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1 Acta Microbiologica Sinica 2018, 58(8): DOI: /j.cnki.wsxb Research Article 研究报告 中药美白成分的复配及复方中药发酵液的功效 李瑜 1, 顾秋亚 1, 刘咏梅 2, 余晓斌 * 摘要 : HPLC g/kg DPPH 5% (W/V) 82.41% 关键词 : [1] [2] [3 4] [5] [6] * 通信作者 Tel xbyu@jiangnan.edu.cn 收稿日期 : 修回日期 : 网络出版日期 :

2 1484 Yu Li et al. Acta Microbiologica Sinica, 2018, 58(8) [7] 1 材料和方法 (Saccharomycopsis fibuligera) (Enterococcus faecalis) YPD MRS (Rhodiola rosea) (Rubifructus) (Green tea) (Eugenia caryophyllata) ( ) (570 U/mg) 1, (DPPH) - (10000 U/g) g 100 ml ph μl - 50 C 3 h 5 min 4 C r/min 10 min 100 ml 50 g/l 4 C [8] 1 PBS L- C 1 C 2 C 3 C 4 37 C 10 min 37 C 20 min 475 nm C 2 C 1 OD C1 T 2 T 1 OD T1 =[1 (OD C1 OD T1 ) OD C1 ] 100% L 9 (3 4 ) 100 ml 200 μl - 50 C 3 h 5 min 4 C r/min 10 min 100 ml SPSS YPD 10% (V/V) YPD 30 C 180 r/min 20 h 2 MRS 5% (V/V) MRS 37 C 20 h 2 表 1. 试剂用量表 Table 1. Table of reagent dosage (ml) Reagent C 1 C 2 T 1 T mol/l (ph 6.80) sodium phosphate buffer Chinese herb extract mg/ml L-Tyrosine U/mL mushroom tyrosinase solution actamicro@im.ac.cn

3 \ \, 2018, 58(8) 1485 表 2. 正交试验因素水平表 Table 2. Factors and level of orthogonal test Level Rhodiola rosea (A)/% Rubifructus (B)/% Green tea (C)/% Eugenia caryophyllata (D)/% CFU/mL 5.0 ml 100 ml CFU/mL 5.0 ml 100 ml 30 C 120 r/min 48 h 4 C r/min 10 min Agrawal [9] 0.5 ml 0.5 ml 1% 0.5 ml PBS 50 C 20 min 0.5 ml 10% 1.0 ml 1.0 ml 1.0 ml 0.1% 10 min 700 nm ( OH) Smirnoff [10] 4 ml 1.0 ml H 2 O 2 (8.8 mmol/l) 1.0 ml FeSO 4 (0.9 mmol/l) 1.0 ml (9 mmol/l) 1.0 ml H 2 O 2 37 C 60 min 510 nm OH OH [1 (A s A b ) A c ] 100% A s A b A c DPPH (DPPH ) Wang [11] 1.0 ml 0.5 ml DPPH (0.1 mmol/l) 1.0 ml 60 min 517 nm DPPH DPPH =[(A 0 A) A 0 ] 100% A 0 A Tian [12] GB/T GB/T ( ) GB/T GB/T Excel 2013 SPSS 20.0 x s P<0.05 P< 结果和讨论

4 1486 Yu Li et al. Acta Microbiologica Sinica, 2018, 58(8) 表 3. 不同质量浓度美白中药对酪氨酸酶的影响 Table 3. Effect of different mass concentration whitening Chinese herbs extract on tyrosinase activity Concentration/% Tyrosinase inhibition rate/% Rhodiola rosea Rubifructus Green tea Eugenia caryophyllata ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± % 40.40% R B >R A >R D >R C > > > A 3 B 3 C 1 D % 0.10% 0.25% 1.00% (P<0.05) A 3 B 3 C 1 D (A 2 B 3 C 1 D 2 ) 6 A 3 B 3 C 1 D 3 A 2 B 3 C 1 D % 68.59% [11,13] A 3 B 3 C 1 D 3 表 4. 正交试验结果 Table 4. Results of orthogonal Factors Group A B C D Tyrosinase inhibition rate/% ± ± ± ± ± ± ± ± ±2.47 k k k R 表 5. 方差分析表 Table 5. Variance analysis Factor SS df MS F Sig.(P) A * B * C D Error Injection: * has significant differences (P<0.05). actamicro@im.ac.cn

5 , 2018, 58(8) 1487 表 6. 验证试验 Table 6. Validation test Group Actual tyrosinase inhibition rate/% A 2 B 3 C 1 D A 3 B 3 C 1 D Theoretical tyrosinase inhibition rate/% 2.3 [14 15] [16] (P<0.01) g/kg C (Vc) ( 2) mg/l Vc (P<0.01) mg/l Vc Vc OH ( 3) OH (P<0.01) OH Vc 1.05 mg/ml OH 54.33% OH DPPH DPPH 4 DPPH DPPH mg/ml DPPH Liquid 表 7. 发酵液和水提液中成分 Table 7. Composition of fermentation broth and water extractive Content/(g/kg) Total polyphenols Total polysaccharides Total flavonoids Total protein Water extractive ± ± ± ±0.04 Fermentation broth ± ± ± ±0.10

6 1488 Yu Li et al. Acta Microbiologica Sinica, 2018, 58(8) 图 1. 水提液和发酵液中游离氨基酸色谱图 (A) 及含量对比 (B) Figure 1. Free amino acids chromatogram (A) and content comparison (B) in water extractive and fermentation broth. 图 2. 复方中药发酵液的总还原力 Figure 2. The total reducing power of compound Chinese herbs fermentation broth. 图 3. 复方中药发酵液的 OH 清除率 Figure 3. The hydroxyl radical scavenging activity of compound Chinese herbs fermentation broth.

7 , 2018, 58(8) 1489 图 4. 复方中药发酵液对 DPPH 的清除能力 Figure 4. DPPH radical scavenging activity of compound Chinese herbs fermentation broth. 图 5. 复方中药发酵液对酪氨酸酶的抑制作用 Figure 5. Effect of compound Chinese herbs fermentation broth on tyrosinase activity. A: 1% Vc; B: 5% Water extractive; C: 5% Black fermentation broth; D: 5% Fermentation broth. (P<0.05) DPPH 0.42 mg/ml DPPH 7.98% 1.47 mg/ml 40.45% 2.5 1% Vc 5% 5 5% 82.41% (P<0.05) 5% 34.20% 5% 1% Vc (86.77%) 3 结论 0.50% 0.10% 0.25% 1.00% 74.53% 4.36 g/kg ( g/kg) [1] Wang YF, Lai JZ, Long XY, Chen WR, Lin WT. Progress of traditional Chinese medicine in skin-whitening mechanism.

8 1490 Yu Li et al. Acta Microbiologica Sinica, 2018, 58(8) Journal of Guangdong Pharmaceutical University, 2014, 30(4): (in Chinese),,,,.., 2014, 30(4): [2] Wang XL, Yao WJ, Yuan L. Effect of the polysaccharides and other active extracts from cynanchum auriculatum on the activity of tyrosinase. Journal of Food Science & Biotechnology, 2013, 32(10): (in Chinese),,.., 32(10): [3] Yuan Y, Luan XN, Rana X, Hassan ME, Dou DQ. Covalent immobilization of cellulase in application of biotransformation of ginsenoside Rb 1. Journal of Molecular Catalysis B: Enzymatic, 2016, 133(S1): S525 S532. [4] Wu TX, Wang N, Zhang Y, Xu XB. Advances in the study on microbial fermentation and transformation of traditional Chinese medicine. African Journal of Microbiology Research, 2013, 7(17): [5] Shi TR, Liu Y, Wang S, Wang Y, Zhu DD, Li D. Research progress of Chinese fermentation technology. Chinese Journal of Traditional Veterinary Science, 2014, (1): (in Chinese),,,,,.., 2014, (1): [6] Su N, Liu HM, Liu J, Zhou HY, Yang L, Wang CT. Preparation, efficacy and safety research of fermented rhodiola liquid cosmetic. Flavour Fragrance Cosmetics, 2017, (2): (in Chinese),,,,,.., 2017, (2): [7] Deng GJ, Liu YT, Ling PX. Study of whitening efficacy on cortex moutan compound extract. Journal of Pharmaceutical Research, 2016, 35(5): (in Chinese),,.., 2016, 35(5): [8] Khazaeli P, Goldoozian R, Sharififar F. An evaluation of extracts of five traditional medicinal plants from Iran on the inhibition of mushroom tyrosinase activity and scavenging of free radicals. International Journal of Cosmetic Science, 2009, 31(5): [9] Agrawal H, Joshi R, Gupta M. Isolation, purification and characterization of antioxidative peptide of pearl millet (Pennisetum glaucum) protein hydrolysate. Food Chemistry, 2016, 204: [10] Smitnoff N, Cumbes QJ. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry, 1989, 28(4): [11] Wang RX, Zhao Z, Zhong Y, Li XD, Wang ZY. Synergistic effect of several kinds of whitening agent on inhibition of tyrosinase activity. China Surfactant Detergent & Cosmetics, 2014, 44(10): (in Chinese),,,,.., 2014, 44(10): [12] Tian F, Li B, Ji BP, Yang JH, Zhang GZ, Chen Y, Luo YC. Antioxidant and antimicrobial activities of consecutive extracts from Galla chinensis: The polarity affects the bioactivities. Food Chemistry, 2009, 113(1): [13] Zhang LX, Cui CC. Study on the recombination of Chinese herb for antifungal activity against fusarium oxysporum. Journal of Anhui Agricultural Sciences, 2012, 40(31): (in Chinese),.., 2012, 40(31): [14] Zou Y, Hu WZ, Liu CH, Chen C, Gu ZX. Production of tyrosinase by Auricularia auricula using low cost fermentation medium. Annals of Microbiology, 2013, 63(2): [15] Wang GH, Chen CY, Lin CP, Huang CL, Lin CH, Cheng CY, Chung YC. Tyrosinase inhibitory and antioxidant activities of three Bifidobacterium bifidum-fermented herb extracts. Industrial Crops & Products, 2016, 89: [16] Zheng GM, Xu LX, Wu P, Xie HH, Jiang YM, Chen F, Wei XY. Polyphenols from longan seeds and their radical-scavenging activity. Food Chemistry, 2009, 116(2):

9 , 2018, 58(8) 1491 Complex of whitening ingredients from different chinese herbs and effectiveness of compound Chinese herbs fermentation broth Yu Li 1, Qiuya Gu 1, Yongmei Liu 2, Xiaobin Yu 1* 1 Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi , Jiangsu Province, China 2 School of Pharmacy, Taizhou Polytechnic College, Taizhou , Jiangsu Province, China Abstract: [Objective] Through complex of whitening ingredients from different Chinese herbs, we studied the active ingredients and effectiveness of compound Chinese herbs fermentation broth. [Methods] The tyrosinase inhibition rate was used as the index, the best compound composition was obtained. The contents of total polyphenols, polysaccharides, flavonoids, and protein in the fermentation broth were measured by UV spectrophotometry, and amino acids compositions were analyzed by HPLC. We evaluated their antioxidant activity in fermentation broth by total reducing power and radical scavenging tests. The whitening efficacy was evaluated by tyrosinase inhibition test. [Results] Rubifructus had the best inhibition of tyrosinase activity by orthogonal designed experiment. The fermentation broth of compound Chinese herbs contained g/kg of total polyphenols, g/kg of polysaccharides, g/kg of flavonoids and g/kg of total protein. The fermentation broth of compound Chinese herbs had strong total reducing power, hydroxyl radical scavenging ability and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. Tyrosinase inhibition rate of 5% (W/V) compound Chinese herbs fermentation broth reached 82.41%. [Conclusion] The fermentation broth of compound Chinese herbs contained multiple active ingredients, with antioxidant activity and whitening efficacy. Keywords: Chinese herb, complex, fermentation, active ingredients, anti-oxidation, whitening ( 本文责编 : 李磊 ) * Corresponding author. Tel: ; xbyu@jiangnan.edu.cn Received: 13 November 2017; Revised: 27 December 2017; Published online: 24 January