CN109468359B - Ginsenoside Rk6Preparation method of (1) - Google Patents

Abstract

The invention relates to the field of bioengineering, in particular to a preparation method of ginsenoside Rk ₆ . The invention ferments Ganoderma lucidum strains and Panax notoginseng, utilizes the complex enzyme system of Ganoderma lucidum to promote the original chemical components in the medicinal materials of Panax notoginseng to undergo structural modification or change of active sites, and convert them into ginsenoside Rk ₆ , the conversion rate is high, and can be used for Bulk preparation of ginsenoside Rk ₆ . It provides raw materials for later drug development and pharmacological research, and has very important research significance and social benefits for the development of new drugs with independent intellectual property rights. At the same time, the method has the advantages of fast reaction, strong specificity, few side reactions, etc., and the reaction conditions are mild and controllable, and it is environmentally friendly and pollution-free.

Description

A kind of preparation method of ginsenoside Rk6

technical field

The invention relates to the field of bioengineering, in particular to a preparation method of ginsenoside Rk ₆ .

Background technique

Panax notoginseng (Panax notoginseng) is the dried root and rhizome of Panax notoginseng, also known as Shan lacquer, Jinbuhuan, Tian lacquer, Tian Sanqi, Tianqi, Shensanqi, blood ginseng, ginseng notoginseng, Diansanqi, etc. , mainly produced in Yunnan, Guangxi and other places in my country. This product is warm, sweet, slightly bitter, non-toxic. The raw products of Panax notoginseng can reduce swelling and relieve pain, disperse blood stasis and stop bleeding, and the cooked products can nourish blood and activate blood. Panax notoginseng has a long history of being used to treat diseases and is one of the traditional and precious medicinal materials commonly used in my country. It has made great contributions to the prosperity of the Chinese nation for thousands of years, and has been deeply concerned and valued by people around the world.

The saponins in Panax notoginseng play a crucial role as its main pharmacodynamic substances. In recent years, scholars have used different extraction methods to separate some more common active saponins from different parts of Panax notoginseng, such as Ginsenoside Rg ₁ , ginsenoside Rb ₁ , ginsenoside Rb3, notoginsenoside R ₁ and the like. However, the types and contents of saponins present in Panax notoginseng are limited, which does not include ginsenoside Rk ₆ . However, ginsenoside Rk ₆ is a rare saponin, and some studies have shown that it has more significant pharmacological activity, and its medicinal and research value is extremely high. The later stage of ginsenoside Rk ₆ drug development and pharmacological research work.

SUMMARY OF THE INVENTION

In view of this, in view of the problem that the existing ginsenoside Rk ₆ cannot be prepared in large quantities, the present invention aims to provide a preparation method of ginsenoside Rk ₆ with a high conversion rate.

For realizing the purpose of the present invention, the present invention adopts following technical scheme:

A preparation method of ginsenoside Rk ₆ , comprising:

Step 1: insert Ganoderma lucidum strain into medicinal medium for cultivation to obtain Ganoderma lucidum fungus seed liquid;

Step 2: drying, pulverizing and sieving the medicinal materials of Panax notoginseng to obtain Panax notoginseng solid medium;

Step 3: Inoculate the Ganoderma lucidum seed liquid described in Step 1 into the Panax notoginseng solid medium described in Step 2 for fermentation to obtain a fermentation product containing ginsenoside Rk ₆ .

The invention provides a preparation method of ginsenoside Rk ₆ , comprising:

Step 1: insert Ganoderma lucidum strain into medicinal medium for cultivation to obtain Ganoderma lucidum fungus seed liquid;

Step 2: drying, pulverizing and sieving the medicinal materials of Panax notoginseng to obtain Panax notoginseng solid medium;

Step 3: inoculate the Ganoderma lucidum fungus seed liquid described in Step 1 into the Panax notoginseng solid medium described in Step 2 for fermentation to obtain a fermentation product containing ginsenoside Rk ₆ ;

Step 4: the fermentation product is extracted, separated and purified to obtain ginsenoside Rk ₆ ;

The extraction is as follows: the fermentation product is extracted by heating under reflux with ethanol, the extract is sequentially extracted with petroleum ether, ethyl acetate and water-saturated n-butanol, and the water-saturated n-butanol layer is collected.

Ganoderma lucidum (Ganoderma lucidum) is a precious medicinal fungus belonging to the phylum Basidiomycota, the family Ganoderma lucidum, and the genus Ganoderma lucidum. Since ancient times, Ganoderma lucidum has been regarded as a "fairy grass", which has the functions of nourishing and strengthening, strengthening the body and strengthening the body.

The preparation method of the invention uses Ganoderma lucidum strain as raw material to prepare Ganoderma lucidum seed liquid, uses Panax notoginseng as raw material to prepare Panax notoginseng solid medium, and then inoculates the Ganoderma lucidum seed liquid into the Panax notoginseng solid medium for fermentation, and uses Ganoderma lucidum complex The enzyme system promotes the conversion of the original chemical components in the Panax notoginseng medicinal materials into ginsenoside Rk ₆ through structural modification or active site change, and the conversion rate is high, which can be used for the mass preparation of ginsenoside Rk ₆ .

The preparation method provided by the present invention can convert the protopanaxatriol type saponins existing in Panax notoginseng into ginsenoside Rk ₆ , and the conversion mechanism diagram is shown in FIG. 1 .

Step 1 of the preparation method of the present invention uses medicinal medium to cultivate Ganoderma lucidum strains, wherein the medicinal medium formula is: glucose 1.9-2.1%, KH ₂ PO ₄ 0.09-0.11%, MgSO ₄ 0.09- 0.11%, peptone 0.45-0.55%, yeast powder 0.18-0.22%, vitamin B ₁ 0.09-0.11%, and the balance is water. In some specific embodiments, the medicinal medium is formulated as follows: glucose 2%, KH ₂ PO ₄ 0.1%, MgSO ₄ 0.1%, peptone 0.5%, yeast powder 0.2%, vitamin B ₁ 0.1%, and the balance is water.

The medicinal culture medium was prepared according to the above formula, packaged in 250ml wide-mouth bottles, placed in a pressure steam sterilizer after bandaging, and sterilized at 121° C. for 30 minutes. After the culture medium is cooled, inoculate Ganoderma lucidum strains in a sterile operating table, and the inoculated Ganoderma lucidum strains are slanted strains.

Wherein, the way of culturing Ganoderma lucidum strains is constant temperature shaking culture, and the constant temperature shaking culture is: under the conditions that the rotating speed is 150r/min, the temperature is 27～29°C, and the humidity is 39～41%, the culture is protected from light for 5～41%. 7 days. In some specific embodiments, the constant-temperature shaking culture is: culturing in the dark for 5-7 days under the conditions of a rotating speed of 150 r/min, a temperature of 28° C., and a humidity of 40%.

In step 2 of the preparation method of the present invention, the medicinal materials of Panax notoginseng are dried at a temperature of 70°C to 75°C, and pulverized to obtain Panax notoginseng powder with a mesh number of 10 to 60, and prepare a Panax notoginseng solid medium.

After drying, pulverizing and sieving the medicinal materials of Panax notoginseng, the steps of adding CaCO ₃ aqueous solution and sterilizing are further included. Specifically: drying the medicinal materials of Panax notoginseng at a temperature of 70 ℃ ~ 75 ℃, pulverizing and sieving to obtain Panax notoginseng powder with a mesh number of 10 ~ 60, adding CaCO ₃ aqueous solution, mixing evenly, and placing it in a triangular flask , 121 ℃ high pressure steam sterilization twice, each 30 minutes, made Panax notoginseng solid medium. Wherein, the water content of the Panax notoginseng solid medium is 50-70%, preferably 60%. Among them, the mass of CaCO ₃ is 1.5% of the mass of Panax notoginseng.

In step 3 of the preparation method of the present invention, the seed liquid of Ganoderma lucidum described in step 1 is inoculated into the solid medium of Panax notoginseng described in step 2 for fermentation, and the complex enzyme system of Ganoderma lucidum is used to promote the original chemical components in the medicinal materials of Panax notoginseng to pass through the structure. The modification or the change of the active site is converted into ginsenoside Rk ₆ . Wherein, in terms of ml/g, the volume-to-mass ratio of Ganoderma lucidum seed liquid and Panax notoginseng solid medium is 0.5-1:1-1.5, and the temperature of fermentation culture is 24-28°C. In some specific embodiments, the volume-to-mass ratio of Ganoderma lucidum seed liquid and Panax notoginseng solid medium is 1:1, and the temperature of fermentation culture is 28°C. In some embodiments, the time for the fermentation culture is 35 to 45 days.

In step 4 of the preparation method of the present invention, the fermentation product is extracted, separated and purified to obtain ginsenoside Rk ₆ . Wherein, the extraction is to use 70% to 75% ethanol for heating and reflux extraction, the extract is desolvated and then suspended in water, the obtained suspension is sequentially extracted with petroleum ether, ethyl acetate and water-saturated n-butanol, and the water-saturated n-butanol is collected. The butanol layer; the quality of the 70% to 75% ethanol is 7 to 8 times that of the fermentation product, and the extraction times are 3 to 4 times.

Wherein, the volume of petroleum ether, ethyl acetate, and water-saturated n-butanol is 1 to 1.2 times the volume of the suspension.

Specifically, the separation and purification includes: taking the water-saturated n-butanol layer and concentrating it, dissolving it in methanol, and then passing it through an MCI chromatographic column and eluting it with ethanol. Elution, silica gel GF254 thin layer detection, 70% methanol as mobile phase, and ginsenoside Rk ₆ is obtained by semi-preparative liquid phase.

Wherein, the relative density of the concentrated to 80°C-85°C is 1.21-1.25.

The diameter of the MCI chromatographic column is 6 cm, and the column height is 75 cm.

The open ODS column is filled with 40-60 um ODS, the chromatographic column diameter is 3 cm, and the column height is 40 cm.

The semi-preparative liquid phase uses 70% methanol as the mobile phase, the flow rate is 1.2 ml/min, the detection wavelength is 203 nm, and the components whose retention time is 126 min are collected.

In a specific embodiment, the separation and purification steps are:

Taking the water-saturated n-butanol layer and concentrating to obtain the extract, taking the extract and adding methanol to dissolve, using MCI chromatographic column to elute with different concentrations of ethanol gradient, the eluent is detected by silica gel GF254 thin layer to obtain ₆ parts rich in ginsenoside Rk . This part was then passed through an open ODS column for gradient elution with different concentrations of methanol (pure water → 30% methanol → 50% methanol → 70% methanol → pure methanol). The part containing ginsenoside Rk ₆ was passed through a semi-preparative liquid phase, and the components with a retention time of 126 min were collected to obtain ginsenoside Rk ₆ .

In the present invention, Ganoderma lucidum strains and Panax notoginseng are fermented, and the complex enzyme system of Ganoderma lucidum is used to promote the original chemical components in the medicinal materials of Panax notoginseng to undergo structural modification or change of active sites, and convert them into ginsenoside Rk ₆ . The Rk ₆ content of ginsenosides in the fermentation products is 0.6481-0.8508%, which provides raw materials for later drug development and pharmacological research, and has very important research significance and social benefits for the development of new drugs with independent intellectual property rights. . At the same time, the method has the advantages of fast reaction, strong specificity, few side reactions, etc., and the reaction conditions are mild and controllable, and it is environmentally friendly and pollution-free.

Description of drawings

Fig. 1 shows the transformation mechanism diagram of Panax notoginseng panaxatriol-type saponins into ginsenoside Rk ₆ ;

Figure 2 shows the semi-preparative high performance liquid chromatogram of ginsenoside Rk ₆ .

Detailed ways

The embodiment of the present invention discloses a preparation method of ginsenoside Rk ₆ . Those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method of the present invention has been described through preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention .

In order to further understand the present invention, the present invention will be described in detail below with reference to the embodiments.

Example 1 Preparation method of the present invention

(1) Preparation of Ganoderma lucidum seed solution: the medium formula is glucose 2%, KH ₂ PO ₄ 0.1%, MgSO ₄ 0.1%, peptone 0.5%, yeast powder 0.2%, vitamin B ₁ 0.1%, balance water, pH nature. The above substances are placed in a large beaker, and an appropriate amount of water is added to make a seed solution. After the seed solution is cooled, it is divided into 250ml wide-mouth bottles. After bandaging, it is placed in a pressure steam sterilizer and sterilized at 121°C for 30 minutes. . After cooling, use an inoculation loop with a diameter of 1 mm to insert 3-4 strains of the preserved Ganoderma lucidum slant in a sterile operating table. Put the liquid culture medium with Ganoderma lucidum strains into a constant temperature shaking incubator, and cultivate at 150r/min, 28°C, and 40% humidity in the dark for 5-7 days. Miscanthus spherules appeared, and Ganoderma lucidum seed liquid was obtained.

(2) Preparation of Panax notoginseng solid medium: take the medicinal materials of Panax notoginseng, dry, pulverize, pass the powder through a 10-mesh sieve but no more than a 60-mesh sieve, add a CaCO ₃ aqueous solution, mix evenly, place it in a conical flask, and sterilize it with high-pressure steam at 121°C The bacteria were carried out twice for 30 minutes each time to prepare a solid medium of Panax notoginseng with a water content of 50% to 70%; wherein, the mass of CaCO ₃ was 1.5% of the mass of Panax notoginseng medicinal material.

(3) Fermentation: inoculate the Ganoderma lucidum seed liquid into the Panax notoginseng solid medium in the aseptic operation table, ferment and cultivate in the constant temperature incubator in the dark, until the mycelium is covered with the bottom of the container, take it out and dry to obtain fermentation product. Wherein, in ml/g, the volume-to-mass ratio of the Ganoderma lucidum seed liquid and the Panax notoginseng solid medium is 0.5-1:1-1.5, and the temperature of the fermentation culture is 24-28°C.

(4) Extraction: The fermented product in step (3) was extracted with 8 times the mass of 70% ethanol by heating and refluxing for 3 times, each time for 2.5 hours, the solvent was recovered and evaporated to no alcohol smell, and then suspended with an appropriate amount of water, and then subjected to 1.2 times Volume petroleum ether, ethyl acetate, and water-saturated n-butanol were sequentially extracted, and the water-saturated n-butanol layer was collected.

(5) Separation and purification: take the water-saturated n-butanol layer and concentrate to obtain an extract with a relative density of 1.21-1.25 (80°C-85°C), add methanol to the extract to dissolve, and use MCI chromatographic column for gradient elution with different concentrations of ethanol , the eluate was detected by silica gel GF254 thin layer to obtain the part rich in ginsenoside Rk ₆ . This part was then passed through an open ODS column for gradient elution with different concentrations of methanol (pure water → 30% methanol → 50% methanol → 70% methanol → pure methanol). The part containing ginsenoside Rk ₆ was passed through the semi-preparative liquid phase, the flow rate was 1.2ml/min, the detection wavelength was 203nm, and the components with a retention time of 126min were collected (see Table 1 No. 24 peak) to obtain the compound. The semi-preparative liquid chromatogram is shown in Figure 2, and the liquid chromatography data is shown in Table 1.

Table 1 Liquid Analysis Results

The compound is white powder (methanol), purplish red in 10% sulfuric acid ethanol solution, positive for Molish reaction and positive for Liebermann-Buchard reaction, so it is presumed to be a triterpenoid saponin compound.

HR-ESI-MS gave: m/z 671.3929 ([M+CI] ⁻ , calc. for 671.3926), and its molecular formula can be inferred to be C ₃₆ H ₆₀ O ₉ in combination with its NMR. ¹ H-NMR (CD ₃ OD, 500MHz) spectrum gives 6 angle methyl proton signals in high field region δ _H : 0.94(3H,s,H-30), 0.99(3H,s,H-19), 1.00 (3H,s,H-29,), 1.13(3H,s,H-18), 1.33(3H,s,H-28), 1.67(3H,s,H-27); on 3 oxycarbons The proton signal δ _C : 3.11 (1H, dd, J=10.0, 5.0), 4.10 (1H, td, J=10.0, 5.0Hz), 3.53-3.63 (1H, m); two pairs of carbon-carbon double bonds, 3 The alkene proton signal δ: 4.70(1H,s), 4.89(1H,s), 5.45(1H,t,J=5.0Hz); the terminal proton signal of 1 sugar δ4.35(1H,d,J= 10.0Hz, H-1').

¹³ C-NMR (CD ₃ OD, 150MHz) spectrum gives 36 carbon signals, and high field region gives 6 angular methyl carbon signals δ _C : 17.2 (C-18), 17.6 (C-19), 13.5 (C-27), 31.1 (C-28), 15.8 (C-29), 16.7 (C-30); 3 oxycarbon signals delta _C : 79.5 (C-3), 80.6 (C-6), 70.1 (C-12); 4 alkene carbon signals δ _C : 155.6 (C-20), 108.3 (C-21), 126.6 (C-24), 135.6 (C-25); wherein δ _C 61.5 (C- 5) is the characteristic signal of triol-type saponins, and the compound is presumed to be dammarane-triol-type triterpenoids. Combined with the corresponding sugar end group signal in ¹ H-NMR, it indicated that there is 1 group of glucose in the compound, the signal is δC: 105.2( _C -1'), 75.2(C-2'), 78.8(C-3'), 71.4 (C-4'), 77.4 (C-5'), 62.6 (C-6').

The carbon spectrum data of the compound is different from the chemical shifts of C-25, C-26 and C-27 of ginsenoside Rk ₃ reported in the literature. It can be seen from the mass spectrum that there is one more oxygen atom than ginsenoside Rk _3. According to the HMQC and HMBC spectra It is inferred that the C-26 position of this compound is connected with an additional hydroxyl group, which changes the surrounding chemical environment. Combined with the HMQC and HMBC spectra of the compounds, it can be seen that there is a long-range correlation between the carbon signals of δ _H 2.20 (H-23) and δ _C 126.6 (C-24), δ _C 135.6 (C-25), and δ _C 68.7 (C-26). ; δ _H 5.45 (H-24) and δ _C 27.0 (C-23), δ _C 68.7 (C-26), δ _C 13.5 (C-27) carbon signals have long-range correlation; δ _H 3.92 (H-26 ) and δC 126.6( _C -24), δC 135.6( _C -25), δC 13.5( _C -27) carbon signals have long-range correlation; δH 1.67( _H -27) and δC 126.6( _C- 24), δC 135.6( _C -25), δC 68.7( _C -26) carbon signals are remotely correlated. In the HMBC spectrum, it can be seen that δ _H 4.35 (d, J=10.0H _Z , H-1') has a long-range correlation with δ _C 80.6 (C-6), indicating that the compound is 6-O-β-D glucose. In summary, the compound is 3β, 6α, 12β, 26-tetrahydroxydammar-20(21), 24(25)(E)-diene-6-O-β-D-Glucopyranoside, namely ginsenoside Rk ₆ , and the molecular formula is C ₃₆ H ₆₀ O ₉ , the structure is shown in formula I,

Table 2 ¹ H-NMR (C ₅ D ₅ N, 500 MHz) and ¹³ C-NMR (C ₅ D ₅ N, 150 MHz) data of the compounds extracted in this example

Example 2 Comparison of RK ₆ content in different bacterial species and Panax notoginseng fermentation products

Thirteen medicinal fungi (Bolete, Fungus, Shiitake 1500, Oyster Mushroom, King Oyster Mushroom, Large Green Mushroom, Bailing Mushroom, Phyllostachys vulgaris, Tree Tongue, Yunzhi, Ganoderma lucidum, Grifola frondosa, Baishuhua) were used to separate Inoculated in Panax notoginseng solid medium for fermentation, other conditions are the same, with reference to the steps (1)～(3) of Example 1, carry out, wherein, Panax notoginseng solid medium water content is 50%, Ganoderma lucidum seed liquid V (ml ): Panax notoginseng solid medium m(g)=1:1, the temperature of fermentation culture is 24℃. The solid medium was observed, and the results were shown in Table 3, and the RK ₆ content in the fermentation product was detected, and the results were shown in Table 4.

Table 3 Fermentation of different strains and Panax notoginseng

strain the first week the second week The third week Boletus not issued not issued not issued fungus few brown nodules Slight increase in bacteria No significant changes Mushroom 1500 few white fimbriae Slow growth of fimbriae Fimbriae are basically covered Oyster mushroom few white fimbriae 5cm diameter flora Fimbriae king oyster mushroom Overgrown with spheroids with needle-like hyphae on the spheroids The flora grows slowly Fimbriae are basically covered big green mushroom sparse white fimbriae few white fimbriae No significant changes Bailing Mushroom few white mycelium Slow growth of fimbriae Fimbriae tree chicken not issued not issued not issued tree tongue Fimbriae grow but do not spread No significant changes No significant changes Yunzhi sparse white fimbriae increased fimbriae Fimbriae Lingzhi dense white fimbriae increased fimbriae The fimbriae are densely covered and spread to the bottom of the bottle maitake not issued not issued not issued white tree flower not issued not issued not issued

Table 4 RK ₆ content% in different strains and Panax notoginseng fermentation products

RK₆ content% Panax notoginseng herbs 0 Shiitake 1500 and Panax notoginseng fermentation products 0 Oyster mushroom and Panax notoginseng fermentation products 0 Fermented products of Pleurotus eryngii and Panax notoginseng 0 Fermented products of Bailing Mushroom and Panax notoginseng 0 Ganoderma lucidum and Panax notoginseng fermentation products 0.6481 Fermented products of Yunzhi and Panax notoginseng 0

Example 3 Optimal process optimization test

The present embodiment adopts orthogonal test to optimize the process conditions of steps (2) to (3), selects A Panax notoginseng solid medium water content (%), B Ganoderma lucidum seed liquid V (ml): Panax notoginseng solid medium m (g), C fermentation culture temperature (℃) is an influencing factor, and the factor level table is shown in Table 5, the orthogonal test result is shown in Table 6, and the comprehensive score variance analysis is shown in Table 7.

Table 5 Factor Level Table

Table 6 Orthogonal experiments and results

Table 7 Comprehensive score analysis of variance table

source of variance deviation sum of squares degrees of freedom F value salience A 0.012 2 7.416 B 0.075 2 47.219 * C 0.011 2 6.653 D 0.015 2 9.708

F _1-0.10 (2,2)＝9.00F _1-0.05 (2,2)＝19.00

It can be seen from the above results that the primary and secondary order of the three influencing factors is: B Ganoderma lucidum seed liquid V (ml): Panax notoginseng solid medium m (g)>A Panax notoginseng solid medium water content %>C fermentation culture temperature ( ℃), among which, factor B: Ganoderma lucidum seed solution V (ml): Panax notoginseng solid medium m (g) had a significant effect on the content of RK ₆ in the product. Comprehensive consideration, the optimal fermentation conditions are determined as: A ₂ B ₂ C ₃ , that is, the water content of Panax notoginseng solid medium is 60%, and the seed liquid of Ganoderma lucidum V (ml): Panax notoginseng solid medium m (g) = 1:1 , the fermentation temperature is 28 ℃.

The preparation of ginsenoside Rk6 under the optimum technological conditions of embodiment 4

(1) Preparation of Ganoderma lucidum seed solution: the medium formula is glucose 2%, KH ₂ PO ₄ 0.1%, MgSO ₄ 0.1%, peptone 0.5%, yeast powder 0.2%, vitamin B ₁ 0.1%, balance water, pH nature. The above substances are placed in a large beaker, and an appropriate amount of water is added to make a seed solution. After the seed solution is cooled, it is divided into 250ml wide-mouth bottles. After bandaging, it is placed in a pressure steam sterilizer and sterilized at 121°C for 30 minutes. . After cooling, use an inoculation ring with a diameter of 1 mm to insert 3-4 strains of the preserved Ganoderma lucidum slant in the aseptic operation table, and put the liquid culture medium with Ganoderma lucidum strains into a constant temperature shaking incubator, at 150 r/ min, 28° C. and humidity 40% for 5-7 days in the dark, and when the seed solution is clarified and a large number of striated fungus balls of uniform size appear, the Ganoderma lucidum seed solution is obtained.

(2) Preparation of Panax notoginseng solid medium: take the medicinal materials of Panax notoginseng, dry, pulverize, pass the powder through a 10-mesh sieve but no more than a 60-mesh sieve, add a CaCO ₃ aqueous solution, mix evenly, place it in a conical flask, and sterilize it with high-pressure steam at 121°C The bacteria were cultured twice for 30 minutes each time to obtain a solid medium of Panax notoginseng with a water content of 60%; wherein, the mass of CaCO ₃ was 1.5% of the mass of the medicinal material of Panax notoginseng.

(3) Fermentation: inoculate 100ml of Ganoderma lucidum seed liquid into 100g of Panax notoginseng solid medium in a sterile operating table, ferment and cultivate in a constant temperature incubator in the dark, until the bottom of the container is covered with mycelium, take it out and dry, A fermentation product is obtained. Wherein, in terms of ml/g, the volume-to-mass ratio of Ganoderma lucidum seed liquid and Panax notoginseng solid medium is 1:1, and the temperature of fermentation culture is 28°C.

(4) Extraction: the fermented product obtained in step (3) was heated and refluxed for 3 times with 8 times the volume of 70% ethanol for 2.5 hours each time. The ester and water-saturated n-butanol were sequentially extracted, and the water-saturated n-butanol layer was collected. The present invention detects the RK ₆ content in each extraction layer, and the results are shown in Table 8.

Table 8 Content (%) of ginsenoside RK ₆ in different extraction solvents

sample Ginsenoside RK₆ Content% Petroleum ether layer 0.0020 Ethyl acetate layer 0.0128 Water saturated n-butanol layer 0.7106

(5) Separation and purification: take the water-saturated n-butanol layer and concentrate to obtain an extract with a relative density of 1.21-1.25 (80°C-85°C), add methanol to the extract to dissolve, and use MCI chromatographic column for gradient elution with different concentrations of ethanol , the eluate was detected by silica gel GF254 thin layer to obtain the part rich in ginsenoside Rk ₆ . This part was then passed through an open ODS column for gradient elution with different concentrations of methanol (pure water → 30% methanol → 50% methanol → 70% methanol → pure methanol). The part containing ginsenoside Rk ₆ was passed through the semi-preparative liquid phase, 70% methanol was used as the mobile phase, and the detection wavelength was 203 nm to obtain the ginsenoside Rk ₆ monomer compound.

After testing, the content of ginsenoside Rk ₆ in the fermentation product of step (3) was 0.8508%.

Example 5 Preparation method of the present invention

(1) Preparation of Ganoderma lucidum seed solution: the medium formula is glucose 2.1%, KH ₂ PO ₄ 0.09%, MgSO ₄ 0.11%, peptone 0.45%, yeast powder 0.22%, vitamin B ₁ 0.11%, and pH is natural. The above substances are placed in a large beaker, and an appropriate amount of water is added to make a seed solution. After the seed solution is cooled, it is divided into 250ml wide-mouth bottles. After bandaging, it is placed in a pressure steam sterilizer and sterilized at 121°C for 30 minutes. . After cooling, use an inoculation ring with a diameter of 1 mm to insert 3-4 strains of the preserved Ganoderma lucidum slant in the aseptic operation table, and put the liquid culture medium with Ganoderma lucidum strains into a constant temperature shaking incubator, at 150 r/ min, 28° C. and humidity 40% for 5-7 days in the dark, and when the seed solution is clarified and a large number of striated fungus balls of uniform size appear, the Ganoderma lucidum seed solution is obtained.

(2) Preparation of Panax notoginseng solid medium: take the medicinal materials of Panax notoginseng, dry, pulverize, pass the powder through a 10-mesh sieve but no more than a 60-mesh sieve, add a CaCO ₃ aqueous solution, mix evenly, place it in a conical flask, and sterilize it with high-pressure steam at 121°C The bacteria were incubated twice for 30 minutes each time to obtain a Panax notoginseng solid medium with a water content of 70%; wherein, the mass of CaCO ₃ was 1.5% of the mass of the Panax notoginseng medicinal material.

(3) Fermentation: inoculate 100ml of Ganoderma lucidum seed liquid into 150g of Panax notoginseng solid medium in a sterile operating table, ferment and cultivate in a constant temperature incubator in the dark, until the bottom of the container is covered with mycelium, take it out and dry, A fermentation product is obtained. Wherein, in ml/g, the volume-to-mass ratio of Ganoderma lucidum seed liquid and Panax notoginseng solid medium is 1:1.5, and the temperature of fermentation culture is 26°C.

(4) Extraction: The fermented product obtained in step (3) was heated and refluxed for 3 times with 8 times the amount of 70% ethanol, each time for 2.5 hours, the solvent was recovered and evaporated to no alcohol smell, and then suspended with an appropriate amount of water, and then subjected to 1.2 Sequential extraction with multiple volumes of petroleum ether, ethyl acetate, and water-saturated n-butanol, and the water-saturated n-butanol layer was collected.

(5) Separation and purification: take the water-saturated n-butanol layer and concentrate to obtain an extract with a relative density of 1.21-1.25 (80°C-85°C), add methanol to the extract to dissolve, and use MCI chromatographic column for gradient elution with different concentrations of ethanol , the eluate was detected by silica gel GF254 thin layer to obtain the part rich in ginsenoside Rk ₆ . This part was then passed through an open ODS column for gradient elution with different concentrations of methanol (pure water → 30% methanol → 50% methanol → 70% methanol → pure methanol). The part containing ginsenoside Rk ₆ was passed through the semi-preparative liquid phase, 70% methanol was used as the mobile phase, and the detection wavelength was 203 nm to obtain the ginsenoside Rk ₆ monomer compound.

After calculation, the content of ginsenoside Rk ₆ in the fermentation product of step (3) is 0.7102%.

The descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (3)

1. a preparation method of ginsenoside Rk ₆ , is characterized in that, comprises: Step 1: insert Ganoderma lucidum strain into medicinal medium for cultivation to obtain Ganoderma lucidum fungus seed liquid; Step 2: drying, pulverizing and sieving the medicinal materials of Panax notoginseng to obtain a solid medium of Panax notoginseng; the water content of the solid medium of Panax notoginseng is 50-70%; the drying temperature is 70° C. to 75° C. ℃, the mesh number of the Panax notoginseng powder after the sieving is 10-60 meshes; Step 3: inoculate the Ganoderma lucidum seed liquid in step 1 into the Panax notoginseng solid medium described in step 2 for fermentation to obtain a fermentation product containing ginsenoside Rk ₆ ; the Ganoderma lucidum seed liquid and the Panax notoginseng solid The volume-to-mass ratio of the culture medium is 0.5 to 1:1 to 1.5; the temperature of the fermentation is 24 to 28°C; Step 4: the fermentation product is extracted, separated and purified to obtain ginsenoside Rk ₆ ; The extraction is to use 70% to 75% ethanol for heating and reflux extraction, the extract is desolvated and then suspended in water, the obtained suspension is sequentially extracted with petroleum ether, ethyl acetate and water-saturated n-butanol, and the water-saturated n-butanol is collected. layer; the quality of the 70%～75% ethanol is 7～8 times of the quality of the fermentation product, and the extraction times are 3～4 times; The separation and purification include: taking the water-saturated n-butanol layer and concentrating it, dissolving it in methanol, passing through an MCI chromatographic column, and eluting with ethanol, and the obtained eluent is successively identified by a thin layer of silica gel GF254, an open ODS chromatographic column, and washed with methanol. The ginsenoside Rk ₆ was obtained by the semi-preparative liquid phase with 70% methanol as the mobile phase. 2. preparation method according to claim 1, is characterized in that, the medicinal medium formula described in step 1 is: glucose 1.9～2.1%, KH ₂ PO ₄ 0.09～0.11%, MgSO ₄ 0.09～0.11%, peptone 0.45~0.55%, yeast powder 0.18~0.22%, vitamin B ₁ 0.09~0.11%, and the balance is water. 3. preparation method according to claim 1 is characterized in that, described MCI chromatographic column aperture is 6 centimeters, column height is 75 centimeters; Described open ODS column is filler with 40-60umODS, and chromatographic column aperture is 3 centimeters, The column height is 40 cm; the semi-preparative liquid phase uses 70% methanol as the mobile phase, the flow rate is 1.2 ml/min, the detection wavelength is 203 nm, and the components whose retention time is 126 min are collected.

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