Topical Use of Cannabis sativa L. Biochemicals

Cannabis sativa L. plant is currently attracting increasing interest in cosmetics and dermatology. In this review, the biologically active compounds of hemp are discussed. Particularly the complex interactions of cannabinoids with the endocannabinoid system of the skin to treat various conditions (such as acne, allergic contact dermatitis, melanoma, and psoriasis) with clinical data. Moreover, the properties of some cannabinoids make them candidates as cosmetic actives for certain skin types. Hemp seed oil and its minor bioactive compounds such as terpenes, flavonoids, carotenoids, and phytosterols are also discussed for their added value in cosmetic formulation.


Introduction
It has already been 6000 years that humans use Cannabis as food, fiber, and medicine [1]. Nowadays, Cannabis has widely spread through the world [1]. It is a predominantly dioecious species, with only male flowers or female flowers [2]. This particularity opened the path to hybridization of the plant, and lead to thousands of cultivars [1]. There is a long taxonomic problem with the classification of the different strains [1]. It is generally accepted to divide the Cannabis sativa L. species into 3 subspecies: "Sativa" refers to strains with a limited amount of THC, "Indica" refers to strains producing principally THC, and "Ruderalis" refers to wild hemp strains [1].
The Cannabis sativa L. plant contains a diversity of bio-active compounds which are promising for topical application in dermatology [3] or as cosmetic ingredients [4,5]. Firstly, because of the high content of cannabinoids, which can modulate diverse inflammatory conditions and immune response via the endocannabinoid system [6]. Secondly, because of the Hemp Seed Oil, which has beneficial properties for the skin [7,8]. And thirdly, because of the diversity of minor bioactive active compounds such as terpenes, flavonoids, carotenoids, phytosterols [9][10][11].
Firstly, this review aims to gather knowledge about the various cannabinoids and their biological actions within and outside the endocannabinoid system. Secondly, to determine the added value of Hemp Seed Oil and its minor constituents compared to other oils for cosmetic formulations or dermatological use.

Cannabis sativa L. Botany
Cannabis sativa L. belongs to the Cannabaceae family. It is an annual herb [1]. The plant can reach a height higher than 5 m in the outdoor 6 months growing season, as shown in Figure 1a [12]. The leaves grow on opposite sides of the stem [13]. The leaves, stems, and bracts of the plants are covered by epidermal protuberances called trichomes [1]. There are two types of trichomes: glandular and non-glandular. The non-glandular trichomes are in the bracts, petioles, stipules, leaves, and stems and serve as a defense mechanism against abiotic and biotic stress. The glandular trichomes are responsible for the synthesis of cannabinoids, secondary metabolites, and terpenes in a viscous resin, as shown in Figure 1b [14]. When the days start to shorten, the inflorescence is triggered and buds of

The Endocannabinoid System
The endocannabinoid system (ECS), in the skin, is implied in cutaneous function such as cell differentiation modulation, growth and survival, inflammatory and immune responses, nociception, and hair growth. Indeed, dysregulation of the ECS seems to be involved in various skin disease conditions [16]. Two G protein-coupled receptors are involved in the ECS's regulation: cannabinoid type 1 receptor (CB1) and cannabinoid type 2 receptor (CB2) [17]. In the skin, CB1 is expressed in hair follicular cells, sensory neurons, immune cells, sebaceous glands, and keratinocytes while CB2 is expressed in sensory neurons, immune cells, sebaceous glands, and keratinocytes [18]. The CB1 main activity is the regulation of pain, of excessive neural activity and the extinction of evasive memories in the central nervous system [19]. It has been recently shown that CB1 regulate inflammatory response in various peripheral organs [20][21][22]. As for the skin, activation of CB1 downregulate the production of pro-inflammatory cytokine in keratinocytes, and protects the skin barrier [19]. Activation of CB2 has anti-inflammatory effects in skin; by inhibiting the macrophage 1 polarizations they downregulate pro-inflammatory cytokines [23]. The orphans G-coupled proteins receptors GPR55 and GPR18 can also be activated by some cannabinoids ligands [24][25][26][27]. Further studies are warranted to determine if GPR55 can be officially considered as a CB3 receptor or not. Depending on the target cell, GPR55 can have a pro-inflammatory or anti-inflammatory effects [27]. Nevertheless, its activation promotes human skin tumors and other squamous cell carcinomas [28]. GPR18 was revealed to have anti-inflammatory and anti-nociceptive activity in case of intestinal inflammation [29]. GPR18 is an active inhibitor of apoptosis in melanoma cells [30].

Secondary Cannabinoid Target
Some of the cannabinoids ligands modulate greatly the response of the ECS by the activation of various transient receptor potential ion channels (TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, and TRPM8) [18], of peroxisome proliferator-activated receptor alpha and gamma (PPARα; PPARγ) transcription factors [16], and of serotonin receptors (particularly 5-HT1A, 5-HT2A, and 5-HT3 receptors) [37]. TRP ion channels permit the transit of various cations in the cells when activated by specific ligands [38]. PPAR enables, upon activation by specific ligands, the proliferation of peroxisomes which regulate inflammatory response and lipid metabolism [39].
The sum of these factors may result in synergistic or antagonistic biological effects. Therefore, it is important to take them into account to predict the pharmacological or cosmetic activity of a cannabinoid ligand. The biodynamic effect of these secondary cannabinoid targets and their location in the skin are detailed in Table 1 and illustrated in Figure 2. Anti-inflammatory activity Reduces chronic pain Slows wound healing [43] TRPV3 [18,41,[44][45][46] Hair follicle, Keratinocytes, Nerve ending, Fibroblasts, Langerhans Cells Promotes epidermal differentiation and hair growth [18]. Pro-inflammatory activity in sebaceous glands and epidermal keratinocytes [44]. Pro-inflammatory activity in case of atopic dermatitis [46].

Cannabinoids
The cannabinoids, which are synthesized in the glandular trichomes [2] of Cannabis Sativa L. are exogenous ligands of the ECS [62]. Therefore, they can interact with endocannabinoid receptors and some of the channels and receptors described below [62].

Cannabinoids
The cannabinoids, which are synthesized in the glandular trichomes [2] of Cannabis sativa L. are exogenous ligands of the ECS [62]. Therefore, they can interact with endocannabinoid receptors and some of the channels and receptors described below [62].
CBD and CBG are transcriptional repressors that can control cell differentiation and proliferation in the skin [96]. CBD induces nuclear export and degradation of BACH1, reducing stress oxidation and skin aging [61,97]. CBG is an agonist of α2-adrenoceptor [73,98], it inhibits the endocannabinoid membrane transporter [50,79,82]. CBC is the most potent agonist of the TRPA1 channel [81,82,99]. CBDV is a partial agonist of dopamine D2-like receptors [100]. It has been shown recently that D2-like receptor agonism in the skin, promotes the recovery of the skin barrier and wound healing [101][102][103].
Some of these phytocannabinoids (mainly THC and CBD) have been tested as a treatment against various skin conditions (see Table 3).  [113] Psoriasis THC and CBD In vitro (human-skin keratinocytes) Inhibition of keratinocytes proliferation [114] CBD and CBG Human trial (2 subjects) 16-33% reduction of lesions after 6 weeks [115] Cannabinoids exhibit frequently antioxidant, antimicrobial activity, and less frequently a photoprotectant (see Table 4).  [123] Because of its wide range of effects, formulation technologies are being developed to ensure better topical delivery of CBD for medical and cosmetic use [124][125][126][127][128].
Hemp Seed Oil, as an oil rich in essential fatty acids (ELA), has an action on atopic dermatitis, psoriasis, and particularly acne. Many studies find conflicting results, which indicates that the actions of ELA are dose-dependant and length-dependant [133]. Both αlinolenic acid and linoleic acids reduce UV damage and hyperpigmentation [134]. Clinical evidence highlights the positive hydrating and anti-aging effect of essentials fatty acids on the skin for oral use [135]. A favorable portion of fatty acids in Hemp Seed Oil improves the gliding of a skin care cream and the smoothness of the skin [4,136]. Moreover, Hemp Seed Oil is a non-comedogenic [137] dry oil that does not leave a greasy and sticky layer on the skin [137]. As a result, formulations have been developed with Hemp Seed Oil as long-term moisturizing patches [138] and stable emulsions in sunscreen cosmetics [5,136]. Hemp Seed Oil's global antioxidant activity can be measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) test [130]. DPPH leads to 60-65% scavenging activity and ABTS leads to 40-88% scavenging activity [130,132].
Hemp Seed Oil extraction leads to byproducts of seed paste which can be recycled as a dermo-cosmetic agent by use of eco-friendly processes: Ultrasound-Assisted Extraction or Supercritical Fluid Extraction. These pastes contain 14 bioactive metabolites: seven cannabinoid acid derivates, four lignamides, two amides, and a phenolic acid. The paste showed more than 80% of inhibition for the collagenase enzyme. The global antioxidant activity was measured by DPPH; it is up to 50% of radical scavenging [9].

Essential Fatty Acids
Linoleic acid, α-linoleic acid and γ-linoleic acid are considered ELA. These compounds are required for the wealth being but not synthesized by our body [139]. Because of the competition of omega-6 and omega-3 family acids for the ∆-6 desaturase enzyme, the ratio of their consumption is important. Hemp Seed Oil fits perfectly in the (ω-6/ω-3) ideal ratio which is between 2:1 to 3:1 this ratio [140].
Linoleic acid is involved in the biosynthesis of leukotrienes, endocannabinoids, and arachidonic acid which is the main precursor of prostaglandins. Linoleic acid is also engaged in β-oxidation in the sebaceous gland to synthesize squalene and wax esters. When skin's linoleic acid levels are low, epidermal barrier function is impaired, the comedone wall becomes permeable to inflammatory substances, resulting in a comedogenic effect [134,141].
α-linoleic acid is a compound in cell and mitochondrial membranes that modifies cell transport and signaling through the lipid layers. The α-linolenic acid metabolites permit the synthesis of the anti-inflammatory prostaglandin and leukotriene [134]. Therefore, α-linoleic acid is involved in barrier function maintenance, the stratum corneum maturation and differentiation, lamellar body formation, lipoxygenase, and pro-inflammatory eicosanoid inhibition, cytokine suppression, inhibition of mast cell degranulation, and modulation of other immune cells [133].
The γ-linoleic acid decreases the production of pro-inflammatory leukotriene B4 by increasing the concentration of dihomo-γ-linoleic acid in the skin [142].

Carotenoids
Hemp Seed Oil contains carotenoids, particularly β-carotene, lutein, and zeaxanthin [7,143]. These carotenoids exhibit antioxidant and UV-filtering properties because of their high solubility in the lipid bilayer membrane [144]. Therefore, β-carotene inhibits the UVBinduced upregulation of pro-inflammatory cytokines, resulting in an anti-inflammatory action [145,146]. Carotenoids improve skin hydration, promote skin regeneration, and stimulate fibroblasts to produce collagen and elastin [137].

Tocopherols
Hemp Seed Oil contains α-tocopherol, β-tocopherol, δ-tocopherol, γ tocopherol [130,147]. γ-tocopherol is the principal isomer with 85-91% of the tocopherols [143]. It is the main antioxidant of the Hemp Seed Oil and is responsible for most of the global antioxidant and anti-aging activities [7].

Phytosterols
Hemp Seed Oil contains phytosterols that inhibit the matrix metalloproteinases. The matrix metalloproteinase action inhibits COL1A1 and COL1A2 genes responsible for collagen synthesis. Therefore phytosterols permit better collagen synthesis and prevent skin aging [148].

Chlorophyll
Chlorophyll is surprisingly high in Hemp Seed Oil, it varies between 100 µg/g to 230 µg/g, depending on the extraction method, which is 11 times higher than in Flax Seed Oil and 88 times higher than in Canola Oil [143]. This component is responsible for the green color of the Hemp Seed Oil. The chlorophyll has beneficial action for woundhealing by promoting tissue growth, and by its antibacterial activity [149]. Therefore, it is an interesting ingredient in topical application against acne [150,151], eczema, and ulcers [152].

Terpene
Biodynamic Effect
Studies consider 4 types of synergies: multi-target effects (each component affects multiple targets), pharmacokinetic effects (components can increase the solubility or the resorption rate of an active), agent interactions affecting bacterial resistance, and modulation of adverse effects and toxicity [164].
The THC and CBD 1:1 combination is the most known case of entourage effect: it alleviates the adverse effects of pure THC [162].
Currently, evidence in favor of an entourage effect of Terpenes or Flavonoids combined with THC is contradictory, further studies are warranted to determine to which extent this concept is relevant [165,166].

Conclusions
The cannabinoids contained in Cannabis sativa L. are of dermatological interest for treating most inflammatory skin conditions as well as skin cancer. We now have a better understanding of the endocannabinoid system of the skin and the possible mode of action of cannabinoids. However, the focus of research remains on CBD and THC, which have demonstrated therapeutic value, but at the expense of studying the action of other cannabinoids.
As for their use in cosmetics, CBD, CBC, THCV and CBDV have potential for formulations for acne-prone skin, while CBG and CBGV are promising for regulating sebum production in dry skin. CBN and CBD also appear to be of interest for sunscreens. Almost all cannabinoids have an antibacterial and antioxidant action which is a good added value. Moreover, the immunoregulatory effects of cannabinoids seem interesting for sensitive skin. It should be noted, however, that their safety for regular use has not yet been demonstrated, apart from CBD. Further studies are warranted in this regard.
Hemp Seed Oil is interesting because of its content of flavonoids, terpenes, carotenoids and phytosterols which ensure its anti-inflammatory and anti-aging action. In addition, its ω-6/ω-3 content is ideal for the skin. It is a rapidly absorbed and non-comedogenic oil. It naturally contains tocopherol and chlorophyll. This oil is therefore of interest for formulations for all skin types, especially sensitive skin, and as a sun cream. It has potential for anti-ageing formulations.

Data Availability Statement:
The data presented in this study are available within the article.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.