Full-spectrum cannabidiol reduces UVB damage through the inhibition of TGF-β1 and the NLRP3 inflammasome.

UOIM conceived, designed, and performed the experiments. VI elaboration of the antioxidant analysis, calculation of the UVB light absorption spectra, and docking of interaction of CBD and CBC with proteins. IMO, performed photography and immunofluorescence analysis . JCBF and EGAM, histopathology of skin mice and mast cell staining. EPEA, analysis of chromatography. FOCM analysis of chromatography. CLYI, management, and sponsorship of sources for the development of the project. ÁAJG, management, and sponsorship of sources for the development of the project.

The FS‐CBD sample was analyzed using GC–MS, and 13 compounds were identified by spectrometric similarity (NIST base; Figure  1 and Table  1 ). The major compound was CBD (52.0223% relative abundance), which showed a similar spectrometric pattern to that of the NIST database, even though the intensities of the ions were the same (Figure  S1 ). The next major compound was CBC (28.0903% relative abundance), which presented a spectrometric pattern reported previously (NIST database; Figure  S1 ). The remaining 11 compounds reported here represent the minority of the sample analyzed (<17.07% relative abundance, 28.7781% relative abundance, II CBD 54.1444% relative abundance). GC–MS data for the FS‐CBD sample. Note : The NIST names of the compounds present in the FS‐CBD mixture are shown. The main compounds were CBD and CBD, and the other 13 compounds detected are presented as percentages. CBC. CBD. The primary ingredient in the mixture, cannabidiol (CBD), is the focus of the analysis of thermodynamic properties conducted in this study. The hydroxyl (OH) groups of cannabidiol, also known as CBD‐OH, are specifically present at the 1′ and 3′ positions, as shown in Figure  2 . These positions are related to the two distinct hydroxyl groups on the cannabinoid molecule, highlighting the structural elements crucial for evaluating its characteristics. Using Gaussian16 software 27 based on density functional theory (DFT) with the B3LYP functional and the 6–31G (d, p) basis set, we aimed to elucidate the impact of hydroxyl group positioning on the antioxidant properties of CBD (Table  2 ). Formal numbering of cannabidiol. The hierarchical list of atoms in the cannabidiol molecule is depicted in this image, emphasizing the particular orientation and labeling of carbon atoms in accordance with the accepted nomenclature. Notably, the hydroxyl (OH) groups—located at the 1′ and 3′ carbon positions—play critical roles in the molecular structure. The hydroxyl (OH) groups were chosen for this investigation because they are particularly fascinating due to their crucial roles in determining the antioxidant qualities of organic compounds such as cannabidiol. Understanding the reactive potential of cannabidiol in scavenging free radicals and its mechanism of action as an antioxidant requires an understanding of the placement of these groups. This image provides a clear reference for the chemical basis upon which the antioxidant calculations were conducted. Calculated thermodynamic properties of CBD. Note : In the analysis of cannabidiol (CBD) as an antioxidant, we considered the potential mechanisms of hydrogen atom transfer (HAT), single electron transfer followed by proton transfer (SET‐PT), and SPLET based on the thermodynamic properties shown in Table  1 for two specific positions on CBD: CBD‐OH3′ and CB‐DOH1′. Abbreviation: H , enthalpy. The results described below were obtained and are displayed in Table  2 . The lower BDE for CDB‐OH1′ suggests a greater antioxidant potential due to the ease of hydrogen donation. This result indicates that the positioning of the OH group at 1′ enhances the radical‐scavenging ability compared with that at the 3′ position. The slightly lower PA for CDB‐OH1′ suggests better radical stabilization after proton capture, implying superior antioxidant capability. The same values of IP and PDE for both derivatives imply that the location of the OH group in these derivatives has little bearing on these attributes. According to this study, both CDB‐OH3′ and CDB‐OH1′ show promise as antioxidants. In contrast, the derivative CDB‐OH1′, which has a hydroxyl group at the 1′ position, has little benefit in terms of proton affinity and bond dissociation energy, which are two crucial factors for antioxidant activity. These findings provide important new information for future experimental studies and possible pharmacological uses in antioxidant therapy by highlighting the significance of hydroxyl group placement in the antioxidant activities of CBD derivatives (Figure  2 ). We calculated the antioxidant capacity of CBD, the main ingredient in our cannabinoid mixture, to transfer hydrogen radicals (H • ) is directly transferred from the antioxidant to the radical during the HAT process. The values for the BDE show how easily a molecule can transfer an atom of hydrogen. For CBD‐OH3′ and CBD‐OH1′, the BDE values are +82.77 and +80.36 kcal/mol, respectively. As a lower BDE would indicate a greater ease of hydrogen donation, these comparatively high values show that CBD is not particularly prone to acting as an antioxidant through the HAT mechanism. In the context of the HAT mechanism, the radical formed would be a phenoxyl radical when CBD donates a hydrogen atom to a peroxyl radical, for example: CBD − OH + ROO • → CBD − O • + ROOH In sequential proton loss electron transfer (SPLET), an electron is transferred to a radical after the antioxidant first contributes a proton (H + ). Here, PA is a crucial metric; higher PA values denote a greater capacity for proton donation. With PA values of +354.98 kcal/mol for CBD‐OH3′ and +352.83 kcal/mol for CBD‐OH1′, CBD may function as an antioxidant through the SPLET mechanism, particularly under conditions that favor proton donation. Furthermore, a very exothermic electron transfer phase is indicated by the negative ETE values (−325.75 kcal/mol for CBD‐OH3′ and −350.60 kcal/mol for CBD‐OH1′), which makes it energetically favorable. The final process (PA + ETE) has a low positive value (+2.24 kcal/mol), indicating that SPLET is a plausible and likely mechanism for the antioxidant effect of CBD. This value is further supported by the positive PA values, which indicate endothermic proton loss. Other authors have also noted that cannabidiol prefers the SPLET mechanism. 43 In SPLET, the process of radical creation starts with the donation of a proton, which generates an anionic radical. Next, the radical is stabilized by electron transfer: CDB − OH → CDB − O − + H + CDB − O − → CDB − O • + e − First, an electron is transferred from the antioxidant to the radical in the SET‐PT pathway, and then a proton is transferred. We can see the viability of this mechanism by adding the IP and the proton dissociation energy (PDE). The results for CBD‐OH1′ and CBD‐OH3′ are the same (+2.23 kcal/mol), indicating that CBD may function as an antioxidant through the SET‐PT pathway with a very low energy barrier. In SET‐PT, the process of radical generation would entail CBD losing a proton after losing an electron to create a cationic radical: CBD − OH → CBD − OH + • + e − CBD − OH + • → CBD − O • + H + The thermodynamic study indicated that the most likely mechanism for the antioxidant action of CBD is SPLET. The positive ETE and PA values imply that CBD can efficiently neutralize free radicals by sequentially transferring electrons and losing protons. Although HAT and SET‐PT have theoretical potential, in physiological situations, SPLET is more plausible because of its thermodynamic properties. This study of the antioxidant mechanism of CBD advances our knowledge of its potential therapeutic uses and directs future investigations into the creation of antioxidant medicines based on CBD. On the other hand, the anticipation that cannabichromene (CBC), which possesses a single hydroxyl (OH) group within its phenolic ring, would exhibit diminished antioxidant capabilities stems from the understanding that OH groups play a pivotal role in the antioxidant function of CBD. The SPLET mechanism, which is integral to this activity, relies on the transfer of both a proton and an electron from an OH group to generate a radical. Consequently, the presence of only one OH group in CBC is likely to lead to a decreased generation of radicals, thereby reducing its antioxidant effectiveness. Electronic transitions related to the UV absorption spectrum of CBD and CBC demonstrate their photoprotective features. In the ultraviolet (UV) spectra, the computed spectra of CBD show three primary absorption peaks at 204.32, 237.52, and 258.19 nm, as illustrated in Figure  3A These values are in good agreement with the experimental values at 209.09, 254.73, and 276.95 nm. 44 The spectrum of CBC shows peaks at 302, 284.5, 251, and 230.5 nm, which are also in good agreement with the experimental values. Calculated UV absorption spectrum. (A) The calculated UV absorption spectra of cannabidiol (CBD) (red) and CBC (blue) using time‐dependent density functional theory (TDDFT), illustrating the characteristic peaks and their respective wavelengths. (B) The electronic transitions between the molecular orbitals of cannabidiol, highlighting the transitions from HOMO to LUMO and from HOMO−1 to LUMO+1, which are critical for understanding the electronic behavior and electronic properties of the CBD molecule; CBC should display similar characteristics. Because of their location in the most important areas of the UV spectrum for sun protection, these absorption peaks are noteworthy. The three main bands of UV light are UVA (320–400 nm), UVB (280–320 nm), and UVC (100–280 nm). The experimental peaks of CBC are located within the UVB zone at 302 and 284.5 nm, while CBD is near this zone with a UV absorption peak at 276.95 nm and is located mainly in the UVC absorption region (Figure  3A ). The calculated natural transition orbitals of CBD depicted in Figure  3B suggest that the observed absorption peaks result from electronic transitions to π→π* orbitals, predominantly via HOMO–LUMO transitions. These transitions occur from the phenolic ring to the double bond and adjacent cycle. Similarly, the peak at 237.52 nm corresponds to the same HOMO–LUMO transition. At 204.32 nm, two primary transitions are noted: one is the HOMO–LUMO transition, and the other, from HOMO‐1 to LUMO+1, is attributed to n→σ* electronic transitions, which are characteristic of alcohol compounds. 45 Cannabidiol may function as an effective photoprotective agent by absorbing and reducing the negative effects of UV radiation, especially in the UVB region, according to the experimental absorption at 276.95 nm. Most importantly, the experimental UV absorption spectrum of CBD was previously published 46 and provided a baseline against which ORCA program computations could be conducted; similarly, the experimental UV spectrum of CBD has also been reported. 47 The computed spectrum and actual data show a strong correlation, indicating the accuracy and dependability of sophisticated computational techniques in predicting the photophysical characteristics of physiologically active substances such as CBD. Ultimately, these results highlight the potential of cannabidiol as an active component in skin care products and sunscreen formulations, providing effective protection against a substantial portion of the UV spectrum. This research contributes to our understanding of the photoprotective qualities of cannabidiol and establishes a foundation for future investigations and implementation in personal care and pharmaceutical products. We determined the photoprotective effect of FS‐CBD using a cell death model in which E. coli was subjected to UVB irradiation for 60 min at different intervals (Figure  4 ). E. coli bacteria (5.0 × 10 10 cells/mL) were protected with FS‐CBD, and the cells died at 30 min, with a mortality rate ( K ) of −0.2867, compared with that of the negative control (MeOH), whose cells died after 10 min ( K  = −0.3028). FS‐CBD significantly protects against cell death. The positive control, Parsol, has a maximum protection threshold of more than 60 min, K  = −0.2198, and thus Parsol is considered a broad‐spectrum photoprotector. Efficacy against UVB‐induced cellular mortality. E. coli were exposed to UVB at different time intervals. Parsol, blue line (positive control); cannabidiol (CBD), red line. The yellow line represents the negative control. The logarithm of the number of survivors decreased with MeOH, full‐spectrum cannabidiol (FS‐CBD) had a photoprotective effect on the number of survivors with respect to MeOH, and Parsol protected the cells for more time than the other treatments. García‐Bores et al. 48 used the same E. coli UVB‐photoprotector model, where Lippia graveolens extract had pronounced photoprotective activity against UVB light (0.60 J/cm 2 ) compared with MeOH, the negative control. L. graveolens protected bacteria from UVB light for 140 min, whereas our extract protected bacteria for 30 min, and thus we did not consider it to be a long‐lasting protector compared with Lippia graveolens . We suggested that phenolic groups of CBD with double bonds absorb UVB photons. 49 Verbascoside has also been reported to be a chemical with notable photoprotective qualities 50 ; it provides broad‐spectrum photoprotection for 440 min. Conversely, CBD has less activity. 51 Because UVB radiation can damage plants, researchers have suggested that plants produce CBD as a protective mechanism against radiation. 52 Recent experiments have shown that plants exposed to UVB radiation do not exhibit an increase in cannabinoid production. CBD plants likely use CBD for secondary metabolic purposes. 53 Studies of cytoprotective effects have indicated that unlike CBG and CBD, CBN and CBC may directly counteract the effects of UVB irradiation. 47 We evaluated HaCaT cell viability after exposure to different UVB intensities (0, 45, 90, 180, 360, 720, 1440, 2880, and 5780 mJ/cm 2 ; Figure  5 ). This experiment was necessary to determine the LD 50 for the photoprotection experiments. We showed that the LD 50 of UVB in HaCaT cells was 1083 to 1436 mJ/cm 2 , equivalent to ~120 s (mJ/cm 2  = 9.03 mW × 120 s; Figure  5A ). UVB killed HaCaT cells at 360 mJ/cm 2 (33% less viable cells), 720 mJ/cm 2 (37%), 1440 mJ/cm 2 (43%), 2880 mJ/cm 2 (74%), and 5760 mJ/cm 2 (88%) compared with untreated cells ( p  < 0.0001 one‐way ANOVA with Tukey's test; Figure  5B ). Cytotoxic UVB effect on the HaCaT keratinocyte cell line. (A) The percentage of surviving cells decreased significantly after UVB irradiation was applied from 360 to 5760 mJ/cm 2 , MTT reduction (*** p  < 0.0001 vs. 0, one‐way ANOVA with Tukey's test). (B) The LD 50 was 1247 mJ/cm 2 according to the log (inhibitor) versus response, variable slope (four parameters) statistical test. (C) The normality of the data was evaluated using a normal QQ plot: normality and lognormality test (alpha = 0.05). UVB is a strong mutagenic agent that induces cell death. Vats et al. induced a form of nonapoptotic programmed cell death called ferroptosis, which is a consequence of excessive peroxidation, in epidermal keratinocytes. 54 UVB exposure induces the death of keratinocytes via DNA damage, such as double‐strand breaks mediated by γ‐H2AX. 55 Melanin (an epidermal pigment) is the first line of defense of the skin against ultraviolet irradiation. 56 Melanin absorbs ultraviolet rays and dissipates ultraviolet radiation. Keratinocytes respond to UVB, inducing apoptosis to prevent malignant transformation. Calabrese et al. 57 described the role of inflammation induced by UVB light exposure and the role of both cell death and transformation to cancer. Photodimer formation in the genome, induced by UV rays, can also induce mutations and ROS generation that alter nucleotides. 58 The viability of HaCaT cells treated with various concentrations of FS‐CBD (0, 1, 3, 6, 12, 25, and 50 μg/mL) was evaluated (Figure  6 ), and this result is essential to determine photoprotective effect of FS‐CBD. The viability decreased at 6 μg/mL (23% decreased viability) and decreased significantly at 12 μg/mL (62%), 25 μg/mL (94%), and 50 μg/mL (98%) ( p  < 0.0001, one‐way ANOVA, Tukey test; Figure  6A ). The IC 50 was 9.7 μg/mL, and we used these data to evaluate the photoprotective effect (Figure  6B ); however, we used doses that did not drastically alter cell viability. For the photoprotection test, we used the maximum nontoxic dose (Figure  6C ); according to this result, we used 10 and 15 mM ( p  < 0.0001, One‐way ANOVA with Tukey's test). Percentages of viable cells after treatment with different concentrations of full‐spectrum cannabidiol (FS‐CBD). (A) The percentage of surviving cells decreased after exposure to 6–50 μg/mL FS‐CBD (*** p  < 0.0001 vs. 0, one‐way ANOVA with Tukey's test). (B) The IC 50 was 9.7 μg/mL, as evaluated by the sigmoidal, 4PL analytical test, where X is the log (concentration). (C) The nontoxic dose was 1.5–3 μg/mL. The use of CBD in the treatment and prevention of skin cancer is due to its ability to induce cell death. 19 The antiproliferative effect of CBD has been well studied. Hasan et al., 59 in 2023, used CBD as an anticancer therapy. Due to the effectiveness of CBD in inducing cancer cell death, the author combined 5‐fluorouracil uracil with CBD and, through the use of nanoparticles, released it into the skin, obtaining excellent results for cancer treatment. Luczaj et al. 60 suggested that CBD induces apoptosis in psoriatic keratinocytes irradiated with UVB, and CBD antioxidants improved the effectiveness of anticancer treatment and protected cells against changes in phospholipid and ceramide profiles induced by UVA. Anticarcinogenic and antiproliferative effects have been widely observed in different cancer cell lines, such as glioma, breast cancer, and hepatocellular carcinoma. 61 Our results agree with the currently established findings regarding the anticancer potential of CBD. We believe that subsequent work will need to use nontransformed cells. Likewise, we believe that studies should be designed to elucidate its effectiveness as a treatment to eradicate skin cancer. Subsequently, various topical therapies could improve the health of people who suffer from skin cancer. The maximum nontoxic dose of FS‐CBD was used, and HaCaT cells were exposed to the LD 50 of UVB at 1080 mJ/cm 2 , while the other two UVB doses were 1620 and 2160 mJ/cm 2 (Figure  7 ). The experimental UVB‐irradiated group without FS‐CBD (positive control) showed less viability at 2 min (~50%) than the untreated HaCaT cell group. The HaCaT cells exposed to 1.5 μg/mL FS‐CBD and 2 min of UVB irradiation were protected from cell death (~58%), and a significantly greater number of viable cells was observed for the cells treated with 3 μg/mL FS‐CBD (67%) than those treated with 1.5 μg/mL FS‐CBD. All cells treated with FS‐CBD were protected from cell death: cells exposed to UVB for 3 min—untreated (38%), 10 mM (43%) or 3 μg/mL M (52%); cells exposed to UVB for 4 min—untreated (32%), 1.5 μg/mL (35%), or 3 μg/mL (43%) ( p  < 0.0001, one‐way ANOVA with Tukey's test). Photoprotective effect of FS‐CBD on UVB irradiation‐induced damage in the HaCaT keratinocyte cell line. The percentage of viable cells was evaluated after exposure to 0, 1.5, and 3 μg/mL FS‐CBD and irradiation with UVB (1080, 1620, and 2160 mJ/cm 2 ). FS‐CBD protected against UVB‐induced cell death, and viability was evaluated using MTT assay ( &&& p  < 0.0001 vs. all groups, ωωω p  < 0.0001 vs. the 0, 1080 mJ/cm 2 group, ψψψ p  < 0.0001 vs. the 0, 1620 mJ/cm 2 group, *** p  < 0.0001 vs. the 0, 2160 mJ/cm 2 group, one‐way ANOVA with Tukey's test). Currently, the photoprotective effects of CBD on UVB damage have been proven. 62 In HaCaT keratinocytes, CBD diminished apoptosis, cytotoxicity, and cell cycle arrest; reduced the expression of γH2AX and the formation of cyclobutene‐pyrimidine dimers; and reduced ROS levels. 63 CBD had a protective effect at a dose of 8 μM, which is consistent with the doses used in our study, which were 1.5 and 3 μg/mL (10–15 μM) CBD. Liu C et al. 64 showed that HaCaT cells treated with CBD were protected from damage induced by UVB radiation, and CBD decreased cell death by modulating the caspase‐1‐IL‐1β axis. The concentration (8 μM) used by Liu et al. 64 coincided with the concentrations established as our maximum nontoxic doses of 1.5 and 3 μg/mL (10 and 15 μM). Luczaj et al. 65 concluded that CBD reduced the procarcinogenic consequences of UVA irradiation in a melanoma cancer model. In another interesting model of psoriasis, CBD accumulated in membrane keratinocytes. CBD reduced the imbalance in redox reactions observed in UV‐irradiated keratinocytes by decreasing ROS levels and increasing the Trx‐dependent system; this effect was increased by the presence of vitamins A and E in the cells. 66 Morakul et al. 67 reported that the presence of CBD in keratinocytes improves photostability and reduces inflammatory processes. CBD decreased p‐AKT levels and reduced apoptotic signaling pathways in cells treated with UVB. 68 Wójcik et al. 68 and Gęgotek A. et al. 51 reported that CBD protected against the effects of UVA/B radiation because it inhibited the expression of the TNFα/NFκB and IκBKB complex. In another study published in the same year, Gęgotek et al. 69 concluded that topically applied CBD may be an effective compound against UVB‐induced oxidative stress. The skin of CD‐1 et/et mice corresponded to that of animals 8–10 weeks of age in our observations. It consists of normal skin, composed of the epidermis, dermis, and hypodermis, with an underlying layer of skeletal striated muscle and a thin layer of connective tissue. The skin of the control group of mice (EtOH) had an epidermis that consisted of a well‐differentiated epithelium of six to eight layers (Figure  8A ). In the dermis, the reticular layer comprises bundles of collagen fibers that form a homogeneous layer. The cellular population primarily comprises fibroblasts, some fibrocytes, and occasional neutrophils. The epithelial component of the hair also has necrotic changes. In the hypodermis, adipocytes exhibit diffuse degenerative and atrophic changes. The hypodermis is characterized by normal adipocytes. Photomicrographs showing representative histological changes in the dorsal skin of mice in each group. (A) In the EtOH group, normal skin characteristics are observed. (B) In the UVB group, all the alterations caused by UV were observed. At the epidermal level, paraqueratosis, hyperplasia, spongiosis, and blisters are mainly recognized. The dermis contains reactive fibroblasts and abundant neutrophilic infiltrates. In the hypodermis, adipocytes with degenerative and necrotic changes are observed. (C) In the FS‐CBD group, normal skin is observed. (D) In the FS‐CBD + UVB group, focal fields with blisters and necrosis in the epidermis are observed; at the dermis level, moderate neutrophilic infiltration is detected. Close‐up boxes show important details for each treatment. H&E staining 100X. Boxes 400X. Ad, adipocytes; Bl, blisters; D, dermis; E, epidermis; H, hypodermis; Hp, hyperplasia; I, neutrophilic infiltrate; Ne, necrosis; P, paraqueratosis; Sp, spongiosis. The skin of mice in the group treated only with extract (FS‐CBD) had a normal structure (Figure  8C ). The skin of mice in the positive control group (EtOH + UVB) exhibited pathological changes in all layers (Figure  8B ), with the most pronounced changes occurring in the epidermal and dermal levels. In these animals, epidermal atrophy, mild hyperplasia, and parakeratosis had a multifocal distribution, and these changes were accompanied by severe spongiosis leading to blister formation. Additionally, multifocal epithelial necrosis was evident. Dermal changes included congestion with hemorrhage and severe neutrophil infiltration, moderate reactive fibroblasts, and multifocal sebaceous gland necrosis. All these changes were indicative of severe diffuse dermatitis. The skin of mice in the experimental group subjected to the extract and UV exposure displayed multifocal mild dermatitis (Figure  8B,D ). At the epidermal level, moderate to severe spongiosis with occasional blistering, parakeratosis, and focal necrosis were observed. Dermal changes included damage to fibroblasts and collagen fibers, edema, and mild neutrophilic inflammatory infiltration. Hypodermal changes manifested as multifocal degenerative and atrophic alterations. In all groups, the population of mast cells was evaluated. In all groups, the mast cells were similar in size and form. No changes in the number of cells were observed. The description of the control group facilitated the establishment of normal histological skin characteristics in the mice (Figure  8A,C ). The UV‐exposed group exhibited all the damage induced by acute ultraviolet light exposure, forming the basis of the histopathological diagnosis. Notably, diffuse necrosis and blister formation were prominent in the observation fields of this group. Evaluation of the experimental group without UV exposure indicated that FS‐CBD alone did not induce significant damage or harmful effects overall, with only focal areas showing shallow dermatitis. Treatment with the extract prior to UV exposure produced a low level of photoprotection because we observed more pathological changes in the skin of the mice in the FS‐CBD + UV group than in the skin of the mice in the UVB treatment‐only group (Figure  8D ). The use of the extract only diminished the extent of pathological changes. UVB produced diffuse changes, while the use of the extract in combination with UVB produced only multifocal changes. Keratinocytes were particularly affected, exhibiting necrosis, spongiosis, and blistering, while the dermis displayed inflammatory infiltrates with edema and mild neutrophil infiltration. A poor effect on the decrease in neutrophilic infiltration likely exists, but this effect is not clear. No differences in the number of mast cells were observed among the different groups (Figure  8D ). Gęgotek et al. (2021) analyzed the effect of CBD on the proteome of keratinocytes irradiated with UVA and UVB in three‐dimensional cultures. Their findings revealed the protective effects of CBD on UVA‐ and UVB‐induced damage, which were similar to our results and were attributed to the downregulation of proinflammatory factors, particularly proteins such as TNFα/NFκB and IκBKB, highlighting the antioxidant and anti‐inflammatory properties of CBD. These findings support further investigations into the long‐term effects of CBD utilization on keratinocytes. The phytocannabinoid CBD has several beneficial effects on skin health, including potential photoaging prevention. However, its mechanisms of action remain incompletely understood. Li et al. (2022) evaluated the photoprotective effects of CBD on acute UVB‐induced damage in human HaCaT keratinocyte cells and murine skin tissue. These findings indicate that CBD attenuates UVB toxicity by reducing reactive oxygen species levels and activating autophagy; moreover, it mitigates photodamage in murine models by ameliorating abnormal proliferative changes in the epidermis and reducing inflammation‐associated cyclooxygenase‐2 (COX‐2) protein expression in UVB‐irradiated skin. These results, along with our findings, suggest that CBD could serve as a beneficial agent for reducing UVB‐induced skin damage and could be employed as an anti‐inflammatory agent. The photoprotective effects of isolated CBD may be attributed to its ability to modulate redox homeostasis and autophagy. Other studies have documented the capacity of CBD to protect against cellular damage during immune responses, warranting further investigation into its potential as an immunomodulatory therapeutic alternative. Compared with our results, we found that the full‐spectrum CBD had low photoprotection, probably due to the sensitivity of our CD‐1 et/et mouse strain. However, we suggest adjusting the radiation intensity according to the sensitivity of this strain. The number of mast cells was determined in the mouse skin histology groups to determine whether FS‐CBD was protected against UVB radiation‐induced inflammatory damage (Figure  9 ). The skin sections from the EtOH group presented a greater number of mast cells (21 cells) than those from the FS‐CBD group (12 cells), but a similar number of mast cells was detected in the UVB group (21 cells). The number of mast cells in the UVB group was significantly greater than that in the FS‐CBD + UVB group (13 cells). Compared with the UVB treatment, FS‐CBD protected against the inflammatory effect of UVB irradiation, decreasing the number of mast cells by 61%. Photomicrographs showing the mast cells in different groups. (A) EtOH‐treated mice; (B) UVB‐treated mice; (C) FS‐CBD‐treated mice; (D) FS‐CBD + UVB‐treated mice. Mast cells are recognized by their violet color. No differences in the size or shape of the cells were observed. Toluidine blue staining, 400X. Graph showing the number of mast cells in skin tissue after UVB irradiation and topical FS‐CBD administration; ** p  < 0.001 versus EtOH, $$ p  < 0.001 versus UVB, and && p  < 0.001 versus UVB + FS‐CBD. Nolasco Ontiveros E., et al. examined, in a photoprotective CD‐1et/et mouse model and showed the anti‐inflammatory effect of L. racemosa , which is a chemophotoprotective plant. The authors reported a decrease in histological damage induced by UVB radiation, based on the low presence and degranulation of mast cells, in addition to the regulation of the expression of IL‐2 and COX‐2. L. racemosa is considered a good photoprotective agent, and FS‐CBD is a low photoprotective agent 70 ; however, FS‐CBD has a similar anti‐inflammatory effect to that of L. racemosa , and thus FS‐CBD has good anti‐inflammatory properties that reduce damage caused by UVB radiation. Another model, utilized mast cells as inflammatory markers, and CBD was shown to induce the degranulation of mast cells. The authors concluded that passive cutaneous anaphylaxis occurs via the reduction of ear swelling in mice caused by the depletion of cannabinoid receptor 1 (CB1) and 2 (CB2). 71 Using an in vivo endometriosis model, CBD exerted an analgesic effect by reducing the recruitment of mast cells to the spinal cord, downregulating the expression of MMP‐9, iNOS, and TGF‐β; reducing cytosolic COX‐2 expression; and reducing NFκB nuclear localization. 72 In an in vivo sclerotic mouse model, the CBD quinol derivative (VCE‐004.8) was shown to have an anti‐inflammatory effect. VCE‐004.8 prevented mast cell degranulation and macrophage infiltration in the skin, and these effects were impaired by the PPARγ antagonist and CB2 antagonist and by the downregulation of genes associated with fibrosis. The author suggested that CBD treatment could be used to manage scleroderma and, potentially, other fibrotic diseases. 73 TGF‐β1 expression was evaluated using immunofluorescence staining. This protein was overexpressed in the UVB group compared with all other groups (Figure  9A ). TGF‐β1 expression in the UVB + FS‐CBD group was similar to that in the control groups that were not irradiated with UVB. Figure  9 shows the nucleus in blue, α‐tubulin in green, and TGF‐β1 in red; the merged signals are shown with Nomarski and merge‐Nomarski. The MFI analysis corroborated a statistically significant decrease in TGF‐β1 expression via FS‐CBD‐mediated photoprotection compared with UVB. MFI showed statistically significant overexpression of TGF‐β1 compared with all groups: FS‐CBD, EtOH, and UVB + FS‐CBD group (Figure  10A,B ). TGF‐β1 expression was detected in both the dermis and epidermis ( p  < 0.0001, one‐way ANOVA with Tukey's test). Overexpression of TGF‐β1 induced by UVB irradiation and the photoprotective effect of FS‐CBD. (A) Histology of mouse skin after UVB exposure showing the overexpression of the inflammatory marker TGF‐β1 in all groups; the FS‐CBD group without TGF‐β1 expression was similar to the untreated UVB group. Blue—nucleus; green—α‐Tubulin; red—TGF‐β1; merge—all colors; Nomarski contour of the tissue and Nomarski + merge. (B) Comparison of TGF‐β1 expression in the UVB group vs. the FS‐CBD + UVB group. Relative florescence units indicate the overexpression of TGF‐β1 in the UVB group ( &&& p  < 0.0001 vs. the UVB group, *** p  < 0.0001 vs. the FS‐CBD group; one‐way ANOVA with Tukey's test). TGF‐β1 is synthesized by many cell lines, such as lymphocytes, macrophages, and dendritic cells, and its expression regulates the differentiation, proliferation, and activation of these cells and many others in an autocrine or paracrine manner. In general, TGF‐β1 has pleiotropic effects on the immune response during the development of infections and neoplastic processes. 74 Thomas et al. 75 and Scola et al. 76 proposed TGF‐β1 as a molecular marker for the diagnosis of skin cancer. CBD has the capacity to inhibit TGF‐β1 expression, and thus we could consider it a protector in the progression of inflammation and the progression to carcinogenesis. By inhibiting TGF‐β1, CBD is a therapeutic target that allows us to suppress processes in premalignant and early malignant lesions. Furthermore, inhibiting TGF‐β1 does not allow the establishment of an immunosuppressive tumor microenvironment by modulating the functions of immune and stromal cells. 77 NLRP3 or the inflammasome was evaluated by performing skin histology and immunofluorescence staining (Figure  11 ). The nonirradiated FS‐CBD and EtOH groups did not exhibit overexpression of the NLRP3 protein (Figure  10A,B ). The UVB group overexpressed the NLRP3 protein. The MFI of NLRP3 was significantly increased in the FS‐CBD, EtOH, and UVB + FS‐CBD groups ( p  < 0.0001, one‐way ANOVA with Tukey's test). Overexpression of NLRP3 (inflammasome) induced by UVB irradiation and the photoprotective effect of FS‐CBD. (A) Histology of mouse skin after UVB exposure showing the overexpression of the inflammatory marker NLRP3 in all groups; the FS‐CBD group without NLRP3 expression was similar to the untreated UVB group. Blue—nucleus; green—α‐Tubulin; purple—NLRP3; merge—all colors; Nomarski contour of the tissue and Nomarski + merge. (B) Comparison of NLRP3 expression in the UVB group with the FS‐CBD + UVB group. Relative fluorescence units show the overexpression of NLRP3 in the UVB group ( &&& p  < 0.0001 vs. the UVB group, *** p  < 0.0001 vs. the FS‐CBD group; one‐way ANOVA with Tukey's test). The anti‐inflammatory effect of FS‐CBD on the UVB + FS‐CBD group was confirmed; inflammation induced by UVB irradiation was inhibited by FS‐CBD (Figure  11A,B ). Figure  10B shows the nucleus (blue), α‐tubulin (green), NLRP3 (purple), and merge; the tissue is shown with Nomarski and merge‐Nomarski. The NLRP3 inflammasome promotes the maturation of caspase‐1 and the proinflammatory cytokines IL‐1β and IL‐18, inducing pyroptosis (which is associated with tumor suppression and suggests antitumor immune responses), as proposed by Tan et al. 78 In this regard, Suryavanshi et al. 79 reported its inhibition by cannabinoids and specifically by CBD. In another study performed by Suryavanshi Set al., 80 CBD and Δ 9 ‐THC inhibited cytokine storms in keratinocytes. CBD also inhibits the phosphorylation of NF‐κB and significantly decreases the levels of IL‐6, IL‐8, and TNF‐α. Currently, according to Martinez Naya et al., the mechanism of action of CBD is the repression of NLRP3. 81 Specifically, CBD has been shown to inhibit NLRP3‐mediated IL‐1β activation, as reported by Liu et al. 82 The simultaneous docking of CBD and CBC was performed because CBD and CBC are among the most abundant cannabinoids in the FS‐CBD; the observation of how CBD and CBC interact with important TGF‐βR1 residues, especially those close to the active site, is quite interesting. Understanding how CBD and CBC may alter the function of the enzyme depends on the residues found in docking research, which include R 240 , R 332 , L 354 , V 356 , I 365 , I 367 , P 369 , N 370 , H 371 , R 372 , G 374 , Y 378 , M 379 , A 380 , V 383 , L 384 , D 386 , I 388 , M 390 , K 391 , F 396 , A 399 , and V 438 . Among these residues, CBD forms two hydrogen bonds with R332, and CBC forms one hydrogen bond with V383. Residue R332 is located within the active site of the enzyme. The potential for the direct regulation of enzyme activity is increased by CBD and CBC placement within the active region. The docking data indicate that strong contact may affect the activity of TGF‐βR1, with an affinity energy of −8.84 kcal/mol (Figure  12A ). Compared to the individual affinity values of CBD and CBC obtained from the virtual screen, they are located at lower affinity energies of −5.0 and −5.9 kcal/mol, respectively, as shown in Table  3 . The simultaneous docking of CBD and CBC showed an affinity energy of −8.868 kcal/mol. This value is higher than the individual affinity energy values, as CBD has an affinity value of −5.7 kcal/mol, while CBC has an affinity value of −5.7 kcal/mol. These results show the importance of the mixture of CBD and CBC in the affinity for the TGF‐βR1 ligand. The residues involved in the interaction between CBD and CBC are D 247 , F 275 , R 276 , W 281 , K 282 , W 283 , H 285 , E 286 , P 287 , K 288 , Y 341 , Y 342 , K 288 , V 343 , and R 345 (Figure  12B ). (A) Molecular docking analysis depicting CBD and CBC in green sticks within the TGF‐βR1 receptor binding site. (B) Simultaneous docking results for the ligand TGF‐β1 with CBD and CBC. Affinity values resulted from a virtual screen of FS‐Mix with TGFβR‐1, indicating the cannabinoids and their affinity in kcal/mol and the digital source where each cannabinoid was identified. The coordinates of the compounds were obtained from the NIST webpage. The coordinates of the compounds were obtained from the PubChem database. Analogs of curcumin have been used in similar TGF‐βR1 inhibition experiments, showing good inhibition rates with high binding energies. This study focused on how TGF‐βR1 inhibition functions as a crucial signal in the development of malignant tumors. 83 The relationship between TGF‐βR1 and cannabidiol suggests possible treatment paths for disorders involving inflammation and skin defense. The possible modulation of TGF‐βR1 activity by CBD represents a new avenue for treating these disorders. However, further experimental research is required to fully understand the precise mechanisms and outcomes of this interaction. The experimental UV absorption maxima of cannabidiol were observed at 276.95, 254.73, and 209.09 nm. According to Ryu et al., 46 it may have a role in UV protection. Given the function of TGF‐βR1 in skin health and repair, the interaction between cannabidiol and TGF‐βR1 may be essential for mediating protective responses against UV‐induced damage. In the case of NLRP3, CBD, and CBC were evaluated simultaneously because they were the most abundant compounds in FS‐CBD. First, the homology of the NLRP3 protein was determined to prepare the protein for the next molecular docking process. This initial step ensured that the NLRP3 protein was accurately and finely represented. A noteworthy interaction was discovered between the 6NPY structure and the CBD and CBC compounds during the docking process. This interaction was measured with an affinity energy of −11.85 kcal/mol. I 149 , E 150 , D 151 , 165 , R 165 , R 165 , Y 166 , Y 166 , L 169 , A 225 , I 228 , K 230 , T 231 , I 232 , L 233 , R 235 , Y 256 , H 258 , R 260 , E 261 , D 300 , D 303 , T 347 , F 371 , Y 379 , L 411 , V 412 , C 413 , W 414 , I 480 , L 481 , R 502 , M 503 , N 504 , F 518 , and M 521 were among the residues implicated in the interaction. Notably, CBD also forms two hydrogen bonds with the R 235 residue. These results point to the potential of these plant compounds to control NLRP3 inflammasome activation, providing exciting new therapeutic options for the treatment of inflammatory illnesses, as shown in Figure  13 . Docking of CBD and CBC with the NLRP3 protein. A 3D model of the NLRP3 protein is depicted. This model, in which CBD and CBC are bound and depicted as green sticks, was developed through homology modeling using YASARA software. A virtual screening was performed with Vina‐2 to evaluate and compare the docking affinities of the different compounds present in the FS‐CBD mixture. O+ for the GPU. The results are shown in Table  4 . Affinity values resulting from the virtual screen of FS‐Mix with NLRP3, which indicates the cannabinoids and their affinity is represented in kcal/mol, and the digital source where each cannabinoid was identified. The coordinates of the compounds were obtained from the NIST webpage. The coordinates of the compounds were obtained from the PubChem database. The affinity determined from the simultaneous docking of CBD and CBC was greater than that of the compounds docked separately. This result indicates a cooperative effect that increases the affinity of the ligands and thus possibly increases the inhibitory effect of CBD and CBC on the NLRP3 protein. The NLRP3 inflammasome is crucial for promoting inflammation. In various models, CBD has been shown to inhibit NLRP3 both in in vivo mouse models and in various cancer cell lines, including HaCaT keratinocytes. 64 , 84 , 85 , 86 Therefore, Chu et al. 87 proposed that CBD is a direct inhibitor of NLRP3, which blocks the NLRP3 inflammatory pathway, pro‐IL‐1β, mature IL‐1β, pro‐caspase‐1, IL‐6, IL‐8, and TNF‐α. Our docking results support the hypothesis proposed by Chu et al. 87 We found that the binding of CBD and CBC to NLRP3 is highly likely because of their high affinity in the docking results. 52 Related studies have been conducted targeting the ATPase activity of the NACHT domain 38 ; similar to our findings, the CBD interaction with G 229 , K 230 , and T 231 indicates potential NLRP3 suppression, as these ligand bonds are essential for ATP binding.

An analysis of the complex FS‐CBD mixture revealed two predominant cannabinoids, CBC and CBD, and other cannabinoids were present at very low concentrations. CBD was more abundant, and thus the effects were mainly attributed to this compound, although the roles of other cannabinoids have not been excluded. Based on time‐dependent density functional theory calculations and corroboration by experimental findings, the electronic transitions observable within the UV absorption spectrum of FS‐CBD imply that CBC plays a pivotal role in mediating the protective effects of UVB radiation‐induced damage. Furthermore, the SPLET mechanism constitutes the principal mechanism sustaining the antioxidant properties of CBD, offering a substantive contribution to the understanding of its biochemical activity. Ultimately, the photoprotective effect of FS‐CBD was evaluated at three different biological levels (bacterial, cellular, and animal), and at all levels, FS‐CBD exhibited low protection against UVB irradiation but in a shallow form. FS‐CBD has the ability to protect bacteria and skin cells from cell death in vitro. FS‐CBD protected the skin of CD‐1 et/et mice from damage caused by UVB‐mediated repression of cyclin TGF‐β1 in vivo, and the inflammasome complex NLRP3 was shown to be a good photoprotector by blocking inflammation. Molecular docking revealed that FS‐CBD is a good inhibitor of TGF‐βR1 and NLRP3, as it binds to the active sites of both proteins, thus indicating the potential of FS‐CBD for treating inflammatory skin conditions.

Solar radiation is classified as UV, visible light, infrared, or radio frequency. The UV spectrum is divided into UVC with wavelengths of 100–280 nm, UVB with wavelengths of 280–315 nm, and UVA with wavelengths of 315–400 nm. 1 UVA irradiation penetrates and alters the deep layer of the dermis. UVB light predominantly acts on the basal cells of the epidermal layer and leads to various adverse effects, such as the generation of reactive oxygen species (ROS), inflammation, immunosuppression, DNA damage, and severe skin cancer. 2 The use of skin products such as solar protectors is beneficial against UV irradiation but has some disadvantages, including environmental pollution; however, adding antioxidant properties to UV protectors represents an additional advantage since antioxidants help to prevent DNA damage. 3 Cannabinoids are a group of terpenophenolic compounds characteristic of Cannabis sativa . 4 The main bioactive forms of cannabinoids detected in the plant are produced after light or heat exposure, which induces a decarboxylation reaction to generate cannabigerol (CBG), Δ 9 ‐tetrahydrocannabinol (Δ 9 ‐THC), and cannabidiol (CBD). 5 , 6 CBD is not a psychoactive component 7 but has other properties, such as antitumor activity in various types of cancers, including pancreatic, 8 , 9 glioblastoma, colorectal, 10 breast, 11 and gastrointestinal cancer. 12 It also has beneficial effects on brain function, metabolism, and pain reduction. 13 , 14 Currently, a variety of derivatives containing CBD have been tested for medicinal purposes. Some of them have been classified as full‐spectrum compounds that contain mainly CBD but also low concentrations of other cannabinoids, such as CBG, Δ 9 ‐THC, cannabinol (CBN), cannabichromene (CBC), and cannabivarine (CBV), 15 and their biological effects are different from those of CBD. 16 However, full‐spectrum CBD might be promising since CBD and the low concentrations of some other compounds could have an entourage effect, 17 indicating a synergistic effect. On the other hand, based on the chemical structures of some compounds present in the full spectrum, CBD could be photoactive, which could be useful against damage induced by UV light. Indeed, the photoprotective effect of isolated CBD has recently been documented. 15 , 16 , 18 In a mouse model, the backs of BALB/c mice were irradiated with UVB (120 mJ/cm 2 ) and treated topically with CBD (0.1 mg/cm 2 ) for 5 days. Epidermal thickening and erythema were less common in the CBD treatment group than in the UVB group, and collagen fiber bundles were more abundant in the UVB group than in the CBD treatment group. 19 Based on the potential entourage effects of CBD and other cannabinoids found in the full‐spectrum preparation, we aimed to evaluate the photoprotective effect of full‐spectrum CBD against UV light‐induced skin damage in an acute mouse CD‐1 et/et model. The absorption spectrum of CBD (220–280 nm) is within the UVC‐UVB light spectrum, 4 , 20 indicating that CBD could function as a photoprotective agent, preventing the interaction of UVC and UVB radiation with the skin. In addition, the antioxidant activity of CBD 21 , 22 prevents free radical damage associated with UVR (ultraviolet radiation). Inhibition of endocannabinoid receptors (CB1/CB2) by cannabinoids reduced tumor growth and increased apoptosis of tumor cells in nude mice inoculated with epidermal tumor cells. 23 The photoprotective effect of CBD on metabolic changes induced by UVA and UVB rays in 2D and 3D cultured skin fibroblasts was determined, and the main changes were associated with proteins involved in the antioxidant and inflammatory responses. 24 , 25 We hypothesized that FS‐CBD is a photoprotective, antioxidant, and anti‐inflammatory agent that inhibits bacterial and HaCaT keratinocyte death. Additionally, FS‐CBD is believed to interact with and repress mouse skin inflammatory markers induced by damage from UVB light exposure. When testing our hypothesis, we aimed to show that the components of FS‐CBD at least partially protect against skin damage induced by UV light exposure. In addition, using in silico strategies, we showed that the reduction in inflammation is related to the interaction between FS‐CBD and the proinflammatory proteins TGF‐β1 and NLRP3.

The FS‐CBD used in the present study was acquired from DESERTFLAVORS® (9920 w camelback rd, Phoenx Arisona 85037). FS‐CBD was extracted with supercritical CO 2 . The methodology used was described by Pattnaik et al., 26 with an analysis certificate and a chromatography test reporting that the extract contained CBD and CBC, along with Δ 9 ‐THC, CBG, CBN, and CBV. Cannabinoid data were corroborated by chromatography–mass spectrometry (Figure  1 ). TIC of the full‐spectrum cannabidiol (FS‐CBD) sample. Major compounds present in FS‐CBD were I is CBC and II is CBD. FS‐CBD (1 mg) was dissolved in HPLC‐grade hexane and subsequently injected (5 μL) into a GC–MS instrument (Agilent Technologies 6850). The following conditions were used to evaluate the presence of cannabinoids in the FS‐CBD mixture: initial temperature, 110 °C; maximum temperature, 325 °C; initial time, 0.00 min; and equilibration time, 0.50 min. The ramp parameters were 1 (20.0 rate, 250 °C, 10 min), 2 (30.0 rate, 290 °C, 5 min), and 3 (0.0, 0 °C, 0 min). An Agilent Technologies model 6850 GC–MS instrument was used. The experimental conditions were: an initial oven temperature of 110 °C, maximum oven temperature of 325 °C, initial time of 0.00 min, equilibration time of 0.50 min, posttemp of 0 °C, post time of 0.00 min, run time of 23.33 min, splitless mode, temperature of 290 °C, initial temperature of 250 °C, pressure of 16.88 psi, purge flow of 60.0 mL/min, purge time of 0.30 min, total flow of 63.2 mL/min, saver flow of 20.0 mL/min, saver time of 2.00 min, and the gas type was helium. The column parameters were as follows: HP‐5MS 5% phenyl methyl siloxane, maximum temperature of 325 °C, length of 30.0 m, diameter of 250.00 μm, film thickness of 0.25 μm, initial flow rate of 1.5 mL/min, initial pressure of 16.88 psi, average velocity of 46 cm/s, and inlet source. MS data were acquired in scanning acquisition mode with a resulting EM voltage of 1894, low mass of 35.0, high mass of 400.0, and threshold of 150. We employed Gaussian16 27 with density functional theory (DFT) and the B3LYP/6‐31G(d,p) functional and basis set to compute the thermodynamic properties of the FS‐CBD radicals, anions, and cations in the gas phase. The focus was on the bond dissociation energy (BDE); the resulting value is the sum of the radical enthalpy plus the hydrogen atom enthalpy, less the enthalpy of the neutral compound, and is represented in kcal/mol. The proton affinity (PA) is the anion enthalpy plus the proton enthalpy, less the neutron enthalpy. The ionization potential (IP) is the sum of the radical cation enthalpy plus the electron enthalpy, less the enthalpy of the neutral compound. Proton dissociation enthalpy (PDE) is the radical enthalpy plus proton enthalpy, less the radical cation, as reported by Messaadia et al. 28 The UV absorption spectrum, as well as the involved orbitals implicated in the electronic transitions, were calculated using ORCA software version 5.0 29 implementing time‐dependent density functional theory (TDDFT) with the B3LYP and def2‐TZVP basis sets to examine the absorption spectrum of cannabidiol. The Escherichia coli (ATCC 25922) strain was cultured in BHI (heart and brain infusion broth, Bioxon‐112) at a density of 10 8 –10 10 (cells/mL), as indicated by an optical density of 0.4 at 550 nm. The bacterial cells were then centrifuged at 6000 rpm for 3 min, resuspended in 1X Ringer's PBS (pH 7.0), and transferred to a quartz cuvette (Pye Unicam B538751 A, 1 mm thickness, 4 mL). 30 FS‐CBD was dissolved in methanol at a concentration of 2 mg/mL and added to a separate quartz cuvette. This cuvette was positioned in front of another cuvette containing the bacterial suspension, thereby creating a single experimental unit. These units were exposed to UVB radiation using a Spectroline EB‐280C lamp (312 nm) 31 with a total irradiation dose of 0.60 J/cm 2 . The survival rate of bacteria was assessed at various intervals postirradiation using the dilution method. The tested substances, FS‐CBD and a positive control organic filter present in the commercial sunscreen octyl methoxycinnamate (Parsol, ISP VAN DIK), were dissolved in ethanol at a concentration of 60 μg/mL, while the respective solvents for each photoprotective substance served as negative controls. HaCaT cells are a transformed noncarcinogenic cell line. These cells are human keratinocytes and conserve differentiation, morphogenesis, and cell surface markers and structure. Human skin‐derived keratinocytes (HaCaT cells; American Type Culture Collection (ATCC PCS‐200‐011) Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; Gibco) and a 1% antibiotic–antimycotic solution (Gibco). The HaCaT cell line was generated from primary cultures of normal, adult human keratinocytes. 32 , 33 , 34 For the cytotoxicity assays, 15,000 HaCaT cells were seeded in 96‐well plates for 24 h and then exposed to 0, 1, 3, 4, 6, 12, 25, or 50 μg/mL FS‐CBD for 24 h or irradiated with UVB lamps (302 nm, UVP. UVM‐26, 9.03 mW) positioned 15 cm above cells at different doses of 0, 45, 90, 180, 360, 720, 1440, 2880, and 5780 mJ/cm 2 (mJ/cm 2  = mW × s). The photoprotective effect of FS‐CBD on UVB irradiation‐induced changes in the keratinocyte cell line was assessed, HaCaT keratinocyte cells were exposed to 0, 1.5, and 3 μg/mL FS‐CBD. After the same cells were immediately irradiated with three UVB doses, namely, 1080, 1620, and 2160 mJ/cm 2 , the cells were washed with 1× PBS, and the culture medium with FS‐CBD was replaced. After 24 h, cell viability was determined by measuring 3‐(4,5‐dimethylthiazol‐2‐ and l)‐2,5‐bromo diphenyltetrazolium (MTT; 0.5 mg/mL) reduction after 2 h of incubation. Formazan crystals were dissolved in isopropanol for 10 min. The absorbance at 540 nm was quantified using an EPOCH plate reader (BioTek). The data are shown as the means ± standard deviations and were graphed with GraphPad Prism software. CD‐1 et/et mice are a good model for studying skin pathologies because these animals are susceptible to skin diseases and are more susceptible to UVB light. For these experiments, the mice were divided into four groups: the FS‐CBD group (without UVB), the EtOH group (vehicle without UVB light), the UVB group (exposure to 302 nm, UVB, UVM‐26, 9.03 mW for 15 min), and FS‐CBD + UVB (exposure to 302 nm, UVB, UVM‐26, 9.03 mW for 15 min). All the treatments were applied to the dorsal part of the mice, and after 24 h, the mice were sacrificed in a CO 2 chamber. The Bioethical Committee/FES Iztacala (CE/FESI/092017/1199, UNAM) approved all the animal protocols, and all the procedures were carried out in accordance with the official Mexican regulation NOM‐062‐ZOO‐1999. Dorsal skin sections (1 cm 2 ) were fixed with 4% paraformaldehyde (pH 7.2) for 24 h in a tissue embedding cassette, dehydrated with a sequence of ethanol solutions (60, 70, 80, 95, and 100%), cleared with xylol and embedded in paraffin. Four‐micron‐thick sections were obtained with a Leica microtome (RM2125) and stained with hematoxylin and eosin (H&E). Histopathological changes were observed in multiple microscopic fields. Photomicrography was performed with a Leica DMC 2900 photomicroscope with 10× and 40× objectives. For the observation of mast cells, sections were dehydrated, cleared, and subsequently stained with toluidine blue. 35 Dorsal skin samples were dehydrated with a sequence of ethanol solutions (100, 96, 80, 70%, and H 2 O) for 5 min in each solution. After rehydration, a goat anti‐mouse TGF‐β1 antibody diluted 1:250 (Abcam, ab92486) was incubated with the samples for 24 h at 4 °C with constant agitation, and a secondary anti‐goat TRITC antibody (Thermo Fisher, A‐16101) was incubated with the samples at 37 °C for 2 h. For NLRP3 detection, the skin samples were incubated with a rabbit polyclonal anti‐NLRP3 antibody (Abcam, ab214185) at 4 °C for 24 h, and a rabbit anti‐mouse Alexa Fluor 647 antibody diluted 1:250 (Thermo Fisher, A‐32733) was used as the secondary antibody. Both primary antibodies were detected with a goat anti‐mouse α‐tubulin conjugated with Alexa Fluor™ 647 antibody diluted 1:250 (Thermo Fisher, A‐21235), and the nuclei were visualized with Hoechst at a dilution of 1:1500 (Thermo Fisher, A‐32733). This study presents a detailed molecular docking analysis of mainly cannabidiol (present in FS‐CBD) with the transforming growth factor β, ALK5 (TGF‐βR1), elucidating potential interactions and implications for therapeutic applications. Molecular docking was performed using the TGF‐βR1 protein structure (PDB ID: 1rw8) obtained from the Protein Data Bank and the cannabidiol structure from PubChem, optimized with Gaussian16, 27 and docked to TGF‐βR1 using YASARA. 36 In this scientific investigation, we focused on the molecular interaction between the NLRP3 protein and the main compounds in FS‐CBD, CBD, and CBC, using the structure represented by the PDB code 6NPY. 37 This structure, which is essential for the role of the NLRP3 inflammasome in the inflammatory response and innate immunity, was subjected to homology modeling analysis using YASARA software. 36 , 38 In both analyses of TGF‐βR1 and NLRP3, once the binding site was identified, simultaneous docking was performed using Vina software, 39 , 40 followed by a virtual screen of the compounds reported in the HPLC analysis to compare the affinity values when both CBD and CBC interacted separately and when they interacted simultaneously. 41 , 42

Figure S1.