CN103720740A - Gynostemma pentaphylla extract and fungus transformation preparation method thereof - Google Patents

Abstract

The invention relates to a Gynostemma Gynostemma extract and a preparation method for fungal transformation thereof. The preparation method of the extract is as follows: take the Gynostemma pentaphyllum ethanol extract, and put it on the fungal culture medium of Trichoderma viride, Absidia cladoides and Aspergillus niger at 27-30°C Fermentation is carried out for 1-6 days, the total fermented Gynostemma pentaphyllum extract is freeze-dried, and ultrasonically extracted with 80% ethanol of 8-10 times the amount (v/w, mL/g) for 2-4 times, each time for 20-40 min. The fermented Gynostemma pentaphyllum ethanol extract is concentrated under reduced pressure and dried to obtain the Gynostemma pentaphyllum extract of the present invention. The content of active ingredients in the Gynostemma pentaphyllum extract prepared by adopting the method of microbial transformation is obviously increased.

Description

A kind of Gynostemma pentaphyllum extract and its fungal transformation preparation method

technical field

The invention relates to a Gynostemma pentaphyllum extract rich in damulin A, damulin B, gypenoside L and gypenoside LI, and a method for preparing the extract through fungal transformation.

Background technique

Saponins are glycosides with a C30 skeleton as the core, and are widely found in the plant kingdom. According to different carbon skeletons, they can be divided into triterpene saponins and steroidal saponins [1]. Among them, triterpene saponins mainly exist in Araliaceae, Fabaceae, Polygalaceae and Cucurbitaceae, and steroidal saponins mainly exist in Dioscoreaceae, Liliaceae and Scrophulariaceae. In addition, saponins also exist in marine organisms such as starfish and sea cucumbers. Saponins have complex physicochemical properties (such as foaming and emulsifying effects), and thus also have a wide range of pharmacological activities, such as anti-tumor, anti-inflammatory, anti-microbial, anti-diabetic, anti-oxidation, immune promotion, and cardiovascular and neurological Protection and other functions [2-7].

Saponins have many different biological activities. For example, ginsenoside Rb1 has sugar groups at C3 and C20, which can protect nerves [8] and improve memory [9]. Ginsenosides Rg3 and Rh2, which only have sugar groups at the C3 position, have strong antitumor activity [10,11]. Previous studies have shown that the gypenosides gypenoside L, gypenoside LI, damulin A and damulin B isolated from heat-treated Gynostemma pentaphyllum products have a strong inhibitory effect on the proliferation of non-small cell lung cancer [12]. But the content of these four compounds in Gynostemma pentaphyllum is very small. Therefore, the rational transformation and structural modification of these low-content saponin active ingredients has become a hot spot in current pharmaceutical research.

Gynostemma pentaphyllum is the whole herb of Gynostemma pentaphyllum (Thunb.) Makino in Cucurbitaceae (Cucurbitaceae) Gynostemma Gynostemma pentaphyllum (Thunb.) Makino [13]. Because Gynostemma pentaphyllum contains dammarane saponins similar to ginseng, it has been dubbed "Southern ginseng" and "Second ginseng" [14].

The biotransformation of organic compounds by microorganisms is one of the effective methods to obtain new types of structures that are difficult to obtain by chemical synthesis methods. The regioselective and stereoselective transformations of triterpenoids by microorganisms have been extensively studied [15,16]. references:

[1] Vincken J P, Heng L, de Groot A, et al. Saponins, classification and °Currence in the plant kingdom[J]. Phyt °Chemistry, 2007, 68(3): 275-297.

[2] Petronelli A, Pannitteri G, Testa U. Triterpenoids as new promising anticancer drugs [J]. Anticancer Drugs, 2009, 20(10): 880-892.

[3]Shin E M, Zhou H Y, Xu G H, et al.Anti-inflammatory activity of hispidol A25-methyl ether, a triterpenoid isolated from Ponciri Immaturus Fructus[J].Eur.J.Pharmacol, 2010, 627( 1-3): 318-324.

[4] Yodsaoue O, Sonprasit J, Karalai C, et al. Diterpenoids and triterpenoids with potentialanti-inflammatory activity from the leaves of Aglaia odorata[J]. Phyt ° chemistry, 2012, 76: 83-91.

[5] Yadava R N, Jharbade J. A new bioactive triterpenoid saponin from the seeds of Lactucascariola Linn [J]. Nat. Prod. Res, 2007, 21(6): 500-506.

[6]Yoon S H, Han E J, Sung J H, et al.Anti-diabetic effects of compound K versus metforminversus compound K-metformin combination therapy in diabetic db/db mice[J].Biol.Pharm.Bull, 2007 , 30(11): 2196-2200.

[7]Kim Y A, Kong C S, Lee J I, et al.Evaluation of novel antioxidant triterpenoid saponins from the halophyte Salicornia herbacea[J].Bioorg.Med.Chem.Lett,2012,22(13):4318-4322 .

[8] Liu Min, Zhang Juntian. Protective effects of ginsenosides Rb1 and Rg1 on primary cultured rat hippocampal neurons [J]. Acta Pharmaceutica Sinica, 1995, 30(9): 674-678.

[9] Wang Q, Sun L H, Jia W, et al. Comparison of ginsenosides Rg1 and Rb1 for their effects on improving scopolamine-induced learning and memory impairment in mice[J]. Phytother. Res, 2010, 24(12): 1748- 1754.

[10] Jiang J W, Chen X M, Chen X H, et al.Ginsenoside Rg3inhibit hepat°cellular carcinoma growth via intrinsic apoptotic pathway[J].World J.Gastroenterol, 2011, 17(31): 3605-3613.

[11] Li B, Zhao J, Wang C Z, et al. Ginsenoside Rh2induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53[J]. Cancer Lett, 2011, 301(2): 185-192.

[12]Piao X L, Wu Q, Yang J, et al.Dammarane-type saponins from heat-pr Cessed Gynostemmapentaphyllum show fortified activity against A549cells[J].Arch Pharn Res, 2013, 36(7): 874-879 .

[13] Liang Qicheng, Zhong Ming, Chinese Pharmacology [M], Guangxi Minzu Publishing House, 2005.

[14] Zhang Tao, Yuan Dishun. Research progress on germplasm resources of Gynostemma pentaphyllum in China [J]. Journal of Yunnan Agricultural University, 2009, 24(3): 459-469.

[15] Huang F X, Yang W Z, Ye F, et al. Microbial trauma of ursolic acid by Syncephalastrum racemosum (Cohn) Schroter AS3.264 [J]. Phyt ° chemistry, 2012, 82: 56-60.

[16] Dong A, Ye M, Guo H, et al. Microbial transformation of ginsenoside Rb1by Rhizopusstlonifer and Curvularia lunata[J]. Biotechnol. Lett, 2003, 25(4): 339-344.

Contents of the invention

The object of the present invention is to propose a kind of Jiaogulan extract rich in damulin A (damulin A), damulin B (damulin B), gypenoside L and gypenoside LI, the preparation method of the extract, and the use of the extract in preparing and treating tumors Uses in medicine. The content of anti-tumor active components in the Gynostemma pentaphyllum extract prepared by adopting the fungal transformation method is obviously increased, thereby enhancing the effect of the Gynostemma pentaphyllum extract of the present invention on treating tumors.

The Gynostemma pentaphyllum extract of the present invention contains the following compounds:

The preparation method of Gynostemma pentaphyllum extract of the present invention is as follows:

Take the ethanol extract of Gynostemma pentaphyllum, ferment it for 1-10 days in the fungal culture medium at 27-31°C, extract the total fermented Gynostemma pentaphyllum extract with an equal volume of n-butanol for 1-6 times, concentrate under reduced pressure, and dry to obtain the present invention. For the Gynostemma pentaphyllum extract, the fungus is selected from Trichoderma viride, Absidia cladoides and Aspergillus niger.

The preferred preparation method is as follows:

Take the ethanol extract of Gynostemma pentaphyllum, ferment it for 2-8 days under the condition of 28-30°C in the fungal culture medium, extract the total fermented Gynostemma pentaphyllum extract with an equal volume of n-butanol for 2-4 times, concentrate under reduced pressure, and dry to obtain the present invention For the Gynostemma pentaphyllum extract, the fungus is selected from Trichoderma viride, Absidia cladoides and Aspergillus niger.

A further preferred preparation method is as follows:

Take the ethanol extract of Gynostemma pentaphyllum, ferment it for 7 days under the condition of 28°C in the fungal culture medium, extract the total Gynostemma pentaphyllum extract after fermentation with an equal volume of n-butanol for 3 times, concentrate under reduced pressure, and dry to obtain the Gynostemma pentaphyllum extract of the present invention , the fungi are selected from Trichoderma viride, Absidia cladoides and Aspergillus niger.

The preparation method of the ethanol extract of Gynostemma pentaphyllum is as follows: take Gynostemma pentaphyllum and use 5-10 times the amount (v/w, mL/g) of 60-95% ethanol to reflux extract for 1-8 times, each time for 1-5h, and concentrate have to;

More preferably: Gynostemma pentaphyllum is extracted with 8 times the amount (v/w, mL/g) of 80% ethanol under reflux for 3 times, each time for 2 hours, and then concentrated.

The present invention identifies 4 active ingredients in the prepared Gynostemma pentaphylla extract according to the literature by using LCMS-IT-TOF technical means: Damulin A (damulin A), Damulin B (damulin B), gypenoside L and gypenoside LI . And using the HPLC-MS analysis method, the content change analysis of each active ingredient in the extract was carried out. in:

The compound is fermented by Trichoderma viride, Absidia cladoides and Aspergillus niger, and the content of the compound is all increased.

The separation and identification of active ingredients in the Gynostemma pentaphyllum extract are as follows:

With silica gel column chromatography, the eluent was eluted in a dichloromethane-methanol ratio from 10:1 to 6:1.

The above methylene chloride-methanol (6:1) eluate was finally identified as gypenoside gypenosdie L, gypenoside LI, Damulin A, and Damulin B through LCMS-IT-TOF technical means and according to the literature.

Since Damulin A, Damulin B, gypenoside L, and gypenoside LI all have strong inhibitory effects on lung cancer, gastric cancer, ovarian cancer, breast cancer and leukemia cells, so in the present invention, we also provide the prepared Use of Jiaogulan extract in preparing medicines for treating tumors.

Detailed ways

Preparation experiment of embodiment 1 extract

We compared the extracts prepared by several different preparation methods, and determined the content of four compounds, Damulin A, Damulin B, gypenoside L, and gypenoside LI. The result is as follows:

1. Preparation of Gynostemma pentaphylla alcoholic extract before fermentation

Take Gynostemma pentaphyllum and use 8 times the amount (v/w, mL/g) of 80% ethanol to reflux extract 3 times, each time for 2 hours, concentrate to obtain Gynostemma pentaphyllum ethanol extract; directly freeze-dry in Trichoderma viride culture medium to obtain fermented Alcoholic extract of Gynostemma pentaphyllum.

2. the preparation of Trichoderma viride, Absidia cladoides and Aspergillus niger fungal fermentation product of Gynostemma pentaphyllum ethanol extract (Gynostemma pentaphyllum prepared by the present invention blue extract)

Take Gynostemma pentaphyllum and use 8 times the amount (v/w, mL/g) of 80% ethanol to reflux extract 3 times, each time for 2h, concentrate to obtain Gynostemma pentaphyllum ethanol extract, which can be used in Trichoderma viride, Absidia cladoides and Aspergillus niger fungi In the culture medium, ferment at 29 DEG C for 7 days, and freeze-dry to obtain the fermentation product of the alcoholic extract of Gynostemma pentaphyllum.

Table 1 The peak area changes of Damulin A, Damulin B, gypenoside L, and gypenoside LI in different extracts

Using the HPLC-MS analysis method, the content of the alcoholic extract of Gynostemma pentaphyllum before fermentation and the fermentation product of Gynostemma pentaphyllum alcoholic extract were compared.

The results show that, as shown in Table 1, in the fermentation product of the Gynostemma pentaphyllum ethanol extract obtained by the technical scheme protected by the present invention (the Gynostemma pentaphyllum extract prepared by the present invention), the content of Gypenoside Damulin A, Damulin B, gypenoside L, and gypenoside LI content increased. Specifically, the transformation effects of C. elegans, Trichoderma longibrachiae, and Penicillium melini were poor, and the contents of Damulin A, Damulin B, gypenoside L, and gypenoside LI all decreased after being transformed by Elegantia xiaograms; while None of the four active ingredients were detected after fermentation by Trichoderma longibrachiae and Penicillium melini.

On the contrary, Trichoderma viride can increase the content of gypenoside L and gypenoside LI, while Absidia cladoides and Aspergillus niger can significantly increase the content of the four active ingredients.

Claims (9)

1. a Gynostemma pentaphyllum extract, is characterized in that, adopts the method for microbial fermentation to prepare, and described microorganism is fungus. 2. An extract of Gynostemma pentaphyllum, it is characterized in that, the preparation method of described Gynostemma pentaphyllum extract comprises the following steps: get the Gynostemma pentaphyllum ethanol extract, carry out fermentation 1-10 days at 27-31 ℃ of conditions of fungal culture medium, after fermenting The total extract of Gynostemma pentaphylla is extracted 1-6 times with an equal volume of n-butanol, concentrated under reduced pressure, and dried to obtain Gynostemma pentaphyllum extract, and the fungus is selected from Trichoderma viride, Absidia cladoides and Aspergillus niger; The extract contains the following compounds: 3. Gynostemma pentaphyllum extract as claimed in claim 2, is characterized in that, the preparation method of described Gynostemma pentaphyllum extract comprises the following steps: get Gynostemma pentaphyllum ethanol extract, carry out fermentation 2-8 in fungal culture medium 28-30 ℃ condition day, the fermented Gynostemma pentaphylla total extract was extracted 2-4 times with an equal volume of n-butanol, concentrated under reduced pressure, and dried. 4. Gynostemma pentaphyllum extract as claimed in claim 2, is characterized in that, the preparation method of described Gynostemma pentaphyllum extract comprises the following steps: get Gynostemma pentaphyllum ethanol extract, ferment 7 days at 28 ℃ of conditions of fungal culture medium, ferment The final Gynostemma pentaphyllum extract was extracted three times with an equal volume of n-butanol, concentrated under reduced pressure, and dried. 5. the Gynostemma extract as described in any one of claim 2-4, is characterized in that, the preparation method of described Gynostemma ethanol extract is: get Gynostemma with 5-10 times of amount (v/w, mL/g ) with 60-95% ethanol for reflux extraction for 1-8 times, each time for 1-5h, and then concentrate. 6. the Gynostemma extract as described in any one of claim 2-4, is characterized in that, the preparation method of described Gynostemma ethanol extract is: get Gynostemma with 8 times of amount (v/w, mL/g) Reflux extraction with 80% ethanol for 3 times, 2 hours each time, and concentrate. 7. the Gynostemma extract as described in any one of claim 2-4, it is characterized in that, the weight ratio that described compound accounts for Gynostemma extract is: damulin A18-30mg/g, damulin B5.5-30mg/g, gypenoside L5-20mg/g, gypenoside LI2.5-10mg/g. 8. The application of the Gynostemma pentaphyllum extract described in any one of claims 2-7 in the preparation of a drug for treating cancer. 9. The application according to claim 8, wherein the cancer is selected from lung cancer, gastric cancer, ovarian cancer, breast cancer or leukemia.