본 연구는 쥐눈이콩열매, 쥐눈이콩잎, 여우콩열매와 여우콩잎 에탄올추출물의 멜라닌합성 촉진효과를 알아보기 위해 melan-a 세포에 시료를 처치하여 멜라닌 합성능 및 tyrosinase 활성능을 평가하였고, tyrosinase, TRP-1, TRP-2, MITF-M 유전자와 단백질 발현량을 측정하였다. 양성대조군은 IBMX를 사용하였다.
항산화능 실험에서 쥐눈이콩열매, 쥐눈이콩잎, 여우콩열매와 여우콩잎 에탄올추출물의 총 폴리페놀 함량은 각각 16.0, 57.7, 365.9, 260.1 mg/g, 총 플라보노이드 함량은 각각 40.4, 91.7, 84.7, 216.5 mg/g, 전자공여능은 1,000 μg/mL에서 각각 32.4, 12.7, 83.5, 84.5 mg/g 이었다.
Melan-a 세포에 대한 쥐눈이콩열매, 쥐눈이콩잎, 여우콩열매와 여우콩잎 에탄올추출물의 최대허용농도는 각각 200, 100, 50, 50 μg/mL으로 확인되었고, 세포 형태 관찰에서 시료처치 농도가 높을수록 멜라닌 생성량과 수지상 돌기가 많이 관찰되었다. 멜라닌 함량 측정 결과, 대조군에 비해 쥐눈이콩열매, 쥐눈이콩잎, 여우콩열매와 여우콩잎 에탄올추출물은 50 μg/mL에서 각각 30.4%, 32.1%, 35.5%, 37.4% 유의하게 증가시켰고(p<0.0l), IBMX는 12.5 μg/mL에서 51.3% 활성을 유의하게 증가시켰다(p<0.0l).
쥐눈이콩열매, 쥐눈이콩잎, 여우콩열매와 여우콩잎 에탄올추출물의 세포내 tyrosinase 활성 측정에서, 대조군에 비해 쥐눈이콩열매, 쥐눈이콩잎, 여우콩열매와 여우콩잎 에탄올추출물은 50 μg/mL에서 각각 18.4%, 21.8%, 21.5%, 21.1% 유의하게 활성을 증가시켰고(p<0.0l), IBMX는 12.5 μg/mL에서 67.4% 유의하게 활성을 증가시켰다(p<0.00l).
Tyrosinase, TRP-1, TRP-2, MITF-M에 대한 유전자 발현 분석결과, 쥐눈이콩잎과 여우콩잎 에탄올추출물은 tyrosinase(p<0.05), TRP-1(p<0.05)과 MITF-M(p<0.01) mRNA 발현을 유의하게 증가시켰다. 여우콩열매 에탄올추출물은 tyrosinase(p<0.05)와 TRP-1(p<0.05) mRNA 발현을 유의하게 증가시켰으나 TRP-2와 MITF-M mRNA 발현에는 영향을 미치지 않았다. 쥐눈이콩열매 에탄올추출물은 TRP-1 mRNA 발현을 유의하게 증가시켰으나(p<0.05) tyrosinase, TRP-2, MITF-M mRNA 발현에는 영향을 미치지 않았다. 한편, IBMX는 tyrosinase, TRP-1, TRP-2, MITF-M mRNA 발현에 모두 영향을 미치지 않았다. 한편, 쥐눈이콩잎 에탄올추출물은 유일하게 TRP-2 mRNA 발현을 유의하게 증가시켰다(p<0.05).
Tyrosinase, TRP-1, TRP-2, MITF-M에 대한 단백질 발현 분석결과, 쥐눈이콩열매, 쥐눈이콩잎, 여우콩잎 에탄올추출물과 IBMX는 tyrosinase(p<0.01), TRP-1(p<0.01), TRP-2(p<0.01)과 MITF-M(p<0.01) 단백질 발현 모두 유의하게 증가시켰다. 반면, 여우콩열매 에탄올추출물은 tyrosinase(p<0.01), TRP-1(p<0.01), MITF-M(p<0.01) 단백질 발현을 유의하게 증가시켰으나 TRP-2 단백질 발현에는 영향을 미치지 않았다.
이상의 실험결과를 종합하면, 쥐눈이콩열매, 쥐눈이콩잎, 여우콩열매와 여우콩잎 에탄올추출물은 melan-a 세포를 이용한 in vitro 시험에서 멜라닌 생성을 유의하게 증가시켰으며 여우콩이 쥐눈이콩보다 잎이 열매보다 높은 효과를 나타냈다. 멜라닌 생성 촉진 작용기전은 멜라닌 생성 주 효소인 tyrosinase 유전자의 전사를 증가시킴으로써 활성을 증가시키는 것으로 해석된다. 반면, IBMX는 멜라닌 생성을 유의하게 증가시켰으며 작용기전은 멜라닌 생성 주 효소인 tyrosinase 유전자의 단백질을 증가시킴으로써 활성을 증가시키는 것으로 해석된다. 이를 근거로 두피와 모발 염색약의 장기간 사용시 나타나는 부작용을 해결하는 백모개선 천연소재로서 실용 가능성이 있을 것으로 사료된다.

This study was conducted to evaluate the melanogenic effect of black soybeans. Melan-a cells were treated with Rhynchosia nulubilis bean ethanol extract (RNBEE), R. nulubilis leaf ethanol extract (RNLEE), R. volubilis bean ethanol extract (RVBEE), and R. volubilis leaf ethanol extract (RVLEE), and melanin synthesis and tyrosinase activity along with mRNA and protein expressions of tyrosinase, TRP-1, TRP-2, and MITF-M were measured. 3-Isobuty-1-methylxanthine (IBMX) was used as a positive control.
Antioxidant activity analysis revealed that RNBEE, RNLEE, RVBEE, and RVLEE had 16.0, 57.7, 365.9, and 260.1 mg/g of polyphenols and 40.4, 91.7, 84.7, and 216.5 mg/g of flavonoids, respectively. The electron-donating abilities of RNBEE, RNLEE, RVBEE, and RVLEE at 1,000 μg/mL were 32.4%, 12.7%, 83.5%, and 84.5%, respectively. Morphological examination of melan-a cells showed that greater melanin accumulation and dendritic development were observed at increased concentration.
RNBEE, RNLEE, RVBEE, and RVLEE at 50 μg/mL significantly increased (p < 0.01) melanin contents by 30.4%, 32.1%, 35.5%, and 37.4%, respectively, compared to the melanin content of the control. IBMX at 12.5 μg/mL also significantly increased (p < 0.01) the melanin content by 51.3%, compared to the control. In comparison with the tyrosinase activity of the control group, the intracellular tyrosinase activity of groups treated with RNBEE, RNLEE, RVBEE, and RVLEE at 50 μg/mL significantly increased (p < 0.01) by 18.4%, 21.8%, 21.5%, and 21.1%, respectively. IBMX at 12.5 μg/mL also significantly increased the tyrosinase activity by 67.4% (p < 0.001).
RNLEE, RVBEE, and RVLEE at 50 μg/mL significantly upregulated tyrosinase mRNA (p < 0.05) and protein expression (p < 0.01), compared to that observed for the control group. On the other hand, RNBEE at 50 μg/mL significantly upregulated tyrosinase protein expression (p < 0.01), but did not affect the mRNA expression, compared to the control. In addition, IBMX at 12.5 μg/mL also significantly upregulated tyrosinase protein expression (p < 0.01), but did not affect the mRNA expression, compared to the control.
RNBEE, RNLEE, RVBEE, and RVLEE at 50 μg/mL significantly upregulated TRP-1 mRNA (p < 0.05) and protein expression (p < 0.01), compared to the TRP-1 mRNA and protein expression of the control group. In contrast, IBMX at 12.5 μg/mL significantly upregulated TRP-1 protein expression (p < 0.001), but did not affect the mRNA expression, compared to the control.
RNLEE at 50 μg/mL significantly upregulated (p < 0.05) TRP-2 mRNA and protein expression, compared to the control. In contrast, RNBEE and RVLEE at 50 μg/mL or IBMX at 12.5 μg/mL significantly upregulated TRP-2 protein expression (p < 0.01), but did not affect the mRNA expression, compared to the control. In addition, RVBEE at 50 μg/mL did not affect TRP-2 mRNA and protein expression.
RNLEE and RVLEE at 50 μg/mL significantly upregulated MITF-M mRNA (p < 0.05) and protein expression (p < 0.01), compared to the MITF-M mRNA and protein expression of the control group. In contrast, RNBEE and RVBEE at 50 μg/mL or IBMX at 12.5 μg/mL significantly upregulated MITF-M protein expression (p < 0.01), but did not affect the mRNA expression, compared to that observed for the control group.
Taken together, the results of this study demonstrated that black soybean ethanol extracts (RNBEE, RNLEE, RVBEE, and RVLEE) promote melanogenesis in melan-a cells; however, it showed that their effects were weaker than IBMX. Among the black soybean ethanol extracts, R. volubilis was found to be more effective than R. nulubilis. Moreover, leaf extract was found to be more effective than bean extract. The potential mechanism underlying the hyperpigmentation effects of black soybeans is the promotion of tyrosinase activity. These hyperpigmentation effects are regulated via increased the tyrosinase transcription and translation levels. RVLEE may be the most effective hyperpigmentation agent with cytotoxicity lower than IBMX.
In conclusion, it can be speculated that RNBEE, RNLEE, RVBEE, and RVLEE could be applied to prevent or improve hair graying. These results also suggest that RNBEE, RNLEE, RVBEE, and RVLEE are found to be effective natural compounds for preventing leukoplakia related to lack of the tyrosinase activities, and for developing materials that alleviate the side effects observed on the scalp and hair owing to the long-term use of hair dye.