CN110742929A - Method for preparing coprophila transplantation capsule containing benzoylaconitine by fermenting monkshood with coprophila - Google Patents

Abstract

The application relates to a method for preparing a coprophila transplanting capsule containing benzoylaconitine by fermenting monkshood with human coprophila. "Fecal transplantation" (FMT) refers to the transplantation of functional flora in the feces of healthy people into the gastrointestinal tract of patients to reconstruct new intestinal flora and realize the treatment of intestinal and parenteral diseases. The clinical findings show that some patients can not achieve the required curative effect after taking the monkshood, and one of the main reasons of individual difference is that the intestinal flora of the patients is deficient, so that toxic substances in the monkshood can not be metabolized normally into nontoxic active ingredients, and the utilization rate of the active ingredients is low, so that the required curative effect of the monkshood can not be exerted. The invention utilizes healthy high-quality excrement of a coprophilous bacterium transplant donor to simulate the conversion action of intestinal microorganisms on monkshood in vitro, converts toxic components such as aconitine in the monkshood into nontoxic metabolites under proper fermentation conditions, and increases the content of active components such as benzoylaconitine and the like. The fecal strain fermentation liquor is prepared into fecal strain capsules for clinical fecal strain transplantation treatment.

Description

Method for preparing coprophila transplantation capsule containing benzoylaconitine by fermenting monkshood with coprophila

Technical Field

The invention belongs to the field of human coproal transplantation, fermentation and traditional Chinese medicine, and relates to a method for preparing coproal transplantation capsules containing phenylformyl aconitine by utilizing human coproal to ferment monkshood.

Background

"Fecal transplantation" (FMT) refers to the transplantation of functional flora in the feces of healthy people into the gastrointestinal tract of patients to reconstruct new intestinal flora and realize the treatment of intestinal and parenteral diseases. Fecal transplantation has been used for the treatment and exploratory studies of various flora-related diseases such as clostridium difficile infection, inflammatory bowel disease, intractable constipation, metabolic disease, intestinal immunodeficiency, intestinal allergy and the like as an effective means for reconstructing intestinal flora, and is considered as a breakthrough medical progress in recent years.

The fecal bacteria feeding way is divided into an upper digestive tract, a middle digestive tract and a lower digestive tract. The upper digestive tract route mainly refers to oral fecal bacteria capsule; the middle digestive tract route comprises passing through a nasointestinal tube, a gastroscope forceps channel hole and a percutaneous endoscopic gastrostomy jejunum tube; the lower gastrointestinal route includes colonoscopy, enema, colostomy, and trans-endoscopic intestinal catheterization (TET), among others. The physician should select the most appropriate input route for the patient according to his condition and the characteristics of the various routes.

The state of the coprophilous fungi transplantation can be divided into three forms of freshness, freezing storage and fermentation. Since the safety of fecal bacteria transplantation is the primary consideration in clinical decision making, in order to isolate a high quality fecal flora without pathogens, the fecal donors are highly demanding, at least within 3-6 months no diarrhea occurs, no antibiotics have been used, and there must be no infectious diseases and gastrointestinal diseases, in addition, there are limitations including age, physiology, pathology, psychology, integrity, time, environment, etc.

The aconite or aconite, which is not only a traditional Chinese medicine material in the Chuan birth canal, but also an important traditional Chinese medicine material in China, has the effect of reviving yang and rescuing from collapse, is highly regarded by the traditional Chinese medicine, but the toxicity is also a clinical concern. The main control indexes in the Chinese pharmacopoeia are limit inspection of diester alkaloids (total amount of neoaconitine, hypaconitine and aconitine) and monoester alkaloids (total amount of benzoylneoaconitine, benzoylaconitine and benzoylhypaconitine), the diester alkaloids are toxic components, and the monoester alkaloids are effective components. The microbial fermentation processing can not only reduce toxic components in the traditional Chinese medicine, but also improve the effective components of the traditional Chinese medicine. In vivo metabolism of aconitine has been reported, and the main metabolites in human serum and urine are benzoylaconitine (benzoylaconine) and aconitine (Aconine). The literature reports that the aconitine and the mesaconitine are greatly reduced after the monkshood is fermented by intestinal microorganisms such as escherichia coli and staphylococcus aureus, and the purpose of attenuation is achieved (Sun Peng, Li Ling, Wuli, Yongxin, attenuation and synergy investigation of monkshood based on biotransformation [ J ]. J.Chinese journal of experimental prescriptions, 2012,18(22): 16-19.). Radix Aconiti lateralis has effects of restoring yang, relieving collapse, invigorating fire, tonifying yang, dispelling cold, and relieving pain. Modern pharmacological studies prove that monkshood has various biological activities of strengthening heart, diminishing inflammation, relieving pain and the like.

However, it is found that some patients may not achieve the due curative effect after taking monkshood, and may have poisoning, one of the main reasons for the individual difference is that the intestinal flora of the patients is deficient, so that monkshood cannot be metabolized into the toxic monoester alkaloids such as benzoylaconine, and the due curative effect of monkshood cannot be achieved.

Disclosure of Invention

The invention aims to provide a method for preparing a coprophilous fungi transplanting capsule by fermenting monkshood with human coprophilous fungi.

The purpose of the invention is realized by the following technical scheme: a method for preparing a fecal bacteria transplanting capsule by fermenting aconite with fecal bacteria transplanting donor feces comprises the following steps:

(1) placing a donor fresh excrement sample in a stirrer, introducing nitrogen, maintaining a low-oxygen environment (the volume ratio of oxygen content is not more than 3%), adding 0.9% sterile NaCl solution, homogenizing at 2000r/min for 3min, and obtaining excrement slurry;

(2) filtering the excrement slurry by a stainless steel filter screen with the thickness of 5.0mm to remove large-particle substances in the excrement slurry, and obtaining excrement slurry after primary filtration;

(3) adding the excrement slurry into the preliminarily filtered excrement slurry ice, transferring the excrement slurry ice to a laboratory, and treating the excrement slurry ice within 2 hours after collection; pouring the fecal slurry and the monkshood powder into a fermentation tank, and carrying out anaerobic fermentation at a feed-liquid ratio of 1:20, a fermentation temperature of 37 ℃, a stirring speed of 200r/min and an initial pH value of 6.8 for 12 hours to obtain fecal bacteria fermented slurry;

(5) filtering the fermented liquid by stainless steel filter screens with diameters of 2.0mm, 1.0mm, 0.5mm and 0.25mm step by step to remove food residues and small particulate matters to obtain coarse fermented liquid;

(6) centrifuging the crude fecal bacteria solution at 6000r/min for 15min, and suspending the fecal bacteria precipitate in 0.9% NaCl solution 100 times of the fecal bacteria weight to obtain colorless and odorless fecal bacteria fermentation liquid containing monoester alkaloids such as benzoylaconine.

(7) Taking the supernatant to carry out HPLC and ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to determine the content of aconitine and benzoylaconine, and determining method reference documents (Sun Peng, Li Ling, Wuli, Yongxin. attenuated, synergistic and investigation of monkshood based on biotransformation [ J ]. J. China journal of experimental prescriptions, 2012,18(22):16-19.) (Xunxinghui, single wenjing, Wujian. ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to simultaneously detect 8 main components [ J ]. Chinese medicine guide, 2017,14(03): 147-;

(8) and (4) filling the fecal strain fermentation liquor in the step (6) into a colon soluble capsule shell to prepare a fecal strain transplanting capsule, and freezing and storing.

Wherein the content of the first and second substances,

the weight ratio of the donor fresh excrement to the NaCl solution in the step (1) is as follows: m is_{Donor fresh feces}∶m_{NaCl solution}＝1∶5。

The monkshood powder in the step (3) is obtained by crushing traditional Chinese medicine monkshood, and the operation steps are as follows: pulverizing radix Aconiti lateralis (commercially available product, purchased from pharmacia of Beijing Hoorentang) with a Chinese medicinal pulverizer, oven drying at 60 deg.C to constant weight, sieving with 40 mesh sieve, sealing, and storing in sealed bag;

step (7) the HPLC assay was carried out using the following instruments: waters 1525 high performance liquid chromatograph (Shimadzu instruments, Japan), Agilent 6460 Mass Spectroscopy Detector.

Chromatographic conditions are as follows: the chromatographic column is Relia-sil C18(250 mm. times.4.6 mm, 5 μm); the detection wavelength is 240nm, and the flow rate is 1.0 mL/min^-1Column temperature 30 deg.C, mobile phase acetonitrile 0.4 mol.L^-1And (3) carrying out gradient elution (0-13 min, 15% A, 13-14 min, 24% A, 17-68 min, 40% A) on an ammonium acetate solution (the pH value of the ammonia test solution is adjusted to 8.0), wherein the sample injection amount is 20 mu L.

The technical principle of the invention is as follows:

the toxic substances in the aconite cannot be metabolized into nontoxic active ingredients normally in the body of some patients with intestinal dysbacteriosis, and the utilization rate of the active ingredients is low. The invention utilizes healthy high-quality excrement of a coprophilous bacterium transplant donor to simulate the conversion action of intestinal microorganisms on monkshood in vitro, converts toxic components such as aconitine in the monkshood into nontoxic metabolites under proper fermentation conditions, and increases the content of active components such as benzoylaconitine and the like. The fecal strain fermentation liquor is prepared into fecal strain capsules for clinical fecal strain transplantation treatment.

Compared with the prior art, the invention has the following advantages and effects:

the high-quality fecal flora from a healthy donor mainly comprises beneficial bacteria including bifidobacterium, lactobacillus, clostridium, AKK bacteria and the like, and a small amount of neutral bacteria and harmful bacteria, so that the high-quality fecal flora does not have great influence on the health of a human body under general conditions. The in vitro fermentation process simulates the metabolism of intestinal tracts in vivo, protects the active ingredients of the traditional Chinese medicine from being damaged, generates new drug effect, ferments the traditional Chinese medicine with the fecal flora of healthy people, can release the active ingredients of the traditional Chinese medicine to the maximum extent, and simultaneously carries out metabolic conversion on toxic substances. At present, reports of adding traditional Chinese medicines into the coprophilous fungi transplanting capsule are not seen temporarily, and the coprophilous fungi transplanting capsule with various biological activities of strengthening heart, diminishing inflammation, relieving pain and the like can be obtained by simulating the degradation and transformation effects of human intestinal tracts on monkshood in vitro.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

It should be understood that the processing equipment or devices not specifically mentioned in the following examples are conventional in the art; all pressure values and ranges refer to absolute pressures.

Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

Wherein the content of the first and second substances,

the concentration of aconitine and benzoylaconine in fecal bacteria liquid is tested by HPLC and ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (Sun Peng, Li Ling, Wuli, Yongxin. biologically transformed attenuated and synergistic investigation of monkshood [ J ]. J. China journal of Experimental formulae, 2012,18(22):16-19.) (Xuxin, monarch, Wujia. ultra-high performance liquid chromatography-triple quadrupole mass spectrometry simultaneously detects 8 main components [ J ] in aconite decoction, 2017,14(03):147 one 151.)

Examples

(1) Pulverizing radix Aconiti lateralis (commercially available product, purchased in pharmacy of Beijing Tongrentang) with a Chinese medicinal pulverizer, oven drying at 60 deg.C to constant weight, sieving with 40 mesh sieve, sealing, and storing in sealed bag;

(2) placing 100 +/-1 g of donor fresh excrement in a stirrer, adding 500mL of sterile 0.9% NaCl solution, introducing nitrogen, maintaining a low-oxygen environment (the volume ratio of oxygen content is not more than 3%), and homogenizing at 2000r/min for 3min to obtain excrement slurry;

(3) filtering the excrement slurry by a stainless steel filter screen with the thickness of 5.0mm to remove large-particle substances in the excrement slurry, and obtaining excrement slurry after primary filtration;

(3) adding the excrement slurry into the preliminarily filtered excrement slurry ice, transferring the excrement slurry ice to a laboratory, and treating the excrement slurry ice within 2 hours after collection; pouring 400mL of excrement slurry and 20g of monkshood powder into a fermentation tank, and carrying out anaerobic fermentation at the stirring speed of 200r/min and the initial pH value of 6.8 in a feed-liquid ratio of 1:20 at the fermentation temperature of 37 ℃ for 12 hours to obtain excrement fermented slurry;

(5) filtering the fecal bacteria fermentation slurry by stainless steel filter screens with the diameters of 2.0mm, 1.0mm, 0.5mm and 0.25mm step by step to remove food residues and small particle substances, and obtaining 300mL of crude fecal bacteria fermentation liquor;

(6) centrifuging the crude fecal strain liquid at 6000r/min for 15min, and suspending fecal strain precipitate in 400mL of 0.9% NaCl solution to obtain colorless and odorless fecal strain fermentation liquid.

(7) And (3) taking the supernatant, and determining the content of aconitine and benzoylaconine by adopting HPLC (high performance liquid chromatography) -triple quadrupole mass spectrometry. The aconitine standard (purchased from China pharmaceutical and biological products institute) is precisely weighed to obtain the standard yeast, and the reduction amount of aconitine and the content of benzoylaconine are measured to be 57 percent and are 0.023 mug/mL.

(8) And (4) filling the fecal strain fermentation liquor in the step (6) into a colon soluble capsule shell to prepare a fecal strain transplanting capsule, and freezing and storing.

It can be seen that monkshood, after fermentation of donor feces, contains high content of benzoylaconitine. The prepared capsule is convenient for fecal bacteria transplantation, is more easily accepted by patients, and has a plurality of biological activities of strengthening heart, diminishing inflammation, relieving pain and the like.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (6)

1. A method for preparing coprophila transplantation capsule containing benzoylaconitine by fermenting monkshood with human coprophila is characterized by comprising the following steps: fermenting radix Aconiti lateralis with fecal bacteria of healthy donor to reduce toxicity of radix Aconiti lateralis, and preparing fecal bacteria transplant capsule containing benzoylaconitine.

2. The method of claim 1, wherein the method comprises fermenting radix Aconiti lateralis Preparata with feces of a fecal bacteria transplant donor to obtain a fecal bacteria transplant capsule, wherein the method comprises: placing a donor fresh excrement sample in a stirrer, introducing nitrogen, maintaining a low-oxygen environment (the volume ratio of oxygen content is not more than 3%), adding 0.9% sterile NaCl solution, homogenizing at 2000r/min for 3min to obtain excrement slurry, and performing subsequent treatment to obtain the excrement liquid containing benzoylaconitine.

3. The method of claim 1, wherein the method comprises fermenting radix Aconiti lateralis Preparata with feces of a fecal bacteria transplant donor to obtain a fecal bacteria transplant capsule, wherein the method comprises: filtering the excrement slurry by a stainless steel filter screen of 5.0mm to remove large-particle substances in the excrement slurry to obtain excrement slurry after primary filtration; transporting the primarily filtered excrement slurry ice to a laboratory and processing the excrement slurry ice within 2 hours after collection; pouring the fecal slurry and the monkshood powder into a fermentation tank, and carrying out anaerobic fermentation at a feed-liquid ratio of 1:20, a fermentation temperature of 37 ℃, a stirring speed of 200r/min and an initial pH value of 6.8 for 12 hours to obtain fecal bacteria fermented slurry; filtering the fermented liquid by stainless steel filter screens with diameters of 2.0mm, 1.0mm, 0.5mm and 0.25mm step by step to remove food residues and small particulate matters to obtain coarse fermented liquid; centrifuging the crude fecal bacteria liquid at 6000r/min for 15min, and suspending the fecal bacteria precipitate in 0.9% NaCl solution 100 times of the fecal bacteria weight to obtain colorless and odorless fecal bacteria fermentation liquid containing effective components of Chinese medicinal materials; and filling the fecal strain fermentation liquor into a colon soluble capsule shell to prepare a fecal strain transplanting capsule, and freezing and storing.

4. The method of claim 1, wherein the method comprises fermenting radix Aconiti lateralis Preparata with feces of a fecal bacteria transplant donor to obtain a fecal bacteria transplant capsule, wherein the method comprises: the weight ratio of the donor fresh excrement to the NaCl solution is as follows: m is_{Donor fresh feces}∶m_{NaCl solution}＝1∶5。

5. The monkshood powder according to claim 3 is obtained by crushing a traditional Chinese medicine monkshood, and the operation steps are as follows: pulverizing radix Aconiti lateralis (commercially available product, purchased from pharmacia of Beijing Hoorentang) with a Chinese medicinal pulverizer, oven drying at 60 deg.C to constant weight, sieving with 40 mesh sieve, sealing, and storing.

6. The HPLC assay according to claim 3, wherein the following apparatus is used: waters 1525 high performance liquid chromatograph (Shimadzu instruments, Japan), Agilent 6460 Mass Spectroscopy Detector.

Chromatographic conditions are as follows: the chromatographic column is Relia-sil C18(250 mm. times.4.6 mm, 5 μm); the detection wavelength is 240nm, and the flow rate is 1.0 mL/min^-1Column temperature 30 deg.C, mobile phase acetonitrile 0.4 mol.L^-1And (3) carrying out gradient elution (0-13 min, 15% A, 13-14 min, 24% A, 17-68 min, 40% A) on an ammonium acetate solution (the pH value of the ammonia test solution is adjusted to 8.0), wherein the sample injection amount is 20 mu L.