CN113230345A

CN113230345A - Composition, extraction method and application of composition in anti-inflammatory field

Info

Publication number: CN113230345A
Application number: CN202110615423.4A
Authority: CN
Inventors: 赵肃清; 李双祁; 梁雨昕; 吴盼盼; 张炳杰
Original assignee: Guangdong University of Technology
Current assignee: Guangdong University of Technology
Priority date: 2021-06-02
Filing date: 2021-06-02
Publication date: 2021-08-10

Abstract

The invention belongs to the technical field of natural medicines, and particularly relates to a composition, an extraction method and application thereof in the field of anti-inflammation. The invention provides a composition, which comprises the following raw materials: garlic, purslane and trifoliate acanthopanax. The invention also provides an extraction method of the composition, and application of the composition or a product obtained by the extraction method in the field of anti-inflammation. In the technical scheme provided by the invention, the natural extract has the advantages of strong antibacterial property, lasting action efficiency and wide action range, and meanwhile, the three extracts have a certain synergistic effect and also have the comprehensive gain effects of clearing away heat and toxic materials, enhancing immunity and resisting fatigue. The composition, the extraction method and the application thereof in the anti-inflammatory field solve the technical defects that the artificially synthesized anti-inflammatory drug has narrow application range, short lasting efficacy, harm stimulation to gastrointestinal tract and easy generation of drug resistance in the prior art.

Description

Composition, extraction method and application of composition in anti-inflammatory field

Technical Field

The invention belongs to the technical field of natural medicines, and particularly relates to a composition, an extraction method and application thereof in the field of anti-inflammation.

Background

The purslane plant of Portulacaceae is widely distributed in temperate and tropical regions, has a long medicinal history, and the herbal medicine is recorded. Herba Portulacae contains alkaloid, flavonoid, coumarin, terpenes, protein, fat, and mineral, has effects of clearing heat, removing toxic substances, cooling blood, and relieving swelling, and can be used for treating dysentery and diarrhea. The existing research shows that the purslane extract has the effects of resisting oxidation, bacteria, fatigue and the like. The purslane extract has high inhibition effect on most gram-negative bacteria and has inhibition effect on part of gram-positive bacteria to different degrees.

Garlic is a semi-annual herb plant of the genus Allium of the family Liliaceae, and is used as a medicine in the form of bulbs. The sulfur-containing compound in the garlic has extremely strong antibacterial and anti-inflammatory effects, has inhibition and killing effects on various cocci, bacilli, fungi, viruses and the like, and is one of the natural plants which are found at present and has the strongest antibacterial effect. Experiments show that the sulfur-containing compound and the selenium-containing compound contained in the garlic also have a certain positive effect on cancer prevention. Modern medical research proves that garlic integrates more than 100 medicinal and health-care components, wherein 43 sulfur-containing volatile matters, 13 sulfurous sulfinic acid (such as garlicin) esters, 9 amino acids, 8 peptides, 12 glycosides and 11 enzymes are contained in the garlic. In addition, alliin is a unique component of garlic, and becomes allicin when entering blood, and the allicin can kill typhoid bacillus, dysentery bacillus, influenza virus and the like instantly even if diluted by 10 ten thousand times.

The Lecai vegetable is widely distributed in the middle and south of China, grows in villages, hillside roads, forest edges and bushes, is vertically distributed from above sea level to 3200 meters, and is also distributed in India, Vietnam and Philippines. According to records of compendium of materia medica, the trifoliate acanthopanax is called as 'relieving hundred toxins', has mellow taste, strong fragrance and endless aftertaste, and has the efficacies of clearing heat, expelling toxin, relieving summer heat, quenching thirst, relieving smoking and drinking, losing weight and resisting fatigue.

At present, anti-inflammatory drugs represented by antibiotics are widely used in the treatment and control of diseases due to their good effects and low price. However, some artificially synthesized anti-inflammatory drugs are effective only against gram-positive bacteria and are prone to develop antibiotic resistance. In addition, antibiotic drugs have problems of noxious stimulation of the gastrointestinal tract and insufficient persistence of efficacy in the body.

Therefore, the development of a composition, an extraction method and an application thereof in the anti-inflammatory field is used for solving the technical defects that the artificial synthetic anti-inflammatory drug has a narrow application range, is easy to generate drug resistance, has noxious stimulation to the gastrointestinal tract and has not lasting efficacy in the prior art, and becomes a problem to be solved by the technical staff in the field.

Disclosure of Invention

In view of the above, the invention provides a composition, an extraction method and an application thereof in the anti-inflammatory field, which are used for solving the technical defects that the artificial synthetic anti-inflammatory drug has a narrow application range, an insufficient lasting effect, and is harmful to the gastrointestinal tract and easy to generate drug resistance in the prior art.

The invention provides a composition, which comprises the following raw materials: garlic, purslane and trifoliate acanthopanax.

Preferably, the raw materials of the composition comprise, by mass: 4-8 parts of garlic, 4-8 parts of purslane and 4-8 parts of trifoliate acanthopanax.

The invention also provides an extraction method of the composition, which comprises the following steps:

step one, garlic, purslane and trifoliate acanthopanax are mixed, dried, crushed and sieved to obtain a first product;

step two, after the first product is degreased, soaking the degreased first product in 75% ethanol for 48 hours to obtain a second product;

step three, after the second product is subjected to ultrasonic treatment, filtering and collecting filtrate, and concentrating the filtrate under reduced pressure to obtain a third product;

and step four, drying the third product to obtain the product.

Preferably, in the first step, the drying temperature is 40-70 ℃, and the drying time is 20-120 min;

in the first step, the sieving is performed by sieving with a 20-40 mesh sieve.

Preferably, in the second step, the feeding ratio of the first product to the ethanol solution is 1 (10-20) g/ml.

Preferably, in the third step, the ultrasonic treatment temperature is 20-45 ℃, and the ultrasonic treatment time is 10-60 min.

Preferably, in the third step, the temperature of the reduced pressure concentration is 30-50 ℃.

Preferably, in step four, the drying method is freeze drying.

Preferably, in the fourth step, the temperature of the freeze drying is-80 to-50 ℃, and the time of the freeze drying is 10 to 12 hours.

The invention also provides application of the composition or the product obtained by the extraction method in the field of anti-inflammation.

In summary, the present invention provides a composition, which comprises the following raw materials: garlic, purslane and trifoliate acanthopanax. The invention also provides an extraction method of the composition, which comprises the following steps: step one, garlic, purslane and trifoliate acanthopanax are mixed, dried, crushed and sieved to obtain a first product; step two, after the first product is degreased, soaking the degreased first product in 75% ethanol to obtain a second product; step three, after the second product is subjected to ultrasonic treatment, filtering and collecting filtrate, and concentrating the filtrate under reduced pressure to obtain a third product; and step four, drying the third product to obtain the product. The invention also provides an application of the composition or the product obtained by the extraction method in the anti-inflammatory field. In the technical scheme provided by the invention, the natural extract has the advantages of strong antibacterial property, lasting action efficiency and wide action range, and meanwhile, the three extracts have a certain synergistic effect and also have the comprehensive gain effects of clearing away heat and toxic materials, enhancing immunity and resisting fatigue. The composition, the extraction method and the application of the composition in the anti-inflammatory field solve the technical defects that the application range of artificially synthesized anti-inflammatory drugs is narrow, drug resistance is easy to generate, gastrointestinal tracts are harmfully stimulated, and the efficacy is not lasting in the prior art.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a plot of the bacteriostatic diameter of different doses of product 1 against Staphylococcus aureus as provided in example 5 of the present application;

FIG. 2 is a graph of the bacteriostatic diameter of product 1 against Staphylococcus albus at different dosages as provided in example 5 of the present application;

FIG. 3 is a plot of the bacteriostatic diameter of E.coli for various doses of product 1 as provided in example 5 of the present application;

fig. 4 is a plot of the bacteriostatic diameter of typhoid bacillus for different doses of product 1 as provided in example 5 of the present application.

Detailed Description

The invention provides a composition, an extraction method and application thereof in the field of anti-inflammation, and aims to overcome the technical defects that in the prior art, an artificially synthesized anti-inflammatory drug has a narrow application range, is easy to generate drug resistance, has noxious stimulation to gastrointestinal tracts and has short lasting effect.

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to illustrate the present invention in more detail, a composition, an extraction method and its application in the anti-inflammatory field provided by the present invention are specifically described below with reference to examples.

Example 1

The garlic, the purslane and the trifoliate acanthopanax are naturally dried in the air after being cleaned, 100g of garlic, 150g of purslane and 150g of trifoliate acanthopanax are mixed, dried for 40min at 70 ℃, crushed and sieved by a 30-mesh sieve, and a first product 1 is obtained.

228g of the first product 1 was degreased and then soaked in 2.28L of 75% ethanol to obtain a second product 1.

And (3) performing ultrasonic treatment on the second product 1 at 45 ℃ for 40min, performing suction filtration to collect filtrate, and performing reduced pressure concentration on the filtrate at 30 ℃ to obtain a third product 1.

And (3) freeze-drying the third product 1 at-50 ℃ for 11h to obtain a product 1.

Example 2

The garlic, the purslane and the trifoliate acanthopanax are naturally dried in the air after being cleaned, 100g of garlic, 150g of purslane and 150g of trifoliate acanthopanax are mixed, the mixture is dried for 40min at the temperature of 50 ℃, and then the mixture is crushed and sieved by a 20-mesh sieve, so that a first product 2 is obtained.

254g of the first product 2 was defatted and then soaked in 2.54L of 75% ethanol to obtain the second product 2.

And (3) carrying out ultrasonic treatment on the second product 2 at the temperature of 30 ℃ for 40min, carrying out suction filtration, collecting filtrate, and carrying out reduced pressure concentration on the filtrate at the temperature of 45 ℃ to obtain a third product 2.

And (3) freeze-drying the third product 2 at-50 ℃ for 10h to obtain a product 2.

Example 3

The garlic, the purslane and the trifoliate acanthopanax are naturally dried after being cleaned, 100g of garlic, 150g of purslane and 150g of trifoliate acanthopanax are mixed, dried for 40min at 40 ℃, crushed and sieved by a 40-mesh sieve, and a first product 3 is obtained.

297kg of the first product 3 was defatted and then soaked in 2.97L of 75% ethanol to obtain a second product 3.

And (3) carrying out ultrasonic treatment on the second product 3 at the temperature of 60 ℃ for 40min, carrying out suction filtration, collecting filtrate, and carrying out reduced pressure concentration on the filtrate at the temperature of 50 ℃ to obtain a third product 3.

And (4) freeze-drying the third product 3 at-50 ℃ for 10h to obtain a product 3.

Example 4

This example is a specific example of measuring the bacteriostatic effect of the products 1-3 prepared in examples 1-3.

The product 1 was formulated to the following concentrations: 1. 2, 4, 8 and 16mg/ml, and respectively measuring the bacteriostatic activity and the minimum bacteriostatic concentration of four stable bacterial colonies (staphylococcus aureus, staphylococcus albus, escherichia coli and typhoid bacillus), wherein the staphylococcus albus and the staphylococcus albus belong to gram-positive bacteria, and the escherichia coli and the typhoid bacillus belong to gram-negative bacteria.

After the activation culture of the test strain, diluting the test strain with sterile water to make the concentration of the bacterial suspension reach 10⁶cfu/mL for use. The sterilized agar medium was heated to completely melt and poured into petri dishes, 10mL per dish, and allowed to solidify. Further, the melted medium was cooled to about 50 ℃ and test bacteria (concentration: 0.2%) were mixed, 5mL of the medium mixed with the bacteria was added to the solidified medium to be solidified, sterile Oxford cups (diameter: 7.8mm) were gently placed in the petri dish with tweezers, and 3 Oxford cups were uniformly placed in the horizontally placed petri dish. Respectively sucking the same amount of the product 1 extract solution with different concentrations, adding into the Oxford cup, preventing the sample solution from overflowing out of the Oxford cup, repeating for 3 times, and using dimethyl sulfoxide (DMSO) with the same amount as a control. Placing the plates added with the extract solutions with different concentrations in a 4-degree refrigerator for diffusion for 4h, culturing bacteria at 37 ℃, and measuring the diameter of the inhibition zone by adopting a cross method.

TABLE 1 inhibition of four bacteria by product 1 and control group

TABLE 2 bacteriostatic Activity of product 1

As can be seen from Table 1, the extract of product 1 has inhibitory effects on all four bacteria and the inhibitory effect is better as the concentration of the composite extract is increased; as can be seen from Table 2, the bacteriostatic activity of product 1 is most significant for Staphylococcus aureus, and has good bacteriostatic effects for Staphylococcus albus, Escherichia coli and Salmonella typhi.

The above experiments were repeated for product 2 and product 3, which all gave similar experimental results and are not described in detail herein.

Example 5

In this example, the bacterial colony diameter is measured during 12-72h to measure the drug effect durability of the sample to the tested strain by the above-mentioned method for determining the antibacterial activity.

As can be seen from fig. 1-4, fig. 1 is a plot of the bacteriostatic diameter of different doses of product 1 against staphylococcus aureus as provided in example 5 of the present application; FIG. 2 is a graph of the bacteriostatic diameter of product 1 against Staphylococcus albus at different dosages as provided in example 5 of the present application; FIG. 3 is a plot of the bacteriostatic diameter of E.coli for various doses of product 1 as provided in example 5 of the present application; fig. 4 is a plot of the bacteriostatic diameter of typhoid bacillus for different doses of product 1 as provided in example 5 of the present application. The efficacy of the extract of product 1 generally showed a decreasing trend with time, but to a different extent for different bacteria. When the drug effect lasts for 48 hours and the concentration of the extract is 1mg/mL, the inhibition effect on the typhoid bacillus is the best, the drug effect is reduced to 17.84%, and the drug effect is reduced to about 20.57% by the staphylococcus aureus. Pseudomonas aeruginosa and Escherichia coli, the inhibitory effect of which is reduced by about 26%. The drug effect lasts for 24 hours, and when the concentration of the extract is 2mg/mL, the inhibition effect on escherichia coli and typhoid bacillus is the best, and is reduced by about 4.5%. The inhibiting effect on staphylococcus aureus and staphylococcus albus is the second time.

Example 6

This example is a specific example of the determination of the effect of product 1 prepared in example 1 on the gastrointestinal tract of mice.

Clean-grade Kunming mouse 60 mice, half male and half female, body weight (18 + -2) g. 20 of the samples were randomly selected as normal control groups, and the other 40 samples were divided into 4 groups, namely a model group, a natural recovery group, a Lizhu Changle treatment group and a product 1 extract treatment group, after the model preparation was successfully carried out by intragastric gavage with lincomycin hydrochloride. The 20 normal control groups were perfused with the same amount of physiological saline, and 40 mice except the normal control group were perfused with lincomycin hydrochloride for 3 days, 2 times a day, 0.3mL each time. Taking the specimen to carry out various index detections.

The results are shown in Table 3, 10 mice were tested, the model group was mice perfused with lincomycin hydrochloride, the control group was mice perfused with physiological saline in equal amount, the mice were continuously perfused with lincomycin hydrochloride for 3 days, and then the ileum and cecum contents were aseptically diluted to 10 degrees^-9100 microliters of each of the cells were inoculated into the selective medium and counted (conventional counting method), and the results are shown in Table 3. The number of lactobacillus is reduced, and the number of enterococcus and enterobacteria is increased; p in comparison with the Normal control group<0.05, indicating that the intestinal tract of the mouse has undergone dysbiosis.

TABLE 3 results of intestinal flora assay in normal control group and model group

Group of	Example number (n)	Lactobacillus strain	Enterobacter	Enterococcus
					Model set	10	10.61±0.85	11.49±1.18	10.44±1.11
Control group	10	11.75±0.91	10.33±0.91	9.21±0.94

The unit of Lactobacillus, Enterobacter, and enterococcus in Table 3 is cfu/mL.

And performing natural recovery, Lizhu Changle intragastric administration treatment and product 1 extract treatment on the constructed model group, and respectively performing intragastric administration by using product 1 extract and Lizhu Changle physiological saline diluent after the model group is constructed, wherein the natural recovery group is infused with the same amount of physiological saline for 2 times every day and each time is 0.3 mL. The mice were sacrificed after continuous gavage for 7 days, and specimens were taken for various index tests. The results are shown in Table 4.

The number of enterobacter livers of the model group mice (11.49 +/-1.18) is increased compared with that of the normal control group (10.33 +/-0.91), and the difference is statistically significant (P < 0.01). The quantity of enterobacteria in the liver of the mice in the product 1 extract treatment group is reduced, the difference has statistical significance (P <0.05) compared with the natural recovery group, and the quantity of enterococcus in the product 1 extract treatment group is reduced more remarkably than that in the lizhu enterole group. The animal model is utilized, and the experimental result shows that the extract can adjust the immune function of mice, promote the proliferation of beneficial bacteria in vivo, and prove that the extract has the function of adjusting intestinal flora, so that the extract does not have the harmful stimulation to gastrointestinal tracts.

TABLE 4 comparison of enterobacter hepatomere translocation assay results in various groups of mice after drug treatment

Group of	Number of examples	Enterobacter cfu/mL	Enterococcus cfu/mL
				Natural recovery group	10	5.42±0.54	7.82±0.83
Lizhu Changle group	10	3.24±0.35	6.43±0.66
				Product 1 extract treatment group	10	3.37±0.29	6.15±0.71

(Note: P <0.05 compared with the Natural recovery group)

The technical scheme can show that the composition, the extraction method and the application thereof in the anti-inflammatory field have the following advantages:

1. the extraction method is time-saving and high in extraction rate, the ultrasonic extraction utilizes mechanical effect and cavitation effect, the cell wall is quickly destroyed, the effective components are dissolved out, the heat effect can also maintain the biological activity of the extract, and the loss of the extract is reduced; the extraction process is simple, easy to operate and especially suitable for large-scale industrial production.

2. Freeze drying to reduce heat consumption, and maintaining the chemical composition of the material without surface tension. The freeze drying can realize drying and crushing at the same time.

3. The obtained product has strong antibacterial property, good stability, wide action range, high efficiency and safety through experimental determination; the raw materials are directly prepared and extracted from plant materials, the source is rich, the fertility is strong, the growth speed is high, the prepared product is low in price and low in production cost, and the method can be widely applied to the production of medicines and health care products.

In summary, the present invention provides a composition, which comprises the following raw materials: garlic, purslane and trifoliate acanthopanax. The invention also provides an extraction method of the composition, which comprises the following steps: step one, garlic, purslane and trifoliate acanthopanax are mixed, dried, crushed and sieved to obtain a first product; step two, after the first product is degreased, soaking the degreased first product in 75% ethanol to obtain a second product; step three, after the second product is subjected to ultrasonic treatment, filtering and collecting filtrate, and concentrating the filtrate under reduced pressure to obtain a third product; and step four, drying the third product to obtain the product. The invention also provides an application of the composition or the product obtained by the extraction method in the anti-inflammatory field. In the technical scheme provided by the invention, the natural extract has the advantages of strong antibacterial property, lasting action efficiency and wide action range, and meanwhile, the three extracts have a certain synergistic effect and also have the comprehensive gain effects of clearing away heat and toxic materials, enhancing immunity and resisting fatigue. The composition, the extraction method and the application thereof in the anti-inflammatory field solve the technical defects that the artificially synthesized anti-inflammatory drug has narrow application range, short lasting efficacy, harm stimulation to gastrointestinal tract and easy generation of drug resistance in the prior art.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A composition characterized in that the raw materials of said composition comprise: garlic, purslane and trifoliate acanthopanax.

2. The composition according to claim 1, wherein the composition comprises the following raw materials in parts by mass: 3-6 parts of garlic, 3-6 parts of purslane and 3-6 parts of trifoliate acanthopanax.

3. An extraction process comprising the composition of claim 1 or 2, characterized in that it is:

and step four, drying the third product to obtain the product.

4. The extraction method according to claim 3, wherein in the first step, the drying temperature is 40-70 ℃, and the drying time is 20-120 min;

in the first step, the sieving is performed by sieving with a 20-40 mesh sieve.

5. The extraction method according to claim 3, wherein in the second step, the feeding ratio of the first product to the ethanol solution is 1 (10-20).

6. The extraction method according to claim 3, wherein in the third step, the temperature of the ultrasonic treatment is 20-45 ℃, and the time of the ultrasonic treatment is 10-60 min.

7. The extraction method according to claim 3, wherein the temperature of the vacuum concentration in the third step is 30 to 50 ℃.

8. The method according to claim 3, wherein in step four, the drying method is freeze drying.

9. The extraction method according to claim 8, wherein the temperature of the freeze-drying is-80 to-50 ℃ in the fourth step, and the time of the freeze-drying is 10 to 12 hours.

10. Use of a composition according to any one of claims 1 to 2 or a product obtained by the extraction process according to any one of claims 3 to 9 in the anti-inflammatory field.