CN114601880A

CN114601880A - Valeriana jatamansi jones compound preparation for resisting piglet diarrhea

Info

Publication number: CN114601880A
Application number: CN202210400053.7A
Authority: CN
Inventors: 李小平; 王一萍
Original assignee: Suzhou Polytechnic Institute of Agriculture
Current assignee: Suzhou Polytechnic Institute of Agriculture
Priority date: 2022-04-15
Filing date: 2022-04-15
Publication date: 2022-06-10

Abstract

The invention discloses a spider aroma compound preparation for resisting piglet diarrhea, which comprises Chinese herbal medicine extracting solutions, wherein the Chinese herbal medicine extracting solutions comprise spider aroma extracting solution, isatis root extracting solution and fructus forsythiae extracting solution with equal mass. The Chinese herbal medicine extract also comprises dyers woad leaf extract with equal mass, and the compound preparation also comprises berberine hydrochloride with equal mass. The invention extracts the valeriana jatamansi jones total flavone by ultrasonic, and the yield of the total flavone can reach 3.410%. The spider aroma compound preparation for resisting piglet diarrhea has a synergistic effect in the aspect of antibacterial activity, can effectively antagonize diarrhea caused by escherichia coli enterotoxin while effectively inhibiting escherichia coli, and has inhibiting and resisting effects on an inflammation process and drug diarrhea.

Description

Valeriana jatamansi jones compound preparation for resisting piglet diarrhea

Technical Field

The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a spider aroma compound preparation for resisting piglet diarrhea.

Background

The design of the extraction process of the Chinese herbal medicine needs to consider the characteristics of the traditional Chinese medicine theoretical basis, the functional indications, the chemical components and the like of the prescription. The design needs to pay attention to the monarch, minister, assistant and guide of the prescription. The major compound and the compound of the three or four classes of medicines should be extracted by compound extraction and classification, such as volatile oil, common class, flavonoid, alkaloid, organic acid and polysaccharide, according to the chemical components of the medicines. The consideration of both the drug effect and the chemical composition is important. The extraction process route is designed to ensure the safety and curative effect of the extract, and the effective components are enriched as much as possible to remove ineffective impurities. The yield of the extract is reduced as much as possible on the premise of ensuring safety and effectiveness.

Chinese herbal medicine is a precious heritage of excellent culture and traditional medicine in China, and is far from the international advanced level in terms of the foundation, accumulation, current situation and overall strength of medicine research in China, but the biological resources in China are rich, and the strengthening of the development of natural medicines is an important decision for reducing the gap and reducing the influence on the veterinary medicine industry brought by the coming-in of the world. The screening and deep systematic research of Chinese herbal medicines are carried out, the veterinary Chinese herbal medicine preparation with strong applicability and good curative effect is developed and developed, the preparation is the most effective way for producing the medicine with the own intellectual property rights of Chinese people, and the preparation has very important significance for fully utilizing rich Chinese herbal medicine resources in China and promoting the production of Chinese veterinary medicine industry and the development of animal husbandry.

The diarrhea is the most common disease in the piglet stage, is a typical multifactorial disease, and causes piglet diarrhea with various pathogenic types and complicated disease properties. The diarrhea can be generally classified into bacterial diarrhea such as yellow scour and white scour of piglets caused by Escherichia coli, red scour of piglets caused by Clostridium welchii, etc., viral diarrhea such as transmissible gastroenteritis, epidemic diarrhea, rotavirus disease, pseudorabies, parasitic diarrhea such as coccidiosis, ascariasis, etc., non-pathogenic diarrhea such as stress (weaning, group transfer, material change, etc.), temperature (sudden cooling of weather), hypoglycemia, excessive crude protein content of daily ration, vitamin and mineral deficiency, electrolyte imbalance, etc. The diarrhea of the piglets causes low feed conversion rate, the survival rate of the piglets is reduced, the growth and development are slow, even the piglets are stagnated (dead pigs), serious people can die, and great economic loss is brought to the pig raising industry, so that how to prevent and treat the diarrhea of the piglets becomes an attractive worldwide problem.

In recent years, a plurality of research institutions at home and abroad turn to extracting effective components from pure natural plants (including Chinese herbal medicines) and developing safe and efficient feed additives, regulating and controlling the internal environment of the digestive tract of livestock and poultry, and replacing the use of feed antibiotics and synthetic antibacterial drugs. However, the current patent products of the Chinese herbal medicine feed additive at home and abroad still have the following defects: (1) the effect of single Chinese herbal medicine and single component is over-emphasized, and dialectical analysis (treatment) can not be carried out according to the actual feeding, so that good feeding effect can not be obtained; (2) the composition research cannot be started from the basic theory of traditional Chinese medicine, and the requirements on various effective components are comprehensively considered; (3) most products still stay at the level of directly feeding Chinese herbal medicines, have large dosage, difficult control of quality indexes and high cost, and are not beneficial to application and popularization in the modern pig-raising mode of intensive scale.

Disclosure of Invention

The invention aims to provide a spider aroma compound preparation for resisting piglet diarrhea, which can safely and efficiently replace the commonly used feed antibiotics and chemically synthesized drug feed additives at present, can be used for symptomatic medicine administration according to the causes of piglet diarrhea, inflammation and the like, and improves the curative effect.

In order to achieve the purpose, the invention provides a spider aroma compound preparation for resisting piglet diarrhea, which comprises Chinese herbal medicine extracting solutions, wherein the Chinese herbal medicine extracting solutions comprise equal mass of spider aroma extracting solution, isatis root extracting solution and forsythia extracting solution.

Furthermore, the Chinese herbal medicine extract also comprises folium isatidis extract with equal mass.

Furthermore, the berberine hydrochloride also comprises berberine hydrochloride with equal mass, and the volume fraction of the berberine hydrochloride is 1 percent.

The beneficial effect who adopts above-mentioned scheme is: berberine hydrochloride, also known as berberine hydrochloride, has effects of clearing away heat and toxic materials, resisting bacteria and diminishing inflammation.

Further comprises valeriana jatamansi extract, isatis root extract, forsythia extract and berberine hydrochloride in a mass ratio of 1:1:1: 1.

Further comprises valeriana jatamansi extract, isatis root extract, forsythia suspensa extract, dyers woad leaf extract and berberine hydrochloride in a mass ratio of 1:1:1:1: 1.

Further, the valeriana jatamansi jones extract is prepared by the following method:

(1) pulverizing radix Pimpinellae Candolleanae medicinal material, sieving with 40-180 mesh sieve, defatting with petroleum ether at a ratio of 1:10 under reflux for 1-3 times, filtering, washing, and drying;

(2) soaking dried radix Pimpinellae Candolleanae powder in solvent, and ultrasonic extracting.

Further, the solvent is ethanol with volume fraction of 60%, and the mass ratio of the valeriana jatamansi jones powder to the ethanol is 1: 25.

The beneficial effect who adopts above-mentioned scheme is: the yield of the total flavone is increased sharply along with the increase of the concentration of the ethanol, and the yield of the total flavone reaches the maximum value when the concentration of the ethanol reaches 60 percent; the excessive solvent consumption brings inconvenience to subsequent concentration and other operations, and simultaneously, the leached flavone has a synergistic extraction effect on the unleached flavone, and the excessive solvent consumption is not beneficial to the synergistic extraction, so the solid-liquid ratio of the invention is preferably about 1: 25.

Further, the soaking time is 24h, and the ultrasonic extraction time is 30 min.

The beneficial effect who adopts above-mentioned scheme is: the total flavone yield is gradually increased along with the prolonging of the soaking time, but the influence on the extraction effect is not large after the total flavone yield exceeds 24 hours, and the soaking time is preferably 24 hours in order to prevent other components from being leached to generate a plurality of impurity components; along with the prolonging of the ultrasonic time, the yield of the total flavone is firstly increased and then decreased, and reaches the maximum value at 30 min.

Further, the folium isatidis extract and the radix isatidis extract are both prepared by a water extraction and alcohol precipitation method.

Further, the forsythia suspense extract is prepared by a double extraction method, which specifically comprises the steps of preparing two extracts by a water extraction and alcohol precipitation method and a distillation method, and mixing the two extracts according to a ratio of 1:1 to prepare the forsythia suspense extract.

In summary, the invention has the following advantages:

1. the method for extracting the valeriana jatamansi jones extract is an ultrasonic method, and the optimal process conditions obtained by single-factor and orthogonal tests are as follows: soaking for 24 hours in 60% ethanol at a solid-to-liquid ratio of 1:20, and performing ultrasonic treatment for 30 min. Under the condition, the extraction rate is highest, and the yield of the total flavone can reach 3.410%.

2. The Chinese herbal medicine preparation provided by the invention is scientific in formula and reasonable in matching, has multiple compatibility possibilities for symptomatic medicines, has good effects of preventing piglet diarrhea and diminishing inflammation, inhibiting bacteria and resisting viruses when being cooperatively applied.

Drawings

FIG. 1 is a graph of standard solution concentrations;

FIG. 2 is a graph showing the effect of soaking time on total flavone extraction efficiency;

FIG. 3 is a graph showing the effect of ethanol concentration on total flavone extraction efficiency;

FIG. 4 shows the effect of solid-liquid ratio on total flavone extraction efficiency;

FIG. 5 is a graph showing the effect of sonication time on total flavone extraction efficiency.

Detailed Description

The green leaves and the isatis roots in the invention are dried leaves and roots of isatis tinctoria of cruciferae. Mainly contains indole compounds such as indirubin, indigo, indoxyl, indolone and the like, tryptanthrin and the like. The effective components of the dyers woad leaf and the dyers woad root are extracted by a water extraction and alcohol precipitation method.

Fructus forsythiae is dried fruit of Forsythia suspensa (Thunb.) Vahl of Oleaceae. Contains lignan and its glycosides, phenylethanoid glycosides, flavonoids, triterpenes, volatile oils, etc. Valerian root is a root and rhizome of valerian of Valerianaceae. Contains volatile oil, glycosides, iridoids and organic acids as main ingredients. Because the effective components of the valeriana jatamansi jones and the forsythia suspense comprise volatile oil, the test adopts a double extraction method, namely a water extraction and alcohol precipitation method and a distillation method are combined to extract the valeriana jatamansi jones and the forsythia suspense, so that the extraction of volatile components and non-volatile components can be considered, and the loss of the effective components can be avoided.

The principles and features of this invention are described below in conjunction with embodiments, which are included to explain the invention and not to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

The embodiment provides a spider aroma compound preparation for resisting piglet diarrhea, which comprises a spider aroma extracting solution, an isatis root extracting solution and a forsythia extracting solution in a mass ratio of 1:1: 1.

Example 2

The embodiment provides a spider aroma compound preparation for resisting piglet diarrhea, which comprises spider aroma extracting solution, isatis root extracting solution, forsythia extracting solution and berberine hydrochloride in a mass ratio of 1:1:1:1, wherein the volume fraction of the berberine hydrochloride is 1%.

Example 3

The embodiment provides a spider aroma compound preparation for resisting piglet diarrhea, which comprises spider aroma extracting solution, isatis root extracting solution, forsythia extracting solution, folium isatidis extracting solution and berberine hydrochloride in a mass ratio of 1:1:1:1:1, wherein the volume fraction of the berberine hydrochloride is 1%.

Example 4

The embodiment provides a spider aroma compound preparation for resisting piglet diarrhea, which comprises a spider aroma extracting solution, an isatis root extracting solution, a fructus forsythiae extracting solution and a folium isatidis extracting solution in a mass ratio of 1:1:1:1: 1.

Test example 1

Selecting 4 factors which may influence the extraction effect, such as ethanol concentration, soaking time, solid-liquid ratio and ultrasonic time, and carrying out single factor test. The yield of the total flavone is taken as a survey index, and the appropriate range of each factor is determined. When the influence of a certain factor on the yield of the valeriana jatamansi jones total flavonoids is examined, other factors and the level of the factors are fixed.

The test method comprises the following steps:

1.1 Standard working Curve formulation

Accurately weighing 0.0051g of hesperidin standard, adding methanol for dissolving, and fixing the volume to 50mL to obtain a hesperidin standard solution with the concentration of 0.102 g/L. The standard solutions were transferred into 5 10mL volumetric flasks at 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL, respectively, and the volume was determined by adding methanol to a constant volume, using methanol as a blank, and measuring the absorbance at 284 nm. And (5) plotting the absorbance A to the concentration C of the hesperidin standard solution to draw a standard working curve.

1.2 determination of Total Flavonoids

Pulverizing radix Pimpinellae Candolleanae medicinal materials, sieving (40-180 mesh), defatting with 1:10 petroleum ether under reflux twice, filtering, washing, and drying in a forced air drying oven at 40 deg.C for about 1 hr.

Weighing 1.0g of degreased valeriana jatamansi jones powder, adding a certain amount of solvent, sealing, shaking up, weighing the mass, performing ultrasonic extraction, taking out, cooling, weighing the mass again, complementing the lost mass with the solvent, shaking up, performing centrifugal separation, taking supernatant (1/5 of the solvent amount), concentrating on a rotary evaporator to dryness, adding 30mL of methanol for dissolving, filtering out insoluble substances, adding methanol into filtrate to fix the volume to 50mL, and thus obtaining a sample solution. 1mL of sample solution was removed, methanol was added to a constant volume of 10mL, and absorbance was measured at 284nm using methanol as a blank. And calculating the yield of the valeriana jatamansi jones total flavonoids according to a linear regression equation.

1.3 Single factor test results

FIGS. 1 to 5 show the effect of the concentration of the standard solution and the soaking time on the extraction efficiency of total flavonoids, the effect of the concentration of ethanol on the extraction efficiency of total flavonoids, the effect of the solid-liquid ratio on the extraction efficiency of total flavonoids and the effect of the ultrasonic time on the extraction efficiency of total flavonoids.

As can be seen from fig. 2: the total flavone yield gradually increases with the increase of soaking time, but the influence on the extraction effect is not large after exceeding 24h, and the soaking time is preferably 24h in order to prevent other components from leaching out to generate a plurality of impurity components.

As can be seen from fig. 3: the method is mainly characterized in that the total flavone yield is increased sharply along with the increase of the ethanol concentration, reaches the maximum value when the ethanol concentration reaches 60 percent, and then gradually becomes lower along with the increase of the ethanol concentration, wherein the main reasons are different ethanol concentrations and different polarities, the polarity of 60 percent ethanol is similar to that of flavonoids, and the leaching of the flavonoids is influenced by the increase of the dissolution of some fat-soluble substances along with the increase of the ethanol concentration.

As can be seen from fig. 4: generally, the larger the using amount of the solvent, the stronger the dissolving capacity of the flavonoid substances, the more favorable the diffusion of the flavonoid substances, and the better the extraction effect. However, the excessive solvent consumption brings inconvenience to subsequent concentration and other operations, and meanwhile, the leached flavone has a synergistic extraction effect on the unleached flavone, and the excessive solvent consumption is not favorable for the synergistic extraction. FIG. 4 shows that the total flavone yield reaches the maximum value when the solid-to-liquid ratio is 1:25, so that the solid-to-liquid ratio is preferably about 1: 25.

As can be seen from fig. 5: along with the prolonging of the ultrasonic time, the yield of the total flavone is firstly increased and then decreased, and reaches the maximum value at 30 min. The reason is that the time is too short, the flavonoid substances are not fully dissolved, the dissolving of the flavonoid substances reaches the balance when the time is 30min, the ultrasonic time is prolonged, some heat-sensitive components are damaged or the solvent is volatilized for too long ultrasonic time, so that the concentration of the ethanol is reduced, and the yield of the total flavone is reduced.

In summary, the optimal process conditions in the present invention are: soaking for 24 hours in 60% ethanol at a solid-liquid ratio of 1:20, and performing ultrasonic treatment for 30 min. Under the condition, the extraction rate is highest, and the yield of the total flavone can reach 3.410%. The process has good experimental reproducibility and has good reference value for further process development.

Test example 2

2.1 preparation of single medicinal material:

extracting Valeriana jatamansi Jones and fructus forsythiae by ethanol extraction and distillation respectively to obtain extractive solutions, and mixing at a ratio of 1:1 to obtain Valeriana jatamansi Jones extractive solution; the folium isatidis and the radix isatidis are extracted with water and precipitated with alcohol to prepare extracting solution.

2.2 reagents

Casein hydrolysate, yeast extract, beef extract, dried egg white, sodium chloride, agar powder, glucose, thimerosal, dipotassium hydrogen phosphate, manganese dichloride, ferric trichloride and the like, wherein the reagents are all domestic analytical purifiers.

2.3 Medium

Preparing a common nutrient agar culture medium and a protein aging water culture medium according to a conventional method, sterilizing, and storing in a refrigerator at 4 ℃ for later use;

CAYE (caseinate-yeast extract) broth;

trace salt mixture: magnesium sulfate 5%, manganese dichloride 0.5%, and iron dichloride 0.5%, sequentially dissolving in 0.001MH₂SO₄In solution;

CAYE culture solution: 20g of casein hydrolysate, 2.5g of sodium chloride, 6g of yeast extract, 11.41g of dipotassium phosphate trihydrate and lmL of trace salt mixture, wherein the reagents are sequentially added into distilled water to be dissolved, the volume is 1000mL, and the pH value is adjusted to 8.5. Subpackaging in conical flasks, and sterilizing at 13.5 pounds for 20 minutes.

2.4 Experimental animals

Japanese big ear white rabbits, 2.2 + -0.2 kg weight, half female and half male, purchased from Shanghai laboratory animal center in batches. The feed is pre-fed for one week and fed with complete feed. And (4) carrying out clinical examination regularly, and if abnormal persons are found, removing and finding out reasons in time, and selecting healthy persons for testing. Fasting for 48h before operation, free drinking, free activity after operation, and fasting for water prohibition.

Preparation of 2.5LT

Inoculating Escherichia coli in common nutrient agar slant culture medium, culturing at 37 deg.C for 12 hr, transplanting in peptone water, and culturing at 37 deg.C for 12 hr. The content of the culture medium components, pH and osmotic pressure were adjusted to match those of the CAYE culture medium without the addition of the valeriana jatamansi jones extract as a control group by aseptically adding the valeriana jatamansi jones extract to the conical flask to which the CAYE culture medium was added at a ratio of 1:0.4 (v/v). The peptone water 12h culture was added at a ratio of 10:1(v/v) while a trace amount of glucose was added to give a final concentration of 2.5 g/L. Then placing on HZS-D water bath oscillator, culturing at 37 deg.C under 170r/min for 18h with continuous aerobic shaking, taking out culture, adding 0.1g/L thimerosal, sterilizing overnight, centrifuging at 4 deg.C 10000r/min for 30min, collecting supernatant, and storing in refrigerator at 4 deg.C (no more than 3D). The concentration of the E.coli inoculum in the test was 1.5X 10⁵one/mL.

2.6 Rabbit ileum ligation assay

Local anesthesia is performed with 1% procaine hydrochloride, the abdominal wall is cut by aseptic operation, the intestines are ligated by No. 4 silk thread in centripetal direction from ileum at the free end of caecum, and the injection section is 4cm, and the interval section is 2 cm. Six intestinal mixes were ligated to each rabbit, and lmL LT +0.4mL of valeriana jatamansi extract or l.4mL of extract culture was injected randomly to each intestinal mix, while 1mL of LT +0.4mL of physiological saline was injected to the control group, each of which required 6 intestinal mixes. After the injection, the abdominal wall incision is sutured. Killing the test rabbits after 11h, taking out and ligating intestines, measuring the length (cm) and liquid accumulation (mL) of each intestine, calculating the ratio (mL/cm) of the two, and judging the strength of the anti-enterotoxin effect of the Chinese herbal medicine compound according to the ratio of the two.

2.7 results of the experiment

2.7.1 the bacteriostatic effect of five medicines and compound compatibility thereof on escherichia coli is shown in table 1.

TABLE 1 results of in vitro bacteriostatic test

As can be seen from Table 1, in the single medicine, Valeriana jatamansi Jones, radix Isatidis, fructus forsythiae and folium Isatidis all show good antibacterial effect, wherein the radix Isatidis has strongest in vitro antibacterial effect, MIC and MBC thereof reach 0.0016g/mL and 0.0032g/mL respectively, and berberine hydrochloride has poorer in vitro antibacterial effect. In the compound compatibility, the in-vitro antibacterial effect of the compound II (valeriana jatamansi jones, fructus forsythiae and radix isatidis) and the compound III (weeping forsythia, radix isatidis and folium isatidis) is equivalent, and the two are superior to the compound I (valeriana jatamansi jones and fructus forsythiae); and the compound IV (valeriana jatamansi jones, fructus forsythiae, isatis root and berberine hydrochloride) has poor in-vitro antibacterial effect.

2.7.2 anti-enterotoxin Effect

2.7.2.1

TABLE 2 Effect of Forsythia suspensa and Valeriana jatamansi jones added to bacterial culture on LT-induced accumulation of ligated gut-mix fluid

Group of	Number of mixed intestines	Liquid accumulation (mL/cm)
			1mL LT +0.4mL physiological saline	6	2.30±0.21^A
1.4mL of Valeriana jatamansi extract culture	6	1.85±0.16^AB
			1.4mL Forsythia suspensa extract culture	6	1.39±0.16^B

Note: the same column data are labeled differently to indicate significant difference, with capital letters different to indicate very significant difference (P <0.01), and lowercase letters different to indicate significant difference (P <0.05), as follows.

As can be seen from Table 2: compared with the control group, the addition of the valeriana jatamansi extract into the bacterial culture solution and the culture of escherichia coli can reduce the accumulation of the intestinal juice caused by LT by 19.57% (P is more than 0.05), but the difference is not obvious. The forsythia suspense extract treated by the same method can remarkably reduce the accumulation amount of intestinal mixed liquid caused by LT (P < 0.01).

2.7.2.2

TABLE 3 influence of Isatis indigotica leaf and Isatis indigotica root added to the bacterial culture solution on LT-induced accumulation of the mixed liquid in the ligated intestine

Group of	Number of mixed intestines	Liquid accumulation (mL/cm)
			1mL LT +0.4mL physiological saline	6	1.55±0.05^a
1.4mL of the extract culture of folium Isatidis	6	1.13±0.09^b
			1.4mL of Isatis root extract culture	6	0.07±0.09^b

As can be seen from Table 3: the extraction liquid of the isatis root or the dyers woad leaf is added into the bacterial culture solution to be cultured together with the escherichia coli, and compared with a control group, the liquid accumulation amount of the ligation intestinal mixture caused by LT can be remarkably reduced (P is less than 0.05) in both cases, but the difference between the two cases is not remarkable.

2.7.2.3

TABLE 4 Effect of combination one addition to bacterial broth on LT-induced accumulation of ligated gut-fluid

Group of	Number of mixed intestines	Liquid accumulation (mL/cm)
			1mL LT +0.4mL physiological saline	6	1.55±0.06^Aa
1.4mL Compound I culture	6	1.14±0.06^Bb
			1.4mL Compound II culture	6	1.26±0.18^ABab
1.4mL Compound III culture	6	1.23±0.05^ABabc
			1.4mL Compound IV culture	6	0.92 soil 0.08^Bc
1.4mL Compound V culture	6	1.21±0.06^ABabc
			1.4mL Compound VI culture	6	1.37±0.04^Aab

From table 4 it can be seen that: the compound extracting solutions prepared by combining valeriana jatamansi, fructus forsythiae, radix isatidis and folium isatidis are respectively added into a bacterial culture solution to culture escherichia coli, and the produced LT is used for rabbit ileum ligation tests, and the test results of the six compound extracting solutions show that compared with a control group, each compound group can reduce the liquid accumulation of mixed ligated intestines to different degrees, wherein the compound IV (valeriana jatamansi and fructus forsythiae) has the strongest effect of antagonizing enterotoxin (P <0.01), and the compound I (fructus forsythiae and radix isatidis) can also obviously reduce the liquid accumulation of mixed ligated intestines caused by LT (P < 0.05).

Compared with the medicine groups, the difference between the compound IV (valeriana jatamansi jones and forsythia suspensa) and the compound VI (valeriana jatamansi jones, isatis root, isatis leaf and forsythia suspensa) is very obvious (P is less than 0.01), and the difference between the compound IV (valeriana jatamansi jones and forsythia suspensa) and the compound II (forsythia suspensa, isatis root and isatis leaf) is obvious (P is less than 0.05). The compound groups (II, III and VI) containing the dyers woad leaf in the table do not show good effects, and the difference is not obvious compared with a control group, so that the dyers woad leaf has small effect of antagonizing LT-induced intestinal mixed ligation liquid accumulation when being compatible with other medicines.

2.7.2.4

TABLE 5 Effect of the addition of combination two to the bacterial broth on LT-induced accumulation of ligated gut-fluid

Group of	Number of mixed intestines	Liquid accumulation (mL/cm)
			1mL LT +0.4mL physiological saline	6	1.40±0.09^Aa
1.4mL Compound I culture	6	1.18±0.06^Ab
			1.4mL Compound II culture	6	0.85±0.05^Bc
1.4mL Compound III culture	6	1.30±0.05^Aab
			1.4mL Compound IV culture	6	0.83±0.07^Bc

The complex formulation of valeriana jatamansi jones, forsythia suspensa, isatis root and berberine hydrochloride (example 4) was added to the culture medium to culture E.coli, and the obtained culture LT was used for rabbit ileum ligation test, the test results are shown in Table 5. Compared with a control group, the compound II (valeriana jatamansi jones, fructus forsythiae and radix isatidis) and the compound IV (valeriana jatamansi jones, fructus forsythiae, radix isatidis and berberine hydrochloride) show extremely obvious enterotoxin antagonistic action (P is less than 0.01 and P is less than 0.01); compound I (Valeriana jatamansi Jones + Forsythia suspensa) also showed significant antagonism (P < 0.05). Compared among the drug groups, the antagonistic action of compound II (valeriana jatamansi jones, forsythia suspense and isatis root) and compound IV (valeriana jatamansi jones, forsythia suspense, isatis root and berberine hydrochloride) on enterotoxin is remarkably superior to that of compound I (forsythia suspense, valeriana jatamansi jones) and compound III (valeriana jatamansi jones, forsythia suspense and berberine hydrochloride) (P is less than 0.01, and P is less than 0.01), but the difference between the two is not obvious.

2.7.2.5

TABLE 6 influence of Lily forsythia fruit and valeriana jatamansi on LT-induced accumulation of mixed liquid in ligated intestine

Group of	Number of mixed intestines	Liquid accumulation (mL/cm)
			1mL LT +0.4mL physiological saline	6	2.29±0.22^a
1.4mL of Valeriana jatamansi extract	6	1.65±0.06^b
			1.4mL of Forsythia suspensa extract	6	1.90±0.14^ab

From table 6, it can be seen that: the valeriana jatamansi jones extract and LT are injected into the ligated intestine together for mixing, and test results show that valeriana jatamansi jones can remarkably (P is less than 0.05) reduce liquid accumulation, while fructus forsythiae extract does not reach a remarkable level.

2.7.2.6

TABLE 7 influence of Isatis indigotica leaf and Isatis indigotica root on LT-induced accumulation of mixed liquid in ligated intestine

Group of	Number of mixed intestines	Liquid accumulation (mL/cm)
			1mL LT +0.4mL physiological saline	6	1.55±0.05
1.4mL of folium Isatidis extract	6	1.53±0.17
			1.4mL of radix Isatidis extract	6	1.53±0.14

As can be seen from table 7, compared with the control group, the folium isatidis extract and the radix isatidis extract had no significant effect on the liquid accumulation in the intestinal tract mixture, and neither of them showed a significant anti-enterotoxin effect.

2.7.2.7

TABLE 8 influence of intestinal fluid accumulation due to ligation of a pair of LT combinations

Group of	Number of mixed intestines	Liquid accumulation (mL/cm)
			1mL LT +0.4mL physiological saline	6	1.55±0.06^a
1.4mL Compound I	6	1.51±0.16^ab
			1.4mL Compound II	6	1.52±0.13^a
1.4mL Compound III	6	1.28±0.07^ab
			1.4mL Compound IV	6	1.28±0.05^ab
1.4mL of Compound V	6	1.18±0.06^b
			1.4mL of compound VI	6	1.53±0.13^a

As can be seen from Table 8: according to the influence of the accumulation amount of the mixed liquid of the ligated intestines caused by LT, the compound V (valeriana jatamansi jones and isatis root) in the six compound combinations has the most obvious effect of inhibiting enterotoxin, and is obviously superior to a control group (P is more than 0.05). And compound III (fructus forsythiae and folium isatidis) and compound IV (fructus forsythiae and valeriana jatamansi jones), which reduce the accumulation of LT-induced ligated intestinal fluid by 17.42% (P > 0.05)). Compared among the medicine groups, the effect of compound V (valeriana jatamansi jones and isatis root) on inhibiting enterotoxin is obviously better than that of compound II (forsythia, isatis root and dyers woad leaf) and compound VI (forsythia, valeriana jatamansi jones, isatis root and dyers woad leaf) (P is less than 0.05, and P is less than 0.05). The compound groups (II and VI) containing the dyers woad leaf antagonize the liquid accumulation amount of the intestinal ligation caused by LT, and the difference is not generated compared with the control group, which shows that the compound groups have no antagonism on enterotoxin.

2.7.2.8

TABLE 9 influence of intestinal fluid accumulation due to ligation by combination of two pairs of LT

Group of	Number of mixed intestines	Liquid accumulation (mL/cm)
			1mL LT +0.4mL physiological saline	6	1.40±0.09
1.4mL Compound I	6	1.34±0.08
			1.4mL Compound II	6	1.26±0.05
1.4mL Compound III	6	1.31±0.05
			1.4mL Compound IV	6	1.29±0.05

As can be seen from Table 9: compared with the control group, the combined two test groups have no obvious difference on the influence of LT-caused accumulation of the mixed liquid of the ligature intestine. Although the effect of compound II (fructus forsythiae + valeriana jatamansi jones + radix isatidis) and compound IV (fructus forsythiae + valeriana jatamansi + radix isatidis + berberine hydrochloride) on inhibiting the liquid accumulation in the intestinal tract is slightly stronger than that of other groups, the liquid accumulation in the intestinal tract is respectively reduced by 10.0% and 7.83%, but the difference is not obvious.

2.7.3 Effect on drug diarrhea

TABLE 10 Effect of several herbal extracts on Castor oil induced diarrhea in mice

As can be seen from table 10: the test result of the Chinese herbal medicine for antagonizing the castor oil-induced diarrhea of mice shows that the Chinese herbal medicine compound extracting solution (fructus forsythiae, isatis root and valeriana jatamansi jones) can obviously reduce the incidence rate and the diarrhea frequency caused by the castor oil. Compared with a control group, the compound group remarkably reduces the diarrhea frequency of mice within 1-3h (P is less than 0.01), and the effect is obviously superior to that of the radix isatidis, fructus forsythiae and berberine hydrochloride groups (P is less than 0.05, and P is less than 0.05). As can be seen from the data in table 10, the incidence rate of diarrhea in valeriana jatamansi jones and the compound group tended to decrease, 40% for 1h, 10% and 30% for 2h, and 30% and 50% for 3h, respectively, compared to the control group in the same time phase. The other single Chinese herbal medicines have no obvious difference in the aspect of resisting the diarrhea of the mice caused by the castor oil.

2.7.4 influence on Small intestine Propulsion movement

TABLE 11 Effect of several herbal extracts on the intestinal ink propulsion of mice

Group of	Number of samples	Propulsion ratio%	Inhibition ratio%
				Control group
	10	0.61±0.01^Aa	-
				Spider incense	10	0.59±0.02^ABa	3.29
Radix Isatidis	10	0.48±0.02^Bb	21.31
				Forsythia fruit	10	0.55±0.03^Abab	9.84
Berberine hydrochloride	10	0.58±0.03^ABa	4.92
				Compound recipe	10	0.52±0.04^Abab	14.75

As can be seen from Table 11: the propulsion rate of the mouse intestinal ink can be reduced to different degrees in each medicine group, wherein the isatis root has the strongest effect, and has the obvious difference (P <0.01) compared with a control group and the obvious difference (P <0.05) compared with a jatamans valeriana group and a berberine hydrochloride group. The second is a compound group (valeriana jatamansi, isatis root and forsythia suspense) and a forsythia suspense group, the inhibition rates are respectively 13.11% and 8.20% (P >0.05), but the inhibition rates do not reach a significant level.

2.7.5 inhibition of inflammatory exudation

TABLE 12 Effect of several herbal extracts on the increase of the permeability of the capillary vessels in the abdominal cavity of mice caused by acetic acid

As can be seen from table 12: each drug group has different degrees of inhibition effects on the increase of the permeability of capillary vessels in the abdominal cavity of a mouse caused by acetic acid. Compared with a control group, the compound group (the valeriana jatamansi jones, the isatis root and the forsythia suspense) can remarkably inhibit the mouse abdominal dye exudation caused by acetic acid (P is less than 0.01), the inhibition rate reaches 59.07%, the valeriana jatamansi jones and the isatis root can remarkably inhibit the mouse abdominal dye exudation caused by acetic acid (P is less than 0.05, and P is less than 0.05), and the inhibition rates are 36.29% and 35.44% respectively.

In summary, the following steps:

(1) the dyers woad leaf, the isatis root, the forsythia and the valeriana jatamansi have good in-vitro antibacterial activity on escherichia coli O149K88, and the minimum bacteriostatic concentration and the minimum bactericidal concentration of a compound I (forsythia and valeriana jatamansi), a compound II (forsythia, valeriana jatamansi + isatis root) and a compound III (forsythia, isatis root and dyers woad leaf) which are composed of the dyers woad leaf, isatis root, valeriana jatamansi leaf and valeriana jatamansi leaf are all enhanced compared with those of a single medicine, and the four Chinese herbal medicines have synergistic effect on the aspect of antibacterial activity.

(2) The antibacterial effect of the compound IV (valeriana jatamansi jones, fructus forsythiae, isatis root and berberine hydrochloride) is relatively poor, and the reason for analyzing the antibacterial effect is probably because the antibacterial activity of the compound is influenced after the berberine hydrochloride is added into the formula, and the effective antibacterial concentration of other three Chinese herbal medicines is reduced.

(3) Anti-enterotoxin effect

Chinese herbal medicine extracting solutions are added into a bacterial culture solution to culture escherichia coli, the produced LT is used for carrying out a rabbit ileum ligation test, and the antagonism of the Chinese herbal medicine on escherichia coli enterotoxin is researched according to the influence of the Chinese herbal medicine extracting solutions on the accumulation of LT-induced ligation intestinal mixed liquid. From the test results in tables 2 and 3, it is understood that the liquid accumulation amount of the ligature intestinal lavage caused by the culture supernatant can be significantly or extremely significantly reduced in the isatis leaf group, isatis root group and forsythia fruit group. The results in table 4 show that, except for the compound group containing folium isatidis, the other compound groups significantly or very significantly reduce the liquid accumulation of the ligated intestine, indicating that the compound combination of radix isatidis, fructus forsythiae and valeriana jatamansi jones has synergistic effect in resisting enterotoxin LT, but folium isatidis is not as good as the single drug formulation, presumably antagonistic action may exist between folium isatidis and the other three drugs in resisting enterotoxin LT, and the combination is not suitable. The forsythia, valeriana jatamansi jones, isatis roots and berberine hydrochloride which have better effects in the first composition are treated by the same method and subjected to rabbit ileum ligation test, so that the liquid accumulation of the intestinal ligature mixture caused by LT is remarkably or extremely remarkably reduced. Wherein, the antagonistic effect of compound II (valeriana jatamansi jones, forsythia suspensa and isatis root) and compound IV (valeriana jatamansi jones, forsythia suspensa, isatis root and berberine hydrochloride) on enterotoxin is remarkably superior to that of compound I (valeriana jatamansi jones, forsythia suspensa) and compound III (valeriana jatamansi jones, forsythia suspensa and berberine hydrochloride). The strong antagonistic action of the compound consisting of the valeriana jatamansi jones, the forsythia suspense and the isatis root and the compound consisting of the valeriana jatamansi jones, the forsythia suspense, the isatis root and the berberine hydrochloride on LT which plays an important role in treating diarrhea caused by escherichia coli shows that the two compounds are the best compound compatibility for treating the escherichia coli diarrhea of piglets.

In the test, the berberine hydrochloride has no bacteriostatic action on escherichia coli at the concentration of l%, but the compound of the berberine hydrochloride, the isatis root, the forsythia and the valeriana jatamansi jones is added into the culture solution to remarkably reduce the liquid accumulation of the intestinal ligation caused by LT, so that the curative effect of the berberine hydrochloride on piglet escherichia coli diarrhea is not dependent on the bacteriostatic action but is due to the antitoxic action of the berberine hydrochloride.

(4) LT resisting effect of Chinese herbal medicine

The Chinese herbal medicine extract or the compound compatible extract and LT are directly injected into the ligation intestinal mixture together, the direct antagonistic action of the Chinese herbal medicine on LT is researched according to the influence of the medicine on the accumulation amount of the ligation intestinal mixture liquid caused by LT, and the test result is shown in tables 6-9. As can be seen from tables 6 and 7, compared with the results of the effect of adding the Chinese herbal medicine extract into the culture solution on the accumulation of the mixed liquid of the ligated intestine caused by LT, in the four single Chinese herbal medicines, the valeriana jatamansi jones can obviously reduce the accumulation of the mixed liquid of the ligated intestine caused by LT (P is less than 0.05), which indicates that the valeriana jatamansi jones have direct antagonistic action on enterotoxin LT of escherichia coli, while the isatis root, the indigowoad leaf and the weeping forsythia are not at a significant level, which indicates that the three Chinese herbal medicines have no direct antagonistic action on LT. The data listed in table 8 show that the compound group consisting of valeriana jatamansi jones and isatis root has a significant effect of inhibiting the accumulation of the mixed liquid of the ligated intestine caused by LT. The result of the effect of the accumulation of the mixed liquid of the ligature intestine caused by combining a pair of LT can prove that the dyers woad leaf can generate antagonism with other two medicines in the aspect of resisting enterotoxin LT, and the dyers woad leaf is not suitable for compatibility, so the dyers woad leaf can be removed. In the four compound combinations consisting of isatis root, valeriana jatamansi, forsythia suspense and berberine hydrochloride, the effect of compound II (valeriana jatamansi + forsythia suspense + isatis root) and compound IV (valeriana jatamansi + forsythia suspense + isatis root + berberine hydrochloride) on inhibiting the liquid accumulation in intestinal tract is slightly stronger than that of other groups.

The comprehensive analysis result of the anti-LT effect of the Chinese herbal medicine extract and the anti-LT effect of the Chinese herbal medicine extract added into the culture solution shows that the fructus forsythiae, the valeriana jatamansi jones, the radix isatidis and the berberine hydrochloride can effectively antagonize liquid accumulation in small intestines caused by escherichia coli heat-sensitive enterotoxin and have strong in-vitro inhibition effect on escherichia coli, and the test result provides an important theoretical basis for clinical application of the Chinese herbal medicine to treatment of piglet escherichia coli diarrhea.

(5) Anti-inflammatory and purgative effects of Chinese herbal medicine

The mouse drug diarrhea model and the mouse small intestine propulsion exercise test are common methods for researching the anti-diarrhea mechanism of the drug. Piglets suffering from colibacillosis diarrhea show a change in acute enteritis, and the main lesion is in the small intestine. In the test, the castor oil-induced small intestinal diarrhea of mice is selected as a test model, and the antagonistic action of the Chinese herbal medicines on the drug-induced diarrhea is researched. The main site of action of castor oil is the small intestine, which promotes the synthesis and release of inflammatory mediators, thus causing effusion in intestinal lumen, accelerating intestinal peristalsis and causing diarrhea. The compound compatibility of the forsythia, the isatis root and the valeriana jatamansi jones can greatly reduce the diarrhea frequency of mice caused by castor oil, and the diarrhea incidence rate is also reduced compared with a control group and other medicine groups, which shows that the compound has better anti-inflammatory and antidiarrheal effects. The compound compatibility has no obvious influence on the small intestine propelling movement of mice, so that the intestinal movement inhibition is not the mechanism of diarrhea resistance.

The test result of the invention shows that the single medicine (valeriana jatamansi jones or isatis root) or the compound medicine (isatis root, forsythia suspense and valeriana jatamansi jones) can obviously or extremely obviously inhibit the increase of the permeability of capillary vessels in the abdominal cavity of a mouse caused by acetic acid, namely, the single medicine, the valeriana jatamansi jones or isatis root and the compound medicine have the effect of inhibiting the acute inflammatory exudation process. The compound compatibility of the valeriana jatamansi jones, the forsythia and the isatis root can play a role in treating the colibacillary diarrhea of piglets through various ways of inhibiting the reproduction of pathogenic bacteria, antagonizing the diarrhea causing effect of heat-sensitive enterotoxin, inhibiting the inflammation process, resisting drug-induced diarrhea and the like. Therefore, when the Chinese herbal medicine exerts the clinical curative effect, the effect of the Chinese herbal medicine is usually multi-target.

And (4) conclusion:

1. the extracts of valeriana jatamansi jones, isatis root, forsythia, dyers woad leaf and the compound thereof have good in-vitro antibacterial activity on main pathogenic bacteria causing colibacillosis diarrhea of piglets, namely Escherichia coli O149K 88.

2. The compound compatibility of the valeriana jatamansi jones, the forsythia suspense and the isatis root and the compound compatibility of the valeriana jatamansi jones, the forsythia suspense, the isatis root and the berberine hydrochloride have extremely obvious antagonistic action on liquid accumulation in small intestines caused by escherichia coli heat-sensitive enterotoxin.

3. The compound compatibility of the valeriana jatamansi jones, the isatis root and the compound preparation consisting of the valeriana jatamansi jones, the isatis root and the forsythia can obviously or extremely obviously inhibit the increase of the permeability of capillary vessels in abdominal cavities of mice caused by acetic acid, namely has an antagonistic effect on the inflammatory process.

4. The compound compatibility of the valeriana jatamansi jones, the isatis root and the forsythia can remarkably reduce the frequency of diarrhea of mice caused by castor oil, and has the efficacy of resisting inflammation and stopping diarrhea.

5. The isatis root can remarkably inhibit the small intestine propulsion movement of the mouse, and other drug groups have no obvious influence on the small intestine propulsion movement of the mouse.

6. The compound compatibility of the valeriana jatamansi jones, the isatis root and the forsythia fruit can effectively inhibit escherichia coli, simultaneously effectively antagonize diarrhea caused by escherichia coli enterotoxin, has the inhibiting and resisting effects on an inflammation process and drug diarrhea, and is the best Chinese herbal medicine compound combination for resisting piglet escherichia coli diarrhea.

While the present invention has been described in detail with reference to the specific embodiments thereof, it should not be construed as limited by the scope of the present patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.

Claims

1. The valeriana jatamansi jones compound preparation for resisting piglet diarrhea is characterized by comprising Chinese herbal medicine extracting solutions, wherein the Chinese herbal medicine extracting solutions comprise valeriana jatamansi jones extracting solution, isatis root extracting solution and fructus forsythiae extracting solution with equal mass.

2. The compound preparation of valeriana jatamansi jones for treating piglet diarrhea as claimed in claim 1, wherein the Chinese herbal medicine extracting solution also includes equal amount of folium isatidis extracting solution.

3. The compound preparation of valeriana jatamansi jones for treating piglet diarrhea as claimed in claim 1 or 2, further comprising berberine hydrochloride in the same amount and in the volume fraction of 1%.

4. The compound preparation of valeriana jatamansi jones for treating piglet diarrhea as claimed in claim 3, which includes valeriana jatamansi jones extract, isatis root extract, forsythia fruit extract and berberine hydrochloride in the weight ratio of 1:1:1: 1.

5. The compound preparation of valeriana jatamansi jones for treating piglet diarrhea as claimed in claim 3, which includes valeriana jatamansi jones extract, isatis root extract, forsythia fruit extract, isatis leaf extract and berberine hydrochloride in the weight ratio of 1:1:1:1: 1.

6. The compound preparation of valeriana jatamansi jones for treating piglet diarrhea as claimed in claim 1, wherein the valeriana jatamansi jones extractive solution is prepared through the following steps:

7. The compound preparation of valeriana jatamansi jones for treating piglet diarrhea as claimed in claim 6, wherein the solvent is ethanol with a volume fraction of 60%, and the mass ratio of valeriana jatamansi jones powder to ethanol is 1: 25.

8. The compound preparation of valeriana jatamansi jones for treating piglet diarrhea as claimed in claim 6, wherein the soaking time is 24 hr and the ultrasonic extraction time is 30 min.