洋虫内产β-葡萄糖苷酶内生菌发酵液转化人参皂苷产物分析及其抗肿瘤活性研究

doi:10.13304/j.nykjdb.2021.0691

摘要/Abstract

摘要：

为提高人参皂苷生物利用度，明确人参皂苷药用洋虫体内生物转化规律及其代谢产物抗肿瘤活性，通过MRS（含七叶苷和柠檬酸铁）培养基筛选洋虫内生菌中高产β-葡萄糖苷酶的菌株，制备复合菌发酵剂液体转化人参皂苷提取物，利用高效液相色谱-串联质谱法分析发酵产物中人参皂苷的化学组成及其动态变化规律，采用四甲基噻唑蓝（methylthiazoletrazolium，MTT）染色法阐明发酵产物低（0.5 mg·L^-1）、中（2 mg·L^-1）、高（5 mg·L^-1）剂量对A549细胞抗肿瘤活性的影响。人参提取物中共检测出Re、Rg1、Rf、Rb1、Rc、Rb2、Rd 7种皂苷，代谢产物中有去糖基化人参皂苷Rh1、Rg3和compoud K产生，发酵产物MTT试验显示高剂量组（5 mg·L^-1）反应72 h抑制肿瘤细胞率可达85%。由此表明，利用洋虫内生菌复合菌液转化人参皂苷提取物可提高中药人参和洋虫的药用价值，为中草药的发酵改性提供了新思路。

关键词: 人参皂苷, 洋虫内生菌, 发酵产物, 抗肿瘤活性, 高效液相色谱质谱

Abstract:

In order to improve the bioavailability of ginsenosides， and clarify the transformated products of ginsenosides in Martianus dermestoides and the anti-tumor activity of their metabolites， the functional strains of high-yield β-glucosidase in the endophytes of Martianus dermestoides were screened by MRS （esculin， ferric citrate） medium to prepare compound fermentation agent liquid fermentation ginsenoside extract. The chemical composition and dynamic changes of ginsenosides in fermentation products were analyzed by HPLC-MS/MS. The anti-tumor activities of the fermentation product in low （0.5 mg·L^-1）， medium （2 mg·L^-1）， and high （5 mg·L^-1） dose groups on A549 cells were clarified using methylthiazoletrazolium （MTT） blue staining method. A total of 7 saponins （Re， Rg1， Rf， Rb1， Rc， Rb2， Rd） were detected in ginseng extract. Among the metabolites， deglycosylated ginsenosides Rh1， Rg3 and compoud K were formed. The MTT experiment showed that the inhibition rate of tumor cells was 85% after treated 72 h in the high does group with the does 5 mg·L^-1. These results suggested that a variety of high-activity stimulating ginsenosides were transformed by endophytic bacteria of Martianus dermestoides， which improved the medicinal value of Chinese herbal medicine ginseng and onion， and provided a new idea for fermentation and modification of Chinese herbal medicine.

Key words: ginsenoside, endophytic fungi of Martianus dermestoides, fermentation products, antitumor activity, HPLC-MS/MS

中图分类号:

R284

胡茜, 王艺凝, 申鹏飞, 李雅倩, 杨阳, 王艳成, 姬文秀, 董微巍. 洋虫内产β-葡萄糖苷酶内生菌发酵液转化人参皂苷产物分析及其抗肿瘤活性研究[J]. 中国农业科技导报, 2023, 25(2): 119-127.

Qian HU, Yining WANG, Pengfei SHEN, Yaqian LI, Yang YANG, Yancheng WANG, Wenxiu JI, Weiwei DONG. Fermentation Products Analysis of Producing β-glucosidase Endophytic Bacteria in Martianus dermestoides Transformated to Ginsenoside and Its Anti-tumor Activity[J]. Journal of Agricultural Science and Technology, 2023, 25(2): 119-127.

图/表 7

表1 人参皂苷样品的回归方程、相关系数、线性范围、检测限、定量限及回收率

Table 1 Regression equation， correlation coefficient， linear range， detection limit， quantification limit and recovery rate of ginsenoside samples

化合物 Compound	回归方程 Regression equation	R²	线性范围 Linear range/ （ng·mL^-1）	检测限 LOQ/ （ng·mL^-1）	定量限 LOD/ （ng·mL^-1）	回收率Recovery rate/%
化合物 Compound	回归方程 Regression equation	R²	线性范围 Linear range/ （ng·mL^-1）	检测限 LOQ/ （ng·mL^-1）	定量限 LOD/ （ng·mL^-1）	100 ng·mL^-1	500 ng·mL^-1	2 000 ng·mL^-1
Re	y=4.301 0x-50.003	0.997 4	100~10 000	9.8	28.8	114.1±6.1	92.2±7.3	105.3±11.9
Rg1	y=1.897 0x-35.112	0.994 6	100~10 000	8.5	31.9	108.9±4.6	86.2±7.3	100.8±9.2
Rf	y=0.700 3x-4.213	0.990 2	100~10 000	12.9	34.2	98.1±7.3	83.9±6.3	110.5±9.2
Rb1	y=2.198 0x+50.013	0.997 3	100~10 000	10.7	30.3	89.9±4.8	81.7±8.4	109.7±8.6
Rc	y=2.603 0x+30.227	0.998 5	100~10 000	8.6	29.8	111.9±7.9	104.8±6.9	99.3±6.4
Rb2	y=7.699 0x+26.989	0.991 7	100~10 000	11.5	37.7	97.2±9.8	102.3±0.7	105.8±2.4
Rd	y=3.494 0x-59.306	0.993 5	100~10 000	12.1	41.7	95.1±7.9	100.3±0.6	96.2±4.7
Rh1	y=0.565 6x+4.012	0.999 2	100~10 000	10.0	33.2	119.3±8.2	94.1±9.4	87.5±5.5
Rg3	y=0.159 8x+2.476	0.992 6	100~10 000	9.6	34.9	101.9±7.6	87.9±6.9	103.9±9.4
Compound K	y=8.983 0x-17.006	0.995 7	100~10 000	11.2	28.9	87.9±9.4	112.1±2.1	76.7±10.0

表2 4株菌的18S rRNA基因相似性对比及鉴定结果

Table 2 Comparison of 18S rRNA gene similarity of 4 strains and identification results

菌株Strain	基因登录号Gene ID	相似性Similarity/%	种Species
AD-01	MZ558029	98.26	烟曲霉 Aspergillus fumigatus
AD-03	MZ558030	99.82	鸽色蓝状菌 Talaromyces columbinus
AD-04	MZ558031	96.53	长枝木霉 Trichoderma longibrachiatum
AD-08	MZ558033	98.75	粉色面包霉菌Neurospora crassa

图1 4株产β-葡萄糖苷酶真菌联合发酵人参皂苷提取液产物分析A：人参皂苷标准品；B：菌液空白对照；C：人参皂苷粗提物；D：菌液与人参皂苷发酵20 d产物

Fig. 1 Analysis of the product of 5 strains of β-glucosidase-producing fungi combined fermentation of ginsenoside extractA： Ginsenoside standard product； B： Bacterial liquid blank control； C： Ginsenoside crude extract； D： Bacterial liquid and ginsenoside fermented product for 20 d

图2 发酵产物EIC色谱图

Fig. 2 EIC chromatogram of fermentation products of ginseng extract

图3 4株产β-葡萄糖苷酶真菌联合发酵产物人参皂苷定量分析A：人参主皂苷；B：Rh1；C：Rg3；D：Compound K

Fig. 3 Quantitative analysis of ginsenosides from the combined fermentation of 4 β-glucosidase-producingA： Ginseng main saponins； B： Rh1； C： Rg3； D： compound K

图4 洋虫内生真菌/人参皂苷20 d发酵产物对肺癌细胞A549的细胞抑制率

Fig. 4 Inhibition rate of 20-day fermentation product of endophytic fungi/ginsenosides of worms on lung cancer cell A549 fermentation

图5 复合菌液-人参皂苷发酵产物对肺癌A549细胞的活性抑制率注：不同小写字母表示不同处理间在P<0.05水平差异显著。

Fig. 5 Inhibition rate of compound bacterial liquid-ginsenoside fermentation product on lung cancer A549 cellsNote： Different lowercase letters indicate significant differences between different treatments at P<0.05 level.

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