CN112370440B

CN112370440B - Thyme-rosemary anti-diarrhea preparation as well as preparation method and application thereof

Info

Publication number: CN112370440B
Application number: CN202011286783.6A
Authority: CN
Inventors: 汪晶; 李润林; 朱伟云
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-11-17
Filing date: 2020-11-17
Publication date: 2023-02-03
Anticipated expiration: 2040-11-17
Also published as: CN112370440A

Abstract

The invention discloses a thyme-rosemary anti-diarrhea preparation, a preparation method and application thereof, wherein the preparation comprises the following components in a concentration ratio of 1-2: 1 a thymol preparation and a rosmarinic acid preparation, the preparation method comprising the steps of: (1) Extracting dry leaves of thyme by a pressure extraction method to prepare a thymol preparation; (2) Extracting rosemary leaves by a solvent extraction method to prepare a rosmarinic acid preparation; (3) Mixing the two preparations, then whirling for 30-60 s, and then placing in a shaking table for 2-3 h to prepare the thyme-rosemary anti-diarrhea preparation; the preparation can be used as feed additive in feed. The preparation is nontoxic and non-drug-resistant, reduces the pollution of livestock and poultry feces to the environment, has strong bactericidal capacity, stronger bacteriostatic capacity, obviously enhanced in-vitro free radical scavenging capacity, improved in-vitro fermentation parameters, promotes microbial fermentation, can effectively improve intestinal micro-ecological balance and improve the body resistance, and therefore can be added into feed.

Description

Thyme-rosemary anti-diarrhea preparation as well as preparation method and application thereof

Technical Field

The invention relates to an anti-diarrhea preparation as well as a preparation method and application thereof, and in particular relates to a thyme-rosemary anti-diarrhea preparation as well as a preparation method and application thereof.

Background

Because a stable micro-ecosystem is not established in the intestinal tract of the piglet, the piglet has low self-resistance, is sensitive to external stimulation and is easily affected by invasion of various pathogenic microorganisms and various stress factors, infectious diarrhea is common for suckling piglets, but diarrhea caused by non-infectious factors such as daily ration antigen allergy, weaning, sudden feed replacement, cold, environmental stress and the like is mainly used for nursing piglets, sick pigs and pigs with viruses are main infection sources, viruses are discharged from excrement to pollute the environment and disseminate and infect, a pig group fed in early winter at the end of autumn and with high humidity, low temperature and high density is more easily infected with the disease, the pigs in the whole field are attacked within a few days, and huge economic loss is brought.

Disclosure of Invention

The invention aims to: the invention aims to provide a thyme-rosemary anti-diarrhea preparation which has small side effect, does not cause adverse reaction of organisms and does not generate drug resistance, and also aims to provide a preparation method and application of the preparation.

The technical scheme is as follows: the thyme-rosemary anti-diarrhea preparation comprises the following components in a concentration ratio of 1-2: 1. thymol preparations and rosmarinic acid preparations.

The preparation method of the thyme-rosemary anti-diarrhea preparation comprises the following steps:

(1) Extracting dry leaves of thyme by a pressure extraction method to prepare a thymol preparation;

(2) Extracting rosemary leaves by a solvent extraction method to prepare a rosmarinic acid preparation;

(3) Mixing the two preparations, then whirling for 30-60 s, and then placing in a shaking table for 2-3 h to prepare the thyme-rosemary anti-diarrhea preparation.

Wherein, step 1 includes the following steps:

(11) Pulverizing dried leaves of herba Thymi, sieving, and bagging;

(12) Adding ethanol solution into the bag, sealing, and maintaining the pressure in a high-pressure device at 350-400 MPa for 5-10 min;

(13) Centrifuging the extracted mixture in the bag, and performing rotary evaporation to obtain thymol;

(14) Dissolving thymol in dimethyl sulfoxide to obtain thymol preparation.

Wherein, the dried thyme leaves in the step 11 are crushed and then screened by a 60-80 mesh sieve, the concentration of the ethanol solution in the step 12 is 83-97%, and the mass ratio of the volume of the added ethanol solution to the dried thyme leaf powder is 40-50 mL:1g of a compound; in step 14, thymol is dissolved in dimethyl sulfoxide and then filtered by a filter membrane with the diameter of 0.22-0.28 μm.

Wherein, step 2 includes the following steps:

(21) Pulverizing herba Rosmarini officinalis leaf, and sieving;

(22) Adding an ethanol solution into the sieved rosemary powder, and leaching to obtain an extract, wherein the leaching time is 6-8 hours;

(23) Filtering the extract with a filter membrane, adding n-butanol, extracting, and recovering n-butanol;

(24) Adjusting the pH value of the recovered n-butyl alcohol to 2-2.5, adding ethyl acetate for extraction, eluting the extract liquor by using an ethanol solution, collecting filtrate by using X-5 macroporous resin, crystallizing at low temperature, filtering and drying to obtain rosmarinic acid;

(25) Dissolving rosmarinic acid in dimethyl sulfoxide to obtain rosmarinic acid preparation.

Wherein, the rosemary leaves in the step 21 are sieved by a sieve of 20 to 30 meshes after being crushed; in the step 22, the concentration of the ethanol solution is 50-60%, and the mass ratio of the volume of the added ethanol solution to the rosemary powder is 15-20 mL:1g, and the leaching times are 2-3 times; adjusting the pH of the extracting solution to 2-3 before adding n-butyl alcohol for extraction in the step 23, wherein the added n-butyl alcohol solution is 1.5-2 times of the volume of the extracting solution, the extraction time is 20-30 min, and the extraction times are 2-4; in the step 24, the ethyl acetate extraction time is 20-30 min, the extraction times are 3-5 times, the usage amount of the ethyl acetate is 1.5-2 times of the volume of the recycled n-butanol, and in the step 25, the rosmarinic acid is dissolved in the dimethyl sulfoxide and then filtered by a filter membrane of 0.22-0.28 μm.

Wherein, the rotating speed of the shaking table in the step 3 is 150-200 rpm, and the temperature is 30-40 ℃.

The thyme-rosemary anti-diarrhea preparation provided by the invention is applied to feed as a feed additive.

The working principle is as follows: the thymol plays a role in bacteriostasis mainly because hydrophobic bonds and hydrogen bond binding membrane proteins contained in the thymol change the permeability of bacterial cell membranes to crack and die bacteria, and the thymol has an enhanced inhibiting effect on escherichia coli, staphylococcus aureus and the like under a low pH condition. Rosmarinic acid is a water-soluble polyphenol hydroxy acid with strong antioxidant and free radical scavenging abilities, which may be related to its catechol hydroxyl group. The weakly acidic and phenolic derivatives of rosmarinic acid can destroy bacterial cell membrane and change cell membrane permeability. The thymol and the rosmarinic acid are matched for use, so that damage to the permeability of a bacterial cell membrane can be enhanced, inhibition to bacterial growth is increased, and the weak acid environment of the rosmarinic acid derivative is more favorable for exerting the bacteriostatic action of the thymol, so that the thymol and the rosmarinic acid possibly have better bacteriostatic ability when being used together. In addition, both of them have strong ability of scavenging free radicals, and the combination of both of them can also enhance the oxidation resistance level. The in-vitro bacteriostatic test result of the thymol and rosmarinic acid combined preparation shows that the thymol and rosmarinic acid combined preparation has strong inhibition capacity on escherichia coli, salmonella, staphylococcus aureus and the like, the FICI value of the combined use of the thymol and rosmarinic acid is less than or equal to 0.5, and the thymol and rosmarinic acid have a synergistic effect, so that the thymol and rosmarinic acid combined preparation has a better effect of inhibiting intestinal pathogenic bacteria, and has a stronger anti-diarrhea effect. Results of in vitro bacteriostatic tests and in vitro antioxidant tests show that the combined preparation has increased inhibition capability on pathogenic bacteria at low dose, and the in vitro free radical scavenging capability is also obviously higher than that of a single plant extract group. Rat experiments also show that the combined preparation of the two under the dosage of 1/2 is superior or obviously superior to a single plant extract group in the aspects of growth performance, diarrhea rate, intestinal antioxidant index, inflammatory factor and the like of LPS challenge rats. The thymol and rosmarinic acid combined preparation not only has the capability of inhibiting pathogenic bacteria, but also has the effect of antibiotics, can improve the oxidation resistance of organisms, inhibit inflammation and the like, and promote the growth and development of animals. The thymol and rosmarinic acid combined preparation can improve in-vitro fermentation parameters, improve or remarkably improve the content of short-chain fatty acids (SCFAs) in a fermentation system, and has the effect of promoting microbial fermentation.

Has the beneficial effects that: compared with the prior art, the invention has the following remarkable advantages: 1. the additive is non-toxic and non-drug resistant, and reduces the pollution of the livestock and poultry manure to the environment; 2. the microbial fermentation liquid has strong bactericidal capacity and bacteriostatic capacity, remarkably enhances the in vitro free radical scavenging capacity, improves in vitro fermentation parameters, and promotes microbial fermentation; 3. can improve intestinal microecological balance and enhance body resistance, and can be used as additive in feed.

Drawings

FIG. 1 is a graph of the change in body weight of rats: (a) a rat weight percent change curve; (b) histogram of daily weight gain of rat;

FIG. 2 is a graph showing the comparison of diarrhea rates in rats;

FIG. 3 is a graph showing the effect of control, thymol preparation, rosmarinic acid preparation and thyme-rosemary anti-diarrhea preparation on the content of intestinal inflammatory factors in LPS challenged rats: (a) the MPO content of the ileum, caecum and colon; (b) ileal IL-6, IL-10 and TNF- α levels; (c) the amount of cecal IL-6, IL-10 and TNF- α; (d) colonic IL-6, IL-10 and TNF- α levels.

Detailed Description

Preparation of thymol preparation:

crushing dried thyme leaves, sieving by a 60-mesh sieve, weighing 100.0g of thyme leaf powder, sealing in a polyethylene bag, adding an ethanol solution with the volume concentration of 42 times of 42 percent, placing the mixture into ultrahigh pressure equipment, extracting under the condition of the pressure of 380 MPa, pressurizing for 6min, centrifuging for 5min at 4000r/min after extraction is finished, immediately rotating and evaporating to remove ethanol to obtain thymol, weighing 4.00g of thymol into a centrifugal tube, adding 10mL of dimethyl sulfoxide, dissolving and uniformly mixing, placing the mixture in a shaking table at 37 ℃ and 150rpm for 2h, taking out the mixture at intervals of 30min, uniformly mixing for 30s, taking out the mixture, fixing the volume to 10mL by sterile water, filtering the mixture through a 0.22 mu m filter membrane to prepare a thymol preparation with the volume of 400mg/mL, and storing the thymol preparation in a refrigerator at 4 ℃ for later use.

Preparation of rosmarinic acid preparation:

the preparation method comprises the following steps of crushing rosemary leaves, sieving the rosemary leaves by a 20-mesh sieve, weighing 100.0g of sieved rosemary leaf powder, sealing the rosemary leaf powder in a polyethylene bag, adding an ethanol solution with the volume concentration of 15 times and 50%, leaching twice, wherein the leaching time is 6 hours, collecting a leaching solution, pretreating the extracting solution by using a microfiltration membrane, filtering for three times, removing suspended particles in the extracting solution, adjusting the pH of filtrate to 3 by using hydrochloric acid, extracting by using 1.5 times of volume of n-butyl alcohol, and extracting under the conditions that: extracting twice, each time being 20min, recovering n-butanol, adding high pure water into the recovered n-butanol, recording the volume, adjusting the pH to 2.0-2.5 by hydrochloric acid, extracting for 3 times by ethyl acetate with the volume ratio of 10. Weighing 4.00g of rosmarinic acid into a centrifuge tube, adding 10mL of dimethyl sulfoxide to dissolve and mix uniformly, placing the mixture on a shaking table for 2h at 37 ℃ and 150rpm, taking out the mixture at intervals of 30min and mixing uniformly for 30s, taking out the mixture, using sterile water to fix the volume to 10mL, filtering the mixture through a 0.22 mu m filter membrane to prepare a rosmarinic acid preparation of 400mg/mL, and storing the rosmarinic acid preparation in a refrigerator at 4 ℃ for later use.

Preparation of thyme-rosemary anti-diarrhea preparation:

diluting the rosmarinic acid preparation and the thymol preparation to 40mg/mL, mixing the two combined preparations according to the concentration of 1.

In vitro bacteriostatic experiments were performed on thymol, rosmarinic acid and thyme-rosemary anti-diarrhea formulations, respectively, and the results are shown in table 1:

TABLE 1 results of in vitro bacteriostatic experiments on rosmarinic acid, thymol and thyme-rosemary anti-diarrhea preparations

The calculation formula of the FICI is as follows: FICI = MIC A (A + B)/MIC A + MIC B (A + B)/MIC B, MIC A (A + B) is the minimum inhibitory concentration of A when A and B are used together; MIC a represents the minimum inhibitory concentration of a when used alone; MIC B (A + B) is the minimum inhibitory concentration of B when A and B are used together; MIC B is the minimum inhibitory concentration for B alone. When the FICI is less than or equal to 0.5, the compounded components show a synergistic effect; when FICI is more than 0.5 and less than or equal to 1, the compound components show the superposition effect; when the FICI is more than 1 and less than 2, the compound components show no relevant effect; when the FICI is more than or equal to 2, the compound components show antagonism. From Table 1, the MIC values of rosmarinic acid to three bacteria are 1.00mg/mL, 0.50mg/mL and 1.00mg/mL respectively; the MIC values of thymol for all three bacteria were 1.00mg/mL. The combined bacteriostasis result shows that when the concentrations of thymol and rosmarinic acid are respectively 0.25mg/mL, the bacteriostasis of the thymol and the rosmarinic acid to three bacteria is shown as a synergistic effect when the thymol and the rosmarinic acid are used together.

The influence of thymol preparation, rosmarinic acid preparation and thyme-rosemary anti-diarrhea preparation on the in vitro fermentation level of pig colon microorganisms was simulated by using the in vitro fermentation method, and the results are shown in table 2:

TABLE 2 simulated fermentation test results of rosmarinic acid, thymol and thyme-rosemary anti-diarrhea preparations

From table 2, it can be seen that the anti-diarrhea preparation significantly improves the in vitro fermentation parameters, significantly reduces the pH value of the fermentation system, and significantly improves the gas production of the fermentation system, indicating that the anti-diarrhea preparation can promote the growth of colonic microorganisms and increase the fermentation power. And the combined preparation can also improve the content of volatile fatty acid (acetic acid, propionic acid and butyric acid) in a fermentation system to a certain extent. The fermentation parameters and the generation amount of volatile fatty acid of the extract anti-diarrhea preparation are both better than those of a single plant extract.

The animal tests of the thymol preparation, the rosmarinic acid preparation and the thyme-rosemary anti-diarrhea preparation are carried out in an animal test center of Nanjing agriculture university for 12 months to 2020 month in 2019 and 1 month in 2020. The test was carried out on 40 weaned SD rats (21 days old and weighing about 50 g) divided into 4 treatments, a control group (Con), a thymol group (Thy), a thymol X rosmarinic acid group (Ros-A + Thy) and a rosmarinic acid group (Ros-A), each of which was treated on 10 rats weighing about days old, half of the male and the female. The gavage amount of the plant extract is shown in table 3, and the plant extract was weighed daily and gavage once. During the test period, the food and the water are taken freely, the pre-test period is 7 days, the formal test period is 14 days, the total period is 21 days, and the food and the water are taken freely during the test process. On the 21 st day of the experiment, all rats were intraperitoneally injected with 1mg/kg LPS, and 8 hours after the injection of lipopolysaccharide, all rats were sacrificed by decapitation and sampled.

TABLE 3 addition amounts of rosmarinic acid, thymol and thyme-rosemary anti-diarrhea formulations

During the test period, the food intake is recorded every day, and the food intake is weighed and recorded at 8 points in the morning on the 1 st, 4 th, 7 th, 10 th, 11 th, 12 th, 13 th and 14 th days of the test respectively; after the LPS is attacked, observing the diarrhea condition of each weaned rat in the 2 nd, 6 th, 8 th, 10 th and 12 th hours of the experiment respectively, and recording the diarrhea index and the diarrhea rate according to the excrement state; after the rats are slaughtered, weighing small intestines (including duodenum, jejunum, ileum and chyme thereof), taking the ileum, the cecum middle section and the colon middle section of each group, performing HE dyeing, measuring and taking the height of villus and the depth of crypt, and calculating the ratio of the height of the villus to the depth of the crypt for evaluating thymus-rosemary anti-diarrhea preparations and the protection effect on intestinal morphology after the administration of LPS; the kit is adopted to measure the contents of SOD, MDA, T-AOC and GSH-px in intestinal mucosa of each group of rats, and is used for evaluating the protection efficacy of thyme-rosemary anti-diarrhea preparation and lipopolysaccharide on an intestinal antioxidant system after attacking toxin; the kit is adopted to measure the contents of IL-6, IL-8, IL-10 and TNF-alpha in intestinal mucosa of each group of rats so as to evaluate the protective effect of the thyme-rosemary anti-diarrhea preparation on the intestinal inflammation system of the rats after the lipopolysaccharide is attacked.

The effect of the control, rosmarinic acid, thymol and thyme-rosemary anti-diarrhea preparations on rat weight gain is shown in fig. 1 (a) and 1 (b), and compared with the control, the growth performance of rats can be improved by using thymol, rosmarinic acid and thyme-rosemary anti-diarrhea preparations, but the growth promoting effect of the thyme-rosemary anti-diarrhea preparation is better than that of the single plant extract treatment after the 7 days of gastric lavage. The daily gain of the thyme-rosemary anti-diarrhea preparation group rats is significantly higher than that of the control group and the rosmarinic acid group. The effect of the control, rosmarinic acid, thymol and thyme-rosemary anti-diarrhea preparations on the diarrhea rate of rats is shown in fig. 2, and the diarrhea rate of weaned rats with gavage plant extracts was reduced compared to the control, wherein the diarrhea rate of the rats with thyme-rosemary anti-diarrhea preparation was significantly lower than that of the control (P < 0.05). Furthermore, the diarrhea rate of the thyme-rosemary anti-diarrhea preparation group rats is also lower than that of the single plant extract group, but does not reach a significant level (P > 0.05). The effects of the control, rosmarinic acid, thymol and thyme-rosemary anti-diarrhea preparations on the intestinal morphological structure of LPS challenged rats are shown in table 4:

TABLE 4 Effect of control, rosmarinic acid, thymol and thyme-rosemary anti-diarrhea preparations on the intestinal morphology of LPS challenged rats

As can be seen from table 4, the gavage plant extract can significantly improve the morphological structure of jejunum, ileum and cecum of rats compared to the control group. Wherein, the thyme-rosemary anti-diarrhea preparation group can obviously improve the villus height of the jejunum and the ileum of the rat and obviously reduce the crypt depth (P < 0.05), and the ratio of the thyme-rosemary anti-diarrhea preparation group to the jejunum and the ileum of the rat is also obviously higher than that of the control group and the single thymol group and rosmarinic acid group (P < 0.05). In addition, the thyme-rosemary anti-diarrhea preparation group rats also had significantly higher cecal villus height and mucosal thickness than the control group and the rosmarinic acid group (P < 0.05), but did not differ significantly from the thymol group (P > 0.05).

The influence of the control group, the rosmarinic acid preparation, the thymol preparation and the thyme-rosemary antidiarrheal preparation on the antioxidant index of the rat attacked by LPS is shown in Table 5, and compared with the control group, the thyme-rosemary antidiarrheal preparation group can obviously improve the T-SOD level (P is less than 0.05) of ileum, jejunum, caecum and colon. The thyme-rosemary antidiarrheal preparation group can also obviously improve the levels of T-SOD, T-AOC and GSH-PX of the liver (P is less than 0.05). And the antioxidant level of the intestinal tract and the liver of the thyme-rosemary anti-diarrhea preparation group rats is slightly higher or obviously higher than that of the single plant extract group.

TABLE 5 Effect of control, rosmarinic acid, thymol and thyme-rosemary anti-diarrhea preparations on the antioxidant index of LPS challenged rats

As can be seen from fig. 3 (a), 3 (b), 3 (c), 3 (d), the rosmarinic acid, thymol, and thyme-rosemary anti-diarrhea formulations all reduced the MPO levels in the ileum, caecum, and colon of LPS challenged rats compared to the control group. Wherein the thyme-rosemary anti-diarrhea preparation group can obviously or extremely obviously reduce MPO content (P < 0.05) of three intestinal segments. Compared with the control group, the three treatment groups can reduce the content of the intestinal inflammatory factors TNF-alpha and IL-6. The thyme-rosemary antidiarrheal preparation group was able to significantly reduce the levels of TNF-alpha and IL-6 in the ileum, caecum, colon (P < 0.05) compared to the control group, and the thyme-rosemary antidiarrheal preparation group was also significantly lower in IL-6 content in the ileum and colon than the thymol group (P < 0.05), but was not significantly different compared to the rosmarinic acid group. The thyme-rosemary anti-diarrhea formulation group was able to significantly increase the IL-10 content in ileum and colon (P < 0.05) compared to the control group.

The total bacteria number of the intestinal tract reflects the steady state of the intestinal tract, and the increase of the total bacteria number indicates that the steady state of microorganisms in the intestinal tract is improved. In the experiment, the total bacteria number in the empty, ilex, blindness and colon sections is obviously higher than that in the control group, and is higher than or obviously higher than that in the thymol preparation group and the rosmarinic acid preparation group, which shows that the thymol and the rosmarinic acid anti-diarrhea preparation can improve the steady state of the intestinal flora of LPS challenge rats. Escherichia coli is a conditional pathogen with a large number in intestinal tracts, and the increase of the number of Escherichia coli or Enterobacter flora can cause diseases such as diarrhea and the like. The lactobacillus is beneficial bacteria in intestinal tract, the lactic acid generated by the metabolism of the lactobacillus can be utilized by organisms, the pH value of the intestinal tract can be reduced, and the growth of harmful bacteria can be inhibited, and the influence of a control group, a rosmarinic acid preparation, a thymol preparation and a thyme-rosemary anti-diarrhea preparation on the number of intestinal flora of LPS challenge rats is shown in Table 6: compared with a control group, the thyme-rosemary anti-diarrhea preparation group can obviously improve the number of lactobacillus in each intestinal section and obviously reduce the number of escherichia coli (P < 0.05), and compared with a single plant extract group, the thyme-rosemary anti-diarrhea preparation group also reduces or obviously reduces the number of escherichia coli and improves or obviously improves the number of lactobacillus. The thyme-rosemary anti-diarrhea preparation group is shown to be capable of increasing the number of beneficial bacteria in the intestinal tract, reducing the number of conditional pathogenic bacteria escherichia coli, adjusting the flora structure of the intestinal tract and improving the health level of the intestinal tract.

TABLE 6 Effect of control, rosmarinic acid, thymol and thyme-rosemary anti-diarrhea preparations on the number of intestinal flora in LPS challenged rats

Claims

1. A thyme-rosemary anti-diarrhea preparation is characterized by comprising components with the concentration ratio of 1:1 and rosmarinic acid preparation.

2. A method of preparing the thyme-rosemary anti-diarrhea preparation of claim 1 comprising the steps of:

(3) Mixing the two preparations, and then swirling for 30 to 60s, and then placing in a shaking table for 2 to 3h to prepare the thyme-rosemary anti-diarrhea preparation.

3. The method of preparing a thyme-rosemary anti-diarrhea preparation according to claim 2, wherein step 1 comprises the steps of:

(11) Pulverizing dried leaves of herba Thymi, sieving, and packaging;

(12) Adding an ethanol solution into the bag, sealing, and putting the bag into high-pressure equipment for 5 to 10min under the pressure of 350 to 400 MPa;

(14) Dissolving thymol in dimethyl sulfoxide to obtain thymol preparation.

4. The preparation method of the thyme-rosemary anti-diarrhea preparation according to claim 3, wherein the dried thyme leaves in the step 11 are ground and sieved through a sieve of 60 to 80 meshes, the concentration of the ethanol solution in the step 12 is 83 to 97 percent, and the mass ratio of the volume of the added ethanol solution to the dried thyme leaf powder is 40 to 50 mL:1g of a compound; in the step 14, the thymol is dissolved in dimethyl sulfoxide and filtered by a filter membrane of 0.22 to 0.28 mu m.

5. The method for preparing thyme-rosemary anti-diarrhea preparation according to claim 2, wherein the step 2 comprises the following steps:

(21) Pulverizing herba Rosmarini officinalis leaf, and sieving;

(22) Adding an ethanol solution into the sieved rosemary powder, and leaching for 6 to 8 hours to obtain an extracting solution;

(24) Adjusting the pH value of the recycled n-butanol to 2-2.5, adding ethyl acetate for extraction, eluting the extract by using an ethanol solution, collecting filtrate by using X-5 macroporous resin, crystallizing at low temperature, filtering and drying to obtain rosmarinic acid;

6. The preparation method of the thyme-rosemary anti-diarrhea preparation according to claim 5, wherein the leaves of rosemary in the step 21 are ground and sieved with a 20 to 30 mesh sieve, the concentration of the ethanol solution in the step 22 is 50 to 60 percent, and the mass ratio of the volume of the added ethanol solution to the rosemary powder is 15 to 20mL:1g, and the leaching times are 2 to 3.

7. The method for preparing the thyme-rosemary anti-diarrhea preparation according to claim 5, wherein the pH of the extracting solution is adjusted to 2 to 3 before the n-butanol is added in the step 23 for extraction, the added n-butanol solution is 1.5 to 2 times of the volume of the extracting solution, the extraction time is 20 to 30min, and the extraction times are 2 to 4 times.

8. The method for preparing the thyme-rosemary anti-diarrhea preparation according to claim 5, wherein the ethyl acetate extraction time in the step 24 is 20 to 30min, the extraction times are 3 to 5, the usage amount of the ethyl acetate is 1.5 to 2 times of the volume of the recycled n-butanol, and the rosmarinic acid in the step 25 is dissolved in dimethyl sulfoxide and then filtered by a 0.22 to 0.28 μm filter membrane.

9. The method for preparing the thyme-rosemary anti-diarrhea preparation according to the claim 2, wherein the shaking table rotation speed in the step 3 is 150 to 200rpm, and the temperature is 30 to 40 ℃.

10. Use of a thyme-rosemary anti-diarrhea preparation according to claim 1 in the preparation of a feed additive.