CN112618598A - Preparation and quality detection of traditional Chinese medicine compound Xiaolinian for preventing and treating cow mastitis - Google Patents
Preparation and quality detection of traditional Chinese medicine compound Xiaolinian for preventing and treating cow mastitis Download PDFInfo
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- CN112618598A CN112618598A CN202011642679.6A CN202011642679A CN112618598A CN 112618598 A CN112618598 A CN 112618598A CN 202011642679 A CN202011642679 A CN 202011642679A CN 112618598 A CN112618598 A CN 112618598A
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Abstract
The invention discloses a preparation and quality detection of a traditional Chinese medicine compound liniment for preventing and treating cow mastitis, which comprises the following raw materials in parts by weight: 1-5g of toad venom, 500-2000g of clove, 5-30g of borneol, 805-20mL of polysorbate, 5-15g of sesbania gum, 5-10g of sucrose stearate glyceride and the balance of solvent added to 1000mL, wherein the solvent consists of ethanol and propylene glycol in a volume ratio of 7: 3. The traditional Chinese medicine compound liniment developed by compounding 3 traditional Chinese medicines has simpler components, lower cost, scientific pharmacology, clear drug effect, stable and controllable preparation quality, obvious effect of preventing and treating the mastitis of the dairy cows, is natural and pollution-free, and is beneficial to popularization and use. Meanwhile, the quantitative analysis is carried out by combining the advantages of a PAD detector with a high performance liquid chromatograph, the contents of 3 components of cinobufagin, resibufogenin and eugenol can be simultaneously measured, and the effect of comprehensively detecting the internal quality and stability of the preparation can be achieved, so that the safety and the effectiveness of the product in medication can be ensured.
Description
Technical Field
The invention belongs to the technical field of veterinary medicines, and particularly relates to a preparation and quality detection method of a traditional Chinese medicine compound liniment for preventing and treating cow mastitis.
Background
The mastitis of the dairy cattle is one of the major diseases in the production of the dairy industry, the etiology of the mastitis has the characteristics of complexity and diversity, and a plurality of difficulties are brought to the prevention and treatment of the mastitis of the dairy cattle. Although research on the prevention and treatment of cow mastitis invests a lot of money and material resources every year, mastitis still belongs to the most difficult disease category. Statistically, among the cows existing all over the world today, more than 30% of cows with various types of mastitis occur. Huge economic losses are caused to the breeding industry every year.
The existing methods for treating cow mastitis include antibiotic treatment, biological agent treatment, gene therapy treatment, traditional Chinese medicine preparation treatment and the like, wherein the antibiotic treatment easily causes secondary infection, the compound rate is high, and the problems of drug resistance and the like are solved; the biological preparation has potential adverse reactions or toxic and side effects, cannot independently exert the biological activity, and needs further tracking research; gene therapy treatment is currently in the growth stage and requires a stage of development. The traditional Chinese medicine preparation has multiple target points of therapeutic drug action and causes less toxic and side effects. The drug resistance is not easy to generate, the efficiency is high, the toxicity is low, no drug residue is generated, and the environment is more green and more environment-friendly compared with chemical drugs.
Although the treatment method of the traditional Chinese medicine preparation has many advantages compared with other treatment methods, the traditional Chinese medicine preparation is extracted and prepared by about 7-10 or more traditional Chinese medicinal materials commonly used in the preparation process, and the increase of the traditional Chinese medicine preparation not only increases the cost of the preparation, but also increases the complexity of the preparation process, and causes the defects of low extraction rate and the like. In order to overcome the defects of the prior Chinese medicinal preparation technology, the Chinese medicinal preparation can achieve the same treatment effect by reducing the use amount of Chinese medicinal materials in the preparation, and meanwhile, the prior method generally detects only one or two index components in the quality detection of the Chinese medicinal preparation, so that the internal quality of the preparation cannot be comprehensively detected, and the safety and the effectiveness of the preparation in use cannot be ensured. Therefore, a method for simultaneously detecting the three components as a reference standard is created, the effect of comprehensively detecting the internal quality and stability of the preparation can be achieved, the method is simple and convenient, the separation degree is good, the reproducibility is good, the recovery rate meets the requirement, and the quantification is accurate. Finally, the quality detection method is used for the quality detection of the traditional Chinese medicine preparation.
Disclosure of Invention
Aiming at the defects, the invention provides preparation and quality detection of a traditional Chinese medicine compound Xiao Ling agent for preventing and treating cow mastitis. The traditional Chinese medicine compound liniment developed by compounding 3 traditional Chinese medicines has simpler components, lower cost, scientific pharmacology, clear drug effect, stable and controllable preparation quality, obvious effect of preventing and treating the mastitis of the dairy cows, is natural and pollution-free, and is beneficial to popularization and use. Meanwhile, the HPLC-PAD quality detection method is simple, convenient and accurate in quantification, and can comprehensively detect the internal quality and stability of the preparation.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a traditional Chinese medicine compound Xiao Ling for preventing and treating cow mastitis comprises the following raw materials in proportion: 1-5g of toad venom, 500-2000g of clove, 5-30g of borneol, 805-20mL of polysorbate, 5-15g of sesbania gum, 5-10g of sucrose glyceride stearate, and the balance of solvent added to 1000 mL.
Further, the solvent consists of ethanol and propylene glycol. Preferably, the volume ratio of the ethanol to the propylene glycol is 7: 3.
The invention also provides a preparation method of the traditional Chinese medicine compound liniment for preventing and treating cow mastitis, which comprises the following steps:
(1) preparing an extracting solution: collecting 1-5g of Bufonis venenum, cleaning, drying, pulverizing into fine powder, adding 300mL of 80% ethanol 150, reflux extracting, mixing extractive solutions, filtering, and refrigerating. 500-2000g of clove medicinal material is treated by removing impurities, screening dust, crushing, adding 6-8 times of water, dipping for 1-3h, heating to 100 ℃ for distillation, collecting volatile oil when oil and water are separated, repeatedly extracting once, standing the collected volatile oil for 24-48 h, filtering and refrigerating for later use;
(2) uniformly mixing sesbania gum and sucrose stearate glyceride, and adding a small amount of water to dissolve the sesbania gum and the sucrose stearate glyceride to obtain a mixed solution;
(3) mixing the venenum bufonis extract and the clove volatile oil, adding 5-20mL of polysorbate 80 and the mixed solution, uniformly mixing, adding 5-30g of borneol under stirring, adding a solvent mixed solvent, stirring to completely dissolve, filtering, and adjusting the total amount of the filtrate to 1000mL by using the solvent mixed solvent to obtain the product.
Preferably, the reflux extraction is performed at 70-80 deg.C for 2-3 times, each time for 1-2 h.
Another object of the present invention is to: a quality detection method of a traditional Chinese medicine compound liniment for preventing and treating cow mastitis measures the contents of three components of Chinese bufogenin, resibufogenin and eugenol of the traditional Chinese medicine compound liniment by an HPLC-PAD method, and comprises the following specific steps:
s1: chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; taking 0.05mol/L citric acid aqueous solution as a mobile phase A phase and acetonitrile as a mobile phase B phase, carrying out isocratic elution, wherein the detection wavelength is 200-300nm, the column temperature is 30-40 ℃, the flow rate is 1-3mL/min, and the sample injection amount is 10-40 mu L;
s2: preparation of a reference solution: precisely weighing appropriate amount of cinobufagin, resibufogenin and eugenol as reference substances, diluting with methanol to desired volume, and mixing to obtain mixed reference substance solution;
s3: preparing a test solution: precisely taking a proper amount of buformin emulsion liniment, placing into a 10mL measuring flask, adding methanol to dilute to scale, shaking, and performing ultrasonic treatment for 15min to obtain a test solution;
s4: and (3) determination: precisely sucking 20 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, recording peak area, and calculating by external standard method.
Preferably, the volume ratio of the mobile phase A to the mobile phase B is 60: 40.
Preferably, each 1mL of the control solution contains 53ug, 50ug and 50ug of cinobufagin, resibufogenin and eugenol.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1. the traditional Chinese medicine compound liniment developed by compounding 3 traditional Chinese medicines has simpler components, lower cost, scientific pharmacology and definite drug effect; the 3 traditional Chinese medicines are extracted and prepared, the effect of preventing and treating the cow mastitis can be achieved, and the advantages of low cost, high extraction rate, simple method, no pollution to the environment and the like are realized.
2. The invention adds polysorbate 80, sesbania gum, sucrose stearate glyceride and propylene glycol as auxiliary materials into the defoaming agent, which not only has the functions of solubilization, emulsification and softening in the defoaming agent, so that the medicine is easy to smear, has good uniformity, better drug permeation and absorption and improved stability, and increases the utilization rate of effective components, thereby achieving better protection and treatment effects.
3. The preparation provided by the invention is convenient to use, can be directly applied to an affected part, has high absorption rate, no stimulation and residue, simple preparation process and lower production cost, and is beneficial to popularization and use.
4. The invention carries out content measurement on three components in the liniment to control the quality of the liniment more comprehensively. The traditional Chinese medicine preparation generally only detects one or two index components, can not comprehensively detect the internal quality of the medicine, carries out quantitative analysis by combining the advantages of a PAD detector and a high performance liquid chromatograph, simultaneously measures the contents of 3 components of cinobufagin, resibufogenin and eugenol by utilizing a method, and can play a role in comprehensively detecting the internal quality and stability of the preparation so as to ensure the safety and effectiveness of the medicine of the product.
Drawings
FIG. 1 is a chromatogram of a reference mixture of cinobufagin, resibufogenin and eugenol;
FIG. 2 is a chromatogram of a test sample of cinobufagin, resibufogenin and eugenol;
in fig. 2: cinobufagin Rt is 10.156 min; resibufogenin Rt is 31.457 min; eugenol Rt is 18.519 min.
FIG. 3 is the standard curve of cinobufagin, resibufogenin and eugenol.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
Example 1
A preparation method of a traditional Chinese medicine compound liniment for preventing and treating cow mastitis comprises the following steps:
(1) preparing an extracting solution: collecting Bufonis venenum 3g, cleaning, drying, pulverizing into fine powder, adding 150mL 80% ethanol, reflux-extracting at 70 deg.C for 2 times, each for 90 min, mixing extractive solutions, filtering, and refrigerating. Removing impurities from flos Caryophylli 1000g, sieving to remove dust, mashing, adding 6 times of water, soaking for 2 hr, heating to 100 deg.C, distilling, collecting volatile oil when oil and water are separated, repeatedly extracting once, standing for 24 hr, filtering, and refrigerating;
(2) uniformly mixing 10g of sesbania gum and 5g of sucrose stearate glyceride, and adding a small amount of water to dissolve the sesbania gum and the sucrose stearate glyceride to obtain a mixed solution;
(3) mixing the venenum bufonis extract and the clove volatile oil, adding 10mL of polysorbate 80 and the mixed solution, uniformly mixing, adding 10g of borneol under stirring, adding a mixed solvent of ethanol and propylene glycol (the volume ratio is 7: 3), stirring to completely dissolve, filtering, and adjusting the total amount of the filtrate to 1000mL by using the mixed solvent of ethanol and propylene glycol (the volume ratio is 7: 3) to obtain the product.
Example 2
A preparation method of a traditional Chinese medicine compound liniment for preventing and treating cow mastitis comprises the following steps:
(1) preparing an extracting solution: collecting Bufonis venenum 4g, cleaning, drying, pulverizing into fine powder, adding 200mL 80% ethanol, reflux-extracting at 70 deg.C for 60 min for 3 times, mixing extractive solutions, filtering, and refrigerating. Removing impurities from flos Caryophylli 2000g, sieving to remove ash, mashing, adding 7 times of water, soaking for 1.5 hr, heating to 100 deg.C, distilling, collecting volatile oil when oil and water are separated, repeatedly extracting once, standing for 36 hr, filtering, and refrigerating;
(2) uniformly mixing 12g of sesbania gum and 8g of sucrose stearate glyceride, and adding a small amount of water to dissolve the sesbania gum and the sucrose stearate glyceride to obtain a mixed solution;
(3) mixing the venenum bufonis extract and the clove volatile oil, adding 20mL of polysorbate 80 and the mixed solution, uniformly mixing, adding 20g of borneol under stirring, adding a mixed solvent of ethanol and propylene glycol (the volume ratio is 7: 3), stirring to completely dissolve, filtering, and adjusting the total amount of the filtrate to 1000mL by using the mixed solvent of ethanol and propylene glycol (the volume ratio is 7: 3) to obtain the product.
Example 3
A preparation method of a traditional Chinese medicine compound liniment for preventing and treating cow mastitis comprises the following steps:
(1) preparing an extracting solution: collecting Bufonis venenum 5g, cleaning, drying, pulverizing into fine powder, adding 300mL 80% ethanol, reflux-extracting at 75 deg.C for 90 min for 2 times, mixing extractive solutions, filtering, and refrigerating. Removing impurities from flos Caryophylli (1500 g), sieving to remove ash, mashing, adding 7 times of water, soaking for 2 hr, heating to 100 deg.C, distilling, collecting volatile oil when oil and water are separated, repeatedly extracting once, standing for 24 hr, filtering, and refrigerating;
(2) uniformly mixing 10g of sesbania gum and 10g of sucrose stearate glyceride, and adding a small amount of water to dissolve the sesbania gum and the sucrose stearate glyceride to obtain a mixed solution;
(3) mixing the venenum bufonis extract and the clove volatile oil, adding 10mL of polysorbate 80 and the mixed solution, uniformly mixing, adding 30g of borneol under stirring, adding a mixed solvent of ethanol and propylene glycol (the volume ratio is 7: 3), stirring to completely dissolve, filtering, and adjusting the total amount of the filtrate to 1000mL by using the mixed solvent of ethanol and propylene glycol (the volume ratio is 7: 3) to obtain the product.
Comparative example
A preparation method of a traditional Chinese medicine compound liniment for preventing and treating cow mastitis comprises the following steps:
(1) preparing an extracting solution: collecting Bufonis venenum 3g, cleaning, drying, pulverizing into fine powder, adding 150mL 80% ethanol, reflux-extracting at 70 deg.C for 2 times, each for 90 min, mixing extractive solutions, filtering, and refrigerating. Removing impurities from flos Caryophylli 1000g, sieving to remove dust, mashing, adding 6 times of water, soaking for 2 hr, heating to 100 deg.C, distilling, collecting volatile oil when oil and water are separated, repeatedly extracting once, standing for 24 hr, filtering, and refrigerating;
(2) mixing the venenum bufonis extract and the clove volatile oil, adding 10mL of polysorbate 80 and the mixed solution, uniformly mixing, adding 10g of borneol under stirring, adding an ethanol mixed solvent, stirring to completely dissolve, filtering, and adjusting the total amount of the filtrate to 1000mL by using the ethanol mixed solvent to obtain the product.
First, acute toxicity test
1. Test drugs: the compound liniment prepared in examples 1-3 and comparative example was subjected to acute toxicity test.
2. Test animals: the common-grade New Zealand rabbits are half female and half male, have the body weight of about 3kg and the month age of 3-4 months, and are provided by the Experimental animal center of Guangxi medical university.
3. Test site: the laboratory of the animal center, general grade, university of Guangxi medical sciences.
4. The test method comprises the following steps: dividing 40 New Zealand rabbits into 4 groups at random, and dividingThe four groups were equally divided into a whole skin group and a broken skin group. Depilating rabbit back spine at both sides of the spine by about 10 × 10cm2And checking whether the unhaired skin is injured due to unhairing 24 hours after unhairing, wherein the injured skin is not suitable for a perfect skin toxicity test, and scratching the skin by using abrasive paper for a damaged skin group until the degree of bleeding is reached.
6mL of the test drugs of examples 1 to 3 and comparative example were uniformly applied to the hair-removed area side of each rabbit in the intact skin group and the damaged skin group, respectively, and the neck of each rabbit was fixed so as not to lick the drug on the back. Each animal was raised in cages. After the tested drugs are given for 24 hours, the residual tested drugs are washed by warm water, the weight, the changes of skin, hair, eyes and mucosa, respiration, central nervous system, limb activities and other toxic manifestations of the animals are observed and recorded every day from 1 hour, 24 hours, 48 hours, 72 hours to 14 days after the removal of the tested drugs, and necropsy and macroscopic observation should be carried out in time if the animals die, and pathological histological examination should be carried out when pathological changes are visible to the naked eyes.
5. Test results
In 14 days after the administration of the medicaments in the examples 1 to 3, the weight of each animal to be tested is not reduced, the skin is complete and has no red swelling, the hair removal area has no hair removal phenomenon, the eye mucosa has no change, the breathing, the intelligence and the activity are normal, other poisoning manifestations do not occur, and no animal dies. Whereas the comparative example showed a partial redness and swelling of the skin after administration. The results show that the compound liniment of examples 1-3 is superior to the compound liniment of comparative example.
Second, topical irritation test of intact skin
1. Test drugs: the compound liniment prepared in examples 1 to 3 and comparative example was subjected to a topical irritation test on intact skin.
2. Test animals: the common-grade New Zealand rabbits are half female and half male, have the body weight of about 3kg and the month age of 3-4 months, and are provided by the Experimental animal center of Guangxi medical university.
3. Test site: the laboratory of the animal center, general grade, university of Guangxi medical sciences.
4. The test method comprises the following steps:
4.1 Single dose irritation test
32 New Zealand rabbits were selected, female and male half. Depilating rabbit back spine at two sides of the spine by about 10 × 10cm2Then, whether the dehaired skins are injured due to dehairing is checked, and white rabbits with damaged skins are removed.
The test uses in-vivo left and right self-comparison. Coating a tested medicine on the hair removal area on the left side: 6mL of the test drug of examples 1-3 and comparative example, 6mL of 75% ethanol was applied to the right defeathered area, and the neck of the rabbit was fixed so as not to lick the back drug. Each animal was raised in cages. After application, the residual drug was washed away with warm water, the skin reaction was visually observed 1, 24, 48, 72 hours after removal of the test drug, and the score, recovery and time of each day were recorded as given in table 1.
4.2 multiple dose irritation test
Another 32 New Zealand rabbits are selected, and the female rabbit and the male rabbit are half female rabbit and half male rabbit. Depilating two sides of spine of rabbit with depilatory, inspecting whether depilated skin is injured due to depilation, and removing skin-damaged white rabbit.
The test uses in-vivo left and right self-comparison. Coating a tested medicine on the hair removal area on the left side: 6mL of the test drug of examples 1-3 and comparative example, 6mL of 75% ethanol was applied to the right depilated area, and the neck of the rabbit was fixed so as not to lick the back drug. Each animal was raised in cages. The administration is continued for 5 days and then stopped for 5 days. In addition to the observation and recording of the daily erythema and edema according to the evaluation given in Table 1, the application site should be observed for pigmentation, bleeding spots, rough skin or thin skin, and the occurrence and regression times thereof should be recorded.
Scoring criteria for degree of epidermal skin irritation response
Table 1: skin irritation response score
TABLE 2 skin irritation Strength scoring criteria
5. Test results
Table 3: single dose skin irritation intensity
Table 4: multiple administration skin irritation intensity
Table 3 shows that, after administration, the skin irritation total scores of the compound anti-inflammatory liniments of examples 1-3 were zero, the skin irritation total score of the compound anti-inflammatory liniments of comparative example was 2 before 24 hours, and the skin irritation total score was 1 at 48 hours, and the compound anti-inflammatory liniments of examples 1-3 were non-irritating according to table 2, and the compound anti-inflammatory liniments of comparative example were slightly irritating before 24 hours. Examples 1-3 the compound liniment has normal animal activity, appetite and defecation. The local skin color was normal at each time interval and no irritant reaction was observed. The comparative example compound Xiaolinian group has the symptoms of erythema, edema and irritation reaction.
Table 4 shows that, after the continuous application days, the skin irritation scores of the compound extinction/liniment groups of examples 1 to 3 are all zero, the skin irritation of the compound extinction/liniment group of comparative example is 2 scores before 48 hours, the compound extinction/liniment groups of examples 1 to 3 according to table 2 are non-irritant, and the compound extinction/liniment group of comparative example is slightly irritant. Examples 1-3 animals were kept normal in activity, diet, and defecation, and had no abnormal secretions in the eyes and nose. No pigmentation and bleeding spot were observed at the site of application. The medicine applying part of the comparative example compound Xiaolinian group has slight pigmentation and bleeding spots.
Third, local irritation test of damaged skin
1. Test drugs: the compound liniment prepared in examples 1 to 3 and comparative example was subjected to a topical irritation test on intact skin.
2. Test animals: the common-grade New Zealand rabbits are half female and half male, have the body weight of about 3kg and the month age of 3-4 months, and are provided by the Experimental animal center of Guangxi medical university.
3. Test site: the laboratory of the animal center, general grade, university of Guangxi medical sciences.
4. The test method comprises the following steps:
4.1 Single dose irritation test
32 New Zealand rabbits are taken, and the female rabbit and the male rabbit are half female rabbit and half male rabbit. Before the test, the two sides of the spine of the back of the rabbit are depilated by depilatory about 10 multiplied by 10cm2Disinfecting, and rubbing with fine sand paper to form local abrasion, wherein the damage degree of the left and right skin is basically consistent with the local blood seepage.
The test uses in-vivo left and right self-comparison. The left depilated area was coated with 6mL of the test drug of examples 1-3 and comparative example, and the right depilated area was coated with 6mL of 75% ethanol, and the neck of the rabbit was fixed so as not to lick the back drug. Each animal was raised in cages. After application, the residual drug was washed away with warm water, the skin reaction was visually observed 1, 24, 48, 72 hours after removal of the test drug, and the score, recovery and time of each day were recorded as given in table 1.
4.2. Multiple dose irritation test
32 New Zealand rabbits are taken, and the female rabbit and the male rabbit are half female rabbit and half male rabbit. Before the test, the two sides of the spine of the back of the rabbit are depilated by depilatory about 10 multiplied by 10cm2Disinfecting, and rubbing with fine sand paper to form local abrasion, wherein the damage degree of the left and right skin is basically consistent with the local blood seepage.
The test uses in-vivo left and right self-comparison. Coating a tested medicine on the hair removal area on the left side: examples 1-3 and comparative example 6mL of drug, 6mL of 75% ethanol was applied to the right defeathered area, and the neck of the rabbit was fixed to avoid licking the back drug. Each animal was raised in cages. The administration is continued for 5 days and then stopped for 5 days. In addition to the observation and recording of the daily erythema and edema according to the evaluation given in Table 1, the application site should be observed for pigmentation, bleeding spots, rough skin or thin skin, and the occurrence and regression times thereof should be recorded.
5. Test results
Table 5: single dose skin irritation intensity
Table 6: multiple administration skin irritation intensity
Table 5 shows that the skin irritation total score of the compound Xiaolinian groups of examples 1-3 was zero after administration, and the skin irritation total score of the compound Xiaolinian group of comparative example was 2 before 24 hours. The compound liniment groups of examples 1 to 3 are judged to be nonirritating according to the table, and the comparative example groups are mild irritations according to the table. Examples 1-3 animals all showed no abnormal response. The animal activity, appetite and defecation are all normal. The local skin color is normal in each time period, and no irritation reaction is seen. The comparative example compound Xiaolinian group has the symptoms of erythema, edema and irritation reaction.
Table 6 shows that, after the days of continuous application, the average skin irritation scores of the compound liniment groups of examples 1 and 3 are 0.05, the average skin irritation score of the compound liniment group of example 2 is 0.1, the total skin irritation score of the compound liniment group of comparative example is 2 after 24 hours, the compound liniment groups of examples 1 to 3 according to table 2 are non-irritant, and the compound liniment group of comparative example is mild reaction irritation. The animal activities, diet, defecation and the like are normal, and abnormal secretion does not appear in eyes, nose and the like. No pigmentation and bleeding spot were observed at the site of application. The comparative example compound Xiaolinian group has slight bleeding point.
IV, in vitro bacteriostasis test
1. Test drugs: the compound liniment prepared in examples 1-3 and comparative example was subjected to in vitro bacteriostatic test.
2. Test strains: escherichia coli CVCC1500, Pasteurella multocida CVCC438, Streptococcus CVCC2282, Salmonella CVCC503, Bacillus cereus CVCC2002, Streptococcus clinical strains, Escherichia coli clinical strains, Pasteurella multocida clinical strains, Escherichia coli ATCC 25922.
3. Test site: pharmacological laboratory of animal science and technology institute of Guangxi university.
4. The test method comprises the following steps:
4.1 preparation of bacterial liquid
(1) Coli: culturing with nutrient broth culture medium, and culturing with Mackanka culture medium for passage. Culturing at 37 deg.C for 15 h.
(2) Pasteurella multocida: culturing with serum broth, and culturing with blood agar medium. Culturing at 37 deg.C for 18 h.
(3) Streptococcus: culturing with serum broth, and culturing with blood agar medium.
Culturing at 37 deg.C for 20 h.
(4) Salmonella: culturing with nutrient broth culture medium, and culturing with SS culture medium for passage. Culturing at 37 deg.C for 24 h.
(5) Bacillus cereus: culturing with nutrient broth culture medium, and culturing with common agar culture medium. Culturing at 37 deg.C for 18 h.
The concentration of the bacterial liquid was measured by the McLeod turbidimetry.
4.2 Minimum Inhibitory Concentration (MIC) assay
And (3) determining the minimum inhibitory concentration MIC by using a test tube double dilution method: taking 12 sterilized test tubes, numbering, arranging the test tubes on a test tube rack according to the number of 1-12, and respectively adding 0.5mL of nutrient broth according to the aseptic operation requirement. Sucking 0.5mL of the liquid medicine, putting the liquid medicine into the 1 st tube, and uniformly mixing. 0.5mL of the solution was aspirated from the 1 st tube, and the solution was put into the 2 nd tube, and 0.5mL of the solution was aspirated after the same mixing, and the solution was put into the 3 rd tube. Dilution was performed tube by tube to tube 10, and 0.5mL was discarded in tube 10. No drug solution was added to tube 11 as a positive control, and no bacteria solution was added to tube 12 as a negative control. 0.5mL of the bacterial suspension was added to each of the first 11 tubes, and a parallel control sample without bacterial suspension was prepared for each sample according to the above method. After being put into an incubator at 37 ℃ for 18 hours, each test tube is taken out to observe the growth condition of bacteria. The lowest concentration of the drug at which the bacteria do not grow is taken as the Minimum Inhibitory Concentration (MIC) of the drug against the bacteria.
5. Test results
Test results show that the compound liniment of examples 1-3 has antibacterial effect on 5 strains, has the strongest antibacterial activity on escherichia coli standard strains and escherichia coli clinical strains, and the minimum antibacterial concentration is 1: 8. The minimum inhibitory concentration to streptococcus, salmonella, pasteurella standard strain and pasteurella clinical strain is 1: 4. The antibacterial activity to the streptococcus clinical strain and the bacillus cereus standard strain is slightly weak, and the minimum antibacterial concentration is 1: 2. The minimum inhibitory concentration of the compound liniment to several standard strains and clinical strains is 1: 2, which is higher than that of the compound liniment in examples 1-3. Compared with the test results in the literature, the compound liniment in the examples 1 to 3 has strong inhibition effect on escherichia coli, pasteurella, streptococcus clinical strains, salmonella and the like, and the inhibition effect is stronger than that of the compound liniment in the comparative example, the compound liniment is superior to the inhibition effect of only adopting a single medicament, even superior to the inhibition effect of compounding clove, tartary buckwheat and dandelion, the dosage is obviously reduced, and the combined application of the clove, the toad venom and the borneol has synergistic effect. Meanwhile, the results of the in-vitro bacteriostasis test of the compound liniment of examples 1-3 are generally better than the test group of the compound liniment of comparative example.
Five, in vitro anti-inflammatory assay
1. Test drugs: the compound liniment prepared in examples 1-3 and comparative example was subjected to in vitro anti-inflammatory tests.
2. Test cells: RAW264.7 macrophages.
3. Test site: pharmacological laboratory of animal science and technology institute of Guangxi university.
4. The test method comprises the following steps:
4.1 MTT method for determining RAW264.7 safety concentration of compound liniment prepared in examples 1-3 and comparative example
Setting Cell control group and drug group (maximum concentration is 0.2 μ L/mL of 10 times of original solution, namely 2 × 10)-4Diluted 11 dose groups with complete medium by 2-fold dilution), for a total of 12 groups, with 8 wells in each group in parallel.
RAW264.7 cells were grown in DMEM high-glucose complete medium (containing 10% fetal bovine serum, 1% glutamine, 100U/mL penicillin and 100U/mL streptomycin). Inoculating cells in logarithmic growth phase into 96-well culture plate, and adjusting concentration to 1 × 105cell/mL, 100 μ L per well, pre-wall cultureAnd culturing for 1 hour. Removing supernatant, adding diluted compound liniment of each concentration 100 μ L/well into the medicinal components, and supplementing culture solution to control group. 37 ℃ and 5% CO2The culture was carried out for 44 hours. mu.L of MTT (5mg/mL) was added to each well, mixed well and cultured for an additional 4 h. Removing supernatant, adding 100 μ L DMSO into each well, mixing by vortex, standing for 10min, and measuring OD with microplate reader490nm。
4.2 Regulation of LPS-induced secretion of proinflammatory cytokines by RAW264.7 cells by the Compound Dislinimentum prepared in examples 1-3 and comparative examples
Test set 6 groups, Cell control group, LPS control group (LPS 10 μ g/mL treatment), LPS + compound liniment group of examples 1-3 (12 dose groups), LPS + compound liniment group of comparative example (4 dose groups), respectively. Each group had 4 wells and 3 replicates were performed.
Taking RAW264.7 cells in logarithmic growth phase to adjust the cell concentration to 1 × 107cells/mL were plated in 24-well plates at 500. mu.L/well. After the cells adhered to the wall, the supernatant was discarded, and each concentration of the drugs 1000 was added to the compound liniment groups of examples 1 to 3 and the compound liniment group of comparative example. After 1 hour of treatment, LPS (final concentration 10. mu.g/mL) was added to the LPS group and incubated for 24 hours. After 24 hours, the cultured cell supernatants were collected and aliquoted into 0.5mL EP tubes and frozen at-20 ℃. The cytokine detection was performed exactly as described for the ELISA test kit.
5. Test results and discussion
5.1 influence of the compound liniment prepared in examples 1-3 and comparative example on RAW264.7 cell Activity by preliminary experiments, we selected the compound liniment prepared in examples 1-3 and comparative example 2 × 10-4The highest dilution was diluted 1/2 times, and the effect of the compound liniment prepared in examples 1-3 and comparative examples on the activity of RAW264.7 cells for 24h and 48h was observed. The results show that the compound Xiaolinian 8 groups prepared in examples 1-3 and comparative example can significantly reduce the cell activity when acting for 24h, while the compound Xiaolinian 9-12 groups can significantly reduce the cell activity; when the compound liniment is acted for 48 hours, the compound liniment 11-12 prepared in the examples 1-3 and the comparative example can remarkably reduce the cell activity. See tables 7-10:
TABLE 7 EXAMPLE 1 Effect of Compound XIAO Liniment on RAW264.7 cell Activity
Table 8 example 2 Effect of Compound Xiaolinian on RAW264.7 cell Activity
TABLE 9 EXAMPLE 3 Effect of Compound XIAO Liniment on RAW264.7 cell Activity
TABLE 10 Effect of comparative example Compound Xiaolinimentum on RAW264.7 cell Activity
Note: p < 0.05 compared to Cell group; p < 0.01 compared to Cell group.
5.2 Regulation of LPS-induced secretion of proinflammatory cytokines by RAW264.7 cells by Compound Dislinimentum prepared in examples 1-3 and comparative examples
Through the experiments of the compound liniment prepared in the examples 1 to 3 and the comparative example on the activity of RAW264.7 cells, the compound liniment prepared in the examples 1 to 3 and the comparative example has a safe concentration dose of 4 to 7 groups of dosage on RAW264.7 cells, and the in vitro anti-inflammatory effect of the compound liniment prepared in the examples 1 to 3 and the comparative example is studied. As can be seen from the results, TNF- α levels were significantly increased 24h after stimulation with LPS (significantly different from the control cell group). After the compound liniment in the examples 1-3 is added, the IL-1 beta and IL-6 levels can be greatly reduced by 6-7 doses (the difference is very obvious compared with a cell control group and an LPS group); the doses 6-7 significantly reduced the IL-2 level (significantly different from the cell control group and LPS group), while the dose 4 significantly reduced the IL-2 level (significantly different from the cell control group and LPS group). After the compound liniment of the comparative example is added, the IL-1 beta and IL-6 levels can be obviously reduced only by the dose 4 (the levels are obviously shown compared with a cell control group and an LPS group). The compound liniment of examples 1-3 is shown to have certain effect of reducing the levels of proinflammatory cytokines IL-1 beta, IL-6 and anti-inflammatory cytokines IL-2. The compound liniment has a certain regulating effect only at high concentration. The results are shown in tables 11-14:
TABLE 11 EXAMPLE 1 Regulation of LPS-induced secretion of proinflammatory cytokines by Compound Dislinimentum (pg/mL) from RAW264.7 cells
TABLE 12 EXAMPLE 2 Regulation of LPS-induced secretion of proinflammatory cytokines by Compound Dislinimentum (pg/mL) from RAW264.7 cells
TABLE 13 EXAMPLE 3 Regulation of LPS-induced secretion of proinflammatory cytokines by Compound Dislinimentum (pg/mL) from RAW264.7 cells
TABLE 14 Regulation of LPS-induced RAW264.7 cell secretion of proinflammatory cytokines (pg/mL) by comparative example compound liniment
Note: p < 0.05 compared to cell group; p < 0.01 in comparison with Cell group
#, P < 0.05 compared to LPS group; #, P < 0.01 in comparison with LPS group
5.3 discussion
The inflammatory response is a protective stress response of the body against the attack of external pathogenic factors. From the viewpoint of inducing inflammation, cytokines can be classified into two types, pro-inflammatory cytokines and anti-inflammatory cytokines. Proinflammatory cytokines such as TNF, IL-1, IL-6, and the like; anti-inflammatory cytokines such as IL-2, IL-4, IL-10, and the like. Generally, the interaction between proinflammatory cytokines and anti-inflammatory cytokines forms an important balance mechanism in the body, i.e., "cytokine balance". After being attacked by stresses such as trauma, infection and the like, the two factors are obviously changed, and at the moment, the body can generate and release excessive proinflammatory cytokines, and the proinflammatory cytokines can induce a chain reaction which is in an uncontrolled state and is amplified step by step through the continuous activation of downstream active substances, so that the self-injury is caused.
The macrophage system is the central cell that initiates the production of inflammatory mediators in vivo and is the primary cell that regulates the inflammatory response. Among the causes of inflammation, LPS is an important trigger for the initiation of an inflammatory response, and LPS stimulates the synthesis and release of numerous endogenous bioactive factors by various cells in the body, particularly macrophages, resulting in a systemic inflammatory response.
The RAW264.7 cell is used as a macrophage, participates in the immune process of an organism, particularly can start the generation of in-vivo inflammation mediators after being interfered by LPS and other external factors, and starts the resisting function to inhibit the generation of inflammation. When activated, cells are capable of producing large amounts of cytokines. This experiment was induced by in vitro establishment of LPSAn in-vitro inflammation model of macrophage of a RAW264.7 mouse researches the in-vitro anti-inflammatory effect of the compound liniment prepared in examples 1-3 and a comparative example on RAW264.7 cells, and experimental results show that the compound liniment prepared in examples 1-3 with extremely low concentration can inhibit LPS (LPS) to induce the RAW264.7 cells to secrete proinflammatory cytokines IL-6 and IL-1 beta; the compound liniment of examples 1-3 is shown to have the function of inhibiting the production of proinflammatory mediators. On the other hand, the compound liniment of examples 1-3 can reduce the level of IL-2 and inhibit the production of anti-inflammatory cytokines, which indicates that the compound liniment of examples 1-3 has the function of regulating the balance of proinflammatory cytokines secreted by cells. MTT experiments prove that the compound liniment in examples 1-3 can generate certain toxic effect on cells due to direct action of the components. But diluted 2X 10 times in 10 times stock solution4The effect of the compound on the viability of RAW264.7 cells in a multiple concentration range is not influenced, and the effect of inhibiting the release of inflammatory mediators is not related to the effect of cytotoxicity. Meanwhile, the balance of proinflammatory and anti-inflammatory cytokines can be adjusted by the compound liniment of examples 1-3 with extremely low concentration, and the compound liniment of examples 1-3 has a certain anti-inflammatory effect. However, the compound liniment of the comparative example can inhibit the proinflammatory production and regulate the balance of proinflammatory cytokines secreted by cells.
In conclusion, the experimental results show that the compound liniment of examples 1-3 has better effects of inhibiting the production of proinflammatory mediators and regulating the balance of cell secretion anti-inflammatory cytokines than the compound liniment of comparative example.
Sixthly, curative effect test
1. Test drugs: the compound liniment prepared in examples 1-3 and comparative example is used for efficacy test.
2. Test animals: the cow breast and milk have no abnormal change visible to naked eyes, but the cow with positive detection by a (CMT) diagnostic solution is determined as recessive mastitis.
3. Test site: a certain dairy farm in Guangxi.
4. The test method comprises the following steps:
4.1 treatment of occult mastitis.
50 cows with positive milk were selected by (CMT) diagnostic test and divided randomly into test groups (examples 1-3, comparative) and control groups. The test group cattle are uniformly sprayed with the medicine in each affected mammary area every day, and the medicine liquid is uniformly sprayed in the whole mammary area without leakage and dripping. The preparation is administered once a day after milking in the morning and evening, and 5 days is a course of treatment. The control group was not treated at all.
The curative effect assessment standard is as follows: before the test, the test group and the control group are sampled at 5 days, 15 days and 30 days after the test, the change condition of the cow milk is detected by a (CMT) method judgment standard, negative is used as recovery, negative and suspicious are used as effectiveness, and positive is used as ineffectiveness. Meanwhile, the total milk yield of the test group and the control group before the test and at the 5 th, 15 th and 30 th days after the test is measured, and the increase or decrease rate of the two groups of cows is calculated by taking the milk yield at the beginning of the test as a reference.
4.2 clinical mastitis treatment test
After the sick cattle are found and diagnosed in a cattle farm, the sick cattle are treated with test medicines immediately, and the medicines are uniformly sprayed on each sick breast area every day, so that the whole breast area is uniformly sprayed with the medicine liquid without leakage or dripping. The preparation is administered once a day after milking in the morning and evening, and 5 days is a course of treatment.
The judgment standard of the curative effect is as follows:
the healing judgment standard is as follows: the appetite is normal, the red, swollen, hot and painful breast areas disappear, the milk is normal when observed by naked eyes, and the milk yield is restored to a normal level.
And (4) invalid judgment standard: after 5 days, the appetite is normal, the red, swollen, hot and painful breast areas disappear unobviously, the milk is still abnormal, and the milk yield is not recovered.
5. Results and discussion
5.1 results of treatment of cow subclinical mastitis with the compound liniment prepared in examples 1-3 and comparative example
5.1.1 results of the compound Xiao Ling prepared in examples 1-3 and comparative example on the treatment of cow subclinical mastitis
TABLE 15 clinical curative test results on occult mastitis
From table 15, it can be concluded that: the compound liniment of examples 1 to 3 has obvious treatment effect on the cow invisible mastitis, and the cure rate is 91.6 to 94.4 percent. The cure rate of the compound Xiaolinian in the comparative example is 77.7%, which is lower than that of the compound Xiaolinian in examples 1-3, and the cow milk is still more positive without any treatment in the control group.
5.1.2 influence of the Compound Xiaolinian preparation prepared in examples 1-3 and comparative examples on milk yield
TABLE 16 influence of the compound liniment prepared in examples 1-3 and comparative example on milk production of cows
Note: the day before the test is 0 day;
the milk yield increase rate is (daily milk yield of the group on the day after the test-daily milk yield of the group on the day before the test) multiplied by 100%
From table 16, the total milk yield of the cows with the compound Xiaolinian in examples 1-3 is higher than that of the comparative example group, the total milk yield of the compound Xiaolinian in examples 1-3 on day 5 is increased by 5.4-5.7%, the total milk yield of the compound Xiaolinian in the comparative example is increased by 2.3%, and the increase rate of the compound Xiaolinian in examples 1-3 is increased to 10-11.3% on day 30; the comparative example of the compound Xiaolinian improves to 5.1%; the total milk yield of the control group dairy cows is always in a descending trend, and is reduced by 3.5% on day 5 and 15.9% on day 30. It can be seen that the compound Xiao Ling preparation of examples 1-3 has significant effects in restoring health of cow breast and increasing milk yield, and is superior to the compound Xiao Ling preparation of comparative example.
5.2 the compound liniment prepared in examples 1-3 and comparative example has the effect of treating clinical mastitis of dairy cows.
TABLE 17 clinical curative effect test results of mastitis
Remarking: if the medicine is continuously taken for 5 days and is not cured, the medicine is combined with antibiotics; the number of the heads of each group is 20, 18 heads (90) of the compound Xiaolinian in the examples 1-3 are cured, 13 heads (65) of the compound Xiaolinian in the comparative examples are cured, 2 heads of the compound Xiaolinian groups in the examples 1-3 are cured by combining with antibiotics, and 7 heads of the compound Xiaolinian group in the comparative examples are cured.
The results in table 17 show that the compound liniment of examples 1-3 has a cure rate of 90.0% and an effective rate of 100% for clinical mastitis in dairy cows, and shows a good treatment effect. The cure rate of the comparative example compound Xiao Ling is 65 percent, which is lower than that of each group in the examples. In the test process, the compound liniment of examples 1-3 is combined with antibiotics to show good synergistic treatment effect.
The compound liniment of examples 1 to 3 has obvious therapeutic effect on the recessive mastitis and can maintain the health of the organism for a long time, which shows that the compound liniment has the functions of enhancing the immune function of the dairy cows, resisting the infection of various pathogenic microorganisms, improving the capability of the dairy cows for resisting various stresses and improving the milk yield of the cattle with the recessive mastitis; it is presumed that the compound liniment of examples 1 to 3 has a therapeutic effect on cow mastitis, which is different from that of antibiotics, and does not exert a therapeutic effect by a simple antibacterial effect, but has pharmacological effects of antipyresis, anti-inflammation, antagonism of bacterial toxin, enhancement of immune function of the body, and the like. The comparative example compound Xiaolinian group has the treatment effect on the recessive mastitis, but the treatment effect is lower than that of the example group. In addition, the compound Xiaolinian in the embodiments 1-3 has good therapeutic action on clinical mastitis; the comparative example compound Xiaolinian has poor treatment effect on clinical breasts, and the cure rate is 65%. Meanwhile, the practice shows that the sterilization liniment has better synergistic effect when being combined with antibiotics, and the compound sterilization liniment group in the embodiment has better effect than the compound sterilization liniment in the comparative example.
The compound liniment of examples 1-3 is prepared by making effective components penetrate breast skin through the action of transdermal enhancer to reach effective drug concentration in mammary gland, and has antibacterial, antiinflammatory, exudation inhibiting, and breast microcirculation improving effects. It is not only convenient to use, but also avoids the liver first pass effect of oral administration, the toxic and side effects of gastrointestinal tract and the stress reaction of breast perfusion administration to the organism; has quick response and good curative effect on mastitis, and is worthy of popularization and application.
Seventhly, method for measuring multiple effective components of bufonid mastitis eliminating liniment by utilizing HPLC-PAD method colleagues
1 test Material
1.1 medicine
Controls of cinobufagin (batch No. 160803-201605), resibufogenin (batch No. 160718-201608), eugenol (batch No. 110725-201512);
the three bufonis venenum Linn liniment has numbers of 20161001, 20161002, and 20161003;
1.2 reagents
Methanol, acetonitrile and other reagents were chromatographically pure.
1.3 instrumentation
Waters e2695 hplc; a diode array detector (PAD); electronic balance Discovery; a numerical control ultrasonic cleaner (model: KQ-250 DE); a water purifier (model: CD-UPT-II-20L);
2 test methods and results
2.1 test methods
2.1.1 preparation of Mixed control solutions
Accurately weighing appropriate amount of cinobufagin, resibufogenin and eugenol as reference substances, and diluting with methanol to obtain mixed reference substance solution containing cinobufagin, resibufogenin and eugenol 53, 50 and 50 μ g per 1 mL.
2.2.2 preparation of test solutions
Precisely taking a proper amount of buformin emulsion liniment, placing into a 10mL measuring flask, adding methanol to dilute to scale, shaking, and performing ultrasonic treatment for 15min to obtain the final product.
2.2 chromatographic conditions
Inertsil ODS-3C 18 (4.6X 250mm, 5 μm); taking 0.05mol/L citric acid aqueous solution as a mobile phase A phase and acetonitrile as a mobile phase B phase, isocratically eluting (60: 40), wherein the detection wavelength is 290nm, the column temperature is 30 ℃, and the sample injection amount is 20 mu L;
2.3 specialization examination
2.3.1 Linear relationship investigation
The standard solutions of cinobufagin, resibufogenin and eugenol are respectively in the ranges of 1.65-15.14 mu g/mL, 1.72-15.71 mu g/mL and 1.57-14.28 mu g/mL, the concentrations and the response values (peak areas) of the concentrations form good linear correlation, and the concentrations (X) and the peak areas (Y) are subjected to linear regression to obtain a linear equation of the cinobufagin, which is as follows: y 11933x-2568.5, and correlation coefficient r 0.9998; the lipobufogenin linear equation is as follows: 19006x-3335.5, and the correlation coefficient r is 0.9994; the eugenol linear equation is: y 11613x 4113.7 and the correlation coefficient r 0.9997.
2.3.2 precision test
Respectively taking 13.25, 12.5 and 12.5 mu g/mL of cinobufagin, resibufogenin and eugenol mixed reference substance solution, carrying out HPLC-PAD determination according to the 2.2 chromatographic condition, carrying out continuous sample injection for 6 times, and determining the peak area. The results are shown in Table 18, and the RSD of cinobufagin, resibufogenin and eugenol is 0.78, 0.76 and 0.73%, respectively. Indicating good precision.
TABLE 18 precision test results
2.3.3 stability test
Weighing 1mL of bufonis liniment, preparing a sample according to a test article preparation method, performing HPLC-PAD determination according to a 2.2 chromatographic condition, and performing sample injection determination for 0, 2, 4, 6, 8 and 12 h. The results are shown in table 19, and RSDs of cinobufagin, resibufogenin, and eugenol are 0.17, 0.34, and 0.18%, respectively, indicating that the sample stability is good.
TABLE 19 stability test results
2.3.4 repeatability test
Weighing 6 parts of a same batch of sample of the bufalin liniment, preparing the sample according to a test preparation method, performing HPLC-PAD determination according to a 2.2 chromatographic condition, and determining the contents of cinobufagin, resibufogenin and eugenol. The results are shown in table 20, and RSDs of cinobufagin, resibufogenin, and eugenol were 0.59, 0.61, and 0.47%, respectively, indicating good reproducibility.
TABLE 20 results of the repeatability tests
2.3.5 sample recovery test
Taking the bufonis eliminating liniment with known content, adding reference substance solution with known concentration, preparing sample according to the preparation method of test sample, performing HPLC-PAD determination according to the '2.2 chromatographic condition', and determining the contents of cinobufagin, resibufogenin and eugenol. The results are shown in table 21, and the average recoveries of cinobufagin, resibufogenin and eugenol were 89.32, 89.72 and 94.33%, respectively, and the RSDs were 0.42, 0.67 and 0.33%, respectively, indicating good recoveries.
TABLE 21 sample recovery test results
2.3.6 sample testing
Weighing 1mL of three batches of bufonis bufonid mastitis eliminating liniment, preparing a sample according to a test article preparation method, performing HPLC-PAD determination according to 2.2 chromatographic conditions, and determining the contents of cinobufagin, resibufogenin and eugenol. The results are shown in Table 22.
TABLE 22 determination of the contents of three batches of Chan Ding Ru Yan Xiao Ling linimentum (n ═ 3)
The table shows that the contents of three toad butyronitrile liniment, namely cinobufagin, resibufogenin and eugenol are respectively (0.16 +/-0.02)%, (0.22 +/-0.01)% and (1.14 +/-0.02)%.
The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and such substitutions and modifications are to be considered as within the scope of the invention.
Claims (8)
1. A traditional Chinese medicine compound Xiaolinian for preventing and treating cow mastitis is characterized in that: the defoaming agent comprises the following raw materials in parts by weight: 1-5g of toad venom, 500-2000g of clove, 5-30g of borneol, 805-20mL of polysorbate, 5-15g of sesbania gum, 5-10g of sucrose glyceride stearate, and the balance of solvent added to 1000 mL.
2. The traditional Chinese medicine compound liniment for preventing and treating cow mastitis according to claim 1, is characterized in that: the solvent consists of ethanol and propylene glycol.
3. The traditional Chinese medicine compound liniment for preventing and treating cow mastitis according to claim 2, is characterized in that: the volume ratio of the ethanol to the propylene glycol is 7: 3.
4. The preparation method of the traditional Chinese medicine compound liniment for preventing and treating cow mastitis according to any one of claims 1 to 3, which is characterized in that: the method comprises the following steps:
(1) preparing an extracting solution: collecting 1-5g of Bufonis venenum, cleaning, drying, pulverizing into fine powder, adding 300mL of 80% ethanol 150, reflux extracting, mixing extractive solutions, filtering, and refrigerating. Removing impurities from 500-2000g of clove medicinal materials, screening out dust, mashing, adding 6-8 times of water, soaking for 1-3h, heating to 100 ℃ for distillation, collecting volatile oil when oil and water are separated, repeatedly extracting once, standing the collected volatile oil for 24-4g of hours, filtering, and refrigerating for later use;
(2) uniformly mixing sesbania gum and sucrose stearate glyceride, and adding a small amount of water to dissolve the sesbania gum and the sucrose stearate glyceride to obtain a mixed solution;
(3) mixing the venenum bufonis extract and the clove volatile oil, adding 5-20mL of polysorbate 80 and the mixed solution, uniformly mixing, adding 5-30g of borneol under stirring, adding a solvent mixed solvent, stirring to completely dissolve, filtering, and adjusting the total amount of the filtrate to 1000mL by using the solvent mixed solvent to obtain the product.
5. The preparation method of the traditional Chinese medicine compound liniment for preventing and treating cow mastitis according to claim 4, is characterized in that: the reflux extraction is carried out at 70-80 deg.C for 2-3 times, each time for 1-2 hr.
6. The quality detection method of the traditional Chinese medicine compound liniment for preventing and treating cow mastitis according to any one of claims 1 to 5, which is characterized in that: the quality determination method is to determine the contents of three components of Chinese bufogenin, resibufogenin and eugenol as the traditional Chinese medicine compound liniment by adopting an HPLC-PAD method, and comprises the following specific steps:
s1: chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; taking 0.05mol/L citric acid aqueous solution as a mobile phase A phase and acetonitrile as a mobile phase B phase, carrying out isocratic elution, wherein the detection wavelength is 200-300nm, the column temperature is 30-40 ℃, the flow rate is 1-3mL/min, and the sample injection amount is 10-40 mu L;
s2: preparation of a reference solution: precisely weighing appropriate amount of cinobufagin, resibufogenin and eugenol as reference substances, diluting with methanol to desired volume, and mixing to obtain mixed reference substance solution;
s3: preparing a test solution: precisely taking a proper amount of buformin emulsion liniment, placing into a 10mL measuring flask, adding methanol to dilute to scale, shaking, and performing ultrasonic treatment for 15min to obtain a test solution;
s4: and (3) determination: precisely sucking 20 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, recording peak area, and calculating by external standard method.
7. The quality detection method of the traditional Chinese medicine compound liniment for preventing and treating cow mastitis according to claim 6, which is characterized in that: the volume ratio of the mobile phase A to the mobile phase B is 60: 40.
8. The quality detection method of the traditional Chinese medicine compound liniment for preventing and treating cow mastitis according to claim 6, which is characterized in that: the contrast solution contains cinobufagin, resibufogenin and eugenol 53ug, 50ug and 50ug respectively per 1 mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1781518A (en) * | 2004-11-30 | 2006-06-07 | 中国农业科学院兰州畜牧与兽药研究所 | Medicinal composition for treating milk cow mammitis and its preparing method |
| CN102188581A (en) * | 2011-05-11 | 2011-09-21 | 张亚东 | Traditional Chinese medicinal composition for treating bovine mastitis |
| CN103251691A (en) * | 2013-05-24 | 2013-08-21 | 西南大学 | Chinese medicine formula for treating bovine mastitis as well as extract and preparation thereof |
| CN109187802A (en) * | 2018-10-18 | 2019-01-11 | 广州悦康生物制药有限公司 | The detection method of dried venom of toads content in pill for promoting coronary circulation is concentrated |
-
2020
- 2020-12-31 CN CN202011642679.6A patent/CN112618598A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1781518A (en) * | 2004-11-30 | 2006-06-07 | 中国农业科学院兰州畜牧与兽药研究所 | Medicinal composition for treating milk cow mammitis and its preparing method |
| CN102188581A (en) * | 2011-05-11 | 2011-09-21 | 张亚东 | Traditional Chinese medicinal composition for treating bovine mastitis |
| CN103251691A (en) * | 2013-05-24 | 2013-08-21 | 西南大学 | Chinese medicine formula for treating bovine mastitis as well as extract and preparation thereof |
| CN109187802A (en) * | 2018-10-18 | 2019-01-11 | 广州悦康生物制药有限公司 | The detection method of dried venom of toads content in pill for promoting coronary circulation is concentrated |
Non-Patent Citations (1)
| Title |
|---|
| 陈斌等: "超临界流体萃取法测定六应丸中的冰片和丁香酚的含量", 《中国中药杂志》 * |
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