CN110564641B

CN110564641B - Complex microbial inoculum fermentation product and application thereof as pholcodine synergist

Info

Publication number: CN110564641B
Application number: CN201910865759.9A
Authority: CN
Inventors: 郑玲英
Original assignee: Nanchang Titan Pharmaceutical Co ltd
Current assignee: NANCHANG TITAN PHARMACEUTICAL Co.,Ltd.
Priority date: 2019-09-12
Filing date: 2019-09-12
Publication date: 2020-08-25
Anticipated expiration: 2039-09-12
Also published as: CN110564641A

Abstract

The invention discloses a complex microbial inoculum, which comprises the following components: the species lactococcus oeni (Oenococcus oeni), lactobacillus delbrueckii subsp. Preparing a composite plant liquid culture medium and fermenting: according to the weight percentage, 10 percent of loquat flower alcohol extract, 10 to 12 percent of ligusticum wallichii alcohol extract, 15 to 18 percent of clematis chinensis alcohol extract, 10 percent of glucose, 2 percent of fructose, and the balance of purified water and 15 percent of mixed bacteria seed liquid are added for fermentation. The fermentation process parameters are as follows: the fermentation time is 10 days, the fermentation temperature is 38 ℃, and the fermentation speed is 130 r/min.

Description

Complex microbial inoculum fermentation product and application thereof as pholcodine synergist

Technical Field

The invention relates to a complex microbial inoculum which can ferment specific plants and effectively modify the specific plants, and a modified fermentation product can be matched with pholcodine to generate a protective effect on cardiac muscle.

Background

The strain is the soul of the fermentation industry, people increasingly recognize the important role of pure strain mixed fermentation along with the continuous maturity of the fermentation industry, and the composition and the proportion of the strain in the fermentation have extremely important roles in fermentation products. Along with the development of scientific technology, the research on the microbial fermentation of traditional Chinese medicinal materials is changed from traditional natural fermentation to pure strain fermentation, and the importance of the research is increasingly and widely concerned; the microbial fermentation research of the traditional Chinese medicinal materials has important significance for relieving the deficiency of traditional Chinese medicine resources and finding novel natural active substances and biotransformation reactions.

The invention aims to provide a compound microbial inoculum, which can ferment specific plants and effectively modify the specific plants, and the modified fermentation product can be matched with the pholcodine to generate a protective effect on cardiac muscle, thereby having important significance.

Disclosure of Invention

The invention aims to provide a complex microbial inoculum, which can ferment specific plants and effectively modify the specific plants, and the modified fermentation product can be matched with the pholcodine to generate a protective effect on cardiac muscle.

The technical problem to be solved by the invention can be realized by the following technical scheme.

The complex microbial inoculum is characterized by comprising the following components:

s. cerevisiae (Oenococcus oeni), L.delbrueckii subsp.

The method for preparing the seed solution by activating the microbial inoculum comprises the following steps:

the wine coccus, the lactobacillus delbrueckii subspecies lactis and the propionibacterium freudenreichii can be purchased, and the three microbial agents in the embodiment of the invention are purchased from northern Navier.

Respectively culturing wine coccus, lactobacillus and Propionibacterium freudenreichii in liquid culture medium, counting bacteria in the liquid culture medium after 10h, mixing the two kinds of lactobacillus according to the CFU ratio of 1:1 to obtain the mixture with the concentration of 1.5 x 10⁷CFU/mL mixed strain seed liquid. The liquid culture medium comprises the following components: corn steep liquor (corn steep liquor) 5%, glucose 2%, K₂HPO₄0.8％，(NH4)₂SO₄0.2 percent of distilled water, and the pH value is 6.8-7.0.

The loquat flower is light in taste and warm in nature, can enter lung to dispel cold, resolve phlegm and relieve cough, and can treat common cold and other symptoms.

The ligusticum wallichii is a characteristic traditional Chinese medicine in Jiangxi, is commonly used for promoting blood circulation and promoting qi circulation, dispelling wind and relieving pain, is pungent, warm, fragrant and dry, can disperse and can reach the vertex; it enters the blood system and goes down to reach the blood sea. Promoting blood circulation and removing blood stasis, and is suitable for various diseases caused by blood stasis; it has good effect in dispelling pathogenic wind and relieving pain, and can be used for treating headache, rheumatalgia, etc.

Clematis root: pungent, salty and warm in nature. It enters bladder meridian. Has effects of dispelling pathogenic wind and removing dampness, and dredging channels and collaterals, and can be used for treating rheumatalgia, numbness of limbs, spasm of tendons and vessels, and difficulty in flexion and extension. The monarch drug in the prescription plays a role in dispelling wind and dispersing lung qi due to the wind-damp dispelling effect of the clematis root, which is very symptomatic for the most common wind evil attacking lung type in cough after cold.

The preparation method of the composite plant liquid culture medium comprises the following steps:

the preparation method of the loquat flower alcohol extract comprises the following steps: collecting flos Eriobotryae powder 1000g, adding 6 times of 75% ethanol, reflux-extracting at 90 deg.C for 2 hr, filtering, repeating for 2 times, mixing filtrates, and concentrating.

The preparation method of the chuanxiong rhizome ethanol extract comprises the following steps: extracting rhizoma Ligustici Chuanxiong powder 1000g with 5 times of 75% ethanol under reflux at 90 deg.C for 3 hr, filtering, repeating for 3 times, mixing filtrates, and concentrating.

The preparation method of the clematis chinensis alcohol extract comprises the following steps: taking 1000g of radix clematidis powder, adding 75% ethanol in an amount which is 6 times that of the radix clematidis powder, carrying out reflux extraction for 4h at 90 ℃, filtering, repeating the extraction for 3 times, combining the filtrates, and concentrating to obtain the radix clematidis powder.

Preparing a composite plant liquid culture medium and fermenting: according to the weight percentage, 10 percent of loquat flower alcohol extract, 10 to 12 percent of ligusticum wallichii alcohol extract, 15 to 18 percent of clematis chinensis alcohol extract, 10 percent of glucose, 2 percent of fructose, and the balance of purified water and 15 percent of mixed bacteria seed liquid are added for fermentation. The fermentation process parameters are as follows: the fermentation time is 10 days, the fermentation temperature is 38 ℃, and the fermentation speed is 130 r/min.

After the fermentation is finished, filtering, heating to 100 ℃ for inactivation, evaporating and concentrating to be half of the original volume, and filtering again to obtain the fermentation concentrated solution.

Pholcodine (named morpholine morphine or focodide) has a chemical name of 17-methyl-3- [2- (4-morpholinyl) ethoxy ] -4,5 alpha-epoxy-7, 8-didehydro morphinan-6 alpha-ol, is a white or off-white crystalline powder or colorless crystal chemical, and has a molecular weight of 398.49500. Is slightly soluble in water, easily soluble in ethanol and acetone, and soluble in inorganic acid. The pholcodine can stop cough, eliminate phlegm and resist nasal mucosa blood-charging medicine, and is mainly used for clinically treating severe dry cough and moderate pain. The effect of the pholcodine is similar to that of dextromethorphan, and the pholcodine also has the cough relieving and pain easing effects similar to those of codeine. Has better oral effect than codeine, especially more effective to dry cough. The toxicity and addiction of the medicament are less than those of codeine; the respiratory depression is weaker than morphine, and the neonates and children have better tolerance to the pholcodine and do not cause constipation or digestive dysfunction. Most of the commercial pholcodine oral solutions are compound preparations, and the components of the oral solutions are as follows: every 5ml oral solution contains 5.0mg of pholcodine, 0.6mg of triprolidine hydrochloride, 15.0mg of pseudoephedrine hydrochloride and 50.0mg of guaifenesin. Can be used for treating cough, excessive phlegm cough, dry cough, sensitive cough, watery nasal discharge, nasal obstruction and pharyngalgia caused by wind, influenza, and irritation of throat and bronchus.

Myocarditis is likely to occur in about 5% of patients after viral infection such as the common cold. According to statistics, the viral myocarditis patients account for 75-80% under 40 years of age and more than 20-30 years of age, and the viral myocarditis patients are one of the important reasons for sudden death of young people. In recent years, the incidence of viral myocarditis has increased significantly. The patients usually have a history of upper respiratory tract infection or intestinal tract infection 1-3 weeks before the onset of the disease. There is a specific incubation period between these infections and the onset of myocarditis, and viral myocarditis is likely to develop or aggravate if it is sustained after viral infection, and if it is stressed, overworked, engaged in heavy physical labor and strenuous exercise, drunk, etc.

Diabetic heart disease refers to heart disease complicated or accompanied with diabetes, including coronary atherosclerotic heart disease (coronary heart disease), diabetic cardiomyopathy, and arrhythmia and cardiac dysfunction caused by autonomic nerve disorder, and also including hypertensive heart disease if hypertension exists. Cardiomyocyte hypertrophy, myocardial fibrosis and intramyocardial arteriolar lesions are characteristic of diabetic cardiomyopathy. The heart of a diabetic patient has coronary atherosclerosis, wide pathological changes of micro-vessels and small and medium-sized vessels, myocardial focal necrosis, fibrosis and myocardial vessel wall thickening, and more fatty calcium salt and glycoprotein deposits, so that the lumen is narrowed, the myocardial blood supply is insufficient, and the heart is not normal.

Patients with diabetic heart disease are more likely to be infected with viral myocarditis during cold, which is likely to cause serious consequences.

The invention unexpectedly discovers that the fermentation concentrated solution can play a synergistic role with the pholcodine, has an auxiliary effect on cough relieving effect, has an unexpected protective effect on cardiac muscle cells, and especially has a remarkable effect on diabetic heart disease.

The invention has the advantages that:

(1) the microbial inoculum of the invention has convenient source, low cost and easy preparation and industrialization.

(2) The fermentation composition has no toxic or side effect and good effect.

(3 the invention is unexpected to find, the fermented concentrated solution of the invention can play a synergistic effect with pholcodine, not merely have auxiliary effects on cough-relieving and cold symptoms, also have protective effects on myocardial cells unexpectedly, especially have more prominent effects on diabetic heart disease.

Drawings

Fig. 1 shows heart tissue after molding has been successful.

FIG. 2 shows heart tissues of normal mice.

FIG. 3 shows heart tissues of rats to which the oral liquid of example 1 was administered.

Detailed Description

The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.

Specific examples of the present invention are described below.

Example 1

s. cerevisiae (Oenococcus oeni), L.delbrueckii subsp.

Preparing a composite plant liquid culture medium and fermenting: adding 10% of loquat flower alcohol extract, 10% of ligusticum wallichii alcohol extract, 15% of radix clematidis alcohol extract, 10% of glucose, 2% of fructose and the balance of purified water according to the weight percentage, and fermenting. The fermentation process parameters are as follows: the fermentation time is 10 days, the fermentation temperature is 38 ℃, and the fermentation speed is 130 r/min.

Dissolving 1g of pholcodine in a proper amount of ethanol, dispersing and dissolving, and adding into 1000ml of the fermentation concentrated solution.

Example 2:

s. cerevisiae (Oenococcus oeni), L.delbrueckii subsp.

Respectively culturing the wine coccus, the lactobacillus and the propionibacterium freudenreichii in a liquid culture medium, counting bacteria in a bacteria liquid after 10 hours, and mixing the two kinds of lactobacillus according to the CFU ratio of 1:1 to obtain a mixed bacteria seed liquid with the concentration of 1.5 x 107 CFU/mL. The liquid culture medium comprises the following components: 5% of corn steep liquor (corn steep liquor), 2% of glucose, K2HPO40.8%, (NH4)2SO 40.2%, and distilled water, wherein the pH value is 6.8-7.0.

Preparing a composite plant liquid culture medium and fermenting: adding 10% of loquat flower alcohol extract, 12% of ligusticum wallichii alcohol extract, 18% of radix clematidis alcohol extract, 10% of glucose, 2% of fructose and the balance of purified water according to the weight percentage, and fermenting. The fermentation process parameters are as follows: the fermentation time is 10 days, the fermentation temperature is 38 ℃, and the fermentation speed is 130 r/min.

Example 3

Taking 60 healthy mice with the weight of 18-22g, half of each of the male and female mice, randomly dividing the mice into 4 groups according to the body mass, 15 mice in each group, and respectively: blank control group (20mg/kg physiological saline), example 1 group (20mg/kg liquid of example 1), example 2 group (20mg/kg liquid of example 2), and pholcodine positive control group (20mg/kg pholcodine solution, 1g of pholcodine dissolved in ethanol and water to 1000ml, with the same pholcodine concentration as in examples 1 and 2).

The administration is carried out 1 time and 3 days continuously every day, 60min after the last administration, the mice are placed into a cough-inducing asthma-inducing instrument added with strong ammonia water, the atomization is carried out for 15s, and the cough latency period (the time from the beginning of the atomization to the cough of the animals, the contraction and the expansion of the abdominal muscles of the mice are taken as cough action indicators) and the cough frequency within 2min are observed and recorded.

The results are set forth in the following table

Group of	Cough incubation(s)	Cough frequency within 2min
			Blank control group	28.12±10.21	58.12±10.21
Example 1	81.23±11.21	2.12±0.93
			Example 2	83.56±10.97	1.98±1.01
Positive control group	75.12±10.52	2.56±1.05

Therefore, the solution of the invention has good cough relieving effect.

EXAMPLE 4 myocardial protection

According to the literature, "yaojieli et al, rat ischemic heart disease model with type 2 diabetes is prepared by STZ combined injection of high-sugar and high-fat diet, and rat diabetic heart disease model is prepared by the method described in china journal of applied physiology, 2014,30 (2)": a male SPF-level Wistar rat of 6 weeks old is fed with high-sugar and high-fat diet (prepared by adding 20% of sucrose, 10% of lard, 10% of egg yolk and 60% of basic feed in proportion) and is subjected to intraperitoneal injection of 40mg/kg of streptozotocin once after being fed for 4 weeks, so that a diabetic rat model is prepared. After the injection of STZ for 48h, fasting blood sugar is measured, and the rat with the fasting blood sugar value of more than 11.1mmol/L is successfully modeled. The electrocardiogram of rats in the 14 th week of the diabetes model group and the ST elevation of 2-lead electrocardiogram show that the rats in the diabetes model group have myocardial ischemia, one rat is taken out and fasted for 12 hours, then 2% sodium pentobarbital is used for abdominal anesthesia according to 40mg/kg, the abdominal aorta is taken for blood collection, the heart is taken, the heart tissue structure is observed, and as shown in figure 1, the heart has obvious defects and serious pathological changes. The normal rat heart is shown in FIG. 2, with intact and homogeneous structure.

50 diabetic model rats were prepared, half each of male and female, and 160-180g in body weight. The rats are randomly divided into 5 groups according to the physical quality, each group comprises 10 rats, and the myocardial ischemia model rats of the 5 groups of rats are respectively as follows: control group (20mg/kg physiological saline), example 1 group (20mg/kg oral liquid of example 1), control group 1(20mg/kg oral liquid of control group 1), control group 2(20mg/kg oral liquid of control group 2), control group 3(20mg/kg oral liquid of control group 3), and control group 4(20mg/kg oral liquid of control group 4). In addition, a group of healthy rats is a blank control group, and the weight of the rats has no obvious difference from that of the rats in the diabetes model group.

In comparison with example 1, the medium of control 1 did not contain the alcoholic extract of loquat flower, and the rest was the same.

In comparison with example 1, the control group 2 contained no chuanxiong alcohol extract in the culture medium, and the rest was the same.

In comparison with example 1, the medium of control 3 contained no ethanol extract of clematis root, and the rest was the same.

Control 4 was the same as example 1 except that it was a fermentation concentrate without pholcodine.

The preparation is administered by gavage for 1 time every day, continuously observed for 6 weeks, fasted for 12h, then anesthetized with 2% sodium pentobarbital at 40mg/kg in abdominal cavity, blood is taken from abdominal aorta, heart is taken, and heart tissue structure is observed.

The role of protein glycosylation end products (AGEs) in diabetic heart disease is increasingly gaining attention, and the accumulation of excessive AGEs in blood and tissues is closely related to the occurrence and progression of various complications of diabetes. Studies have shown that AGEs can not only directly affect cell and tissue function. It is also involved in disease production, and may react with specific receptor binding to alter protein and cell functions, resulting in pathological changes in the body. Inhibition of AGEs levels is an effective way to prevent and treat diabetic complications. Cardiac AGEs was determined by Monnier's method: the heart was minced (frozen) 200mg, homogenized in a homogenizer for 60s with 5mL of cold phosphate buffer (PBS, pH 7.4), centrifuged at 6500r/min at 4 ℃ for 30min, the supernatant removed, the pellet washed 3 times with deionized water, the supernatant removed, 2.5mL of chloroform-methanol (2:1) added to the pellet, shaken gently, and overnight at 4 ℃ to remove the fat. 1mL of methanol and 250. mu.L of water were added, hydrated, allowed to settle naturally, centrifuged at 6500r/min at 4 ℃ for 30min, the supernatant removed, and the precipitate washed 2 times with 2.5mL of methanol, 3 times with 2mL of water, 2 times with 0.02MHEPES containing 0.1M CaCl2 (pH 7.5), and overnight at 4 ℃. The supernatant was centrifuged and 1mL of 0.1MCaCl2 (containing 150 units of type VII collagenase) was added, and 1. mu.L chloroform and 1. mu.L toluene were added to inhibit bacterial growth, while a blank of 0.1MCaCl2 buffer containing type VII collagenase was shaken gently at 37 ℃ for 24h, and then centrifuged at 8000r/min at 4 ℃ for 5min to obtain a supernatant for use. The supernatant was measured for fluorescence at 370/440nm (excitation wavelength/emission wavelength) using a fluorescence spectrophotometer. The fluorescence value of 0.1MCaCl2 buffer is taken as 1 arbitrary fluorescence unit (AUF), and the fluorescence intensity of the sample is converted according to the fluorescence value. The glycosylation end product content in the heart is expressed in fluorescence units per mg protein (AUF/mg.

The results are set forth in the following table

As can be seen, the ischemia was significantly improved in the heart tissue of the group of example 1 as shown in FIG. 3. From biochemical indexes, the heart AGES of the group in example 1 is obviously reduced, the heart AGES is not obviously different from that of a blank control group, the heart AGES of the group in example 1 is slightly reduced from that of the control group 1 to 4, and the heart disease is obviously different from that of the blank control group, which indicates that the fermentation concentrated solution containing the loquat flower alcohol extract, the ligusticum wallichii alcohol extract and the clematis chinensis alcohol extract and the pholcodine synergistically protect the diabetic heart disease. Has unexpected protective effect on myocardial cells, especially has more obvious effect on diabetic heart disease, thereby having important significance on preventing and treating diabetic heart disease complications, and the specific action mechanism of the traditional Chinese medicine is to be further deeply researched.

It is to be understood that the foregoing is only a preferred embodiment of the invention and that modifications, variations and changes may be made in the invention without departing from the spirit or scope of the invention as defined in the appended claims.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for obtaining a fermentation concentrated solution by fermenting plant extract with a complex microbial inoculum is characterized by comprising the following steps:

the complex microbial inoculum comprises the following components:

s. oenococcus (oenococcus soeni), lactobacillus delbrueckii subsp. lactis, propionibacterium freudenreichii;

the fermentation steps are as follows:

(1) preparing a composite plant liquid culture medium and fermenting: adding 10% of loquat flower alcohol extract, 10-12% of ligusticum wallichii alcohol extract, 15-18% of radix clematidis alcohol extract, 10% of glucose, 2% of fructose and 15% of compound bacteria agent into the activated mixed bacteria seed liquid respectively, and adding purified water into the mixture for fermentation;

the concentration of the mixed strain seed liquid after the composite microbial inoculum is respectively activated is 1.5 x 10⁷CFU/mL；

The fermentation process parameters are as follows: the fermentation time is 10 days, the fermentation temperature is 38 ℃, and the fermentation speed is 130 r/min;

the preparation method of the loquat flower alcohol extract comprises the following steps: taking 1000g of loquat flower powder, adding 75% ethanol in an amount which is 6 times that of the loquat flower powder, performing reflux extraction at 90 ℃ for 2 hours, filtering, repeating the extraction for 2 times, combining filtrates, and concentrating to obtain the loquat flower powder;

the preparation method of the ligusticum wallichii ethanol extract comprises the following steps: extracting rhizoma Ligustici Chuanxiong powder 1000g with 5 times of 75% ethanol under reflux at 90 deg.C for 3 hr, filtering, repeating for 3 times, mixing filtrates, and concentrating;

the preparation method of the clematis chinensis alcohol extract comprises the following steps: taking 1000g of radix clematidis powder, adding 75% ethanol in an amount which is 6 times that of the radix clematidis powder, carrying out reflux extraction for 4 hours at 90 ℃, filtering, repeating the extraction for 3 times, combining the filtrates, and concentrating to obtain the radix clematidis powder;

(2) after the fermentation is finished, filtering, heating to 100 ℃ for inactivation, evaporating and concentrating to be half of the original volume, and filtering again to obtain the fermentation concentrated solution.

2. A fermentation concentrate produced by the method of claim 1.

3. Use of the fermentation concentrate of claim 2 as a pholcodine potentiator.