CN101376669B - Preparation of 6-O-beta-D- glucosyl-3,6,16,25-tetrahydroxy cycloartane - Google Patents
Preparation of 6-O-beta-D- glucosyl-3,6,16,25-tetrahydroxy cycloartane Download PDFInfo
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Abstract
The invention discloses a method for preparing 6-O-Beta-D-glucosyl-3,6,16,25-tetrahydroxycycloartane. The method comprises the following steps: inoculating pharmaceutical fungi in a solid culture medium containing Chinese traditional medicinal materials or residue thereof; fermenting under a certain fermentation condition; collecting the solid fermented material; and drying, pulverizing and extracting and separating to obtain the product. The method has the advantages that the process is simple, the waste reutilization is implemented, during the extracting and separating process, the macro porous resin method is not adopted to enrich the compound so that the repeated activation of the macro porous resin is obviated, the mixed n-butyl alcohol extraction solution is washed with sodium hydroxide solution to remove a great amount of phenolic acid impurities and color pigments, a neutral alumina chromatographic column is adopted for the purification so that the pigment impurities with the polarity similar to that of the target component is effectively removed so that the pure product can be directly obtained without need of the re-crystallization, the yield of the pure product is improved, and the method is worth extending and applying..
Description
Technical Field
The invention relates to a preparation method of 6-O-beta-D-glucosyl-3, 6,16, 25-tetrahydroxy cycloartane, belonging to the technical field of medicines.
Background
The compound 6-O-beta-D-glucosyl-3, 6,16, 25-tetrahydroxy cycloartane (hereinafter referred to as compound 1) is shown in a chemical formula (I):
A novel (bidirectional) solid fermentation engineering (bidirectional fermentation for short) for medicinal fungi is characterized in that a fermentation substrate in a fermentation combination formed by fermentation strains and the fermentation substrate is changed into a nutrient substrate (medicinal mycoplasm) formed by agricultural and sideline products (bagasse, wheat bran and the like) which are rich in nutrients such as carbon, nitrogen and the like in the past, and a Chinese medicinal material with certain active ingredients is adopted as a medicinal substrate to ensure that the nutrient required by the growth of the fungi can be provided and the tissues and the ingredients of the Chinese medicinal material can be changed due to enzymes generated by the fungi, so that new flavor functions are generated, and therefore, the medicinal fungi have the bidirectional property. The medicinal mycoplasm can be added with better medicinal effect than the fungus or the medicinal matrix or both, and is mainly reflected in the aspects of synergism, expansion, detoxification and the like. The invention discloses a method for preparing a medicament and a feed additive for livestock and poultry breeding by adopting mixed traditional Chinese medicinal materials and mixed strains through bidirectional fermentation, wherein the application number is 200610038850.6, and the invention is named as a traditional Chinese medicine multi-strain mixed solid fermentation technology and an invention patent application publication specification of the application thereof; the patent No. ZL 200510038072.6, entitled feed additive and its preparation process and application in preparing feed for preventing avian influenza, the invention patent specification discloses a preparation method of feed additive by mixing and fermenting whole substrate and medicinal fungi.
The report of preparing pure compounds by using a bidirectional fermentation method is not reported yet.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for preparing 6-O-beta-D-glucosyl-3, 6,16, 25-tetrahydroxy cycloartane by applying a biotransformation mode.
The invention is realized by the following technical scheme: a method for preparing 6-O-beta-D-glucosyl-3, 6,16, 25-tetrahydroxy cycloartane, which comprises the following steps:
1. preparing a solid fermentation product by a biotransformation mode:
(1) pulverizing Chinese medicinal materials, sieving, adjusting to appropriate water content, and sterilizing to obtain solid culture medium containing Chinese medicinal materials;
(2) inoculating medicinal fungi 5-15% of the dry weight of the solid culture medium into the solid culture medium, and fermenting at 24-30 deg.C under 45-60% humidity for 30-45 days to obtain solid fermented product;
(3) collecting solid fermented product, drying at 50-70 deg.C, and pulverizing; wherein,
the traditional Chinese medicine in the step (1) is astragalus or astragalus residue;
the medicinal fungus in the step (2) is any one of ganoderma lucidum, hericium erinaceus, grifola frondosa, corious versicolor, trametes robinii and lentinus edodes, preferably ganoderma lucidum;
2. extracting and separating compounds from the dried and crushed solid fermentation product:
(1) taking a proper amount of dry solid fermentation product powder particles obtained in the step 1, carrying out ethanol reflux extraction, and recovering a solvent to obtain an extract;
(2) dissolving the extract with water, extracting with water saturated n-butanol, and evaporating to remove solvent to obtain extract;
(3) subjecting the extract to silica gel column chromatography, eluting with chloroform-methanol mixture (v/v:10:0-10:5), combining the same fractions, and volatilizing the solvent;
(4) adding neutral alumina, performing dry chromatography with alumina column, eluting with chloroform-methanol mixture (v/v:10:0-10:5), and volatilizing solvent to obtain compound of formula (I); wherein,
the ethanol concentration in the step (1) is 70%, and the reflux extraction is carried out for 2 times, 2 hours each time;
in the step (2), after the water saturated n-butanol is extracted, washing the extract by using a 1% NaOH solution and the water saturated n-butanol;
in the steps (3) and (4), the chromatography method is dry chromatography.
The invention has the beneficial effects that: the method uses the waste Chinese medicinal materials of astragalus dregs and ganoderma lucidum mycelia to carry out solid fermentation to prepare the target compound, is simple and easy to implement, is environment-friendly, and can change waste into valuable. The compound is not enriched by adopting a macroporous resin method in the extraction and separation process, and the step of repeatedly activating and treating the macroporous resin is omitted; the combined normal definite alcohol solution is washed by 1 percent sodium hydroxide solution, so that a large amount of phenolic acid impurities and colored pigments can be removed; and finally, purifying by using a neutral alumina chromatographic column, effectively removing pigment impurities, directly obtaining a pure product without recrystallization, improving the yield of the pure product, and being worthy of popularization and application.
Drawings
FIG. 1 is a mass spectrum of Compound 1 of the present invention;
FIG. 2 is a graph showing an ultraviolet absorption spectrum of Compound 1 of the present invention;
FIG. 3 is an IR (KBr) spectrum of Compound 1 of the present invention;
FIG. 4 is H of Compound 1 of the present invention1-NMR spectrum.
Detailed Description
The following detailed description of embodiments of the invention will be made with reference to the accompanying drawings:
the weight of the components of each of the following examples is calculated on a dry basis.
Example 1 preparation of solid fermentate:
pulverizing radix astragali residue, sieving with 10 mesh sieve, adding water to make water content of radix astragali residue 52%, placing into 10 culture bottles, and sterilizing at 120 deg.C for 2 hr; cooling, inoculating 10% Ganoderma, adjusting temperature to 27 deg.C, and fermenting for 32 days to obtain solid fermented product; collecting solid fermented product, oven drying at 60 deg.C, measuring water content to 8%, and pulverizing.
Example 2 preparation of solid fermentate:
pulverizing radix astragali residue, sieving with 10 mesh sieve, adding water to make water content of radix astragali residue be 45%, placing into 10 culture bottles, and sterilizing at 121 deg.C for 1.5 hr; cooling, inoculating 5% Ganoderma, adjusting temperature to 25 deg.C, and fermenting for 45 days to obtain solid fermented product; collecting solid fermented product, oven drying at 50 deg.C, and pulverizing with water content of 10%.
Example 3 preparation of solid fermentate:
pulverizing radix astragali residue, sieving with 10 mesh sieve, adding water to make radix astragali residue water content be 60%, placing into 10 culture bottles, and sterilizing at 121 deg.C for 2 hr; cooling, inoculating 15% of Auricularia, adjusting temperature to 30 deg.C, and fermenting for 40 days to obtain solid fermented product; collecting solid fermented product, oven drying at 70 deg.C, and pulverizing to obtain the final product with water content of 6%.
The dried and pulverized solid fermented material obtained in example 1 was extracted and separated:
taking 1kg of solid fermentation product powder, adding 70% ethanol extract, reflux-extracting for 2 times, each time for 2 hr, mixing the 2 times of extracts, and recovering ethanol under reduced pressure to obtain soft extract. Dissolving the extract with appropriate amount of water, extracting the water solution with water saturated n-butanol for 3 times, mixing n-butanol solutions, washing with 1% NaOH solution for 2 times, washing with n-butanol saturated water for 1 time, and evaporating n-butanol solution to obtain extract. Mixing the residue with silica gel, loading onto silica gel column by dry method, eluting with chloroform-methanol mixture (V/V10: 2), collecting 8 ml of each fraction, mixing the same fractions, standing, volatilizing solvent, mixing with neutral alumina, loading onto alumina column by dry method, eluting with chloroform-methanol mixture (V/V10: 3), mixing the same fractions, standing, and volatilizing solvent to obtain pure compound 1 (100 mg), with yield of 40.1%.
The astragalus medicine dregs are residues after the astragalus medicine is extracted and are generally treated as waste, but the medicine dregs often contain active ingredients which are not completely extracted, the astragalus medicine dregs used as raw materials contain astragaloside IV components, and according to the results in a table 1, the compound 1 is obtained from the biotransformation of the astragaloside IV. Astragaloside IV is the primary glycoside of radix astragali and its residue, and Compound 1 is the secondary glycoside generated after hydrolysis of Astragaloside IV loses one molecule of sugar. That is, the astragalus residue produces enzyme which can hydrolyze astragaloside IV and obtain the compound 1 in the fermentation process with the ganoderma lucidum strain, so that the compound 1 can be produced by only the strain which generates the hydrolase with the astragalus medicinal material or the astragalus residue in the fermentation process.
TABLE 1 content of astragaloside IV and Compound 1 in each sample
TABLE 2 content of Compound 1 obtained by extraction of different kinds of samples
Astragaloside is an effective monomer component in astragalus or dregs thereof, and the compound 1 is derived from the conversion of astragaloside, so theoretically, the greater the specific gravity of the astragalus in the solid culture medium, the higher the content of the astragaloside, and the greater the content of the compound 1, which is also illustrated by the experimental data provided in table 4.
Physical and chemical characteristics and spectral data of the compound:
white powder, m.p.181.5-182.5 ℃, FABMS (fig. 1): m/z675[ M + Na ]]+，m/z653[M+H]+，m/z691[M+K]+Determining the molecular formula as C36H60O10。
Uv absorption spectrum shows (fig. 2): the maximum absorption wave λ max is 206 nm.
Ir (kbr) (fig. 3): vmax(cm-1): 3388.7(OH), 2968.6 (cyclopropylalkyl), 2933.5 (CH)2)，2872.4(CH3)，1454.1(CH2)，1378.1(CH3)。
H1-NMR(500MHz，CD3OD): see table 3.
Table 3: process for preparation of Compound 11H and13C NMR data
By combining the above spectral analyses, the structure of the compound was determined to be: 6-O-beta-D-glucosyl-3, 6,16, 25-tetrahydroxy cycloartane.
The present invention has been disclosed in terms of the preferred embodiment, but it is not intended to be limited to the embodiment, and all technical solutions obtained by substituting or converting the equivalent embodiments fall within the scope of the present invention.
Claims (4)
1. A preparation method of tetrahydroxy cycloartane triterpenoid saponin compounds with a structure shown as a formula (I) comprises the following steps:
(1) preparing a solid fermentation product by a biotransformation mode:
(a) pulverizing Chinese medicinal materials, sieving, adjusting to appropriate water content, and sterilizing to obtain solid culture medium containing Chinese medicinal materials;
(b) inoculating medicinal fungi 5-15% of the dry weight of the solid culture medium into the solid culture medium, and fermenting at 24-30 deg.C under 45-60% humidity for 30-45 days to obtain solid fermented product;
(c) collecting solid fermented product, drying at 50-70 deg.C, and pulverizing;
(2) extracting and separating compounds from the dried solid fermentation:
(a) taking a proper amount of dry solid fermentation product powder particles obtained in the step (1), carrying out reflux extraction by using ethanol, and recovering a solvent to obtain an extract;
(b) dissolving the extract with water, extracting with water saturated n-butanol, and evaporating to remove solvent to obtain extract;
(c) subjecting the extract to silica gel column chromatography, eluting with chloroform-methanol mixture, mixing the same fractions, and volatilizing solvent;
(d) adding neutral alumina, performing alumina column chromatography, eluting with chloroform-methanol mixed solution, and volatilizing solvent to obtain compound of formula (I);
wherein, the traditional Chinese medicine in the steps (1) and (a) is astragalus or astragalus residue;
in the steps (1) and (b), the medicinal fungus is selected from any one of ganoderma lucidum, hericium erinaceus, grifola frondosa, coriolus versicolor, sophora fungus and lentinus edodes.
2. The method according to claim 1, wherein in the steps (1) and (b), the medicinal fungus is preferably Ganoderma lucidum.
3. The production method according to claim 1, wherein, in the step (2),
the ethanol concentration in the step (a) is 70%, and the reflux extraction is carried out for 2 times, 2 hours each time;
in the step (b), after the water saturated n-butanol is extracted, washing the extract by using NaOH solution and the water saturated n-butanol;
in the steps (c) and (d), the chromatography method is dry chromatography, and the volume ratio of the two in the chloroform-methanol mixture is 10:0-10: 5.
4. The method of claim 3, wherein in step (2) (b), the concentration of the NaOH solution is 1%.
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