CN104138417A - Tinospora crispa medicine composition with effect of blood sugar reduction and preparation method thereof - Google Patents
Tinospora crispa medicine composition with effect of blood sugar reduction and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a tinospora crispa medicine composition and a preparation method thereof. The tinospora crispa medicine composition is prepared from the following raw materials in percentage by mass: 5-30% of lucid ganoderma and 70-95% of tinospora crispa. The preparation method comprises the following steps: inoculating a lucid ganoderma strain with a liquid seed culture medium for culture under an aseptic condition so as to obtain liquid lucid ganoderma seeds, meanwhile drying and crushing the stem of fresh tinospora crispa, putting the stem into a plurality of fermentation bottles for sterilization under high pressure so as to obtain a medical tinospora crispa matrix, inoculating the medical tinospora crispa matrix with the liquid lucid ganoderma seeds in bottles, putting the bottles in a constant temperature fermentation chamber for culture, continuously culturing after the hyphae grow all over the bottles, thereby finally obtaining the tinospora crispa medicine composition. The tinospora crispa medicine composition has the beneficial effect that the toxicity of tinospora crispa is still well controlled under the condition that the contents of total diterpenoid componds Rumphioside Ac-D and Borapetoside B with the blood sugar reduction effect are very high.
Description
Technical field
The invention belongs to bitter rattan medicine manufacture method technical field, relate to one and there is blood sugar lowering and imitate bitter rattan pharmaceutical composition (Rumphioside Ac-D) and Borapetoside B) and preparation method thereof.
Background technology
Wrinkle Tinospora crispa (bitter rattan) Learn name: Tinospora crispa (L.) Miers section belongs to: Menispermaceae Menispermaceae. black ox Bile Genus Tinospora. Do name: tumor Stems rattan, Green bag rattan, Green rattan, little Lai rattan, the cold rattan of Hair, Caulis Tinosporae crispae, golden Chicken Satisfied rattan, overnightly look for ma, Stems tumor rattan.Yuan Productivity ground: the ground such as India, Sri Lanka, Burma, East south Asia.
It has significance effect to type 2 diabetes mellitus discovered in recent years, but also shows liver toxic and side effects to a certain degree simultaneously.The toxicity that how to reduce bitter rattan has become the focus that we pay close attention to.Commonly use at present and remove root bark part, the control decocting time that toxicity is the strongest; The methods such as the different compatibilities of Chinese medicine or compound recipe associating coupling reduce the toxicity of bitter rattan.By activated monomer conversion technology, bitter rattan is detoxified and also obtained certain progress.Although these methods can reach the effect of removing toxic substances, can not fundamentally solve the toxicity problem of bitter rattan.
So far, Chinese scholars has been isolated the multiple compounds such as diterpene, triterpene, alkaloid and glycoside from bitter rattan, and in bitter rattan, main hypoglycemic activity composition is diterpene-kind compound and alkaloid compound.The toxicity of bitter rattan derives from its complicated chemical composition and physiologically active ingredient, and toxicity also mainly comes from diterpene-kind compound, is secondly alkaloid, mainly damages liver.Do not have at present a kind of effective bitter rattan process for preparing medicine can in the situation that controlling bitter rattan toxicity, ensure well effective ingredient (Rumphioside Ac-D and the Borapetoside B) total diterpene of maximum.
Summary of the invention
Object of the present invention is providing a kind of bitter rattan pharmaceutical composition (Rumphioside Ac-D) and Borapetoside B), solve the existing bitter rattan pharmaceutical composition content that contains total diterpene (Rumphioside Ac-D and Borapetoside B) as much as possible in the situation that controlling bitter rattan toxicity well.
Another object of the present invention is to provide the manufacture method of bitter rattan pharmaceutical composition.
The technical solution adopted in the present invention is Ganoderma lucidum (Leyss. Ex Fr.) Karst. fermentation manufacturing technique, comprises the condition of culture medium and cultivation.Formed by following raw material by mass percentage: Ganoderma content 5-30%, bitter rattan content 70-95%.
Further, Ganoderma content 30%, bitter rattan content 70%.
Further, described bitter rattan pharmaceutical composition or its extract are effective medicinal ingredient (Rumphioside Ac-D-) and Borapetoside B, separately or with pharmacy in the common composition of acceptable auxiliary element there is the medicine of blood sugar lowering and effect of weight reducing.
Further, described bitter rattan pharmaceutical composition or its extract are active component, separately or with food in the common composition of other composition of acceptable there is the functional food of blood sugar lowering and effect of weight reducing.
A kind of preparation method of bitter rattan pharmaceutical composition, Ganderma lucidum strain being accessed under aseptic condition to liquid seed culture medium cultivates, obtain ganoderma lucidum liquid seed, fresh bitter rattan portion is dried and pulverized simultaneously, and be loaded on the sterilizing of multiple fermentation flask mesohigh, obtain bitter rattan medicinal mycoplasma, ganoderma lucidum liquid seed is inoculated in the bitter rattan medicinal mycoplasma in bottle, is placed on ferment at constant temperature chamber and cultivates, make mycelia cover with full bottle, covering with continuation cultivation after mycelia, obtain final bitter rattan pharmaceutical composition.
Further, described mycelial growth can be divided into four-stage: (1) laundering period (0-10d): mycelial growth is slower, and mycelia is more slim and frahile; (2) animated period (ll-35d): mycelia vigor is stronger, and metabolism is vigorous, growth is fast; (3) decrement phase (35-50d): mycelial growth rate sharply declines; (4) phase of decline (50-70d): the decline of mycelia vigor, mycelia aging, starts to secrete yellowish-brown pigment.
The invention has the beneficial effects as follows the toxicity of well having controlled bitter rattan in the situation that total diterpene content is high.
Brief description of the drawings
Fig. 1 is Tinospora crispa n-butanol portion liquid chromatogram;
Fig. 2 is the total ion current figure of Tinospora crispa n-butyl alcohol portion;
Fig. 3 is the mass spectrum of Rumphioside Ac-D;
Fig. 4 is the chemical structural formula of Rumphioside Ac-D;
Fig. 5 is the mass spectrum of Borapetoside B;
Fig. 6 is the chemical constitution of Borapetoside B;
Fig. 7 be the impact of Tinospora crispa fermentation part ethanol extraction on normal mouse (A) and type 2 diabetes mellitus mice (B) oral glucose tolerance (
n=12) cartogram.
Detailed description of the invention
1 materials and methods
1.1 material
Ganoderma Ganoderma lucidum strain, bitter rattan Tinospora crispa (L.) Miers raw medicinal herbs, is provided by the century-old ecological park GAP of Malaysia planting base.
1.2 method
Preparation method is that the bitter rattan of the plant taking water content as gross weight 30%～70% is as culture medium, the original seed of Ganoderma is seeded in this culture medium, under the condition of suitable Ganoderma growth after solid fermentation, collect and comprise Ganoderma mycelium and medium component at interior whole mycoplasma mixture.
1.2.1 strain: the activation of slant strains: cut 0.5cm from the female inclined-plane of planting of Ganoderma under aseptic condition
2truffle, be transferred on PDA slant medium, be placed in 28 ± 1 DEG C, under relative humidity 60% condition, cultivate 7d, select mycelia stalwartness, pure white, pollution-free, the strain that covers with test tube slant, 4 DEG C of Refrigerator stores are for subsequent use.
The preparation of liquid spawn: under aseptic condition, the slant strains of activation is accessed to liquid seed culture medium (glucose 20g/L, yeast powder 5g/L, KH2PO41.5g/L, MgSO40.75g/L, CaCO32.5g/L, VB10.1g/L), under 28 DEG C, 120r/min dark condition, cultivate 7d, select liquid clarification, evenly person is for subsequent use as the liquid spawn of two-way solid fermentation for fungus ball size.Scale 7.5g coarse rice powder+150ml water (two heavy Complex) is placed in 250ml shaking flask and stirs, and carries out 121 DEG C, and sterilizing in 20 minutes after sterilization is placed in shaking flask aseptic operating platform and is cooled to after room temperature for subsequent use.Every bottle graft enters the preactivated lucidum bacteria liquid of 10ml, at 30 DEG C, 150rpm Zhen Swing incubator is cultivated two days access liquid seed culture mediums (glucose 20g/L, yeast powder 5g/L, KH2PO41.5g/L, MgSO40.75g/L, CaCO32.5g/L, VB10.1g/L), under 28 DEG C, 120r/min dark condition, cultivate 7d, select liquid clarification, evenly person is for subsequent use as the liquid spawn of two-way solid fermentation for fungus ball size.
1.2.2 medicinal mycoplasma: by the fresh bitter rattan gathering, the stem of getting its belt leather, is placed in 60 DEG C of electric drying oven with forced convections and dries, and pulverizes with pulverizer, sieves, and mixes, and is sub-packed in fermentation flask, and 121 DEG C of autoclaving 120min are for subsequent use.
1.2.3 two-way fermentation: ganoderma lucidum liquid seed quantitative is inoculated in to bitter rattan medicinal mycoplasma (substrate that contains bitter rattan composition in bottle), cultivate in ferment at constant temperature chamber, condition of culture is temperature 25-28 DEG C, dark condition, the growing state of tight observation every day Ganoderma, record half bottle of phase, full bottle phase, vigorous degree etc.
1.2.4 mycoplasma post processing: by Ganoderma mycoplasma, mycoplasma wherein refers to that Ganoderma is grown to serve as new medicinal fungi (abbreviation mycoplasma) in bitter rattan medicinal mycoplasma, by the sampling of different fermentations natural law G0, G10, G15, G20, G25, G30, G40, G50, G60, G70, G80, G90, (G0 mycelia is covered with full bottle in the situation that temperature is suitable, within general 48 hours, just can see mycelium germination.About 35 days, can cover with full bottle).G10 represents that mycelia covers with 10d after full bottle, the like).After going out sample, each mycoplasma group fully mixes, weigh mycoplasma weight in wet base, 60 DEG C of oven dry in electric drying oven with forced convection (dry to water content be 5% ± 1%), weigh mycoplasma dry weight, calculate the conversion ratio of Ganoderma to bitter rattan medicinal mycoplasma, consumption rate, computing formula is: conversion ratio=mycoplasma dry weight/substrate gross dry weight × 100% consumption rate=1-conversion ratio, this test is by the HPLC Monitoring on Dynamic Change (seeing Fig. 1) of mycoplasma total diterpene compounds content in sweat, provide theoretical foundation in conjunction with the acute toxicity test of mycoplasma for the attenuation holding effect research of the bitter rattan of Ganoderma solid fermentation.Two-way fermentation is (to be mostly aspergillosis in air according to Chinese medicine by some fungus, the miscellaneous bacterias such as penicillium sp) pollute to go mouldy and cause the principle of herbal nature pharmacodynamic change after (fermentation), by modern science and technology, medicinal fungi fermented bacterium being formed to fermentation with the vegetable drug with certain active component as medicinal mycoplasma combines, ferment under given conditions, substrate can be changed tissue by the enzyme of fungus again when conk desired nutritional is provided, composition, thereby produce the medicinal fungal substance (abbreviation mycoplasma) of new nature,taste and action, this medicinal fungal substance is made new medical material.It is mainly used in potentiation, expands the aspects such as use, removing toxic substances.
The present invention has studied the biotechnology processing of bitter rattan, is adopted as " three layers of optimal seeking method of fermentation combination " that medicinal fungi novel (two-way) solid fermentation engineering is set up, and filtering out effective fermented bacterium is Ganoderma.Use " sweat dynamic comparison " further to study Ganoderma and be inoculated in the variation of total diterpene content (seeing Fig. 2), acute toxicity and the immunologic function of bitter rattan medicinal mycoplasma top fermentation different time gained mycoplasma herein, set up the fermentation technology of the two-way solid fermentation of bitter rattan removing toxic substances holding effect, for further carrying out bitter rattan biotechnology removing toxic substances holding effect, improve function of reducing blood sugar research and lay the foundation.
1.2.5 the mensuration of different fermentations time mycoplasma total diterpene: the Ganoderma mycoplasma powder 0.5g that takes bitter rattan crude drug, sterilizing crude drug and different fermentations time, 95% ethanol ultrasonic extraction 3 times, each ethanol consumption is 40mL, time is 20min, merge 3 times extracting solution, the extractum of reclaim under reduced pressure 95% ethanol, after adding distil water 50mL dissolves, be extracted with ethyl acetate 3 times, each ethyl acetate is 30mL, combined ethyl acetate extract reclaim under reduced pressure to odorlessness obtains the thick total diterpene of bitter rattan, and the bitter thick total diterpene of rattan of dissolve with methanol is also settled to 50mL.Accurately draw sample preparation liquid 7mL and be placed in tool plug test tube, add respectively 2.5%3,5-dinitrobenzoic acid 1.5mL and 10%KOH1.5mL, shake up, leave standstill 10min, to add sample preparation liquid as contrast, measure the content of total diterpene at wavelength 510nm.
1.2.6 the acute toxicity test of different fermentations time mycoplasma: the acute toxicity of measuring G0, G10, G15, G20, G25, G30, G40, G50, G60, G70, G80 mycoplasma according to conventional methods.Each medicine group is respectively got 60 (body weight 18-22g of healthy Kunming mouse, purchased from Jiangsu University's experimental animal center, ♀ ♂ half and half, sub-cage rearing, be divided at random 6 groups, 10 every group, wherein 5 administration groups, 1 blank group (normal saline), gavage (i.g.) administration.0% and the 100% dead dosage range obtaining according to trial test, by 0.4mL/10g administration volume, ratio between 1:0.75 or 1:0.8 agent, the Ganoderma mycoplasma extractum of i.g. corresponding dosage, 1d administration 2 times, is total to administration 0.8mL/10g in 1d.Fasting 16h before administration, freely drinks water.After first administration, fasting 4h tight ordinary circumstance and death toll of observing mice, observe 1 every day, and Continuous Observation 7d, with median lethal dose(LD 50) software for calculation (Bliss method) calculating mice LD50.
1.2.7 the immunologic function of different fermentations time mycoplasma test: get 54 of Kunming mouses, male and female half and half, are divided into 9 groups at random: distilled water group, Dexamethasone group, G30 group, G35 group, G40 group, G50 group, G60 group, G70 group, G80 group, 6 every group.Press 0.2mL/10g/d administration according to Mouse Weight, the concentration of each mycoplasma group is its LD50 1/6, and the concentration of dexamethasone is 0.0075g/kg, and distilled water group is by body weight i.g. equivalent distilled water, i.g.7d continuously.The 7th day de-neck of mice administration put to death, and under aseptic condition, wins spleen, is prepared into the splenocyte suspension of 2 × 106/mL concentration.On 96 orifice plates, every hole adds 100 μ L cell suspension, adds respectively ConA (10 μ g/mL) and the each 100 μ L of LPS (20 μ g/mL).Each sample is established 3 repeating holes.Put 37 DEG C, 5%CO2 and cultivate after 48h, every hole adds 10 μ L5mg/mLMTT, hatches 4h for 37 DEG C, 1,000r/min, and centrifugal 10min, abandons supernatant, more every hole adds 150 μ L dimethyl sulfoxide (DMSO), and vibration 10min, dissolves purple crystal completely.Measure OD value with 492nm wavelength by automatic microplate reader.
2 results and analysis
The growing state Ganoderma of 2.1 Ganodermas on bitter rattan medicinal mycoplasma well-grown in bitter rattan medicinal mycoplasma, mycelial growth is vigorous, and mycelium is dense to be white in color, and cannot separate with bitter rattan medicinal mycoplasma.It is 13.6d that mycelium covers with average time of half bottle, and be 17.68d the average time of covering with full bottle.Be designated as the 0th day of fermentation to cover with the time of full bottle, by consumption rate and the conversion ratio result of different fermentations time Ganoderma to bitter rattan medicinal mycoplasma, known, glossy ganoderma fermentation extends with fermentation time the conversion of bitter rattan substrate, and conversion ratio reduces gradually; And extend with fermentation time, it is large that the consumption rate of bitter rattan medicinal mycoplasma becomes gradually.
2.2 different fermentations time mycoplasma total diterpene content
Adopting ultraviolet spectrometry degree meter method to measure bitter rattan mycoplasma shows in medicinal fungi fermentation different time total diterpene changes of contents (Fig. 2) result, bitter rattan crude drug after glossy ganoderma fermentation in mycoplasma the changes of contents of total diterpene be evident regularity, Ganoderma mycelium covers with bottle and rises in the time of 30d after extremely full bottle, with the prolongation of fermentation time, total diterpene content declines, in the time of fermentation 30d, total diterpene content is minimum, and content rises in the time of fermentation 35d, content is 0.65%, content is more steady afterwards, but occurs again during to 70d declining.Total diterpene changes of contents in mycoplasma presents first and reduces afterwards and raise during the fermentation, the rule that then content keeps relative stability, and this may be because in the early stage of fermentation, Ganoderma mycelium metabolic activity is vigorous, and the diterpene-kind compound in bitter rattan is decomposed; And along with the prolongation of fermentation time, the secondary metabolite that Ganoderma itself can produce may with bitter rattan in some composition reaction generated diterpene-kind compound; In the fermentation later stage, Ganoderma mycelium vital movement weakens, and metabolism is slow, and the diterpene content in mycoplasma has kept relatively stable.In whole fermentation period, the total diterpene content in the 30th day mycoplasma reaches minimum.
2.3 different fermentations times mycoplasma acute toxicity tests application Ganoderma carries out the mycoplasma the acute toxicity tests of gained after two-way fermentation to bitter rattan and shows, the toxicity of bitter rattan has obtained great reduction.
After glossy ganoderma fermentation, the LD50 of gained mycoplasma is all higher than bitter rattan crude drug and sterilizing medical material, and at first 30 days of fermentation, along with the prolongation of fermentation time, LD50 increased gradually, within the 30th day, reaches maximum, is 28.46g/kg; After the 30th day, along with the prolongation of time, the LD50 of mycoplasma reduces again gradually, ferments the 80th day time, and the LD50 of mycoplasma is down to 17.81g/kg.Result of the test shows that amphicheirality ferments to reach and reduces the object of bitter rattan toxicity, and the acute toxicity of each mycoplasma group reduces compared with crude drug group, and the toxicity of G20 group, G30 group is lower, and especially minimum with G30 group toxicity, after fermentation, the 30th day gained mycoplasma toxicity is minimum.
The carbohydrate tolerance impact of 2.4T.crispa on normal mouse and diabetic mice.
In order to measure the impact of T.crispa fermented product extract (T.crispa fermentation extract-is called for short TFE) on carbohydrate tolerance, this test is taking normal ICR mice and diabetic mice as experimental subject.In normal group (as shown in Figure 7), normal ICR mice is respectively 6.68 ± 0.64mmol/L and 7.91 ± 0.36mmol/L at the blood glucose giving after TFE and 5% sodium carboxymethyl cellulose (CMC) solution; After gavage glucose solution 60min, the mouse blood sugar value that gives 5% carboxymethylcellulose sodium solution reaches 20.31 ± 1.20mmol/L, and the blood glucose value that gives the mice of TFE is 16.53 ± 0.65mmol/L, this shows, compared with giving 5% carboxymethylcellulose sodium solution mouse blood sugar, TFE has significantly reduced the blood sugar level of normal mouse; Giving after glucose solution 120min and 150min, the blood sugar level of TFE mice is still low than giving 5% carboxymethylcellulose sodium solution mouse blood sugar, and has significant difference (P<0.05).In type 2 diabetes mellitus mice, mice is respectively 13.00 ± 0.91mmol/L and 15.39 ± 1.04mmol/L (as shown in Fig. 4 .2.B) at the blood glucose giving after TFE and 5% carboxymethylcellulose sodium solution, the mouse blood sugar value that gives 5% carboxymethylcellulose sodium solution after gavage glucose 60min reaches 22.59 ± 0.92mmol/L, and the blood glucose value that gives the mice of TFE is 18.74 ± 0.75mmol/L, result shows, compared with giving the type 2 diabetes mellitus mice of 5% carboxymethylcellulose sodium solution, oral administration gavage 2g/kg D/W 60, 120, after 150min, TFE has significantly reduced plasma glucose levels.Oral glucose tolerance experimental result shows that TFE has significantly improved the utilization rate of type 2 diabetes mellitus mice glucose, improves its Glucose Tolerance.In addition compared with giving the AUC of 5%CMC, give TFE and significantly reduced the AUC of normal group and model group, be respectively: 17.93% and 18.69% (Fig. 7).
2.5 test method
2.5.1 ferment preparation and the qualification of afterproduct need testing solution;
After getting the appropriate dissolve with methanol of Tinospora crispa fermentation afterproduct n-butanol portion dried powder, use 0.45um microporous filter membrane to filter, for subsequent use.
2.5.2HPLC analyze the Establishment and optimization of T.crispa n-butanol portion condition;
To Tinospora crispa n-butanol portion RP-HPLC analytical method, mobile phase condition is: acetonitrile: 0.02% trifluoroacetic acid aqueous solution gradient elution (0-5min, 2%:98%～6%:94%; 5-20min, 6%:94%～15%:85%; 20-25min, 15%:85%; 25-30min, 16%:84%; 30-35min, 17%:83%; 35-40min, 17%:83%～20%:80%; 43-53min, 20%:80%～38%:62%; 53-60min, 38%:62%).Analyzing and compare its main chemical compositions of document by LC-MS, is 2 compounds, is respectively: Rumphioside Ac-D and Borapetoside B.(seeing Fig. 4,6)
2.5.3 LC-MS analysis;
After setting up T.crispa fermentation afterproduct n-butanol portion PR-HPLC analytical method, adopt LC-MS to analyze its chemical composition, and infer compound chemical structure formula according to mass spectrum molecular ion peak and fragment peak.Mass spectrum parameter: ESI ion source, ion source temperature 350oC, capillary voltage 4.5kv, cation detects pattern, nitrogen flow rate 30L/min.Sweep limits: m/z100～1000.(seeing Fig. 3,5).
Claims (6)
1. a bitter rattan pharmaceutical composition, is characterized in that being made up of following raw material by mass percentage: Ganoderma content 5-30%, bitter rattan content 70-95%.
2. according to a kind of bitter rattan pharmaceutical composition described in claim 1, it is characterized in that Ganoderma content 30%, bitter rattan content 70%.
3. according to a kind of bitter rattan pharmaceutical composition described in claim 1, it is characterized in that: described bitter rattan pharmaceutical composition or its extract are effective medicinal ingredient Rumphioside Ac-D and Borapetoside B, separately or with pharmacy in the common composition of acceptable auxiliary element there is the medicine of blood sugar lowering and effect of weight reducing.
4. according to a kind of bitter rattan pharmaceutical composition described in claim 1, it is characterized in that: described bitter rattan pharmaceutical composition or its extract are active component, separately or with food in the common composition of other composition of acceptable there is the functional food of blood sugar lowering and effect of weight reducing.
5. according to the preparation method of a kind of bitter rattan pharmaceutical composition described in claim 1, it is characterized in that: Ganderma lucidum strain is accessed under aseptic condition to liquid seed culture medium and cultivate, obtain ganoderma lucidum liquid seed, fresh bitter rattan portion is dried and pulverized simultaneously, and be loaded on the sterilizing of multiple fermentation flask mesohigh, obtain bitter rattan medicinal mycoplasma, ganoderma lucidum liquid seed is inoculated in the bitter rattan medicinal mycoplasma in bottle, being placed on ferment at constant temperature chamber cultivates, make mycelia cover with full bottle, covering with continuation cultivation after mycelia, obtain final bitter rattan pharmaceutical composition.
6. according to the preparation method of a kind of bitter rattan pharmaceutical composition claimed in claim 5, it is characterized in that: described mycelial growth can be divided into four-stage: (1) laundering period (0-10d): mycelial growth is slower, and mycelia is more slim and frahile; (2) animated period (ll-35d): mycelia vigor is stronger, and metabolism is vigorous, growth is fast; (3) decrement phase (35-50d): mycelial growth rate sharply declines; (4) phase of decline (50-70d): the decline of mycelia vigor, mycelia aging, starts to secrete yellowish-brown pigment.
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Application publication date: 20141112 |