- About MBL
- For Contributors
Fermentation Microbiology (FM) | Applied Microbiology
Microbiol. Biotechnol. Lett. 2023; 51(3): 250-256
Taehyun Kim1†, Jin-Woo Kim1,2†, Huitae Min1, Jisu Park1,3, Taejung Kim1,4, Geun-Hyeong Kim5, Byung-Joon Park5, Jeong Kook Kim6, Young-Tae Park1*, Jin-Chul Kim1,4*, and Jungyeob Ham1,4,6*
1Natural Product Research Center, Korea Institute of Science and Technology (KIST), Gangneung 25451, Republic of Korea
2Interdisciplinary Program in Agricultural Genomics, Seoul National University, Seoul 08826, Republic of Korea
3Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea
4Division of Bio-Medical Science and Technology, University of Science and Technology (UST), Daejeon 34113, Republic of Korea
5Kolmar Korea Co., Ltd., Seoul 06800, Republic of Korea
6NeoCannBio Co., Ltd., Seoul 02792, Republic of Korea
Correspondence to :
Jin-Chul Kim, firstname.lastname@example.org
Young-Tae Park, email@example.com
Jungyeob Ham, firstname.lastname@example.org
Cannabis sativa (CS) has been in the spotlight not only for its medical uses but also as a raw material for cosmetics. As fermented cosmetics are known to have various health benefits, they have been extensively researched. Here, we investigated the characteristics of CS stems fermented using various gut microbes. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay and melanin content analysis revealed that melan-a cells containing CS stems fermented with Weissella paramesenteroides (CSWP) showed considerably reduced melanin content. Additionally, CSWP downregulated the expression of several melanogenesis factors, tyrosinase-related protein-1, and tyrosinase-related protein-2. This study suggests that the anti-melanogenic effect of CSWP could provide a new basis for the development of skin-lightening agents.
Keywords: Cannabis sativa, fermentation, Weissella paramesenteroides, anti-melanogenesis
Light skin tones are associated with beauty and have long been of great interest. Skin pigmentation is caused by the accumulation of melanin synthesized by melanocytes to protect human skin from ultraviolet radiation . Furthermore, excessive production of melanin can cause melasma, a skin condition characterized by brown or blue-gray patches or freckle-like spots . Tyrosinase and tyrosinase-related protein-1 (TRP-1)—crucial regulators of melanogenesis—convert tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) to melanin in a series of enzymatic catalytic reactions . The signaling pathway of melanogenesis in melanocytes can activate the microphthalmia-associated transcription factor (MITF) , a key transcription factor in melanocyte development that regulates various genes, including tyrosinase and TRP-1 . Most whitening agents in the cosmetic industry inhibit these enzymatic reactions or reduce the melanogenesis pathway [12, 13], and it is well known that they (e.g., hydroquinone) cause various side effects . Therefore, there is a high demand for natural or fermented skin-whitening agents in the cosmetic industry, and research on them is actively being conducted .
Recent studies have attempted to identify natural compounds derived from plants or bacteria with antimelanogenic effects . Many types of bacteria are used to ferment dairy products and cosmetic ingredients. Lactic acid bacteria, such as the genera
CS stems (2 kg) collected in Andong, Gyeongsangbukdo were pulverized and extracted three times with 40 L of EtOH at room temperature. The EtOH extract was dried using a rotary evaporator and lyophilized to obtain a dark green powder (37.5 g), which was then dissolved in MeOH (78.3 mg/ml) and incubated at room temperature for 3 days. For fermentation, the Gifu Anaerobic Medium (GAM), MRS , and Nutrient Broth (all obtained from Kisan Bio., Korea) were used. The indicated bacteria were grown at 37 or 30℃ in each broth overnight. Subsequently, 1 ml was inoculated into 100 ml of GAM with 1 ml of MeOH. These samples were then fermented for 3 days at 37℃ or 30℃ with shaking at 200 rpm. The samples were then centrifuged at 855 ×
The bacteria used here were obtained from the Korean Collection for Type Cultures (KCTC) and the Korean Agricultural Culture Collection (KACC) and are listed in Table 1. The cells were cultured according to the KCTC or KACC protocols. Melan-a cell lines from American Type Culture Collection (ATCC) were cultured in a Roswell Park Memorial Institute 1640 (RPMI) medium (Gibco, USA) containing 10% fetal bovine serum (HyClone Laboratories, USA), 1% penicillin/streptomycin (HyClone Laboratories), and 200 nM phorbol 12-myristate 13-acetate (PMA). Melan-a cells (1 × 105) were cultured in a 50φ dish plate and then incubated at 37℃ and 5% CO2 condition.
Melan-a cells (4 × 104) were seeded in each well of a 96-well plate in a RPMI medium containing 200 nM PMA and cultured for 24 or 72 h. The medium was then removed and RPMI media containing 0.5 mg/ml of 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide were added. After the cells were incubated for 1 h, 200 μl dimethyl sulfoxide was added, and the absorbance was measured at 560 nm using a microplate spectrophotometer (Multiskan Sky, Thermo Fisher Scientific, USA). The cell viability was calculated using the following formula:
Melan-a cells (4 × 104) were seeded in each well of a 96-well plate in a RPMI medium containing 200 nM PMA and cultured for 72 h. The medium was then removed, RPMI media containing 0.5 mg/ml 6N NaOH were applied, and the samples were incubated for 30 min at 85℃. The absorbance was measured at 490 nm using a microplate spectrophotometer. Arbutin was used for the positive control group . The melanin content in live melan-a cells was calculated as follows:
L-DOPA was used as a substrate to determine tyrosinase inhibition activity. After preheating the sample of L-DOPA mixture and tyrosinase at 37℃ for 10 min, the preheated tyrosinase was added to the samples. They were incubated at 37℃ for 15 min and detected at 490 nm using a microplate spectrophotometer. Tyrosinase inhibition was calculated as follows:
Melan-a cells (1.6 × 106) were seeded in a 50Φ dish with RPMI media containing PMA (200 nM) and then incubated for 24 h. CS stems fermented with
Although CS is banned from general use in many countries owing to its addictive properties, it is well known for its use in various industries, including medicine and cosmetics [20−22]. Since fermented cosmetics have various health benefits and few side effects, we fermented CS to obtain new cosmetic functions. As described in the Materials and Methods, CS stems were extracted and fermented with various probiotics (Table 1). Many lactic acid bacteria, such as those listed in Table 1, have been reported to have various health benefits such as the antioxidant, anticancer, antiinflammatory, and antibiotic activities of
It is widely accepted that CS is utilized as a cosmetic material . Inhibition of the expression of whiteningrelated markers, such as tyrosinase and MITF, was observed in microbial extracts of probiotics, such as
Tyrosinase is involved in various early melanogenesis pathways. For example, tyrosinase catalyzes the biological conversion of tyrosine to dopaquinone using dioxygen at its binuclear copper active site under physiological conditions [9, 10]. Therefore, we speculated that the anti-melanogenic effect of CSWP might depend on the regulation of tyrosinase activity. To verify this, we measured tyrosinase activity in melan-a cells treated with EB, CSWP, kojic acid, and arbutin (Fig. 2). Kojic acid inhibits and prevents the formation of tyrosine, an amino acid necessary for melanin production . As expected, tyrosinase activity was inhibited in melan-a cells treated with kojic acid. However, melan-a cells containing EB or CSWP showed no inhibition of tyrosinase activity. Therefore, we conclude that the anti-melanogenic effect of CSWP does not depend on tyrosinase activity.
MITF is the main transcription factor for melanocyte development and regulates the expression of related genes, such as tyrosinase, TRP-1, and TRP-2 . Since we confirmed that CSWP does not affect tyrosinase activity, we assumed that its anti-melanogenic effect depends on the regulation of MITF or its related genes, such as TRP-1 and TRP-2. To verify this assumption we performed western blotting analysis to detect the expression levels of MITF, TRP-1 and TRP-2, as described in the Materials and Methods (Fig. 3). Melana cells containing arbutin showed significantly reduced levels of MITF, TRP-1 and TRP-2. Although melan-a cells containing EB showed a slightly decreased level of MITF, the expression level of TRP-2 rarely changed. However, the expression levels of TRP-1 and TRP-2 in melan-a cells containing CSWP were comparable to those in melan-a cells containing arbutin. Therefore, we found that the anti-melanogenic effect of CSWP occurred through the inhibition of MITF, TRP-1 and TRP-2 expression.
We examined the anti-melanogenic effects of CS stem extracts fermented by various bacteria. We found CSWP showed a notable whitening effect, similar to that of arbutin. CS has been used in medicine and textiles for thousands of years . Recently, owing to its various skin beautifying effects, it has also been used as a cosmetic raw material . Nevertheless, CS stems have little medicinal effect and have been used mainly in the textile industry [3, 22]. Our results show the whitening effect of CSWP, suggesting the possibility of improving the industrial applicability of CS stems.
Probiotics are effective against allergies and cancer, and also reduce blood cholesterol levels [17−19, 29]. They beneficially improve the health of the host and are abundantly found in fermented foods [23, 24, 30]. For example,
The whitening effect of ferulic acid—present in large amounts in CS seeds—has often been reported , and several cannabinoids have also shown promise as whitening agents . Although these substances are rarely present in the CS stems , our study demonstrated the non-toxic whitening effect of CSWP (Fig. 1 and Table 3). Furthermore, its whitening effect was clearly superior to that of the CS stems fermented by the other probiotics listed in Table 1 (Table 2). Contrary to previous studies on probiotic whitening activity, CSWP did not cause tyrosinase activity changes (Fig. 2). Although this study does not elucidate the precise mechanism of the whitening effect of CSWP, we infer that it inhibits MITF, TRP-1, and TRP-2 in an unconventional way. Therefore, further studies and investigations are required to understand its mechanism and increase its industrial applicability.
This research was supported by intramural grants (2V09620, 2Z06822) from the Korea Institute of Science and Technology (KIST), the Promotion (NO : P0016080) of Innovative Businesses for Regulation-Free Special Zones funded by the Ministry of SMEs and Startups (MSS, Korea) and the Ministry of Science and ICT (MSIT, Korea) (support program: 2021-DD-UP-0379).
The authors have no financial conflicts of interest to declare.
Ji Yeon Yoo, Dong Sin Kim, Tae Jin Kim, Yun Ji Kang, Min Jae Kim, and Jeong Hwan KimMicrobiol. Biotechnol. Lett. 2023; 51(2): 147-156 https://doi.org/10.48022/mbl.2303.03001
Lieu My Dong*, Doan Trung Nam, Tran Thi Phuong, and Dang Kim ThuyMicrobiol. Biotechnol. Lett. 2021; 49(2): 201-209 https://doi.org/10.48022/mbl.2101.01003
Dan-Hee Yoo, Min-Jeong Oh, Hyeon-Ji Yeom and Jin-Young LeeMicrobiol. Biotechnol. Lett. 2020; 48(4): 556-563 https://doi.org/10.48022/mbl.2006.06003