50]. Thus, CBD can also indirectly modify the activation of CB1 and CB2
receptors through AEA and vanilloid receptor (TRPV1) [51]. Published literature data
indicate that PEA may reduce the expression and the levels of inflammatory cytokines in
skin diseases through this mechanism [52]. Regarding the effect of cannabinoids on
fibroblasts, there are still few publications on this subject. Various studies do not show the
Molecules 2021, 26, 802 10 of 23
cytotoxic effects of cannabinoids on skin cells, but there is still little evidence to show a
significant cell proliferation of fibroblasts so it needs to extend research [53,54].
2.4. Assessment of Matrix Metalloproteinases Inhibition
Elastin and collagen are the major skin building proteins. They are responsible for an
adequate strength, flexibility, elasticity as well as hydration of the skin. These proteins
play an important role not only in the skin aging process, but also in wound healing and
skin regeneration. Elastase and collagenase enzymes are responsible for a degradation of
elastin and collagen structure in the skin. Their activity may be induced by free radicals
and UV radiation,as well as internal and genetic conditions [55]. In these studies, the effect
of hemp extracts on elastase and collagenase enzymes activity was performed. Results are
shown in Figures 7 and 8.
Figure 7. Influence of hemp extracts on elastase inhibition. The inhibitor used was Methyl-4-[[(2S) -1-[[(2S)-1-[(2S) -2-
[[(3S)-1-chloro-4-methyl-2-oxopentane -3-yl] carbamoyl] pyrrolidin-1-yl]-1-oxopropan-2-yl] amino] -1-oxopropan-2-yl]
amino] -4-oxobutanoate. Data are the mean ± SD of three independent experiments, each of which consists of three
replicates per treatment group. ** p < 0.0012, *** p < 0.0004, **** p < 0.0001 versus the control.
Molecules 2021, 26, 802 11 of 23
Figure 8. Influence of hemp extracts on collagenase inhibition. Data are the mean ± SD of three
independent experiments, each of which consists of three replicates per treatment group. ** p <
0.001 versus the control.
It was found that hemp extracts decreased activity of elastase and collagenase and
have the ability to inhibit these enzymes. In the case of elastase activity, addition of
increased concentration of hemp extracts resulted in an increase in elastase inhibition.
Extract concentration of 100 μg/mL was characterized by a low ability to elastase
inhibition. The results for these extracts were about 10%. At extract concentration of 1000
μg/mL, the ability to elastase inhibition increased significantly up to 30%. It was not
observed the significant influence of an extraction method on the analysed parameter
(Figure 7). The extraction method had a significant influence on the collagenase inhibition.
Activity of this enzyme also depended on the extract concentration. The ultrasound
extract had much stronger properties to collagenase inhibition. At a concentration of 250
μg/mL, the MAE and UAE extract showed the ability to reduce collagenase activity at the
level of about 25% and 30%, respectively. As concentrations rise, the increase of
collagenase inhibition by extracts was observed. At 1000μg/mL, the value of the analyzed
parameter was about 55% for the MAE extract. For UAE extract, collagenase inhibition
was significantly stronger and was about 80% (Figure 8). The effect of cannabis extracts
on the activity of collagenase and elastase enzymes has not yet been studied. However
numerous literature data, based on clinical and animal studies, show the significant
influence of cannabinoids (e.g., THC, CBD) derived from hemp in wound and burns
healing process, skin regeneration, delays of skin ageing and relieving of pruritus or pain.
Cannabinoids have also strong anti-inflammatory and antibacterial properties [55–58]. In
addition to supporting wound healing, cannabinoids may be an effective ingredient in the
treatment of dermatoses, psoriasis, atopy, skin allergies and skin melanoma [55–57]. This
effect is attributed to an endocannabinoid system (ECS) formed by endocannabinoids and
their CB1 and CB2 receptors. Recent studies showed that CB1 and CB2 receptors have
endogenous ligands located in the skin. It may indicate that the skin has its own ECS
system, which is responsible for skin health by maintaining a skin homeostasis.
Cannabinoids can act as a stimulant or an inhibitory agent for ECS, e.g., by inhibiting or
promoting proliferation of keratinocytes, sebum production or inhibition of inflammatory
promoters [56–59]. The regenerating effect of cannabinoids and their positive effect on the
skin condition may therefore be a result of ECS action and ability of cannabinoids to
inhibit activity of collagenase and elastase.
2.5. Assessment of Hydrogel Properties
In the case of many dermatological problems like atopy, dermatosis, psoriasis, as well
as in the process of wound healing, it is extremely important to keep an adequate level of
skin moisture. Due to many external factors, drinking too small amounts of water, dietary,
and above all, cleansing cosmetics used every day, the level of skin moisture may
decrease. Cleansing cosmetics may also disturb the hydrolipid skin barrier, which can
increase the amount of water that evaporates from the skin. It may impede and slow down
processes of wounds, scars and burns healing [54]. Therefore, the influence of hemp
extracts (in the form of hydrogels) on skin moisture and transepidermal water loss
(TEWL) was evaluated. Before application of hydrogels the forearm skin was washed with
1% SLS solution. SLS is one of the most popular cleaning agents used in formulations of
cleansing cosmetics and it has a strong ability to dry the skin and damage the skin’s lipid
barrier [55–60]. The results of the corneometric and TEWL study are shown in Figure 9. In
these studies, a hydrogel based on hydroxyethylcellulose (base sample) and the same
hydrogel with addition of analysed hemp extracts was applied on the skin of 15
volunteers. 90 and 300 min. after the hydrogels application, changes in skin moisture and
TEWL were measured.
Molecules 2021, 26, 802 12 of 23
Figure 9. Influence of hemp extracts on skin hydration (A) and TEWL (B). Different letters on the charts indicate significant
differences between groups (p < 0.05). The determinations were made in 5 replicates. MAE_0.5 and MAE_1.0 are hydrogels
containing 0.5 and 1.0% of the dry extract obtained using traditional method and UAE_0.5 and UAE_1.0 are hydrogels
containing 0.5 and 1.0% of the dry extract obtained by ultrasound-assisted extraction.
Application of hemp extracts in the hydrogel form have a positive effect on the skin
condition. After the skin cleaning process with 1% of SLS level of the skin moisture was
decreased. In related to a control field without treatment with any samples, after
application of SLS solution it was noted decrease in the skin hydration by about 14% and
11% (after 1.5 h and 5 h, respectively). The negative value of a skin moisture change means
a strong ability of this surfactant to dry the skin. Analogically, SLS also had a strong ability
to disturb the skin hydrolipid balance as evidenced by the positive value of TEWL change
1.5 and 5 h after SLS application. 5 h after SLS application it was observed that skin did
not return to its physiological state and the moisture level was lower and the TEWL value
was higher than values observed for the control field. Drying effect and damaging of skin
barrier by surfactants can make it difficult to wound healing and skin regeneration, as
well as exacerbate symptoms of atopy, psoriasis and sensitive skin and induce skin
irritations. After application of hydrogels containing hemp extracts on sodium lauryl
sulfate (SLS)-treated skin, the skin condition improved significantly. Hemp hydrogels
eliminated adverse effect of analyzed surfactant on the skin moisture and TEWL. The use
of hydrogels without extracts (base sample) also improved the skin condition, but the
effect of base sample was not so strong. Hydrogels containing 0.5% of extracts acted as
skin moisturizer. 5h after application of these hydrogels, the skin returned to a
physiological state, and the change in skin moisture was close to the value observed for
the control field. For MAE_0.5 i UAE_0.5 hydrogels it was not observed a significant
influence of hemp extraction method on the skin moisture (p < 0.05). Samples with 1.0%
of the extract in the formulation had a stronger skin moisturizing potential and for the
UAE extract the long-lasting,moisturizing properties were significantly better than for the
MAE extract. After 5 h, the increase in skin moisture (relating to the control field) was
about 10% for UAE_1.0 sample and 5% for MAE_1.0 hydrogel. TEWL analysis showed
that addition of hemp extracts in the formulation of hydrogels have an influence to
restoring of the hydrolipid balance and to rebuilding of the hydrolipid barrier of the skin,
which are damaged in a cleaning process by SLS. Analysis of results indicated that the
influence of the hemp extraction method and concentration of the extract on hydrogels
properties was not significant. Similar TEWL results were obtained for all samples
containing the extract in the formulation. Change of TEWL after application of these
Molecules 2021, 26, 802 13 of 23
samples was about 20–30% lower than for SLS treated skin, which indicates the protective
effect of hemp hydrogels against water loss from the epidermis. Plant extracts are a source
of many substances that moisturize the skin. These are mainly substances with hydroxyl
groups in their molecules that can form a hydrogen bond with water, thus binding water
in the epidermis. The active components of plant extracts are mainly proteins,
carbohydrates, as well as polyphenols and flavonoids, containing several hydroxyl
groups in the molecule [55]. Hemp extracts are rich in this kind of substances and they are
responsible for the moisturizing of the skin [25]. Proteins and carbohydrates due to their
high molecular weight may act as an occlusive film on the skin surface causing decrease
of transepidermal water loss. Cannabinoids because of their hydrophobicity and a low
molecular weight may act on the skin surface as an occlusive film and also penetrate into
deeper layers of the epidermis, retaining water and providing a long-lasting moisturizing
effect [55].
3. Materials and Methods
3.1. Plant Material and Extraction Procedure
Plant material was purchased from a local herbal store. Cannabis sativa L. herb were
collected on controlled and ecological plantations. No chemical fertilizers nor plant
protection products were used for the cultivation. As part of this work, two types of hemp
extracts were prepared using ultrasound assisted extraction (UAE) and magnetic stirrer
assisted extraction (MAE). The extract was prepared by extracting 15 g of Cannabis sativa
L. herb in a 100 g water-ethanol solution (20:80). UAE was obtained according to the
method described by Yang et al. in an ultrasonic bath (Digital Ultrasonic Cleaner, Berlin
Germany) equipped with a time controller [61]. The mixtures were extracted at room
temperature for 60 min (6 cycles of 10 min). When the extract temperature reached 25 °C
the extract was rapidly cooled in the ice to 22–23 °C. MAE extracts were made on a
magnetic stirrer at 300 rpm (RCT basic, IKA, Staufen, Germany). Extraction was carried
out for 60 min. The obtained extracts were then collected and filtered three times through
Whatman No. 1 filter paper using vacuum filtration. After filtration, the UAE and MAE
extracts were evaporated under reduced pressure at 40 °C. A stock solution at the
concentration of 100 mg/mL was prepared from the dried extracts, and was stored in the
dark at 4 °C until further analysis.
3.2. Determination of Biologically Active Compounds
3.2.1. Total Phenolic Content Determination
The concentration of total phenolic compounds in Cannabis sativa L. extracts was
determined spectrophotometrically using the Folin-Ciocalteu method described by
Singleton et al. with some modifications [62]. Gallic acid (GA) was used as standard. For
this purpose, 300 μL of hemp extract sample at various concentrations was mixed with
1500 μL of Folin Ciocalteu 1:10 reagent. After 6 min of incubation, 1200 μL of a 7.5%
sodium carbonate solution was added to the analyzed samples. Samples were mixed and
incubated in the dark at room temperature (about 22 °C) for 2 h. Absorbance was read at
λ = 740 nm on an Aquamate Helion spectrophotometer (Thermo Scientific, Waltham, MA,
USA). To calculate the total concentration of phenols in hemp extracts (both UAE and
MAE), a gallic acid (GA) calibration curve (in the 10–100 mg/mL concentration range) was
used. As a negative control was used ethyl alcohol. The measurements were made in
triplicate and the results obtained were averaged.
3.2.2. Total Flavonoids Content Determination
The concentration of flavonoids in the analyzed hemp extracts was measured
spectrophotometrically using aluminum nitrate nonahydrate. For this purpose, the
method described by Matejić et al. with modifications was used [63]. 2400 μL of the
previously prepared reaction mixture consisting of 80% C2H5OH, 10% Al (NO3)3 ×9 H2O
Molecules 2021, 26, 802 14 of 23
and 1M C2H3KO2 were mixed with 600 μL of the tested sample of the extract at various
concentrations. After 40 min incubation at room temperature (about 22 °C) in the dark,
the absorbance of the prepared mixtures at λ = 415 nm was measured using a FilterMax
F5 AquamateHelion spectrophotometer (Thermo Scientific). The total flavonoid
concentration in the analyzed samples was calculated from the calibration curve for
quercetin (Qu) hydrate (in the concentration range of 10–100 mg/mL). Measurements
were made in triplicate for each sample.
3.2.3. Determination of Chlorophyll Content
The chlorophyll content of hemp extracts was determined by spectrophotometry.
Stock solution of dry MAE and UAE extracts at a concentration of 100 μL/mL in 80%
acetone was prepared. The absorbance of the solutions was measured at λ = 645 nm and
λ = 663 nm using UV-Vis spectrophotometer Filter Max 5 (Thermo Scientific). The results
were expressed as the content of chlorophyll a and b and the total content of chlorophyll
(a + b), calculated on 1 g of dry hemp extract (DWE) and hemp herb (DWH). The final
result is the average of three independent determinations.
3.2.4. Determination of Cannabinoids
The test was performed in an external accredited laboratory. A Hewlett Packard
HPLC system (DionexUltiMate 3000 RS, Thermo Fisher Scientifilic, Sunnyvale, CA, USA)
equipped with an integrator and a UV/VIS detector as well as a C18 column filled with
silica gel was used in the tests. The study was conducted in a reverse phase system. The
mobile phase was mixing methanol and phosphoric acid (75:25). Analysis conditions: 1
mL / min, 35 °C, detection at λ = 230 nm. Cannabinoid concentration was read from the
standard curve using external standards.
3.3. Assessment of Antioxidant Activity
3.3.1. DPPH Radical Scavenging Assay
The ability of the extracts obtained to scavenge free radicals was determined using
the stable DPPH radical. For this purpose, the methodology described by Brand-Williams
et al. has been applied [64]. Briefly, 33 μL of cannabis extracts tested at various
concentrations (0.1–10%) were mixed with 167 μL methanol solution of DPPH (4 mM) and
transferred to a 96 well plate. The analyzed samples were mixed thoroughly by shaking.
In the next step, the absorbance of the samples at λ = 517 nm was measured.
Measurements were made every 5 min for 30 min on a UV-ViS Filter Max 5
spectrophotometer (Thermo Scientific). Three independent replicates were performed for
each concentration. Water-ethanol solution (20:80) with a DPPH solution was used as a
control. The antioxidant capacity of Cannabis sativa L. extracts was expressed as a
percentage of DPPH inhibition using the following equation:
% DPPH scavenging = Abs control − Abs sample
Abs control × 100%
where Abs control is the absorbance of the control sample (containing DPPH and water-
ethanol solution), Abs sample is the absorbance of the test sample (containing DPPH and
test extract)
3.3.2. Detection of Intracellular Levels of Reactive Oxygen Species (ROS)
In order to determine the ability of the analyzed UAE and MAE hemp extracts to
generate the intracellular production of reactive oxygen species in HaCaT and fibroblast
cells, a fluorogenic H2DCFDA dye was used. After passive diffusion of this compound
into the cells, it is deacetylated by intracellular esterases to a non-fluorescent compound.
Molecules 2021, 26, 802 15 of 23
In the presence of reactive oxygen species it is oxidized and transformed into highly
fluorescent DCF [65].
To determine the intracellular level of ROS in HaCaTs and fibroblasts, cells were
seeded in 96 well plates at a density of 1 × 104 cells per well. Then, cells were cultured in
an incubator for 24 h. DMEM medium was removed and replaced with 10 μM H2DCFDA
(Sigma Aldrich, Sant Louis, MO, USA) dissolved in serum free DMEM medium. HaCaT
and BJ cells were incubated in H2DCFDA for 45 min and then incubated with Cannabis
sativa L. extracts in the concentration range of 1–1000 μg/mL. Cells treated with 1 mM
hydrogen peroxide (H2O2) were used as positive controls. The control samples were cells
untreated with the tested extracts. DCF fluorescence was measured every 30 min for 90
min using a FilterMax F5 microplate reader (Thermo Fisher Scientific) at a maximum
excitation of 485 nm and emission spectra of 530 nm.
3.3.3. Determination of Superoxide Dismutase (SOD) Activity
Colorimetric Superoxide Dismutase Activity Assay kit (ab65354, Abcam, Cambridge,
UK) was used to determine the impact of the cannabis extracts on the activity of the
antioxidant enzyme involved in the defense system against reactive oxygen species. MAE
and UAE Cannabis sativa L. extracts in concentrations of 100, 250 and 1000 μg/mL were
used for the analysis. Recombinant human Superoxide Dismutase 1 protein (ab112193,
Abcam) was used to prepare the standard curve. Samples were prepared in 96-well plates
(clear bottoms) and the analysis was performed according to the manufacturer’s
instructions. Initially, 200 μL of WST working solution was added to each well. Then, test
samples were prepared by adding 20 μL Enzyme Working Solution and 20 μL of hemp
extracts (with a final concentration 100, 250, 1000 μg/mL) to these wells. Three different
blank samples were also prepared as recommended. Blank 1 was prepared by adding 20
μL Enzyme Working Solution and 20 μL dd H2O to the wells. To blank 2 20 μL Dilution
Buffer and 20 μl hemp extracts (with a final concentration 100, 250, 1000 μg/mL) was
added. 20 μL of Dilution Buffer and 20 μL dd H2O were added to blank 3. All samples
were mixed thoroughly by shaking and incubated at 37 °C for 20 min. The absorbance of
the samples was then measured at λ = 450 nm using a microplate reader (FilterMax F5,
Thermo Fisher). All samples were prepared in duplicate according to the manufacturer’s
instructions. The ability to inhibit SOD activity by the analyzed samples was calculated
from the equation:
% SOD Activity = (Ablank1– Ablank3) – (Asample– Ablank2)
(Ablank1 – Ablank3) × 100 %
3.4. Cell Culture
In this work, two skin cell lines were used. HaCaT cells (normal human
keratinocytes) were purchased from CLS Cell Lines Service (Eppelheim, Germany), while
BJ cells (fibroblasts, ATCC®CRL-2522 ™) were obtained from the American Type Culture
Collection (Manassas, VA, USA). Both cell lines were maintained in DMEM (Dulbecco’s
Modification of Eagle’s Medium, Biological Industries, Cromwell, CO, USA) with L-
glutamine, 4.5 g/l glucose and sodium pyruvate. The medium was additionally
supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, Waltham, MA, USA) and
1% (v/v) antibiotics (100 U/mL penicillin and 1000 μg/mL streptomycin, Gibco). Cells were
kept in an incubator at 37 °C in a humid atmosphere of 95% air and 5% carbon dioxide
(CO2).
3.5. Cell Viability Assay
After the cultured cells (HaCaT and BJ) reached the appropriate confluence, the
DMEM culture medium was removed from the culture plate (VWR, Radnor, PE, USA)
and the cells were washed twice with sterile PBS (phosphate buffered saline, Gibco). The
Molecules 2021, 26, 802 16 of 23
cell layer was trypsinized using Trypsin/EDTA (Gibco), and then the cells were
suspended in a fresh medium. In the next step, cells were plated into 96 well plates
(separate plates for both cell types). After the attachment of HaCaT and fibroblasts to the
bottom of the plates, the cells were incubated with various concentrations (1, 100, 250, 500
and 1000 μg/mL) of Cannabis sativa L. extracts. The cells were cultured in an incubator for
24 h. Controls were cells cultured in DMEM medium without the addition of extracts.
3.5.1. Neutral Red Uptake Assay
The neutral red uptake test (Sigma Aldrich) was used in the studies to assess the
viability of skin cells treated with the tested extracts. This test is based on the protocol
described by Borenfreund et al. [66]. After exposure to MAE and UAE extracts from
Cannabis sativa L., cells were incubated for 2 h with a neutral red dye (40 μg/mL) which
was dissolved in serum-free medium (DMEM). After incubation, the cells were washed
with phosphate buffered saline (PBS) and 150 μL decolorizing buffer
(C2H5OH/CH3COOH/H2O2, 50/1/49%) was added to each well. After shaking the test cells
for 15 min, the absorbance of dissolved dye at λ = 540 nm was determined using a
FilterMax F5 Multi-Mode microplate reader (Thermo Fisher). The average optical density
of the control cells was set to 100% viability and was used to calculate the percentage of
viable cells in the experimental samples. The experiments were repeated three times using
four wells for each concentration of extracts.
3.5.2. Alamar Blue Assay
To assess the cytotoxicity of the tested extracts and check their effect on cell viability
the Alamar Blue assay (R7017, Sigma) was used. This assay is based on the initial protocol
described by Page et al. [67]. After exposure of the cells to individual concentrations of the
analyzed hemp extracts (1–1000 μg/mL), a solution of resazurin with a final concentration
of 60 μM was added to the wells and incubated for 2 h at 37 °C in the dark. Fluorescence
was measured at λ = 570 nm using a microplate reader (FilterMax F5, Thermo Fisher).
Controls were cells cultured in DMEM medium without the addition of extracts. The
experiments were carried out in three independent experiments in which the fluorescence
of cells in four wells was measured for each extract concentration. Results are expressed
as percent of cell viability compared to control (100%).
3.5.3. Lactate Dehydrogenase (LDH) Cytotoxicity Assay
Another test used to assess cytotoxicity of the tested hemp extracts was a high-
throughput, reliable colorimetric method for quantifying cellular cytotoxicity. The activity
of LDH in the studied extracts was determined using a commercially available kit
(Cytoscan™ LDH Cytotoxicity Assay) from G-Biosciences (A Geno Technology, St. Louis,
MO, USA). The assay is based on the conversion of lactate to pyruvate in the presence of
LDH with parallel reduction of NAD. The test was carried out according to the
instructions provided with the reagents. Analyzes were performed by seeding cells
(keratinocytes and fibroblasts) into 96 well plates in DMEM medium. After attachment of
the cells to the bottom of the wells, the plates were treated with MAE and UAE extracts at
concentrations of 1–1000 μg/mL (Compound Treated). To prepare Spontaneous LDH
Activity Control, sterile, ultrapure water was added to the wells instead of the tested
extracts. To obtain Maximum LDH Activity Control, according to the instructions, 10 μL
of Lysis Buffer was added to the wells. Following exposure the extracts diluted in DMEM,
medium was removed and then the culture supernatant was collected and incubated with
50 μL reaction mixture. After incubation at room temperature for 30 min, the reaction was
stopped by adding 50 μL Stop Solution. To determine LDH activity, absorbance at λ = 490
nm and λ = 680 nm was measured. Cytotoxicity of the analyzed extracts was calculated
using the following equation:
Molecules 2021, 26, 802 17 of 23
% Cytotoxicity = Compound Treated −Spontaneous LDH Activity
Maximum LDH release −Spontaneous LDH Activity × 100 %
3.6. Assessment of Matrix Metalloproteinases Inhibition
3.6.1. Determination of Anti-Collagenase Activity
The ability of the tested Cannabis sativa L. extracts to inhibit collagenase activity was
analyzed using a fluorometric Collagenase Inhibitor Screening Kit (ab211108, Abcam).
UAE and MAE extracts in concentrations of 100, 250 and 1000 μg/mL were used for the
analysis. Samples were prepared for analysis in a 96 well plate with clear flat bottom. In
the first step, collagenase (COL) was dissolved in Collagenase Assay Buffer (CAB). The
test samples were prepared by mixing the analyzed hemp extracts with COL and CAB.
Inhibitor control samples were prepared by mixing inhibitor (1,10-Phenanthroline (80
mM)) with diluted collagenase and CAB buffer. Enzyme control wells were prepared by
mixing diluted COL with CAB. The CAB buffer was used as background control. The
samples were incubated for 15 min at room temperature. In the meantime, a reaction
mixture was prepared by mixing the collagenase substrate with CAB. Then, the reaction
mixture was added to the prepared samples and mixed thoroughly. The fluorescence was
immediately measured with an excitation wavelength of λ = 490 nm and emission λ = 520
nm using a microplate reader (FilterMax F5, Thermo Fisher). The measurement was made
in kinetic mode, for 60 min at 37 °C. All samples were prepared in duplicate according to
the manufacturer’s instructions. The ability to inhibit COL activity by the analyzed
samples was calculated from the equation:
% relative COL inhibition = enzyme control −sample
enzyme control × 100%
3.6.2. Determination of Anti-Elastase Activity
Fluorometric Neutrophil Elastase Inhibitor Screening Kit (ab118971, Abcam) was
used to determine the ability of the extracts to inhibit the activity of the neutrophil elastase
(NE) enzyme. MAE and UAE Cannabis sativa L. extracts in concentrations of 100, 250 and
1000 μg/mL were used for the analysis. Samples were prepared in 96-well black plates
(clear bottoms) for fluorometric assay and the analysis was performed according to the
manufacturer’s instructions. Briefly, neutrophil elastase enzyme solutions, NE substrate
and inhibitor control (SPCK) were prepared as initially as recommended. The neutrophilic
elastase inhibitor used in the analyzes was the chemical compound with the formula
C22H35ClN4O7 (CAS number 65144-34-5). It is a strong, irreversible elastase inhibitor that
was part of a kit designed to measure the activity of this enzyme. Then diluted NE solution
was added to all wells. Test samples, inhibitor control and enzyme control (Assay Buffer)
were applied to subsequent wells. All samples were prepared in duplicate according to
the manufacturer’s instructions. The samples were mixed thoroughly on a shaker and the
plate incubated at 37 °C for 5 min. The fluorometric reaction mix was then prepared by
mixing Assay Buffer and substrate. The prepared reaction mixture was added to each
sample and mixed thoroughly. The fluorescence was immediately measured with an
excitation wavelength of λ = 400 nm and emission λ = 505 nm using a microplate reader
(FilterMax F5, Thermo Fisher). The measurement was made in kinetic mode, for 30 min at
37 °C protected from light. The ability to inhibit NE activity by the analyzed samples was
calculated from the equation:
% relative NE activity =
∆RFU tes
t
inhibitor
∆ RFU Enzyme control × 100%
Molecules 2021, 26, 802 18 of 23
3.7. Hydrogel Preparation
The base hydrogel was a 1.2% aqueous solution of hydroxyethyl cellulose (HEC).
HEC was added to water and mixed on a mechanical stirrer (Chemland O20, Hamburg,
Germany) using a propeller stirrer and stirring speed of 250 rpm. The polymer solution
was heated to 60 °C and then cooled to room temperature while stirring to cross-link of
HEC. Hydrogels containing hemp extracts were prepared by an analogous method. Hemp
extracts were added to HEC hydrogel after cooling. Dry cannabis extracts were added to
the hydrogel as a 100 mg/mL solution, using 80% solution of 1,3-propanediol to dissolve
them. Four hemp hydrogels were obtained: MAE_0.5 and MAE_1.0, containing 0.5 and
1.0% of the dry extract obtained using traditional method and UAE_0.5 and UAE_1.0,
containing 0.5 and 1.0% of the dry extract obtained by ultrasound- assisted extraction.
3.8. Transepidermal Water Loss (TEWL) and Skin Hydration Measurements
TEWL and skin hydration measurements were conducted using a TEWAmeterTM 300
probe and Corneometer CM 825 probe connected to MPA adapter (Courage + Khazaka
Electronic, Köln, Germany). The study was conducted on 15 volunteers in the age of 28–
36. Before the study, each volunteer was informed about the study procedure, research
material and contraindications. Each of the volunteers was healthy and signed a
declaration of voluntary participation in the study.
Six areas (2 × 2 cm in size) were marked on the forearm skin. 0.2 mL of 1% SLS
solution was applied to 5 fields. One field (control field) was not treated with any sample.
The SLS solution was gently spread over every skin fragment, and then rinsed with
distilled water and dried with a paper towel. After 10 min, 0.2 g of hydrogels were applied
on 4 fields of skin treated with SLS. Dry-out hydrogels were removed from the skin with
a paper towel 30 min after their application. After 90 and 300 min, the hydration and
TEWL measurements were taken. The final result was the arithmetic mean (from each
volunteer) of 5 independent measurements (skin hydration) and 20 measurements
(TEWL). The change in skin hydration and the change in TEWL were calculated by the
formulas:
% ∆ Skin moisture = M1 −M0
M0 × 100%
% ∆ TEWL = TEWL1 −TEWL0
TEWL0 × 100%
where M1 (TEWL1)—mean skin hydration (TEWL) after t (90 or 300 min) time for the test
field. M0 (TEWL0—mean skin hydration (TEWL) after t (90 or 300 min) time for the
control field
3.9. Statistical Analysis
Values of different parameters were expressed as the mean ± standard deviation (SD).
The two-way analysis of variance (ANOVA) and Bonferroni posttest between groups
were performed at the level p value of < 0.05 to evaluate the significance of differences
between values. Statistical analyses were performed using GraphPad Prism 8.4.3
(GraphPad Software, Inc., San Diego, CA, USA) and Statistica 9.0 (StatSoft, CA, USA)
using One-way ANOVA and Tukey’s test.
4. Conclusions
As part of this work, the unusual properties of extracts obtained from Cannabis sativa
L. were shown. Comparing both extraction methods, it can be concluded that the UAE is
a more efficient method, leading to obtaining more organic compounds in the tested plant.
The analysis confirmed the ultrasonic extraction resulted in a higher concentration of
cannabinoids, phenolic compounds, flavonoids and chlorophyll in extracts of Cannabis
sativa L. In addition to the previously known antioxidant properties of the tested extracts,
Molecules 2021, 26, 802 19 of 23
which can have a positive effect on the structure and condition of skin cells, this work also
shows other benefits of hemp extracts. Due to the constantly growing popularity of
hydrogel preparations in cosmetology and dermatology, the results presented in this
work may contribute to the development of new hydrogels containing hemp extracts or
individual compounds isolated from Cannabis sativa L herb. The abilities of inhibiting
matrix metalloproteinases, collagenase and elastase, presented for the first time in this
work, as well as proven antioxidant properties make these extracts valuable ingredients
for the production of a wide range of products that can be used in the treatment and care
of the skin. Due to the high demand for preparations that inhibit the aging processes of
the skin, the effect of hemp extracts on skin hydration and the possibility of preventing
the degradation of collagen and elastin fibers presented here indicates the value of these
extracts. The lack of a negative effect on the metabolic activity and viability of skin cells
indicate the legitimacy of including hemp extracts in the recipes of skin care cosmetics as
well as medicinal preparations. However, further research is obviously needed to
determine the detailed mechanisms of action of these extracts, however the results
presented in this paper seem to be promising.
Author Contributions: Conceptualization: M.Z.-D., T.B., A.Z. and Z.N.-Ł.; methodology: M.Z.-D.,
T.B., A.Z. and Z.N.-Ł.; formal analysis: M.Z.-D., T.B., A.Z. and Z.N.-Ł.; investigation: M.Z.-D., T.B.,
A.Z. and Z.N.-Ł.; writing—original draft preparation M.Z.-D., T.B., A.Z. and Z.N.-Ł.; writing—
review and editing, M.Z.-D., T.B., A.Z. and Z.N.-Ł.; visualization: M.Z.-D., T.B. and A.Z.;
supervision M.Z.-D., T.B., A.Z. and Z.N.-Ł.; All authors have read and agreed to the published
version of the manuscript.
Funding: This work was founded by the 503-07-01-34 Statutory Project of the University of
Information Technology and Management in Rzeszow, Poland.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the
design of the study; in the collection, analysis, or interpretation of data; in the writing of the
manuscript, or in the decision to publish the results.
Sample Availability: Samples of the compounds used and the plants extracts tested are available
from the authors.
References
1. Beg, S.; Swain, S.; Hasan, H.; Barkat, M.A.; Hussain, M.S. Systematic review of herbals as potential anti-inflammatory agents:
Recent advances, current clinical status and future perspectives. Pharmacogn Rev. 2011, 5, 120–137, doi:10.4103/0973-7847.91102.
2. Cowan, M.M. Plant products as antimicrobial agents. Clin. Microbiol Rev. 1999, 12, 564–582.
3. Mahlo, S.M.; Chauke, H.R.; McGaw, L.; Eloff, J. Antioxidant and antifungal activity of selected medicinal plant extracts against
phytopathogenic fungi. Afr. J. Tradit. Complement. Altern. Med. 2016, 13, 216–222, doi:10.21010/ajtcam.v13i4.28.
4. Pisoschi, A.M.; Pop, A.; Cimpeanu, C.; Predoi, G. Antioxidant Capacity Determination in Plants and Plant-Derived Products: A
Review. Oxid. Med. Cell Longev. 2016, 9130976, doi:10.1155/2016/9130976.
5. Sultana, B.; Anwar, F. Flavonols (kaempferol, quercetin, myricetin) contents of selected fruits, vegetables and medicinal plants.
Food Chem. 2008, 108, 879–884, doi:10.1016/j.foodchem.2007.11.053.
6. Sen, S.; Chakraborty, R.; Sridhar, C.; Reddy, Y.R.S.; De, B. Free radicals, antioxidants, diseases and phytomedicines: Current
status and future prospect. Int. J. Pharm. Sci Rev. Res. 2010, 3, 91–100.
7. Elser, P.; Maibach, H. Cosmeceuticals and Active Cosmetics; Taylor & Francis Group: New York, NY, USA, 2000.
8. Nizioł-Łukaszewska, Z.; Furman-Toczek, D.; Zagórska–Dziok, M. Antioxidant activity and cytotoxicity of Jerusalem artichoke
tubers and leaves extract on HaCaT and BJ fibroblast cells. Lipids Health Dis. 2018, 17, 1–12, doi:10.1186/s12944-018-0929-8.
9. Cavalcanti, R.; Forster-Carneiro, T.; Gomes, M.; Rostagno, M.; Prado, J.; Meireles, M. Uses and applications of extracts from
natural sources. In Natural Product Extraction: Principles and Applications; Royal Society of Chemistry: Cambridge, UK, 2013; pp.
1–3.
10. Faccio, G. Plant Complexity and Cosmetic Innovation. iScience 2020, 23, 101358, doi:10.1016/j.isci.2020.101358.
11. Bujak, T.; Wasilewski, T.; Nizioł-Łukaszewska, Z. Role of macromolecules in the safety of use of body wash cosmetics. Colloids
Surf. B Biointerfaces 2015, 135, 497–503, doi:10.1016/j.colsurfb.2015.07.051.
Molecules 2021, 26, 802 20 of 23
12. Barel, M.; Paye, M. Handbook of Cosmetic Science and Technology, 4th ed.; Taylor & Francis Group: Boca Raton, FL, USA, 2014; pp.
353–65.
13. Rock, E.M.; Goodwin, J.M.; Limebeer, C.L.; Breuer, A.; Pertwee, R.G.; Mechoulam, R.; Parker, L.A. Interaction between non-
psychotropic cannabinoids in marihuana: Effect of cannabigerol (CBG) on the anti-nausea or anti-emetic effects of cannabidiol
(CBD) in rats and shrews. Psychopharmacology 2011, 215, 505–512, doi:10.1007/s00213-010-2157-4.
14. Brenneisen, R. Chemistry and analysis of phytocannabinoids and other cannabis constituents. In Marijuana and the Cannabinoids
(ElSohly MA Ed.); Humana Press Inc.: Totowa, NJ, USA, 2007; pp. 17–50.
15. Thomas, B.F.; Elsohly, M. Quality Assessment, Assurance, and Regulation of Medicinal Marijuana and Cannabinoid
Preparations. Biosynthesis and Pharmacology of Phytocannabinoids and Related Chemical Constituents. In The Analytical
Chemistry of Cannabis; Elsevier: Amsterdam, The Netherlands, 2016, Volume 2, pp. 27–41, doi:10.1016/B978-0-12-804646-3.00002-
3.
16. Russo, E.B. Taming THC: Potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. Br. J. Pharmacol. 2011,
163, 1344–1364, doi:10.1111/j.1476-5381.2011.01238.x.
17. Mańkowska, G.; Grabowska, L. Genetic resources of Cannabis sativa at the Institute of Natural Fibres and Medicinal Plants.
Herba Pol. 2009, 55, 178–184.
18. Borges, R.; Batista, J.; Viana, R.; Baetas, A.; Orestes, E.; Andrade, M.; da Silva, A. Understanding the molecular aspects of
tetrahydrocannabinol and cannabidiol as antioxidants. Molecules 2013, 18, 12663–12674.
19. Elsohly, M.A.; Slade, D. Chemical constituents of marijuana: The complex mixture of natural cannabinoids. Life Sci. 2005, 78,
539–548, doi:10.1016/j.lfs.2005.09.01.
20. Sharma, P.; Murthy, P.; Bharat, M.M.S. Chemistry, metabolism, and toxicology of Cannabis: Clinical implications. Iranian J.
Psychiatry 2012, 7, 149–156.
21. Whiting, P.F.; Wolff, R.F.; Deshpande, S.; Di Nisio, M.; Duffy, S.; Hernandez, A.V.; Keurentjes, J.C.; Lang, S.; Misso, K.; Ryder,
S.; et al. Cannabinoids for medical use a systematic review and meta-analysis. JAMA 2015, 313, 2456–2473,
doi:10.1001/jama.2015.6358.
22. Grotenhermen, F. Pharmacology of cannabinoids. Neuro Endocrinol. Lett. 2004, 25, 14–23.
23. Morales, P.; Hurst, D.P.; Reggio, P.H. Molecular Targets of the Phytocannabinoids: A Complex Picture. Prog. Chem. Org. Nat.
Prod. 2017, 103, 103–131, doi:10.1007/978-3-319-45541-9_4.
24. Fukumoto, L.R.; Mazza, G. Assessing antioxidant and prooxidant activities of phenolic compounds. J. Agric. Food Chem. 2000,
48, 3597–3604, doi:10.1021/jf000220w.
25. Pollastro, F.; Minassi, A.; Fresu, L.G. Cannabis Phenolics and their Bioactivities. Curr. Med. Chem. 2018, 25, 1160–1185,
doi:10.2174/0929867324666170810164636.
26. Koch, W.; Zagórska, J.; Marzec, Z.; Kukula-Koch, W. Applications of Tea (Camellia sinensis) and Its Active Constituents in
Cosmetics. Molecules 2019, 24, 4277, doi:10.3390/molecules24234277.
27. Izzo, L.; Castaldo, L.; Narváez, A.; Graziani, G.; Gaspari, A.; Rodríguez-Carrasco, Y.; Ritien, A. Analysis of Phenolic Compounds
in Commercial Cannabis sativa L. Inflorescences Using UHPLC-Q-Orbitrap HRMS. Molecules 2020, 25, 631,
doi:10.3390/molecules25030631.
28. Maqsood, A.; Aatika, S.; Mazher, I.; Ansar, J.; Mingshan, I.; Qin, P.; Yuyang, L.; Zumin, G. Phytochemical screening, Total
phenolics and Flavonoids Content and Antioxidant Activities of Citrullus colocynthis L. and Cannabis sativa L. Appl. Ecol. Environ.
Res. 2019, 17, 6961–6979, doi:10.15666/aeer/1703_69616979.
29. Dan, J.; Dai, K.; Xie, Z.; Chen, J. Secondary Metabolites Profiled in Cannabis Inforescences, Leaves, Stem Barks, and Roots for
Medicinal purposes. Sci. Rep. 2020, 24, 3309, doi:10.1038/s41598-020-60172-6.
30. Mkpenie, V.; Essien, E.; Udoh, I. Effect of extraction conditions on total polyphenol contents, antioxidant and antimicrobial
activities of Cannabis sativa L. Elec. J. Environ. Agricult. Food Chem. 2012, 11, 300–307.
31. Ferrante, C.; Recinella, L.; Maurizio, R.; Menghini, L.; Brunetti, L.; Chiavaroli, A.; Leone, S.; Iorio, L.; Carradori, S.; Tirillini, B.;
et al. Multiple pharmacognostic characterization on hemp commercial cultivars: Focus on inflorescence water extract activity.
Food Chem. Toxicol. 2019, 125, 452–461.
32. Agarwal, C.; Mathe, K.; Hofmann, T.; Csóka, L. Ultrasound-Assisted Extraction of Cannabinoids from Cannabis Sativa L.
Optimized by Response Surface Methodology. J. Food Sci. 2018, 83, 700–710.
33. Hacke, A.C.M.; Lima, D.; de Costa, F.; Deshmukh, K.; Li, N.; Chow, A.M.; Marques, J.A.; Pereira, R.P.; Kerman, K. Probing the
antioxidant activity of Δ9-tetrahydrocannabinol and cannabidiol in Cannabis sativa extracts. Analyst. 2019, 144, 4952–4961,
doi:10.1039/c9an00890j.
34. Moldzio, R.; Pacher, T.; Krewenka, C.; Kranner, B.; Novak, J.; Duvigneau, J.C.; Rausch, W.D. Effects of cannabinoids Δ(9)-
tetrahydrocannabinol, Δ(9)-tetrahydrocannabinolic acid and cannabidiol in MPP+ affected murine mesencephalic cultures.
Phytomedicine 2012, 19, 819–824, doi:10.1016/j.phymed.2012.04.002.
35. Allen, R.G. Oxidative stress and superoxide dismutase in development, aging and gene regulation. Age 1998, 21, 47–76,
doi:10.1007/s11357-998-0007-7.
36. Konca, Y.; Cimen, B.; Yalcin, H.; Kaliber, M.; Beyzi, S.B. Effect of Hemp Seed (Cannabis sativa sp.) Inclusion to the Diet on
Performance, Carcass and Antioxidative Activity in Japanese Quail (Coturnix coturnix japonica). Korean J. Food Sci. Anim. Resour.
2014, 34, 141–150, doi:10.5851/kosfa.2014.34.2.141.
Molecules 2021, 26, 802 21 of 23
37. Coskun, Z.M.; Bolkent, S. Evaluation of Δ(9)-tetrahydrocannabinol metabolites and oxidative stress in type 2 diabetic rats. Iran.
J. Basic Med. Sci. 2016, 19, 154–158.
38. Repetto, G.; Sanz, P. Neutral red uptake, cellular growth and lysosomal function: In vitro effects of 24 metals. Altern. Lab. Anim.
1993, 21, 501–507.
39. Nemes, Z.; Dietz, R.; Luth, J.B. The pharmacological relevance of vital staining with neutral red. Experientia 1979, 35, 1475–1476.
40. Nakayama, G.R.; Caton, M.C.; Nova, M.P.; Parandoosh, Z. Assessment of the Alamar Blue assay for cellular growth and
viability in vitro. J. Immunol. Methods 1997, 204, 205–208.
41. O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of
mammalian cell cytotoxicity. Eur. J. Biochem. 2000, 267, 5421–5426, doi:10.1046/j.1432-1327.2000.01606.x.
42. Park, M.V.; Lankveld, D.P.; van Loveren Hand de Jong, W.H. The status of in vitro toxicity studies in the risk assessment of
nanomaterials. Nanomedicine 2009, 4, 669–685, doi:10.2217/nnm.09.40.
43. Patravale, V.; Dandekar, P.; Jain, R. Nanotoxicology: Evaluating toxicity potential of drug-nanoparticles. Nanoparticulate Drug
Delivery 2012, 4, 123–155, doi:10.1533/9781908818195.123.
44. Navarro, G.; Reyes-Resina, I.; Rivas-Santisteban, R.; de Medina, V.S.; Morales, P.; Cassano, S.; Ferreiro-Vera, C.; Lillo, A.;
Aguinaga, D.; Jagerovic, N.; et al. Cannabidiol skews biased agonism at cannabinoid CB1 and CB2 receptors with smaller effect
in CB1-CB2 heteroreceptor complexes. Biochem. Pharmacol. 2018, 157, 148–158, doi:10.1016/j.bcp.2018.08.046.
45. Pastar, I.; Stojadinovic, O.; Yin, N.C.; Ramirez, H.; Nusbaum, A.G.; Sawaya, A.; Tomic-Canic, M. Epithelialization in wound
healing: A comprehensive review. Adv. Wound Care 2014, 3, 445–464, doi:10.1089/wound.2013.0473.
46. Marina, M.E.; Roman, I.I.; Constantin, A.M.; Mihu, C.M.; Tataru, A.D. VEGF involvement in psoriasis. Clujul Med. 2015, 88, 247–
252, doi:10.15386/cjmed-494.
47. Hanel, K.H.; Cornelissen, C.; Luscher, B.; Baron, J.M. Cytokines and the skin barrier. Int. J. Mol. Sci. 2013, 14, 6720–6745,
doi:10.3390/ijms14046720.
48. Siurkus, J.; Peciura, R. The Topical Composition with Active Compounds from Cannabis sativa and Calendula Officinalis for
Reduction of Skin Lesions. U.S. Patent WO2017175126, 12 October 2017.
49. Bisogno, T.; Hanus, L.; De Petrocellis, L.; Tchilibon, S.; Ponde, D.E.; Brandi, I.; Moriello, A.S.; Davis, J.B.; Mechoulam, R.; Di
Marzo, V. Molecular targets for cannabidiol and its synthetic analogues: Effect on vanilloid VR1 receptors and on the cellular
uptake and enzymatic hydrolysis of anandamide. Br. J. Pharmacol. 2001, 134, 845–852, doi:10.1038/sj.bjp.0704327.
50. Jarocka-Karpowicz, I.; Biernacki, M.; Wroński, A.; Gęgotek, A.; Skrzydlewska, E. Cannabidiol Effects on Phospholipid
Metabolism in Keratinocytes from Patients with Psoriasis Vulgaris. Biomolecules 2020, 10, 367, doi:10.3390/biom10030367.
51. Sangiovanni, E.; Fumagalli, M.; Pacchetti, B.; Piazza, S.; Magnavacca, A.; Khalilpour, S.; Melzi, G.; Martinelli, G.; Dell’Agli, M.
Cannabis sativa L. extract and cannabidiol inhibit in vitro mediators of skin inflammation and wound injury. Phytother. Res. 2019,
33, 2083–2093, doi:10.1002/ptr.6400.
52. Petrosino, S.; Verde, R.; Vaia, M.; Allarà, M.; Iuvone, T.; Di Marzo, V. 2-Pentadecyl-2-Oxazoline, the Oxazoline of Pea, Modulates
Carrageenan-Induced Acute Inflammation. Front. Pharmacol. 2017, 8, 308, doi:10.3389/fphar.2017.00308.
53. Song, R.; Bian, H.N.; Lai, W.; Chen, H.D.; Zhao, K.S. Normal skin and hypertrophic scar fibroblasts differentially regulate
collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor.
Braz. J. Med. Biol Res. 2011, 44, 402–410, doi:10.1590/S0100-879X2011007500041.
54. Rawal, S.Y.; Dabbous, M.Kh.; Tipton, D.A. Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism:
MMP production and activity, and production of fibronectin and transforming growth factor β. J. Periodontal Res. 2012, 47, 320–
329, doi:10.1111/j.1600-0765.2011.01435.x.
55. Leyden, J.J.; Rewlings, A.V. Skin Moisturization. Cosmetics Science and Technology Series; Marcel Dekker: New York, NY, USA,
2002; pp. 223–45.
56. Avila, C.; Massick, S.; Kaffenberger, B.H.; Kwatra, S.G.; Bechtel, M. Cannabinoids for the Treatment of Chronic Pruritus: A
Review. J. Am. Acad. Dermatol. 2020, 82, 1205–1212, doi:10.1016/j.jaad.2020.01.036.
57. Sheriff, T.; Lin, M.J.; Dubin, D.; Khorasani, H. The potential role of cannabinoids in dermatology. J. Dermatol. Treat. 2019, 8, 1–7,
doi:10.1080/09546634.2019.1675854.
58. Eagelston, L.R.M.; Yazd, N.K.K.; Patel, R.R.; Flaten, H.K.; Dunnick, C.A.; Dellavalle, R.P. Cannabinoids in dermatology: A
scoping review. Dermatol. Online J. 2018, 24, 1–17.
59. Oláh, A.; Tóth, B.I.; Borbíró, I. Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes. J. Clin. Investig.
2014, 124, 3713–3724, doi:10.1172/JCI64628.
60. Seweryn, A. Interactions between surfactants and the skin—Theory and practice. Adv. Coll. Int. Sci. 2018, 256, 242–255,
doi:10.1016/j.cis.2018.04.002.
61. Yang, B.; Liu, X.; Gao, Y. Extraction optimization of bioactive compounds crocin, geniposide and total phenolic compounds
from Gardenia Gardenia jasminoides Ellis fruits with response surface methodology. Innov. Food Sci. Emerg. Technol. 2009, 10, 610–
615.
62. Singleton, V.L.; Rossi, J.J. Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am. J. Enol.
Viticult. 1965, 16, 144−158.
63. Matejić, J.S.; Džamić, A.M.; Mihajilov-Krstev, T.M.; Ranđelović, V.N.; Krivošej, Z.D.; Marin, P.D. Total phenolic and flavonoid
content, antioxidant and antimicrobial activity of extracts from Tordylium maximum. J. Appl. Pharm. Sci. 2013, 3, 55–59,
doi:10.7324/JAPS.2013.30110.
Molecules 2021, 26, 802 22 of 23
Molecules 2021, 26, 802 23 of 23
64. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT—Food Sci.
Technol. 1995, 28, 25–30.
65. Sharma, O.P.; Bhat, T.K. DPPH antioxidant assay revisited. Food Chem. 2009, 113, 1202–1205, doi:10.1016/j.foodchem.2008.08.008.
66. Borenfreund, E.; Puerner, J.A. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol.
Lett. 1985, 24, 119–124. doi:10.1016/0378-4274(85)90046-3.
67. Page, B.; Page, M.; Noel, C. A new fluorometric assay for cytotoxicity measurements in-vitro. Int. J. Oncol. 1993, 3, 473–476
