CN113425770A - Preparation method of biotransformation monkshood - Google Patents

Abstract

The invention discloses a preparation method of biotransformation monkshood, which comprises the following steps of (A) crushing: pulverizing radix Aconiti lateralis, and sieving; (II) preparing a culture medium: weighing quantitative crushed raw monkshood powder, putting into a culture container, adding water into the culture container, and stirring and mixing; (III) sterilizing; (IV) inoculation: taking out the culture container from the sterilization container, and inoculating solid slant fungus block prepared from edible and medicinal fungi Poria into the culture container; (V) culturing: performing static culture on the inoculated culture medium for 10-40 days in a culture device at the temperature of 25-35 ℃ to finish the culture; (VI) drying: drying and crushing to obtain the biotransformation aconite; the invention reduces the toxic and side effect of the medicine and enhances the curative effect of the medicine by introducing microorganisms for fermentation culture; meanwhile, no chemical reagent is added, green manufacturing is realized, the production cost of processing monkshood is reduced, and the method is convenient to popularize and apply.

Description

Preparation method of biotransformation monkshood

Technical Field

The invention relates to the field of traditional Chinese medicine, in particular to a preparation method of biotransformation monkshood.

Background

The Chinese medicine aconite root is derived from Aconitum carmichaeli Debx (Aconitum carmichaeli Debx) of Ranunculaceae, about 167 species in China, and the lateral root (son root) is called aconite root, is pungent and sweet in taste, strong in nature, hot and toxic, is praised as ' good general after going around the world, the first item for restoring yang and rescuing from collapse, the first essential medicine for tonifying true fire of Mingmen's life ', and has the functions of restoring yang, expelling cold and expelling wind-damp. It can be used for treating diseases such as yang exhaustion due to excessive sweat, cold pain of limbs, kidney yang asthenia, lumbago and knee pain, cold and loempe, listlessness, pain due to wind-cold-dampness, and tinea pedis. Later generations are averted by their "toxicity". Some say that it is the most useful and difficult to use. The monkshood has wide prospect in treating critical severe diseases in recent years as a key medicine for clinical restoration of yang and rescue of collapse, and has pharmacological effects of analgesia, inflammation diminishing, heart strengthening, cancer resistance and the like.

The main components of radix Aconiti lateralis include alkaloids including diester alkaloids (aconitine, hypaconitine, and mesaconine), monoester alkaloids (benzoylaconitine, benzoylhypaconitine, and benzoylmesaconine), ammonia alcohol alkaloids (aconitine, hypaconitine, and mesaconine), and other alkaloids (such as mesaconine and racemic norcoclaurine). The toxicity of the alkaloids is very different, wherein the toxicity of diester alkaloids is very high, and the mouse LD50 of aconitine, mesaconine and hypaconitine is 1.8mg/kg, 1.9mg/kg and 5.8mg/kg respectively. Hydrolysis of diester alkaloid can generate monoester alkaloid with low toxicity.

Because unprocessed radix aconiti lateralis has high toxicity, clinically applied radix aconiti lateralis is regarded as a processing method of radix aconiti lateralis. In order to achieve the purpose of reducing toxicity and keeping efficacy of monkshood, more than 70 monkshoods have been processed and processed in the Han Dynasty so far, such as charcoal frying method, honey roasting method, fire-processing method, paper-wrapped roasting method, ginger preparing method, salt preparing method, licorice decoction frying method and the like. The traditional processing principle of the monkshood is that during the processing process, ester bonds are hydrolyzed to lose acetyl firstly and generate benzyl substances, or the hydrolysis is continued and the toxicity is eliminated. Pharmacological research shows that diester aconitine has unstable chemical property, can be converted into monoester aconitine after hydrolysis, and has toxicity reduced to 1/200-1/500 of diester aconitine; then hydrolyzing into aminoalcohol alkali, and reducing toxicity to 1/2000-1/4000 of diester aconitine. However, the traditional processing method of the traditional Chinese medicine monkshood aims at reducing the toxicity of monkshood, and a large amount of active ingredients of monkshood can be lost in the processing process.

In order to reform the traditional monkshood processing technology, scientifically control the hydrolysis reaction degree of diester alkaloids such as aconitine and the like, avoid loss and waste of monkshood active ingredients, improve the utilization rate of traditional Chinese medicine resources, and prepare to inoculate strains to obtain the biotransformation monkshood. In the patent of processing monkshood by adopting a mixed liquid of various probiotics reported at present, additives such as chemical reagents (sodium hydroxide, calcium hydroxide and the like), alpha-amylase and the like need to be added in the process of preparing monkshood, particularly, the addition of acid-base chemical reagents is often unacceptable to consumers, and the processing production cost of monkshood is high and is limited by factors such as environmental evaluation and the like, so that the monkshood is difficult to popularize and apply.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a preparation method of biotransformation monkshood, which reduces the toxic and side effects of the medicine and enhances the curative effect of the medicine by introducing microorganisms for fermentation culture; meanwhile, no chemical reagent is added, green manufacturing is realized, the production cost of processing monkshood is reduced, and the method is convenient to popularize and apply.

In order to solve the technical problem, the invention adopts the following technical scheme:

a preparation method of biotransformation monkshood is characterized by comprising the following steps:

(I) crushing: pulverizing radix Aconiti lateralis, and sieving;

(II) preparing a culture medium: weighing quantitative crushed raw monkshood powder, putting into a culture container, adding water into the culture container, and stirring and mixing;

(III) sterilizing;

(IV) inoculation: taking out the culture container from the sterilization container, and inoculating solid slant fungus block prepared from edible and medicinal fungi Poria into the culture container;

(V) culturing: performing static culture on the inoculated culture medium for 10-40 days in a culture device at the temperature of 25-35 ℃ to finish the culture;

(VI) drying: drying and crushing to obtain the biotransformation aconite.

The step (III) is specifically as follows: the culture vessel is placed into a sterilization vessel and sterilized for 20-30min at the temperature of 115 ℃ and 121 ℃.

And (IV) the size of the solid slant bacterium block in the step (IV) is 0.5 multiplied by 1cm-2 multiplied by 2 cm.

The culture device in the step (five) is an incubator.

The drying temperature in the step (VI) is 50-80 ℃.

Preferably, the step (one) is to crush the raw monkshood and pass through a 10-mesh sieve but not a 20-mesh sieve;

weighing quantitative crushed raw monkshood powder, subpackaging the raw monkshood powder in a plurality of test tubes, adding water accounting for 15-30% of the volume of each test tube, and stirring and mixing.

Preferably, the step (one) is to crush the raw monkshood and pass through a 10-mesh sieve but not a 30-mesh sieve;

weighing quantitative crushed raw monkshood powder, subpackaging the raw monkshood powder in a plurality of triangular flasks, adding water accounting for 15-30% of the volume of the triangular flasks into each triangular flask, and stirring and mixing.

Preferably, the step (one) is to crush the raw monkshood and pass through a 20-mesh sieve, but not pass through a 30-mesh sieve;

weighing quantitative crushed raw monkshood powder, placing the raw monkshood powder in a bioreactor, adding water accounting for 15-30% of the volume of the bioreactor into the bioreactor, and stirring and mixing.

The invention has the positive effects that:

the preparation method provided by the invention has a great positive effect on the processing process of monkshood by introducing microorganisms for fermentation culture, and specifically comprises the following steps:

firstly, the microbial flora has the advantages of high propagation speed, strong substrate utilization specificity, mild growth conditions and the like;

secondly, the traditional Chinese medicine is processed through microbial transformation, and compared with the conventional physical or chemical processing means, the traditional Chinese medicine processing method can greatly enhance or adjust the medicine property, thereby more effectively improving the curative effect of the traditional Chinese medicine, reducing the toxic and side effects and expanding the indications of the traditional Chinese medicine.

The method does not add any chemical reagent, completes the absorption, utilization and conversion of the effective components of the monkshood along with the growth of microbial flora in the fermentation process, realizes the green manufacture of monkshood processing, has simple operation steps and low energy consumption, reduces the production cost of monkshood processing, and is convenient for popularization and application.

Drawings

FIG. 1 is a liquid chromatogram showing changes in the contents of active ingredients before and after fermentation in Experimental example 1 of the present invention.

Detailed Description

For a better understanding and appreciation of the invention, the invention will be further illustrated with reference to the accompanying drawings and specific examples.

Example 1

A method for preparing biotransformation monkshood by test tube solid fermentation is characterized by comprising the following steps:

(I) crushing: pulverizing radix Aconiti lateralis, and sieving with 10 mesh sieve but 20 mesh sieve;

(II) preparing a culture medium: weighing 4g of the raw monkshood powder, subpackaging the raw monkshood powder into 10 clean test tubes with the specification of 18 multiplied by 180, adding 1.8mL of drinking water into each test tube, stirring and mixing, and maintaining a natural pH value;

(III) sterilization: placing the 10 test tubes into a sterilization container, and sterilizing at 121 deg.C for 30 min;

(IV) inoculation: after cooling, taking out 10 test tubes from the sterilization container, and respectively inoculating 1 solid slant fungus block prepared from Poria for both medicine and food into 10 test tubes, wherein the size of the solid slant fungus block is 2 × 2 cm;

(V) culturing: standing the inoculated 10 test tubes in an incubator at the temperature of 30 ℃ for 21 days until the culture is finished;

(VI) drying: and taking the 10 test tubes out of the incubator, drying at the low temperature of 70 ℃, and crushing to obtain the biotransformation monkshood.

Example 2

A method for preparing biotransformation monkshood by triangular flask solid fermentation is characterized by comprising the following steps:

(I) crushing: pulverizing radix Aconiti lateralis, sieving with 10 mesh sieve, and sieving with 30 mesh sieve;

(II) preparing a culture medium: weighing 20g of the raw monkshood powder, subpackaging the raw monkshood powder into 20 triangular flasks with the volume of 100mL, adding 22mL of distilled water into each triangular flask, stirring and mixing, and maintaining a natural pH value;

(III) sterilization: placing the 20 triangular bottles into a sterilization container, and sterilizing at 121 deg.C for 30 min;

(IV) inoculation: after cooling, taking out 20 triangular flasks from the sterilization container, and respectively adding 5 solid slant fungus blocks prepared from Poria for both medicine and food into 20 triangular flasks, wherein the size of the solid slant fungus block is 2 × 2 cm;

(V) culturing: standing the inoculated 20 triangular flasks in an incubator at 27 ℃ for 12 days until the culture is finished;

(VI) drying: and taking the 20 triangular flasks out of the incubator, drying at the low temperature of 60 ℃, and crushing to obtain the biotransformation monkshood.

Example 3

A method for preparing biotransformation monkshood by solid fermentation of a bioreactor is characterized by comprising the following steps:

(I) crushing: pulverizing radix Aconiti lateralis, and sieving with 20 mesh sieve but 30 mesh sieve;

(II) preparing a culture medium: weighing 400g of the raw monkshood powder, subpackaging the raw monkshood powder in a bioreactor with the volume of 1L, adding 240mL of distilled water into the bioreactor, stirring and mixing, and maintaining the natural pH value;

(III) sterilization: placing the bioreactor into a sterilization container, and sterilizing at 121 deg.C for 30 min;

(IV) inoculation: after cooling, taking the bioreactor out of the sterilization container, and inoculating 20 solid slant fungus blocks prepared from edible and medicinal fungi Poria into the bioreactor, wherein the size of the solid slant fungus blocks is 2 × 2 cm;

(V) culturing: standing the inoculated bioreactor in an incubator at 32 ℃ for 21 days until the culture is finished;

(VI) drying: and taking the bioreactor out of the incubator, drying at the low temperature of 60 ℃, and crushing to obtain the biotransformation aconite.

Experimental example 1

Purpose of the experiment: the contents of diester-type alkaloids and monoester-type alkaloids in the biotransformed monkshood prepared in examples 1 to 3 were determined by the following methods, respectively, comparing the changes of synergy and attenuation after processing monkshood.

Step one, configuration of single reference substance

Accurately weighing aconitine 10mg, hypaconitine 10mg, mesaconine 10mg, benzoylaconine 10mg, benzoylhypaconine 10mg, and benzoylmesaconine 10 mg. Adding isopropanol: the dichloromethane (1:1) mixed solution is respectively added to a constant volume of 50mL to prepare a single reference solution, and the single reference solution is stored in a refrigerator at 4 ℃ for later use.

Step two, preparation of mixed reference substance

2mL of each of the six single control solutions was taken and placed in a 25mL volumetric flask and the volume was determined with acetonitrile. Making into mixed standard solution, and storing in refrigerator at 4 deg.C.

Step three, preparation of test solution

2g of unprocessed radix aconiti lateralis powder (20 meshes and 40 meshes are not needed) before fermentation is precisely weighed, placed in a conical flask with a plug, added with 3mL of ammonia test solution for infiltration, and kept stand for 30 min. Adding isopropanol: and (2) carrying out volume fixing on the mixed solution of ethyl acetate (1:1) to 50mL, carrying out ultrasonic treatment (power of 300w, frequency of 60khz and water temperature of 25 ℃), treating for 30min, standing for 24h, filtering, carrying out volume fixing again, shaking up, filtering, precisely weighing 25mL of filtrate, carrying out reduced pressure recovery at 40 ℃ until the filtrate is dry, transferring the residue acetonitrile into a 2mL volumetric flask to carry out volume fixing, and filtering with a microporous filter membrane (0.45μm) to obtain a crude monkshood sample to be detected.

Step four, chromatographic conditions of HPLC

On the basis of preliminary experiments and related documents, the content determination method of 3 kinds of aconitine and 3 kinds of aconine is determined as follows: akzo Nobel Kromasil C₁₈Chromatography column (4.6 mm. times.250 mm, 5 μm); mobile phase: a is slowFlushing liquid (0.2% glacial acetic acid, pH value of which is adjusted to 6.20 by triethylamine), wherein B is acetonitrile; gradient elution, elution program 0-44min, A-B (79:21) → (65: 35); 44-65min, A-B (65:35) → (60: 40); 65-85min, A-B (20: 80); flow rate: 1.0 mL/min; sample introduction amount: 20 mu l of the mixture; detection wavelength: 240 nm; column temperature: at 30 ℃.

The liquid chromatogram of the content change of effective components before and after fermentation is shown in figure 1, and the liquid chromatogram in figure 1 sequentially comprises the following components from top to bottom: a liquid chromatogram of the mixed standard substance, a liquid chromatogram of the fermented mixture of the fungus and the medicine of tuckahoe in example 1, and a liquid chromatogram of alkaloid in raw aconite.

The contents of diester-type alkaloids and monoester-type alkaloids in the biotransformed aconite prepared in examples 1 to 3 are shown in table 1.

TABLE 1

As can be seen from fig. 1 and table 1: toxic components (diester alkaloid) in the poria cocos fermented biotransformed monkshood are obviously reduced, effective components (monoester alkaloid) are increased, and the conversion from the toxic components (diester alkaloid) to the effective components (monoester alkaloid) can be effectively realized. In examples 1 to 3, the conversion rates of the active ingredients (monoester alkaloids) were 90.90%, 90.66%, and 90.22%, respectively, and the conversion rates were 90% or more in different levels, so that the industrial production with high conversion rates in large scale was realized, and the method was easy to popularize and apply widely.

Experimental example 2

Purpose of the experiment: acute toxicity experiments of mice are carried out, and whether the biotransformation aconite has attenuation effect or not is comparatively observed.

Materials for experiments:

(1) unprocessed radix aconiti lateralis: provided by Chann Hospital, Laishan Yangtai, produced in Hanzhong City of Shaanxi province.

(2) Preparing monkshood by using medicine: provided by Yantai Laishan Chann Hospital, produced by pharmaceutical Limited company of pharmaceutical Potential in Anguo city, and having the batch number: 1904005, execution criteria: the first part of the year 2015 in the Chinese pharmacopoeia.

(3) Biotransformation of monkshood: the biotransformed monkshood prepared in example 1.

The experimental steps are as follows:

40 mice were selected, half male and half female, and randomly divided into 4 groups according to sex and body weight: the traditional Chinese medicine comprises a normal group, a raw monkshood group, a prepared monkshood and a biotransformation monkshood group, wherein each group is administered with 50g raw monkshood/kg. The observation was performed once a day for 14 consecutive days after the administration. The mice were recorded for gross color, mental, mobility, eating, drinking, general symptoms of intoxication and death. Dead mice were dissected immediately. After the experiment, the mouse was dissected and the gross pathological changes of the important organs such as heart, liver, spleen, lung and kidney were observed. The effect of each sample on mouse body weight is shown in table 2.

TABLE 2

*: p is less than 0.05 compared with the normal control group.

The experimental results are as follows:

during the experiment, the hair color, stool and the like are normal within 14 days after the mice in the unprocessed radix aconiti lateralis group are used, the mice (6/10) have different degrees of symptoms such as prostrate immobility and the like, 2 mice die, and the weight of the mice is remarkably reduced compared with that of a control group (P is less than 0.05).

During the experiment, the hair color, stool and the like are normal within 14 days after the mice in the monkshood group are processed with the traditional Chinese medicine, the symptoms such as the prostrate immobility and the like of individual mice (2/10) are generated, the mice are not dead, but the weight is obviously reduced compared with the control group (P is less than 0.05).

During the experiment, the hair color, the stool and the like are normal within 14 days after the aconite group mice are biologically converted, no obvious toxic symptom appears, and the body weight is not abnormal compared with the control group.

No abnormality was found in each group of mice at necropsy.

As can be seen from Table 2: the biotransformation of radix Aconiti lateralis has an obvious attenuation effect compared with unprocessed radix Aconiti lateralis and processed radix Aconiti lateralis.

Claims (8)

1. A preparation method of biotransformation monkshood is characterized by comprising the following steps:

(I) crushing: pulverizing radix Aconiti lateralis, and sieving;

(II) preparing a culture medium: weighing quantitative crushed raw monkshood powder, putting into a culture container, adding water into the culture container, and stirring and mixing;

(III) sterilizing;

(IV) inoculation: taking out the culture container from the sterilization container, and inoculating solid slant fungus block prepared from edible and medicinal fungi Poria into the culture container;

(V) culturing: performing static culture on the inoculated culture medium for 10-40 days in a culture device at the temperature of 25-35 ℃ to finish the culture;

(VI) drying: drying and crushing to obtain the biotransformation aconite.

2. The method for preparing transformed aconite according to claim 1, wherein the step (three) is specifically: the culture vessel is placed into a sterilization vessel and sterilized for 20-30min at the temperature of 115 ℃ and 121 ℃.

3. The method for preparing transformed aconite according to claim 1, wherein: and (IV) the size of the solid slant bacterium block in the step (IV) is 0.5 multiplied by 1cm-2 multiplied by 2 cm.

4. The method for preparing transformed aconite according to claim 1, wherein: the culture device in the step (five) is an incubator.

5. The method for preparing transformed aconite according to claim 1, wherein: the drying temperature in the step (VI) is 50-80 ℃.

6. The method for preparing transformed aconite according to any one of claims 1 to 5, wherein:

the step (one) is specifically that the raw monkshood is crushed and sieved by a sieve with 10 meshes but not 20 meshes;

weighing quantitative crushed raw monkshood powder, subpackaging the raw monkshood powder in a plurality of test tubes, adding water accounting for 15-30% of the volume of each test tube, and stirring and mixing.

7. The method for preparing transformed aconite according to any one of claims 1 to 5, wherein:

the step (one) is specifically that the raw monkshood is crushed and sieved by a sieve with 10 meshes, but not 30 meshes;

weighing quantitative crushed raw monkshood powder, subpackaging the raw monkshood powder in a plurality of triangular flasks, adding water accounting for 15-30% of the volume of the triangular flasks into each triangular flask, and stirring and mixing.

8. The method for preparing transformed aconite according to any one of claims 1 to 5, wherein:

the step (one) is specifically that the raw monkshood is crushed and sieved by a 20-mesh sieve, but not a 30-mesh sieve;

weighing quantitative crushed raw monkshood powder, placing the raw monkshood powder in a bioreactor, adding water accounting for 15-30% of the volume of the bioreactor into the bioreactor, and stirring and mixing.

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