A strain of Panax notoginseng rhizosphere fungus and a method for preparing rare ginsenoside CK using the same

Abstract

The present invention provides a strain of Panax notoginseng rhizosphere fungus and a method for preparing rare ginsenoside CK using the strain, belonging to the field of medical technology. The present invention uses a fungus isolation method to obtain a strain of Panax notoginseng rhizosphere fungus, namely, Pink Spiral Polysporus ( Clonostachys rosea ), and fermented it under various culture conditions. The rare compound ginsenoside CK was obtained from the pink spiral polysporus fermentation fungus by using natural product chemical research methods such as organic solvent reflux extraction, column chromatography separation and purification, HPLC preparation technology, countercurrent chromatography technology, and spectral structure analysis techniques such as nuclear magnetic resonance, mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy, circular dichroism, etc. The present invention directly produces rare ginsenoside CK by fungal fermentation. The method has the characteristics of simple production steps, short preparation time, energy saving and environmental protection in the preparation process.

Description

Technical field:

[0001] The present invention belongs to the field of medical technology, and specifically relates to a method for preparing rare ginsenoside CK by fermenting Panax notoginseng rhizosphere fungi, and its application in preparing medicines and pharmaceutical compositions for preventing or treating diseases such as skin aging, cancer, inflammation and myocardial injury. Background technology:

[0002] Rare ginsenoside CK [20-O-β-D-glucosyl-20(S)-protopanaxadiol] is the main absorption form and pharmacodynamic entity of protopanaxadiol ginsenosides in the human body. This substance has the effect of inducing apoptosis on a variety of tumor cell lines, but has no obvious effect on normal cells, so that rare ginsenoside CK can effectively reduce the adverse reactions of chemotherapy drugs in the combined application of anticancer drugs, enhance the effects of radiotherapy and chemotherapy, and reduce the drug resistance of drug-resistant cell lines. In addition, rare ginsenoside CK also has significant anti-allergic and anti-inflammatory, neuroprotective, anti-diabetic and anti-skin aging effects. In recent years, with the in-depth study of the pharmacological activity and mechanism of action of rare ginsenoside CK, the development and application of this compound has been accelerated, and the demand for this substance has increased.

[0003] Rare ginsenoside CK is present in very small amounts in nature and is mainly obtained by microbial or enzyme transformation of natural diol ginsenosides Rb1, Rb2, Rc, etc. However, the method of preparing rare ginsenoside CK by biotransformation not only requires the preparation of a large amount of diol ginsenosides as precursors, but also requires the screening of strains or biological enzymes with strong reaction ability. In addition, there are disadvantages such as harsh biological reaction conditions, complex reaction products, and difficulty in separation and purification, which to some extent hinder the further development of the rare ginsenoside CK industry.

[0004] The production of active natural products using microbial metabolism is a green, environmentally friendly and efficient preparation method, which has been widely used in the field of medicine and health. Clonostachys rosea is a fungus of the Bionectriaceae family, Clonostachys rosea, which is often parasitic or saprophytic in the root area of ​​plants. It has a parasitic effect on plant root pathogens or root nematodes, and is often developed and used as a biocontrol agent for crop root diseases.

[0005] However, to date, there has been no development and utilization of pink spiral polysporus derived from the rhizosphere soil of Panax notoginseng in the prior art, and no reports on the method of fermenting rare ginsenoside CK using pink spiral polysporus derived from the rhizosphere soil of Panax notoginseng and its application. Summary of the invention:

[0006] The purpose of the present invention is to provide a strain of Panax notoginseng rhizosphere fungus, namely a strain of Clonostachys rosea, a method for preparing rare ginsenoside CK by fermenting Panax notoginseng rhizosphere fungus, a pharmaceutical composition with it as an active ingredient, and its application in the preparation of drugs for preventing or treating diseases such as skin aging, cancer, inflammation and myocardial damage. The preparation method of the present invention does not require the preparation of precursors, does not rely on the production and cultivation of medicinal plants, and does not occupy land resources; and the fungus in the preparation method has a fast proliferation rate, a relatively single secondary metabolite, and is easy to separate and purify the target product, which is easy to industrialize and produce, and can effectively reduce the pollution or ecological damage caused by traditional preparation methods.

[0007] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0008] A strain of Panax notoginseng rhizosphere fungus, Clonostachys rosea, was isolated and purified from the rhizosphere soil of Panax notoginseng and has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with the deposit number CGMCC NO.40327. Figure 1 shown.

[0009] The preparation method of ginsenoside CK utilizes the Panax notoginseng rhizosphere fungi to ferment and culture under specific culture conditions to metabolize and produce rare ginsenoside CK. The specific culture conditions refer to artificial solid and liquid fermentation conditions, and the solid culture matrix is ​​wheat, barley, rice, millet, sorghum, corn, and glutinous rice.

[0010] The preparation method of ginsenoside CK utilizes the Panax notoginseng rhizosphere fungus to metabolize and produce rare ginsenoside CK under specific culture conditions. The method comprises the following steps: fermenting and culturing the Panax notoginseng rhizosphere fungus, namely, pink spiral polysporus, and then leaching the rice or wheat fermentation culture of pink spiral polysporus with 3 volumes of ethyl acetate for 5 times, each time for 5 days, and concentrating the extract under reduced pressure to obtain a crude ethyl acetate extract, subjecting the crude extract to silica gel column chromatography, and eluting with an organic solvent to obtain a petroleum ether elution part, fraction A; continuing to elute the chromatography column with a gradient of chloroform: methanol 500: 1-10: 1 to obtain 6 main parts, fractions BG; wherein, fraction D is chromatographed on an RP-18 column, and eluted with a gradient of methanol: water 1: 9-9: 1 to obtain 6 subcomponents, fractions D1-D6; wherein, fraction D2 is chromatographed on a silica gel column, and chloroform: methanol 20: 1 to obtain 6 parts, fractions D2.1-2.6; D2.4 was subjected to RP-18 semi-preparative column chromatography and eluted with acetonitrile:water 55:45. The eluate was decompressed, concentrated and dried to obtain the monomer compound rare ginsenoside CK.

[0011] According to the preparation method of ginsenoside CK, the fermentation culture of the pink spiral polysporon is as follows: first prepare a potato-rice solid culture medium, wash, peel, cut 1000g of potatoes, place them in 4000mL of pure water and boil them for 20min, cool, filter with three layers of gauze, collect the filtrate and dilute it to 5000mL as potato extract for standby use; then weigh 100g of rice and place it in a 500mL wide-mouth culture bottle, add 150mL of potato extract to each bottle, seal it with a breathable membrane, and sterilize it with high-pressure steam for 30min at 121°C and 0.105Mpa, and cool it for standby use; inoculate the activated pink spiral polysporon into the potato-rice solid culture medium, and ferment and culture it for 40d at 25±2°C in the dark or in alternating dark and light conditions.

[0012] According to the preparation method of ginsenoside CK, the fermentation culture of the pink spiral polysporon is as follows: 4 kg of potatoes are washed, peeled, cut into pieces, and then placed in 12 L of pure water for boiling for 20 minutes, cooled, and filtered through three layers of gauze; 4 kg of wheat are boiled in 8 L of pure water for 2 hours, filtered through three layers of gauze, the potato filtrate and the wheat filtrate are mixed and the volume is fixed to 20 L, 400 g of glucose is dissolved in the mixed solution, and the pH value is adjusted to 6.8 to obtain a modified potato-wheat liquid culture medium, the prepared liquid culture medium is divided into triangular bottles, sealed with a breathable membrane, and sterilized with high-pressure steam for 30 minutes at 121° C. and 0.105 MPa, and the activated pink spiral polysporon is inoculated into the cooled liquid culture medium, and suspended and cultured at 25±2° C. for 30 days.

[0013] Application of any preparation method of ginsenoside CK of the present invention in the preparation of rare ginsenoside CK.

[0014] The use of any preparation method of ginsenoside CK of the present invention in the preparation of drugs for preventing or treating skin aging, cancer, inflammation, and myocardial damage.

[0015] The rare ginsenoside CK prepared by any of the preparation methods of ginsenoside CK of the present invention is used in the preparation of drugs for preventing or treating skin aging, cancer, inflammation, and myocardial damage.

[0016] The rare ginsenoside CK prepared by any of the preparation methods of ginsenoside CK of the present invention has the following structural formula:

[0017]

[0018] The method of preparing rare ginsenoside CK by fermenting Panax notoginseng rhizosphere fungi of the present invention is to use Panax notoginseng rhizosphere fungi, namely Clonostachys rosea, which is derived from Panax notoginseng rhizosphere soil and has a preservation number of CGMCCNO.40327, to metabolize and produce rare ginsenoside CK under certain culture conditions. The specific culture conditions of the fungi refer to artificial solid and liquid fermentation culture. The solid culture matrix includes wheat, barley, rice, millet, sorghum, corn, glutinous rice, etc.

[0019] The isolation and identification of rhizosphere fungi of Panax notoginseng were carried out as follows: 5 g of rhizosphere soil of Panax notoginseng was weighed and placed in a conical flask containing 45 mL of sterile water and shaken for 20 min. -1 Soil suspension; gradually diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 Afterwards, take each dilution and spread it on the separation plate, and add ampicillin (100 mg / L) to the separation plate to inhibit the growth of bacteria. Invert and culture at 28°C for 7-14 days. According to the morphological characteristics of the colonies and hyphae of pink spiral polysporon, pick the hyphae and inoculate them on the PDA plate, invert and culture at 28°C for 7-10 days, and repeat the transfer for 2-3 generations. By extracting DNA from the single strain obtained, using ITS universal primers for amplification and sequencing, the sequencing results were compared and analyzed using the NCBI database, and the single strain obtained was determined to be pink spiral polysporon (Clonostachys rosea).

[0020] The medicine for preventing or treating diseases such as skin aging, cancer, inflammation and myocardial damage contains the rare ginsenoside CK prepared by the method of the invention.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The present invention adopts the Panax notoginseng rhizosphere fungus (pink spiral polysporus) derived from the rhizosphere soil of Panax notoginseng to ferment the bacteria to directly produce the rare ginsenoside CK. The preparation method does not need to prepare precursors, does not rely on the production and cultivation of medicinal plants, and does not occupy land resources. In addition, the fungus in the preparation method has a fast proliferation rate, the secondary metabolites are relatively single, the target product is easy to separate and purify, and it is easy to industrialize production, which can effectively reduce the pollution or ecological damage caused by traditional preparation methods. Description of the drawings:

[0023] Figure 1 It is the rhizosphere fungus of Panax notoginseng of the present invention, namely the pink spiral polysporon colony. Specific implementation method:

[0024] The essential contents of the present invention are further illustrated by the following embodiments of the present invention. These embodiments are only for describing the preferred embodiments of the present invention, and do not limit the protection scope of the present invention in any way.

[0025] Embodiment 1:

[0026] Step 1, separation and purification of pink spiral polysporus.

[0027] Weigh 5 g of the rhizosphere soil of Panax notoginseng and put it into a conical flask containing 45 mL of sterile water. Oscillate for 20 min. -1 Soil suspension; gradually diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 Afterwards, take each dilution and spread it on the separation plate, and add ampicillin (100 mg / L) to the separation plate to inhibit the growth of bacteria. Invert and culture at 28°C for 7-14 days. According to the morphological characteristics of the colonies and hyphae of pink spiral polysporon, pick the hyphae and inoculate them on the PDA plate, invert and culture at 28°C for 7-10 days, and repeat the transfer for 2-3 generations. By extracting DNA from the single strain obtained, using ITS universal primers for amplification and sequencing, the sequencing results were compared and analyzed using the NCBI database, and the single strain obtained was determined to be pink spiral polysporon (Clonostachys rosea).

[0028] The above-mentioned Panax notoginseng rhizosphere fungus isolated and purified from the rhizosphere soil of Panax notoginseng, namely Clonostachys rosea, has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with the deposit number of CGMCCNO.40327. Figure 1 shown.

[0029] Step 2, fermentation culture of pink spiral polysporus.

[0030] Wash, peel and cut 1000g potatoes into 4000mL pure water and boil for 20min, cool and filter through three layers of gauze, collect the filtrate and dilute to 5000mL for potato extract. Weigh 100g rice and place it in a 500mL wide-mouth culture bottle, add 150mL potato extract to each bottle, seal with a breathable membrane and sterilize with high pressure steam for 30min (121℃, 0.105Mpa), cool and set aside. Inoculate the activated pink spiral polysporus into the potato rice solid culture medium and ferment and culture for 40d at 25±2℃ in the dark (or dark and light alternately).

[0031] Step 3, preparation of rare ginsenoside CK.

[0032] The rice culture of pink spiral polysporus (30kg) was extracted with 3 volumes of ethyl acetate for 5 times (5d / time), and the extract was concentrated under reduced pressure to obtain 390g of ethyl acetate crude extract. The crude extract was chromatographed on a silica gel column and eluted with an organic solvent to obtain a petroleum ether elution fraction A: 300g. The chromatography column was further eluted with a gradient of chloroform: methanol 500:1 to 10:1 to obtain 6 main fractions, namely fractions BG. Among them, fraction D (16.4g) was chromatographed on an RP-18 column, and eluted with a gradient of methanol: water 1:9 to 9:1 to obtain 6 subcomponents, namely fractions D1-D6. Among them, fraction D2 (1.2g) was chromatographed on a silica gel column, and chloroform: methanol 20:1 to obtain 6 fractions, namely fractions D2.1-2.6. Fraction D2.4 (37 mg) was subjected to RP-18 semi-preparative column chromatography and eluted with acetonitrile:water 55:45. The eluate was decompressed, concentrated and dried to obtain the monomer compound (20 mg).

[0033] Step 4: Identification of the chemical structure of the monomer compound.

[0034] The compound is ginsenoside CK, which has been identified as 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol. The chemical structure of ginsenoside CK is shown below:

[0035]

[0036] Its physicochemical and spectral data are as follows: white amorphous powder; IRν max 3450-3150(br),1025(br)cm -1 ; UV(MeOH)λ max (Abs)203(2.45)nm; ESIMS m / z645[M+Na] + , molecular formula C 36 H 62 O8; 1H NMR(C5D5N,500MHz): 0.900(3H,s,Me-19),0.956(3H,s,Me-30),0.997(3H,s,Me-18),1.049(3H,s,Me-29),1.239(3H, s,Me-28),1.604(3H,s,Me-27),1.607(3H,s,Me-26),1.640(3H,s,Me-21),3.43(1H,m,H-3α),3.94(1H,ddd,J=9.15,5 .53,2.64,H-5′),4.02(1H,t,J=8.2,H-2′),4.18(1H,t,J=8.9,H-4′),4.18(1H,m,H-12α),4.25(1H,t,J=8.7,H-3′),4 .34(1H,dd,J=11.7,5.2,H-6′),4.51(1H,dd,J=11.7,2.4,H-6′),5.21(1H,d,J=7.8,H-1′),5.26(1H,t,J=6.8,H-24); 13 C NMR(C5D5N,125MHz):130.69(C-25),125.75(C-24),98.05(C-1'),83.08(C-20),79.10(C-3'),78.07(C-5'),77.83(C-3),74.92(C -2'),71.45(C-4'),69.96(C-12),62.68(C-6'),56.14(C-5),51.39(C-17),51.21(C-14),50.09(C-9),49.30(C-13),39.86(C-8),3 9.34(C-4),39.19(C-1),37.14(C-10),34.95(C-7),30.74(C-15),30.56(C-11),28.47(C-28),28.05(C-2),26.42(C-16),25.54(C- 26),22.13(C-21),35.96(C-22),22.98(C-23),18.55(C-6),17.54(C-27),17.18(C-30),16.14(C-29),16.14(C-19),15.81(C-18).

[0037] Embodiment 2:

[0038] Step 1, separation and purification of pink spiral polysporus.

[0039] Weigh 5 g of the rhizosphere soil of Panax notoginseng and put it into a conical flask containing 45 mL of sterile water. Oscillate for 20 min.-1 Soil suspension; gradually diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 Afterwards, take each dilution and spread it on the separation plate, and add ampicillin (100 mg / L) to the separation plate to inhibit the growth of bacteria. Invert and culture at 28°C for 7-14 days. According to the morphological characteristics of the colonies and hyphae of pink spiral polysporon, pick the hyphae and inoculate them on the PDA plate, invert and culture at 28°C for 7-10 days, and repeat the transfer for 2-3 generations. By extracting DNA from the single strain obtained, using ITS universal primers for amplification and sequencing, the sequencing results were compared and analyzed using the NCBI database, and the single strain obtained was determined to be pink spiral polysporon (Clonostachys rosea).

[0040] Step 2, fermentation culture of pink spiral polysporus.

[0041] Wash, peel and cut 1000g potatoes into 4000mL pure water and boil for 20min, cool and filter through three layers of gauze, collect the filtrate and dilute to 5000mL for potato extract. Weigh 100g wheat and place it in a 500mL wide-mouth culture bottle, add 150mL potato extract to each bottle, seal with a breathable membrane and sterilize with high pressure steam for 30min (121℃, 0.105Mpa), cool and set aside. Inoculate the activated pink spiral polysporus into potato rice solid culture medium and ferment and culture for 40d at 25±2℃ in dark (or dark and light alternately).

[0042] Step 3, preparation of rare ginsenoside CK.

[0043] The wheat culture of pink spiral polysporus (30kg) was extracted with 3 volumes of ethyl acetate for 5 times (5d / time), and the extract was concentrated under reduced pressure to obtain 460g of ethyl acetate crude extract. The crude extract was chromatographed on a silica gel column and eluted with an organic solvent to obtain a petroleum ether elution fraction A: 350g. The chromatographic column was further eluted with a gradient of chloroform: methanol from 500:1 to 10:1 to obtain 6 main fractions, namely fractions BG. Among them, fraction D (27.4g) was chromatographed on an RP-18 column, and eluted with a gradient of methanol: water from 1:9 to 9:1 to obtain 6 subcomponents, namely fractions D1-D6. Among them, fraction D2 (1.9g) was chromatographed on a silica gel column, and chloroform: methanol was 20:1 to obtain 6 fractions, namely fractions D2.1-2.6. Fraction D2.4 (71 mg) was subjected to RP-18 semi-preparative column chromatography and eluted with acetonitrile:water 55:45. The eluate was decompressed, concentrated and dried to obtain the monomer compound (40 mg).

[0044] Step 4: Chemical structure identification of the monomer compound is the same as in Example 1.

[0045] Embodiment 3:

[0046] Step 1, separation and purification of pink spiral polysporus.

[0047] Weigh 5 g of the rhizosphere soil of Panax notoginseng and put it into a conical flask containing 45 mL of sterile water. Oscillate for 20 min. -1 Soil suspension; gradually diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 Afterwards, take each dilution and spread it on the separation plate, and add ampicillin (100 mg / L) to the separation plate to inhibit the growth of bacteria. Invert and culture at 28°C for 7-14 days. According to the morphological characteristics of the colonies and hyphae of pink spiral polysporon, pick the hyphae and inoculate them on the PDA plate, invert and culture at 28°C for 7-10 days, and repeat the transfer for 2-3 generations. By extracting DNA from the single strain obtained, using ITS universal primers for amplification and sequencing, the sequencing results were compared and analyzed using the NCBI database, and the single strain obtained was determined to be pink spiral polysporon (Clonostachys rosea).

[0048] Step 2, fermentation culture of pink spiral polysporus.

[0049] Wash, peel and cut 4kg potatoes into pieces and boil them in 12L pure water for 20min, cool and filter through three layers of gauze; boil 4kg wheat in 8L pure water for 2h and filter through three layers of gauze. Mix the potato filtrate with the wheat filtrate and adjust the volume to 20L. Dissolve 400g glucose in the mixture and adjust the pH value to 6.8 to obtain the improved potato-wheat liquid medium (PWL). The prepared PWL was divided into 1L brown triangular bottles (500mL / bottle), sealed with a breathable membrane and sterilized with high pressure steam for 30min (121℃, 0.105Mpa). Inoculate the activated pink spiral polysporus into the cooled PWL medium and culture it in suspension at 25±2℃ for 30d.

[0050] Step 3, preparation of rare ginsenoside CK.

[0051] The culture fluid of the pink spiral polysporus culture obtained in step 2 was extracted with ethyl acetate, the mycelium was dried and extracted with methanol, and then the extracts were combined to obtain 46g. The crude extract was chromatographed on a silica gel column with a gradient elution of chloroform: methanol 500:1 to 10:1 to obtain 6 main parts, namely fractions BG. Among them, fraction D (2.3g) was chromatographed on an RP-18 column, and eluted with a gradient elution of methanol: water 1:9 to 9:1 to obtain 6 subcomponents, namely fractions D1-D6. Among them, fraction D2 (1.7g) was chromatographed on a silica gel column, chloroform: methanol 20:1 to obtain 6 parts, namely fractions D2.1-2.6. Fraction D2.4 (68mg) was chromatographed on an RP-18 semi-preparative column, eluted with acetonitrile: water 55:45, and the eluate was decompressed, concentrated, and dried to obtain a monomer compound (33mg).

[0052] Step 4: Chemical structure identification of the monomer compound is the same as in Example 1.

[0053] Formulation Example 1:

[0054] According to the methods of Examples 1, 2 and 3, rare ginsenoside CK was prepared, and injection solution was prepared by adding injection water, fine filtering, filling and sterilization according to the conventional preparation method.

[0055] Formulation Example 2:

[0056] Firstly prepare rare ginsenoside CK according to the method of Examples 1, 2 and 3, dissolve it in sterile water for injection, stir it to fully dissolve it, filter it with a sterile suction funnel, filter it with sterile fine filtration, pack it into 2 ampoules, freeze-dry it at low temperature, and aseptically seal it to obtain a powder injection.

[0057] Formulation Example 3:

[0058] According to the example, the rare ginsenoside CK was first prepared according to the method of Examples 1, 2 and 3, and excipients were added in a weight ratio of 1:5-1:10 to the excipients, and granulated and tableted.

[0059] Formulation Example 4:

[0060] According to the examples, the rare ginsenoside CK was first prepared according to the methods of Examples 1, 2 and 3, and then prepared into an oral liquid according to a conventional oral liquid preparation method.

Claims

1. A Panax notoginseng rhizosphere fungus, characterized in that: The rhizosphere fungus of Panax notoginseng is a pink spiral polysporus isolated and purified from the rhizosphere soil of Panax notoginseng ( Clonostachys rosea ), which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Committee with the deposit number of CGMCC NO. 40327.

2. Use of a strain of Panax notoginseng rhizosphere fungus as claimed in claim 1 in fermentation to prepare rare ginsenoside CK.

Images