CN102899378A - Method for biotransformation of Panax notoginseng (Burk.) F. H. Chen medicinal material by using Monascus purpureus - Google Patents
Method for biotransformation of Panax notoginseng (Burk.) F. H. Chen medicinal material by using Monascus purpureus Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 17
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Abstract
The invention discloses a method for biotransformation of Panax notoginseng (Burk.) F. H. Chen medicinal materials by using Monascus purpureus. The method mainly comprises the following operating steps of: 1) preparation of Monascus purpureus strains; 2) preparation of Panax notoginseng (Burk.) F. H. Chen culture medium; and 3) fermentation and culture to obtain fermentation products containing active components, product post-treatment and detection of major efficacy components. By using the method for fermenting the Panax notoginseng (Burk.) F. H. Chen by using the Monascus purpureus provided by the invention and by directly using Panax notoginseng (Burk.) F. H. Chen raw medicinal materials as fermentation substrate, through one-step fermentation process, medicinal components, i.e. Panax notoginseng (Burk.) F. H. Chen and Monascus purpureus are simultaneously obtained, the culture is enabled to have dual effects of the Panax notoginseng (Burk.) F. H. Chen and the Monascus purpureus, the products contain lovastatin which is a lipid-decreasing active component according to experiments, the type and the content of saponin are obviously changed, and rare saponin with higher pharmacological activity is produced.
Description
Technical field
The present invention relates to a kind of monascus carries out bio-transformation to pseudo-ginseng method, can obtain to contain the product of activeconstituents by the method.
Background technology
Pseudo-ginseng [
Panax notoginseng(Burk.) F. H. Chen] be the araliaceae ginseng plant, main product is in Yunnan, main active ingredient be Radix Notoginseng total arasaponins (
Panax notoginsengSaponins, PNS).So far, from pseudo-ginseng, isolated more than 70 and planted ginsenoside, all belonged to dammarane type four-ring triterpenoid saponin.Can be subdivided into two classes again by its sapogenin type: 20 (S)-protopanoxadiol types [20 (S)-protopanaxadiol] and 20 (S)-Protopanaxatriol types [20 (S)-protopanaxatriol].Wherein protopanoxadiol type saponin(e comprises Rb1, Rb2, Rc, Rd, Rh2, Rg3, Rb3, C-K, PPD; Protopanaxatriol's type saponin(e comprises Rg1, Rg2, Re, R1, Rh1, F1, PPT.That content is higher in the natural saponin(e is Rg1, Rb1, Rd, R1, Rc, Re; Content rare saponin(e very little or that do not contain is Rd, Rg2, Rg3, Rh1, Rh2, F1, F2, PPD, PPT, C-K.Rare saponin(e shows preferably pharmacologically active at treatment cardiovascular and cerebrovascular diseases, anti-tumor aspect, and is unique to effects such as cardiovascular and cerebrovascular, neural system, immunity systems such as the Ginsenoside Rd, might become the Novel clinical treatment medicine for the treatment of cerebral apoplexy.Ginsenoside Rd's content in plant is few, and chemosynthesis is not yet successful so far, only can extract from the root of former plant, stem, leaf at present, and efficient is low, and cost is high.
Radix Notoginseng total arasaponins absorbs relatively poor in gi tract, slight hydrolysis reaction only occurs in saponin(e under the gastric juice effect, under the effect of meta-bolites enzyme or bacterium in intestinal juice, glycosidic link progressively ruptures, be degraded into rare saponin(e and just really bring into play pharmacological action, this has not only increased the stomach and intestine burden, and has reduced bioavailability.Rare saponin(e in pseudo-ginseng, do not contain or content very little, can transform be obtained by Radix Notoginseng total arasaponins by biological process external, and because its efficient is high, pollution-free, and have preferably application prospect.
Monascus (
Monascus purpureus) be a kind of medicinal fungi, contain the compound that lovastatin, Stigmasterol etc. have Lipid-lowering activities in the tunning of monascus.At present, the medicine take the Fermentation Condition of Monascus spp thing as main component is remarkable because of effect, and safety has no side effect, but the characteristics of long-term taking have occupied the maximum capture of Reducing Blood Lipid by Chinese medicine medicine.
The main effect of pseudo-ginseng is prevention and treatment cerebral apoplexy, and the monascus culture is mainly used in prevention and treatment hyperlipidemia and hypercholesterolemia, and they the two all be the specifics of control cardiovascular and cerebrovascular diseases, but differ from one another aspect indication.In order to reach the purpose of comprehensive control cardiovascular and cerebrovascular diseases, usually take simultaneously pseudo-ginseng and red colouring agent for food, also used as a Chinese medicine, but stomach and intestine are not high to absorption, conversion and the utilization ratio of effective constituent.The present invention is take pseudo-ginseng as the fermentation culture based component, directly use monascus (
Monascus purpureus) pseudo-ginseng is carried out bio-transformation, obtain simultaneously medicinal rare ginsenoside, lovastatin isoreactivity composition.Produced in the crude drug in the fermenting process and do not detected, but the active substance that can directly absorb and utilize, improved the bioavailability of medicine, fermenting culture has pseudo-ginseng and red colouring agent for food, also used as a Chinese medicine drug effect concurrently, might become the candidate new medicine for the control cardiovascular and cerebrovascular diseases.Above summary of the invention has no domestic and international correlative study report.
Summary of the invention
The invention provides a kind of monascus pseudo-ginseng carried out the method that bio-transformation obtains activeconstituents, the method utilize the medicinal fungi monascus (
Monascus purpureus) the fermentation pseudo-ginseng, obtain simultaneously fungi and pseudo-ginseng active pharmaceutical ingredients.
The operation steps of the method is as follows:
(1) bacterial classification preparation: with monascus (
Monascus purpureus) be inoculated into the PPDA slant medium, in 30 ℃ of constant temperature culture, to growing bacterium colony;
(2) be the aqueous suspensions that 80-100 purpose Radix Notoginseng powder adds the ratio preparation Radix Notoginseng powder of 6-8 mL water in 1 g granularity, 121 ℃ of sterilization 30 min namely get the pseudo-ginseng substratum, and are for subsequent use;
(3) in the pseudo-ginseng substratum of the bacterial classification access step (2) for preparing in the step (1) being prepared, under 15-35 ℃, 120 r/min shaking tables are cultivated 2-7 d, namely obtain containing the tunning of activeconstituents, and this product can be used for preparing the medicine of preventing and treating cardiovascular and cerebrovascular diseases.
Liquid fermentate aftertreatment among the present invention: with cultured liquid fermentate suction filtration in the step (3), clean filter residue 2 times with pure water, merging filtrate, centrifugal 30 min under 4000 r/min, get supernatant liquor and add equal-volume water-saturated n-butanol ultrasonic extraction 1 h, behind the standing demix, collect 60-70 ℃ of upper strata propyl carbinol phase and revolve steaming to dry, namely get the solid that contains the saponin(e activeconstituents; The filter residue mycelium is dried to constant weight at 60-70 ℃, and the ratio of adding 10-15 mL methyl alcohol in every gram mycelium adds methyl alcohol, and then supersound extraction 30 min use 0.24 μ m membrane filtration, namely get the filtrate that contains lovastatin.
Key point of the present invention is: use medicinal fungi that vegetable drug is carried out bio-transformation, obtain simultaneously red colouring agent for food, also used as a Chinese medicine and pseudo-ginseng effective component by the one time fermentation process, and the medicinal saponin constituent of pseudo-ginseng is compared with crude drug, considerable change has appearred in the content of main saponin(e, produced simultaneously and wanted not detect in the medicinal material, but had more the rare saponin constituent of pharmacologically active.Owing to biotransformation, improved the bioavailability of medicinal material, reduced gastrointestinal reaction and the side effect of medicine.The present invention shows, tunning has red colouring agent for food, also used as a Chinese medicine and the pseudo-ginseng medicinal ingredients of comprehensive control cardiovascular and cerebrovascular diseases.By the controlled fermentation condition, can reconstruct medicinal ingredients kind and content, and then realize directed change and adjust the human intervention of drug effect.
Embodiment
By the following examples the present invention is described in further detail, but protection domain of the present invention is not limited to described content.
Embodiment 1: monascus carries out the method for bio-transformation to pseudo-ginseng, concrete operations are as follows:
(1) bacterial classification preparation: with monascus (
Monascus purpureus) be inoculated into the PPDA slant medium, in 30 ℃ of constant temperature culture 3 d, to growing bacterium colony; Monascus specie is from Chinese common micro-organisms culture presevation administrative center's acquisition (CGMCC 3.991);
(2) pseudo-ginseng substratum: accurately take by weighing Radix Notoginseng powder that 3 g cross 80 mesh sieves in 100 mL triangular flasks, add 20 mL water, natural pH, 121 ℃ of sterilization 30 min namely get the pseudo-ginseng substratum, and are for subsequent use;
(3) liquid fermentation and culture: the bacterial classification for preparing in the step (1) is accessed in the pseudo-ginseng substratum of step (2) preparation, and respectively at 15 and 35 ℃, 120 r/min cultivate 4 d, namely obtain containing the tunning of activeconstituents;
(4) liquid fermentate aftertreatment: with cultured liquid fermentate suction filtration in the step (3), clean filter residue 2 times with 10 mL pure water, merging filtrate, centrifugal (4000 r/min) 30 min get supernatant liquor and add equal-volume water-saturated n-butanol ultrasonic extraction 1 h, behind the standing demix, collect the upper strata propyl carbinol and revolve mutually steaming (60 ℃) to dry, namely get the product that contains the saponin(e activeconstituents, with 5 mL70% dissolve with methanol residues, adopt the saponin constituent in the HPLC detection culture; The filter residue mycelium is dried to constant weight at 60 ℃, and the ratio of adding 12 mL methyl alcohol in every gram mycelium adds 25 mL methyl alcohol, and then supersound extraction 30 min use 0.24 μ m membrane filtration, detects lovastatin content in the filtrate with HPLC.See Table 1.
Table 1: different culture temperature are on the impact of tunning effective constituent kind and content
Above experimental result shows: the various saponin constituents of the product that the Fermentation Condition of Monascus spp pseudo-ginseng obtains in containing crude drug, also having synthesized in the crude drug does not have, but can the rare saponin(e medicinal ingredients with utilizing directly absorbed by the body.Simultaneously, contain the lovastatin of reducing blood-fat, decreasing cholesterol effect in the culture, demonstrated fully the medicinal efficacy of red colouring agent for food, also used as a Chinese medicine.Therefore, tunning is except the drug effect that has pseudo-ginseng and red colouring agent for food, also used as a Chinese medicine concurrently, also because of the synthetic bioavailability of having improved medicinal ingredients of rare saponin(e.
Embodiment 2: monascus carries out the method for bio-transformation to pseudo-ginseng, concrete operations are as follows:
(1) bacterial classification preparation: with monascus (
Monascus purpureus) be inoculated into the PPDA slant medium, in 30 ℃ of constant temperature culture, to growing bacterium colony; Monascus specie is from Chinese common micro-organisms culture presevation administrative center's acquisition (CGMCC), preserving number: 3.991;
(2) pseudo-ginseng substratum: accurately take by weighing Radix Notoginseng powder that 3 g cross 90 mesh sieves in 100 mL triangular flasks, add 24 mL water, natural pH, 121 ℃ of sterilization 30 min;
(3) liquid fermentation and culture: the bacterial classification for preparing in the step (1) is accessed in the pseudo-ginseng substratum of step (2) preparation, and in 30 ℃, 120 r/min cultivate 2 d and 7 d, namely obtain containing the tunning of activeconstituents;
(4) liquid fermentate aftertreatment: with cultured liquid fermentate suction filtration in the step (3), clean filter residue 2 times with 10 mL pure water, merging filtrate, centrifugal (4000 r/min) 30 min get supernatant liquor and add equal-volume water-saturated n-butanol ultrasonic extraction 1 h, behind the standing demix, collect the upper strata propyl carbinol and revolve mutually steaming (70 ℃) to dry, namely get the product that contains the saponin(e activeconstituents, with 5 mL70% dissolve with methanol residues, adopt the saponin constituent in the HPLC detection culture; The filter residue mycelium is dried to constant weight at 70 ℃, and the ratio of adding 10 mL methyl alcohol in every gram mycelium adds 22 mL methyl alcohol, and then supersound extraction 30 min use 0.24 μ m membrane filtration, detects lovastatin content in the filtrate with HPLC.See Table 2.
Table 2: different incubation times are on the impact of tunning effective constituent kind and content
Above experimental result shows: the various saponin constituents of the product that the Fermentation Condition of Monascus spp pseudo-ginseng obtains in containing crude drug, also having synthesized can the rare saponin(e medicinal ingredients with utilizing directly absorbed by the body.The lovastatin that contains reducing blood-fat, decreasing cholesterol effect in the culture has demonstrated fully the medicinal efficacy of red colouring agent for food, also used as a Chinese medicine.Therefore, tunning is except the drug effect that has pseudo-ginseng and red colouring agent for food, also used as a Chinese medicine concurrently, also because of the synthetic bioavailability of having improved medicinal ingredients of rare saponin(e.The change of experiment condition can affect the content of each effective constituent,
Embodiment 3: monascus carries out the method for bio-transformation to pseudo-ginseng, concrete operations are as follows:
(1) bacterial classification preparation: with monascus (
Monascus purpureus) be inoculated into the PPDA slant medium, in 30 ℃ of constant temperature culture, to growing bacterium colony; Monascus specie is from Chinese common micro-organisms culture presevation administrative center's acquisition (CGMCC), preserving number: 3.991;
(2) pseudo-ginseng substratum: accurately take by weighing Radix Notoginseng powder that 3 g cross 100 mesh sieves in 100 mL triangular flasks, add 18 mL water, natural pH, 121 ℃ of sterilization 30 min;
(3) liquid fermentation and culture: the bacterial classification for preparing in the step (1) is accessed in the pseudo-ginseng substratum of step (2) preparation, and in 25 ℃, 120 r/min cultivate 5d;
(4) liquid fermentate aftertreatment: with cultured liquid fermentate suction filtration in the step (3), clean filter residue 2 times with 10 mL pure water, merging filtrate, centrifugal (4000 r/min) 30 min get supernatant liquor and add equal-volume water-saturated n-butanol ultrasonic extraction 1 h, behind the standing demix, collect the upper strata propyl carbinol and revolve mutually steaming (65 ℃) to dry, namely get the product that contains the saponin(e activeconstituents, with 5 mL70% dissolve with methanol residues, adopt the saponin constituent in the HPLC detection culture; The filter residue mycelium is dried to constant weight at 65 ℃, and the ratio of adding 15 mL methyl alcohol in every gram mycelium adds 28 mL methyl alcohol, and then supersound extraction 30 min use 0.24 μ m membrane filtration, detects lovastatin content in the filtrate with HPLC.See Table 3.
Table 3: tunning effective constituent kind and content
Above experimental result shows: the various saponin constituents of the product that the Fermentation Condition of Monascus spp pseudo-ginseng obtains in containing crude drug, also having synthesized in the crude drug does not have, but can the rare saponin(e medicinal ingredients with utilizing directly absorbed by the body.Simultaneously, contain the lovastatin of reducing blood-fat, decreasing cholesterol effect in the culture, demonstrated fully the medicinal efficacy of red colouring agent for food, also used as a Chinese medicine.Therefore, tunning is except the drug effect that has pseudo-ginseng and red colouring agent for food, also used as a Chinese medicine concurrently, also because of the synthetic bioavailability of having improved medicinal ingredients of rare saponin(e.By the controlled fermentation condition, can the Effective Regulation culture system in composition and the content of medicinal composition, and then realize artificial design and intervention to the culture drug effect.
Claims (2)
1. monascus carries out bio-transformation to pseudo-ginseng a method is characterized in that the method comprises the steps:
(1) will produce bacterial strain is that monascus is inoculated in the PPDA slant medium, in 30 ℃ of constant temperature culture, to growing bacterium colony;
(2) be the aqueous suspensions that 80-100 purpose Radix Notoginseng powder adds the ratio preparation Radix Notoginseng powder of 6-8 mL water in 1 g granularity, 121 ℃ of sterilization 30 min namely get the pseudo-ginseng substratum, and are for subsequent use;
(3) in the pseudo-ginseng substratum of the bacterial classification access step (2) for preparing in the step (1) being prepared, under 15-35 ℃, 120 r/min shaking tables are cultivated 2-7 d, namely obtain containing the tunning of activeconstituents.
2. the described monascus method of pseudo-ginseng being carried out bio-transformation according to claim 1, it is characterized in that: the tunning that contains activeconstituents is carried out suction filtration, filter residue cleans 2 times with pure water, merging filtrate, centrifugal 30 min of filtrate 4000 r/min, supernatant liquor add equal-volume water-saturated n-butanol ultrasonic extraction 1 h, behind the standing demix, collect 60-70 ℃ of upper strata propyl carbinol phase and revolve steaming to dry, namely get the product that contains the saponin(e activeconstituents; The filter residue mycelium is dried to constant weight at 60-70 ℃, and the ratio of adding 10-15 mL methyl alcohol in every gram mycelium adds methyl alcohol, and then supersound extraction 30 min use 0.24 μ m membrane filtration, namely get the filtrate that contains lovastatin.
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