CN116898921B

CN116898921B - Preparation method of cattail and dyer woad compound traditional Chinese medicine preparation

Info

Publication number: CN116898921B
Application number: CN202311004549.3A
Authority: CN
Inventors: 曹小英; 黄凯锋; 江润蓓; 刘高艺; 管蕴康; 刘达
Original assignee: JIANGXI NEW CENTURY MINXING ANIMAL HEALTH PRODUCTS CO Ltd
Current assignee: JIANGXI NEW CENTURY MINXING ANIMAL HEALTH PRODUCTS CO Ltd
Filing date: 2023-08-10
Publication date: 2024-05-31
Anticipated expiration: 2043-08-10

Abstract

The invention discloses a preparation method of a cattail blue compound traditional Chinese medicine preparation, which comprises four traditional Chinese medicines of dandelion, isatis root, bunge corydalis herb and baical skullcap root, wherein a certain modifier is added to the traditional Chinese medicine raw materials by a pressurized percolation extraction method for improving the absorption of lipophilic substances in flavone components, and the antibacterial and anti-inflammatory effects are improved by adding proper substances in the fermentation process to enhance the absorption of medicines without being metabolized, and the stability of the preparation is improved by enhancing the structure of volatile substances.

Description

Preparation method of cattail and dyer woad compound traditional Chinese medicine preparation

Technical Field

The invention relates to the field of traditional Chinese medicine preparations, in particular to extraction of traditional Chinese medicine raw materials and improvement of active ingredients, and specifically relates to a preparation method of a cattail-basket compound traditional Chinese medicine preparation.

Background

The cattail herba is composed of four traditional Chinese medicines of dandelion, radix isatidis, herba violae and radix scutellariae, and has the effects of clearing heat, detoxicating, resisting inflammation and reducing swelling. Dandelion herb and Chinese medicine name. Aliases herba Violae, grandma, hua Hualang, etc. Perennial herb of Compositae. Bitter, sweet and cold in nature. It is mainly used for treating furuncle, acute mastitis, scrofula, conjunctival congestion, pharyngalgia, pulmonary abscess, appendicitis, jaundice due to damp-heat, stranguria due to heat, etc.

Radix Isatidis is the dry root of Isatis tinctoria L of Brassicaceae. Mainly treats influenza, epidemic encephalitis B, pneumonia, erysipelas, heat toxin spots, coma, epistaxis, pharyngeal swelling, mumps, fire eyes, sore rash, dark purple tongue, pharyngitis, throat sore and skin disease, plague of large head, carbuncle and swelling; can be used for preventing and treating epidemic encephalitis B, acute and chronic hepatitis, epidemic parotitis, and osteomyelitis.

The herba Violae is herba Violae of Viola; the bag of Leguminosae plant Rice, semen Setariae bag or root-carrying whole plant of Gentiana lutea of Gentianaceae. Clearing heat and promoting diuresis, removing toxic substances and relieving swelling. For furuncle, carbuncle, swelling, scrofula, jaundice, dysentery, diarrhea, conjunctival congestion, pharyngitis and snake bite.

Radix Scutellariae, called radix Camelliae Japonicae, is perennial herb of Scutellaria of Labiatae; the radix scutellariae grows on sunny grass slopes. It has the effects of clearing heat, eliminating dampness, purging pathogenic fire, removing toxic substances, stopping bleeding, and preventing miscarriage. Is used for treating warm diseases, upper respiratory infection, cough due to lung heat, jaundice due to damp-heat, pneumonia, dysentery, hemoptysis, conjunctival congestion, fetal irritability, hypertension, carbuncle, furuncle, and sore. The radix scutellariae has good clinical antibacterial property and does not generate drug resistance.

The commercial Pu Di lan is clinically used, and although the curative effect is definite, the curative effect is slow, mainly because the concentration of the effective components in the commercial Pu Di lan is low, the efficacy is still to be further improved, so that the effective components are respectively improved by adopting a proper extraction mode for the traditional Chinese medicine raw materials, and the improvement of the absorption and the efficacy of the traditional Chinese medicine is the focus of the research of the invention.

Disclosure of Invention

In order to solve the technical problems, the specific preparation process of the invention comprises the following steps:

(1) Pressurized percolation

A. cleaning 9-13 parts of dandelion and 9-13 parts of bunge corydalis herb, drying, crushing and sieving with a 100-mesh sieve;

b. wetting and expanding the crushed materials and 70% ethanol solvent according to the mass ratio of 1:1, stirring for 10-20min, standing for 15-30min, filling the wetted materials into a percolating tank in pressurized percolating equipment, adding 10-12 parts of 50% ethanol solution into the percolating tank, regulating the pressure to 5-7Mpa, the percolating flow rate to 4-8ml/min, and collecting bottom effluent filtrate into the percolating tank through a filter;

c. Heating the percolate tank in 70-80deg.C water bath for 30-40min, volatilizing alcohol, adding into a microwave tank, adding 1-3 parts of modifier, reacting at 40-60deg.C and microwave power of 10-15w for 10-15min to obtain filtrate A;

wherein the modifier is acetyl L-carnitine hydrochloride;

(2) Fermentation extraction

D. Taking 8-12 parts of radix isatidis and 6-8 parts of radix scutellariae, cleaning with deionized water for 4-6 times, draining off water, and drying at 50-60 ℃ until the weight is constant to obtain a fermentation material;

e. preparing a compound bacterial liquid: firstly, adding 1-3 parts of additive into 50 parts of water, mixing to prepare a solvent, then adding 1 part of lactobacillus, 1 part of saccharomycete and 1 part of bacillus licheniformis, and uniformly stirring to obtain a compound bacterial liquid;

f. Placing the fermentation material into a compound bacterial solution, fermenting for 3-5d under the condition of pH=4-7 at 40-50 ℃, and filtering and sterilizing to obtain a fermentation filtrate B;

wherein the additive is L-ornithine hydrochloride

(3) Mixing

Mixing the filtrate A and the fermentation filtrate B according to the mass ratio of 1:1, preserving for 12-16 hours at the low temperature environment of 4-10 ℃, then adopting ultrasonic treatment to combine with a nozzle circulation system, dropwise adding 0.8-1.2 parts of accelerator into the mixture, wherein the ultrasonic power is 100-150w, the circulation flow rate is 25-40L/min, the reaction time is 40-60min, and recovering to room temperature to obtain the traditional Chinese medicine preparation;

wherein the promoter is N-t-butoxycarbonyl-D-proline.

The invention has the beneficial effects that:

1) The dandelion and the bunge corydalis herb raw materials are subjected to a pressurized percolation extraction method, so that the solvent can easily infiltrate into the tissue of the medicinal materials under the action of a large pressure to dissolve the internal components of the medicinal materials, the concentration in the tissue is large, the outward diffusion speed is also high, and the extraction of the components is facilitated; the microwave treatment can promote the dissolution of partial insoluble components through the thermal effect and the physical effect, and can accelerate the dissolution of active components in the traditional Chinese medicine preparation, thereby improving the bioavailability; absorption and transport of the drug is also more efficient when it dissolves and is released into the body more rapidly.

2) The extracting solution is modified in a microwave heating mode, on one hand, acetyl L-carnitine hydrochloride preferentially reacts with lipophilic substances, so that flavonoid lipophilic substances in the traditional Chinese medicine component are connected with acetyl and hydroxyl in the acetyl L-carnitine hydrochloride, and the absorption of the flavonoid lipophilic substances in the traditional Chinese medicine component is facilitated; on the other hand, the modified substance promotes the transportation of CRP and P-gp transport proteins of the intestinal epithelial cells, thereby improving the absorption rate and rapidly achieving the therapeutic effect.

3) The method has the advantages that the contents of active ingredients in the isatis roots and the baikal skullcap roots can be improved firstly by using a fermentation mode, the synergistic effect between the active ingredients in the isatis roots and the baikal skullcap roots can be promoted, the drug effect can be improved, more polypeptides and amino acids are generated in the fermentation process by adding L-ornithine hydrochloride in the fermentation process, and the synergistic effect of various active substances can lead cytokines such as IL-6, TNF-alpha and the like to polarize TAMs through NF- κB signal paths and convert the polarized TAMs into M1, so that the functions of anti-inflammatory, antibacterial and immunoregulation are enhanced, and the synergistic effect on a plurality of targets can avoid drug resistance.

4) The L-ornithine hydrochloride can play a competitive inhibition role, and can play a role in inhibiting CYP3A and CYP2C in isozymes with the most abundant CYP450 enzyme content in the small intestine, so that the CYP3A4 and P-gp are inhibited to increase the probability of medicament metabolism in a synergistic mode, the bioavailability of the medicament is reduced, the circulation of the medicament in the body is accelerated, and the treatment effect is quickly and effectively achieved through blood circulation.

5) The stability of the N-t-butoxycarbonyl-D-proline is high, the activity is good in a low-temperature environment, the generation of organic acid can be catalyzed by combining an improved process in the low-temperature environment, the pH value of the preparation is reduced, on one hand, the organic acid inhibits the growth of harmful microorganisms, the multiple double bonds and the rigid structure in the N-t-butoxycarbonyl-D-proline can also participate in improving the volatile oil component in the low-temperature environment, and the organic acid component, the N-t-butoxycarbonyl-D-proline and the volatile oil form a stable structure of ester or acid salt, so that the biological activity is improved, the volatile oil component is retained to the greatest extent, the activity and stability of the volatile oil component are improved, and the curative effect and tolerance of the medicine are improved; the organic acid component also gives the special taste and texture characteristics to the traditional Chinese medicine fermentation liquor; on the other hand, the tert-butoxycarbonyl in N-tert-butoxycarbonyl-D-proline is a good protecting group, and can also protect the active ingredients in the preparation from being damaged and deactivated when the acidity in the preparation is higher.

6) The preparation in low temperature environment can be firstly crushed by shaking macromolecular substances or salt substances which are separated out in low temperature, on the other hand, the occurrence of side reaction is reduced in low temperature environment, and more generated organic acid can play a role in stabilizing the volatile oil unstable components, so that the reaction interface is increased by ultrasonic treatment, the energy transfer between the volatile oil unstable components is promoted, and the stability of the preparation is improved.

Detailed Description

The present invention will be described in further detail with reference to examples.

All the traditional Chinese medicine raw materials used in the embodiment of the invention are purchased by Jiangxi Jie Chengjie medicinal wholesale Limited company, and the rest raw materials or chemical reagents are obtained through conventional commercial paths if no special description exists.

Example 1

(1) Pressurized percolation

A. cleaning, drying and crushing 11 parts of dandelion and 11 parts of bunge corydalis herb, and sieving the mixture through a 100-mesh sieve;

b. Wetting and expanding the crushed materials and 70% ethanol solvent according to the mass ratio of 1:1, standing for 22min, filling the wetted materials into a percolating tank in pressurized percolating equipment, adding 11 parts of 50% ethanol solution into the percolating tank, regulating the pressure to 6Mpa, percolating at the flow rate of 6ml/min, and collecting bottom effluent filtrate into the percolating tank through a filter;

c. heating the percolate tank in a water bath at 75 ℃ for 35min, adding the percolate tank into a microwave tank after alcohol volatilizes, adding 2 parts of acetyl L-carnitine hydrochloride at 50 ℃ and microwave power of 12w, and reacting for 12min to obtain a filtrate A;

(2) Fermentation extraction

D. taking 10 parts of radix isatidis and 7 parts of radix scutellariae, washing with deionized water for 5 times, draining off water, and drying at 55 ℃ until the weight is constant to obtain a fermentation material;

e. Preparing a compound bacterial liquid: firstly, adding 2 parts of L-ornithine hydrochloride into 50 parts of water, mixing to prepare a solvent, then adding 1 part of lactic acid bacteria, 1 part of saccharomycetes and 1 part of bacillus licheniformis, and uniformly stirring to obtain a compound bacterial solution;

f. Placing the fermentation material in the compound bacterial liquid, fermenting for 4d under the condition of pH=6 at 40-50 ℃, and filtering and sterilizing to obtain fermentation filtrate B;

(3) Mixing

Mixing the filtrate A and the fermentation filtrate B according to the mass ratio of 1:1, preserving for 14 hours at the low temperature environment of 6 ℃, then adopting ultrasonic treatment to combine with a nozzle circulation system, dropwise adding 1 part of N-tert-butoxycarbonyl-D-proline into the mixture, wherein the ultrasonic power is 120w, the circulation flow rate is 32L/min, the reaction time is 50min, and recovering to room temperature to obtain the traditional Chinese medicine preparation;

Example 2

(1) Pressurized percolation

A. Cleaning 9 parts of dandelion and 13 parts of bunge corydalis herb, drying, crushing and sieving with a 100-mesh sieve;

b. wetting and expanding the crushed materials and 70% ethanol solvent according to the mass ratio of 1:1, standing for 30min, filling the wetted materials into a percolating tank in pressurized percolating equipment, adding 12 parts of 50% ethanol solution into the percolating tank, regulating the pressure to 5Mpa, percolating at the flow rate of 4ml/min, and collecting bottom effluent filtrate into the percolating tank through a filter;

c. Heating the percolate tank in water bath at 70 ℃ for 30min, adding the percolate tank into a microwave tank after alcohol volatilizes, adding 3 parts of acetyl L-carnitine hydrochloride at 40 ℃ and microwave power of 15w, and reacting for 10min to obtain filtrate A;

(2) Fermentation extraction

D. Taking 12 parts of radix isatidis and 6 parts of radix scutellariae, washing with deionized water for 4 times, draining off water, and drying at 50 ℃ until the weight is constant to obtain a fermentation material;

e. Preparing a compound bacterial liquid: firstly, adding 1 part of L-ornithine hydrochloride into 50 parts of water, mixing to prepare a solvent, then adding 1 part of lactic acid bacteria, 1 part of saccharomycetes and 1 part of bacillus licheniformis, and uniformly stirring to obtain a compound bacterial solution;

f. placing the fermentation material in a compound bacterial solution, fermenting for 3d at 40 ℃ and pH=7, and filtering and sterilizing to obtain a fermentation filtrate B;

(3) Mixing

Mixing the filtrate A and the fermentation filtrate B according to the mass ratio of 1:1, preserving for 12 hours at the low temperature environment of 10 ℃, then adopting ultrasonic treatment to combine with a nozzle circulation system, dropwise adding 0.8 part of N-t-butoxycarbonyl-D-proline into the mixture, wherein the ultrasonic power is 150w, the circulation flow rate is 40L/min, the reaction time is 60min, and the mixture is recovered to the room temperature to obtain the traditional Chinese medicine preparation;

Example 3

(1) Pressurized percolation

A. cleaning 13 parts of dandelion and 9 parts of bunge corydalis herb, drying, crushing and sieving with a 100-mesh sieve;

b. Wetting and expanding the crushed materials and 70% ethanol solvent according to the mass ratio of 1:1, standing for 15min, filling the wetted materials into a percolating tank in pressurized percolating equipment, adding 10 parts of 50% ethanol solution into the percolating tank, regulating the pressure to 7Mpa, controlling the percolating flow rate to 8ml/min, and collecting bottom effluent filtrate into the percolating tank through a filter;

c. Heating the percolate tank in water bath at 80 ℃ for 40min, adding the percolate tank into a microwave tank after alcohol volatilizes, adding 1 part of acetyl L-carnitine hydrochloride at 60 ℃ and microwave power of 10w, and reacting for 15min to obtain filtrate A;

(2) Fermentation extraction

D. taking 8 parts of radix isatidis and 8 parts of radix scutellariae, washing with deionized water for 6 times, draining off water, and drying at 60 ℃ until the weight is constant to obtain a fermentation material;

e. Preparing a compound bacterial liquid: firstly, adding 3 parts of L-ornithine hydrochloride into 50 parts of water, mixing to prepare a solvent, then adding 1 part of lactic acid bacteria, 1 part of saccharomycetes and 1 part of bacillus licheniformis, and uniformly stirring to obtain a compound bacterial solution;

f. placing the fermentation material in a compound bacterial solution, fermenting for 5 days at 50 ℃ and under the condition of pH=4, and filtering and sterilizing to obtain a fermentation filtrate B;

(3) Mixing

Mixing the filtrate A and the fermentation filtrate B according to the mass ratio of 1:1, preserving for 16 hours at the low temperature environment of 4 ℃, then adopting ultrasonic treatment to combine with a nozzle circulation system, dropwise adding 1.2 parts of N-t-butoxycarbonyl-D-proline into the mixture, wherein the ultrasonic power is 100w, the circulation flow rate is 25L/min, the reaction time is 40min, and recovering to room temperature to obtain the traditional Chinese medicine preparation;

Comparative example 1

This comparative example differs from example 1 in that the acetyll-carnitine hydrochloride in step (1) is palmitic acid, and the other embodiments are the same as example 1.

Comparative example 2

This comparative example differs from example 1 in that the acetyll-carnitine hydrochloride in step (1) is methylformamide, and the remaining embodiments are the same as example 1.

Comparative example 3

This comparative example differs from example 1 in that the L-ornithine hydrochloride in step (2) is hydrochloric acid, and the remaining embodiments are the same as example 1.

Comparative example 4

This comparative example differs from example 1 in that L-ornithine hydrochloride in step (2) is an amino acid, and the other embodiments are the same as example 1.

Comparative example 5

This comparative example differs from example 1 in that the N-t-butoxycarbonyl-D-proline in step (3) is hydroxypropyl methylcellulose and the remaining embodiments are the same as example 1.

Comparative example 6

This comparative example differs from example 1 in that the N-t-butoxycarbonyl-D-proline in step (3) is sodium carboxymethyl cellulose and the remaining embodiments are the same as example 1.

The evaluation method comprises the following steps:

(1) Chromatographic content determination: the main component of Pu Di lan was measured by high performance liquid chromatography (Chinese pharmacopoeia 2020 edition). Octadecylsilane chemically bonded silica is used as a filler, acetonitrile-water (volume ratio is 32:68) is used for isocratic elution; the theoretical plate number should be not less than 2000 calculated on the astragaloside peak.

Preparation of a control solution: taking appropriate amount of astragaloside and chicoric acid reference substances, precisely weighing, and adding methanol to prepare a solution containing 0.4mg per 1 mL.

Preparation of test solution: precisely measuring 20mL of the product, extracting with water saturated n-butanol for 5 times under shaking, each time 25mL, mixing n-butanol extract, washing with ammonia test solution for 3 times, each time 20mL, recovering n-butanol extract, dissolving residues in methanol, transferring to a 10mL measuring flask, adding methanol to scale, shaking, centrifuging, and collecting supernatant.

Assay: respectively precisely sucking 20 μl of control solution and 20 μl of sample solution, and injecting into liquid chromatograph for measurement to obtain the contents of astragaloside and chicoric acid. The results of the measurement of the active ingredients are shown in Table 1.

TABLE 1 determination of the content of Astragaloside and chicoric acid

Group of	Astragaloside content mg/mL	Chicoric acid content mg/mL
			Example 1	52.82	0.67
Example 2	49.55	0.64
			Example 3	48.83	0.63
Comparative example 1	40.73	0.56
			Comparative example 2	42.54	0.47
Comparative example 3	35.76	0.41
			Comparative example 4	32.62	0.49
Comparative example 5	38.71	0.42
			Comparative example 6	35.28	0.41

(2) Pharmacological experimental study for resisting acute inflammation of mice

80 KM mice, male, weighing about 25g, were prepared, and randomly divided into 10 groups of 8 animals each, blank groups, examples 1-3 groups, and comparative examples 1-6 groups. The respective liquid medicines of 0.21mL/10g body weight were administered to the groups of examples 1 to 3 and the groups of comparative examples 1 to 6, and the blank group was administered with an equal amount of physiological saline. Each group was administered 1 time a day, 3d continuously, and after 1h last administration, the mice of each group except the blank group were uniformly smeared with 0.04mL of xylene on the front and back sides of the left ear, and the right ear was used as a control. Mice were sacrificed 30min after inflammation, two ears were cut off along the auricle edge, the same positions were punched out with a direct 9mm punch, the analytical balance was weighed, the difference in weight of the left and right ears was the swelling degree, and the swelling inhibition rate was calculated and expressed as the anti-inflammatory strength of the drug. The results are shown in Table 2 below.

Inhibition ratio = (difference in weight of both ears of blank group-difference in weight of both ears of administration group)/difference in weight of both ears of blank group×100%

Table 2 effect of drug on ear swelling in mice due to xylene (x±s, n=10)

Group of	Left ear weight/mg	Right ear weight/mg	Weight difference of two ears/mg	Inhibition/%
					Blank group	24.0±1.8	9.3±0.8	14.7±1.9
Example 1	14.9±1.6	8.7±0.7	6.2±1.9**	57.8
					Example 2	15.9±1.4	9.1±0.8	6.8±1.8**	53.7
Example 3	16.1±1.3	8.9±0.6	7.2±1.1*	51.0
					Comparative example 1	18.2±0.9	9.1±0.8	9.1±1.6	38.0
Comparative example 2	17.4±1.1	9.2±0.5	8.2±1.2	44.2
					Comparative example 3	18.1±1.2	9.4±0.7	8.7±1.5	40.8
Comparative example 4	17.2±1.0	9.3±0.8	7.9±1.2	46.2
					Comparative example 5	17.5±0.9	9.2±0.7	8.3±1.0	43.5
Comparative example 6	18.3±1.2	9.3±0.8	9.0±1.1	38.7

P <0.01, P <0.05 compared to the blank.

Claims

1. A preparation method of a cattail-basket compound traditional Chinese medicine preparation is characterized by comprising the following steps:

The preparation process comprises the following steps:

(1) Pressurized percolation

(2) Fermentation extraction

(3) Mixing

the modifier in the step (1) is acetyl L-carnitine hydrochloride;

the additive in the step (2) is L-ornithine hydrochloride;

the promoter in the step (3) is N-t-butoxycarbonyl-D-proline.