Originating from central Asia, Cannabis sativa is an annual herbaceous flowering plant. Although used medicinally for centuries,1, 2 it has experienced a significant resurgence in interest, becoming a recent buzzword in beauty.3 The reasons behind this are the richness of chemical compounds produced by the plant on one hand, and the significant opening up of regulatory markets on the other hand.4
Cannabis plants contain more than 500 known compounds. In addition to phyto-cannabinoids, the plant contains saccharides, phenolic acids, flavonoids, stilbenes, lignans, fatty acids and terpenes.5-8 Several compounds of these classes are known to impart relevant biological activities in mammalian cells, including modulating appetite, pain sensation, mood, memory, inﬂammation and energy metabolism.9-10
In relation, plant cell and tissue cultures are an appropriate alternative to whole-plant cultivation both for studying and producing secondary metabolites of cosmetic interest. Moreover, plant cell cultures support the push toward sustainable sourcing methods to reduce land exploitation and minimize impact on the environment. At the same time, they represent a standardized, contaminant-free source of compounds for cosmetic applications on an industrial scale.11 Interestingly, many investigators have established callus cultures from the explants of different C. sativa organs (i.e., roots, hypocotyls, epicotyls, cotyledons, petioles, leaves and immature flower buds). However, scientific reports on secondary metabolite extraction and on the biological properties of cannabis culture extracts are limited.
Taken together, this study examines and reports the properties and activities of a C. sativa cell culture extract. For example, cell suspension cultures of the botanical are prepared in a water/ethanol extract and tested for the capacity to modulate neurogenic inflammation in human cells. The term neurogenic inflammation describes the mechanism by which sensory nerves contribute to local inflammation by releasing inflammatory neuropeptides such as the Calcitonin Gene-Related Peptide (CGRP) and Substance P (SP).12 CGRP is the most abundant neuropeptide produced by the sensory nerves in the various skin layers13 and plays a striking role in the pathogenesis of inflammatory skin diseases such as psoriasis and atopic dermatitis.14-16
Thanks to its capacity to switch off neurogenic inflammation and modulate the production of neurotransmitters and neuropeptides in nerve cells, the cannabis extract has the potential for alternative applications to cosmetics.
In the skin, the release of CGRP from the nerve fibers stimulates keratinocytes to produce pro-inflammatory cytokines including interleukins IL-1α, IL-6 and IL-8.17 In macrophages, it induces the release of the pro-inflammatory mediator histamine, which in turn triggers neuropeptide synthesis, thus establishing a bidirectional loop between sensory nerves and epidermis cells.18
While CGRP represents a pro-inflammatory neuropeptide, other types of neuropeptides such as β-endorphin produce an opposite effect, having analgesic and pain-relieving properties.19 β-Endorphins are mainly produced by nerve cells however, a fully functional β-endorphins/receptor system is also present in skin keratinocytes, where it is involved in epithelization, tissue regeneration and pain relief.20, 21
Described herein are tests to assess the effects of C. sativa cell culture extract on CGRP in neuronal cells, and on histamine production in CGRP-treated macrophages. In addition, the extract’s activity is measured on the expression of genes involved in skin inflammation, such as those of the pro-inflammatory cytokines IL-1α, IL-8 and TNFα, and of the β-endorphin precursor POMC. Lastly, clinical studies examine its potential to soothe skin and reduce erythema.
In vitro Materials and Methods
C. sativa cell cultures and extract: C. sativa stem cells were obtained from leaves of the variety Carmagnola and grown in Gamborg B5 basal mediuma at a pH 5.7 supplemented with sucrose, myo-inositol, phytohormones and growth elicitors. After four weeks, the cells were collected and extracted using three volumes of ice-cold ethanol (96%). The obtained supernatant was distilled by a low-pressure evaporation process and lyophilized until obtaining a powder, which was then dissolved in water and used for the analyses and assays described at the required concentration.
Chemical analysis: A sample of cannabis extract was dissolved in water to a final concentration of 0.25 mg/mL. The analysis was performed by NP-HPLCb. The detection was performed by light scatteringc using the following settings: 45°C, gas flow 1.5 L/min, gain factor 1.
Cell cultures and reagents for bioassays: SHSY5Y neuroblastoma cells, HaCaT keratinocytes and macrophages RAW 264.7 were maintained in DMEM mediumd supplemented with 10% FBSe. Normal human epidermal keratinocytes (nHEK) were maintained in EpiLifed supplemented with human keratinocyte growth supplement (HKGS). All cell types were grown under 5% CO2 at 37°C. Additional materials procured included: capsaicin, capsazepine, T0901317 and cetirizine dihydrochloridef; the human neuropeptide CGRPg; and the CGRP Enzyme Immunoassay kit Bertin Pharmah.
Gene expression analysis: For gene expression analyses of CGRP and POMC, 1.8 × 106 SHSY5Y or 1.5 × 106 NHEK, respectively, were seeded in six-well plates and treated with C. sativa extract or capsaicin for 6 hr. For the proinflammatory cytokines, 1.5 × 106 HaCaT cells were seeded in six-well plates pretreated with C. sativa extract for 16 hr, then stressed with 1 nM CGRP for 1 hr.
After the treatments, cells were collected and RNA was extractedj. The RNA was first treated with DNase I to eliminate contaminating genomic DNA and then cDNA was synthesizedk. RT-PCR was performed using gene specific primers and the QuantumRNA 18S internal standardm. The amplification reactions were performed according to the following scheme: 2 min at 94°C, followed by 35 cycles of 94°C for 30 sec, annealing temperature (specific for each gene) for 30 sec, and 72°C for 30 sec with a 10 min final extension at 72°C.
The obtained PCR products were loaded onto 1.5% agarose gel and the amplification bands, visualized and quantifiedn, were normalized to the amplification band corresponding to the 18S. The average values, obtained from three independent experiments, were converted into percentage values by considering the measurement of the untreated control as 100%.
CGRP measurements: The amount of CGRP neuropeptide produced was measured using the ELISA assay by seeding 1 × 105 SHSY5Y in a 96-well plate and treating the samples with the extract for 4 hr before adding 50 μM capsaicin alone or in the presence of capsazepine. After 24 hr, the cell media was collected and transferred to anti-CGRP monoclonal antibody-coated plates (CGRP human) and the assayp was performed according to the manufacturer’s instructions.
Histamine measurements: Macrophages RAW264.7 were seeded at a density of 5 × 104 cells/well in 96-well plates and treated with C. sativa extract for 2 hr. The cells were then treated with 10 nM CGRP and the amount of histamine was measured after 16 hr by adding a solution of 0.4 M NaOH and 1 mg/mL of O-Phthaldialdehyde (OPA) in methanol. After 10 min, 0.1 M HCl was added to each well to stop the reaction and the fluorescence intensity—proportional to the amount of released histamine—was measured at 443 nm by a Multi-plate Reader.
Targeting CGRP release represents a novel approach to reduce nerve-induced inflammation and soothe inflamed skin.
Statistical analysis: All in vitro experiments were conducted in triplicate and repeated three times. The data was expressed as the mean ± the standard deviation of the values obtained by three independent experiments. Statistical comparisons between controls and treated groups were performed according to the student’s t-test using the softwareq; p values lower than 0.05 were considered statistically signiﬁcant. The number of asterisks in the graphs indicate the level of signiﬁcance (*** = p value < 0.001; **p < 0.01 and *p < 0.05).
Clinical Test Protocols
Skin erythema index: A total of 20 healthy volunteers between the ages of 20 and 65 with normal skin were enrolled in this study and gave their written informed consent. Three different sites on their forearms, referred to as A, P and C, were identified where erythema was induced by applying a solution of 30% sodium laureth sulfate. Skin erythema was quantified instrumentallyr.
First, the baseline erythema index was calculated, then study products were applied on the different areas, including: a formulation (see Formula 1) containing cannabis extract on A, a placebo cream on P and nothing in the C area (control area). After 30 min, the erythema index was measured to evaluate the soothing effect of the tested products. Three sequential measurements on each area were taken and the data was expressed as a mean value variation % from the baseline.
TEWL and corneometers hydration index: The rate of transepidermal water loss (TEWL, g/hm2) and corneometer hydration indices are the most important parameters for evaluating the efficiency of skin barrier function. The volunteers were directed to apply each formulation to the three different areas twice daily, in the morning and evening, for 14 consecutive days. Skin hydration levelss and TEWL valuest were assessed at the baseline, after erythema induction and post-application for 7 and 14 days.
Statistical analysis: For the clinical tests, the student’s t-test was performed using softwareu; a p value < 0.05 was considered significant.
Results: Cell Culture Extract and Chemical Analysis
Starting from Cannabis sativa plants, cell suspension cultures were developed using a specific growth medium, optimized to obtain the highest yield of cell biomass per volume. The biomass was extracted in ethanol 96% (1:3 w/v ratio), and the obtained extract contained flavonoids (including cannflavin B), phenolic acids and their glucosides, spiro-compounds (such as cannabispirol), vitamins and terpenes, as determined by mass spectrometry analysis.
Concentrations of cannabinol (CBN), cannabidiol (CBD) and cannabidiolic acid (CBDA) lower than 1 mg/100 g of dried extract were detected, in agreement with previous studies reporting that cannabis callus cultures produce undetectable or very low levels of cannabinoids, irrespective of the elicitors or growth regulators used in the culture medium.22 As expected, due to the plant variety used for obtaining the cells, the psychoactive compounds Δ9-tetrahydrocannabinol (THC) and Δ9-tetrahydrocannabinolic acid (THCA) were absent from the extract.
Results: In vitro Activity on Neurogenic Inflammation
Since CGRP is important to the skin, targeting its release represents a novel approach to reduce nerve-induced inflammation and soothe inflamed skin. SHSY5Y nerve cells were treated with C. sativa extract at two concentrations and with capsaicin for comparison, which is known to induce CGRP synthesis.23 CGRP gene expression showed the extract reduced the basal expression level of CGRP by ~35%—differently from capsaicin, which increased the neuropeptide expression by 32% (see Figure 1a).
The amounts of CGRP produced by the cells after treatments also were assessed by ELISA assay. To overcome problems related to detection limits, cells were stressed with 50 μM capsaicin to induce the production of CGRP after the treatment with C. sativa extract. Furthermore, in place of the extract, some samples were treated with capsazepine, a capsaicin antagonist, used as positive control in the assay. As shown in Figure 1b, capsaicin increased the synthesis of CGRP, which was inhibited by approximately 30% by the C. sativa extract as well as the capsazepine.
To evaluate the capacity of the C. sativa extract to inhibit the CGRP-induced inflammation in skin cells, keratinocytes were treated with the extract or with the anti-inflammatory drug T0901317, used as a positive control,24 and then with 1 nM of purified peptide CGRP. The expression analysis of three pro-inflammatory cytokines was performed 1 hr after the neuropeptide treatment. Results showed the extract significantly inhibited the expression of IL-1α, IL-8 and TNFα analogously to the compound T0901317 (see Figure 2).
Both concentrations of the extract increased POMC expression level by 40%, suggesting the extract’s potential to reduce the pain sensation and impart skin-soothing activity.
Besides cytokines, histamine is another inflammatory mediator induced by CGRP. Thus, the capacity of C. sativa extract to modulate histamine production was evaluated in macrophages after treatment with GCRP 10 nM. Analysis performed by a fluorescent assay showed that the extract, at both the concentrations, inhibited CGRP-induced histamine synthesis by approximately 25-30% (see Figure 3), similarly to the cetirizine dihydrochloride, a known drug with antihistamine activity.25 These results suggested the potential of C. sativa extract to reduce neuropeptide-induced inflammation in immune system cells.
To assess whether the C. sativa extract was able to stimulate the production of β-endorphin, epidermal keratinocytes were treated with the extract and the gene expression level Pro-OpioMelanoCortin (POMC)—a 241 amino acid precursor polypeptide that generates β-endorphin peptides under cleavage—was assessed. Both concentrations of the extract increased POMC expression level by 40%, suggesting the extract’s potential to reduce the pain sensation and impart skin-soothing activity (see Figure 4).
Results: Clinical Testing
To evaluate the activity of C. sativa extract in ameliorating skin properties in vivo, measurements were made of the skin erythema index, transepidermal water loss (TEWL) and corneometer index as described previously. Results reported in Figure 5 show that treatment with a cream containing C. sativa extract at 0.002% reduced the erythema value by a statistically significant 24.3%, while the placebo cream did not.
The principle of skin inflammation is often associated with altered skin barrier permeability and an increase in TEWL. Treatment of the skin for two consecutive weeks with the twice daily application of a C. sativa extract-containing emulsion significantly reduced TEWL values by 16% (see Figure 6a). At the same time, the corneometer index increased by 18%, indicating higher skin hydration (see Figure 6b). Overall, the results of the clinical tests indicated that C. sativa extract demonstrated significant anti-inflammatory and skin-moisturizing properties, suggesting its use as an active ingredient for soothing face and body skin care formulations.
This study shows that a water-ethanol extract derived from C. sativa cell suspension cultures effectively inhibited the expression and the release of inflammatory neuropeptides, therefore modulating skin neurogenic inflammation. Moreover, by acting on the release of the calcitonin gene related peptide (CGRP), it reduced inflammatory cytokine expression and histamine release, resulting in a global soothing and moisturizing effect on the skin in vivo.
Several ingredients obtained from plant cell cultures are drawing interest for cosmetics thanks to their characteristics of safety, efficacy and sustainability.26 The extract described here is the first ingredient derived from cannabis cell cultures and developed and characterized for cosmetic use—and with promising effects. In addition, thanks to its capacity to switch off neurogenic inflammation and modulate the production of neurotransmitters and neuropeptides in nerve cells, the cannabis extract has the potential for alternative applications to cosmetics.27
Chemical characterization of the extract revealed the presence of bioactive compounds that have been linked to an anti-inflammatory activity, such as phenolic acids, flavonoids and terpenes. In particular, cannflavins and methylated isoprenoid flavones unique to cannabis28 have been studied for their neuro-protective and anti-cancer properties.7-29 Thanks to their capacity to significantly inhibit the in vivo production of pro-inflammatory mediators such as prostaglandin E2 and the leukotrienes,30 cannflavins have previously demonstrated anti-inflammatory action 30 times greater than that exerted by the common drug aspirin.31
Today, consumer demand for cannabis-based products has exponentially increased. The use of cell culture systems represents a convenient solution to address this demand, as demonstrated here by the described C. sativa cell culture extract. This suitable source supports product claims not only for the presence of cannabis compounds, but also their efficacy.
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