CN113332245B - Compound sophora flavescens spray for treating cow foot rot, and preparation method and application thereof - Google Patents

Compound sophora flavescens spray for treating cow foot rot, and preparation method and application thereof Download PDF

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CN113332245B
CN113332245B CN202110729785.6A CN202110729785A CN113332245B CN 113332245 B CN113332245 B CN 113332245B CN 202110729785 A CN202110729785 A CN 202110729785A CN 113332245 B CN113332245 B CN 113332245B
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radix sophorae
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于文会
姜晓文
马隽
刘金松
冯国峰
王海彬
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Northeast Agricultural University
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Abstract

A compound radix sophorae flavescentis spray for treating cow foot rot and a preparation method and application thereof belong to the technical field of animal external drugs. In order to obtain an external medicine capable of effectively treating the cow foot rot and not polluting the environment, the sophora flavescens, the phellodendron amurense and the smilax glabra are compatible, prepared into superfine powder, extracted by using ethanol, and then boric acid, salicylic acid and a color developing agent brilliant blue are added into the extract, so that the external medicine for treating the cow foot rot, which can obviously improve clinical symptoms, shorten symptom improvement time and cure time, is obtained. Compared with the conventional treatment method for cow foot rot, the medicine has high cure rate and no environmental pollution.

Description

Compound sophora flavescens spray for treating cow foot rot, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of animal external medicines, and particularly relates to a compound radix sophorae flavescentis spray for treating cow foot rot, and a preparation method and application thereof.
Background
Cow foot rot is one of three common diseases (mastitis, endometritis and foot rot) faced by cow breeding all over the world, and belongs to the invasion of damp-heat toxin and the collection of damp-heat on the hoof to cause the necrosis of the hoof tissue. For the treatment of cow foot rot, currently, in domestic clinical treatment, antiseptic disinfectant formalin is mainly used for disinfecting hooves (sonyada, sun-limna, fangchen, etc., cow foot rot spray treatment research [ J ]. Chinese cows 2010(4):36-38) or heavy metal ion hardened hoof boxes such as copper sulfate and zinc sulfate (payfish, prevention and treatment of cow foot rot [ J ], animal husbandry veterinarians, 2015(3): 152). There are also clinical cases where cow foot rot is treated with traditional Chinese medicines, such as the observation of the effect of cow foot rot treated with "cow foot health bath lotion" in combination with traditional Chinese medicines [ J ], Chinese herbivorous animals 2006, 26(6):51-52), where methanol, formaldehyde, copper sulfate solution are combined with traditional Chinese medicines to treat cow foot rot. Whether formalin, heavy metal copper sulfate and zinc sulfate can cause certain pollution to the environment. Especially, the discharge of heavy metal ions such as copper sulfate, zinc sulfate and the like is prohibited according to the current environmental protection method, so that the application of heavy metal salts such as copper sulfate, zinc sulfate and the like in the traditional hoof bath is limited. Therefore, a medicine which can effectively prevent and treat the cow foot rot and has no pollution to the environment is urgently needed to be found.
Disclosure of Invention
The invention provides a compound sophora flavescens spray for treating cow foot rot, aiming at solving the problem of environmental pollution of the existing cow foot rot treatment medicine or method, the compound sophora flavescens spray comprises the following raw materials: sophora flavescens, phellodendron amurense, rhizoma smilacis glabrae, developer brilliant blue, boric acid and salicylic acid.
In one embodiment of the present invention, the ratio of the sophora flavescens, the phellodendron amurense and the smilax glabra in the compound sophora flavescens spray is as follows: 2-3 parts of radix sophorae flavescentis, 1-1.5 parts of golden cypress and 2-3 parts of rhizoma smilacis glabrae.
In one embodiment of the present invention, the ratio of the sophora flavescens, the phellodendron amurense and the smilax glabra in the compound sophora flavescens spray is as follows: 2 parts of radix sophorae flavescentis, 1 part of cortex phellodendri and 2 parts of rhizoma smilacis glabrae.
The invention also provides a preparation method of the compound sophora flavescens spray as claimed in any one of claims 1 to 3, which comprises the following steps:
1) weighing radix Sophorae Flavescentis, cortex Phellodendri and rhizoma Smilacis Glabrae according to weight parts, mixing, and making into Chinese medicinal micropowder;
2) according to the weight ratio of 1 kg: 2L-1 kg: adding a material-liquid ratio of 2.5L into the traditional Chinese medicine micro powder obtained in the step 1), leaching for 20-28 h by using 70% ethanol solution, filtering, centrifuging, and taking supernatant to obtain leaching liquor;
3) adding boric acid and salicylic acid into the leaching liquor obtained in the step 2), and fully stirring;
4) adding a color developing agent brilliant blue into the leaching liquor obtained in the step 3), fully stirring, subpackaging, sealing and storing.
In one embodiment of the invention, the particle size of the Chinese medicinal micropowder in the step 1) is 300-400 meshes.
In one embodiment of the present invention, the ratio of the material to the liquid in step 2) is 1 kg: 2.5L, and the leaching time is 24 h.
In one embodiment of the present invention, the boric acid and the salicylic acid in step 3) are added in an amount of 10 g/L.
In one embodiment of the invention, the addition amount of the developer brilliant blue in the step 4) is 0.15-0.20 g/L.
The invention also provides an application of the compound sophora flavescens spray as claimed in any one of claims 1 to 3 in preparing a medicine for treating cow foot rot.
In one embodiment of the present invention, the compound sophora flavescens spray as claimed in any one of claims 1 to 3 is sprayed onto the affected part 2 times a day for 5 to 7 days.
The invention has the beneficial effects that:
the invention provides a compound radix sophorae flavescentis spray capable of effectively treating cow foot rot, which utilizes the traditional veterinary medicine theory, takes clinical differentiation of cow foot rot as a formula principle, takes radix sophorae flavescentis as a monarch drug and cortex phellodendri as a ministerial drug, has the functions of clearing heat and drying dampness, mainly clears damp-heat toxin and mutually reinforces to play a synergistic effect and increase the curative effect. Rhizoma smilacis glabrae is an adjuvant drug, has the effects of detoxifying and dehumidifying, can dissolve pathogenic qi such as furuncle, carbuncle swelling, scrofula and the like, is combined with radix sophorae flavescentis and cortex phellodendri for application, enhances the functions of removing putrefaction and promoting granulation of the prescription and improves the treatment effect of the cow foot rot while improving the functions of clearing heat and eliminating dampness of the radix sophorae flavescentis and the cortex phellodendri. By using 70% ethanol for extraction, active ingredients in the traditional Chinese medicine formula can be extracted to the maximum extent, and the salicylic acid which is insoluble in water is dissolved at the same time. In addition, 70% ethanol has good disinfection effect as an external medicine. The addition of boric acid and salicylic acid can promote the repair of damaged tissues. The addition of brilliant blue can facilitate the positioning of the application site. By using 70% ethanol for extraction, active ingredients in the traditional Chinese medicine formula can be extracted to the maximum extent, and the salicylic acid which is insoluble in water is dissolved at the same time. In addition, 70% ethanol has good disinfection effect as an external medicine. The addition of boric acid and salicylic acid can promote the repair of damaged tissues. The addition of brilliant blue can facilitate the positioning of the application site. The compound sophora flavescens spray prepared by the method not only has the antibacterial and antiseptic effects, but also has the effect of promoting skin injury repair, has no toxic or side effect on skin tissues, and does not cause environmental pollution. Veterinary clinical experiment results show that the compound radix sophorae flavescentis spray has a remarkable treatment effect on cow foot rot.
Drawings
FIG. 1 shows a process for preparing a compound radix Sophorae Flavescentis spray;
fig. 2 is a diagram of the stability detection result of the compound sophora flavescens spray.
Detailed Description
The present invention will be further described in detail with reference to the following specific examples and accompanying drawings, and the preparation process of the compound sophora flavescens spray of the present invention is shown in fig. 1.
Example 1: preparation of compound sophora flavescens spray
1) Weighing 0.8kg of radix sophorae flavescentis, 0.4kg of cortex phellodendri and 0.8kg of rhizoma smilacis glabrae, mixing and crushing into traditional Chinese medicine micro powder of 300 meshes;
2) according to the weight ratio of 1 kg: adding 70% ethanol solution into the Chinese medicinal micropowder obtained in step 1) at a feed-liquid ratio of 2.5L, leaching for 24 hr, filtering, centrifuging, and collecting supernatant to obtain leaching solution;
3) adding 40g of boric acid and 40g of salicylic acid into the leaching liquor obtained in the step 2), and fully stirring;
4) adding 0.64g of developer brilliant blue into the leaching liquor obtained in the step 3), fully stirring, subpackaging, sealing and storing.
Example 2: preparation of compound sophora flavescens spray
1) Weighing 1.5kg of radix sophorae flavescentis, 0.5kg of cortex phellodendri and 1.5kg of rhizoma smilacis glabrae, mixing and crushing into 325-mesh traditional Chinese medicine micro powder;
2) according to the weight ratio of 1 kg: adding 70% ethanol solution into the Chinese medicinal micropowder obtained in step 1) at a feed-liquid ratio of 2L, leaching for 28h, filtering, centrifuging, and collecting supernatant to obtain leaching solution;
3) adding 60g of boric acid and 60g of salicylic acid into the leaching liquor obtained in the step 2), and fully stirring;
4) adding 1g of developer brilliant blue into the leaching liquor obtained in the step 3), fully stirring, subpackaging, sealing and storing.
Example 3: preparation of compound sophora flavescens spray
1) Weighing 2kg of radix sophorae flavescentis, 1.5kg of golden cypress and 2kg of rhizoma smilacis glabrae, mixing and crushing into 400-mesh traditional Chinese medicine micro powder;
2) according to the weight ratio of 1 kg: adding 70% ethanol solution into the Chinese medicinal micropowder obtained in step 1) at a feed-liquid ratio of 2L, leaching for 20h, filtering, centrifuging, and collecting supernatant to obtain leaching solution;
3) adding 100g of boric acid and 100g of salicylic acid into the leaching liquor obtained in the step 2), and fully stirring;
4) adding 1.5g of developer brilliant blue into the leaching liquor obtained in the step 3), fully stirring, subpackaging, sealing and storing.
Example 4: application of compound radix sophorae flavescentis spray in treatment of cow foot rot
In this example, the compound spray of sophora flavescens obtained in example 1 was used as a sample for examination.
1. Stability detection of compound sophora flavescens spray
Bottling and sealing the compound radix Sophorae Flavescentis spray, storing in shade for 6 months, and detecting the properties of the spray every other month, with the detection results shown in Table 1 and figure 2. The results show that the preparation has no precipitate and floccule in 6 months, and the color, smell and pH of the preparation have no change. The stability test result of the compound lightyellow sophora root spray shows that the quality of the product is stable at different time in the storage process and meets the requirements.
TABLE 1 stability test results
Figure BDA0003138902140000031
Figure BDA0003138902140000041
2. Pharmacological experiment of compound sophora flavescens spray
(1) Detection of antibacterial experiments
The MIC and the MBC are detected by adopting a two-fold dilution method, and the experimental result shows that the MIC of the compound sophora flavescens spray to escherichia coli is 62.5mg/mL, and the MBC is 125 mg/mL; the MIC for Staphylococcus aureus was 31.25mg/mL, and the MBC was 62.5 mg/mL. The results of the experiment are shown in table 2.
TABLE 2 MIC and MBC of the compound radix Sophorae Flavescentis spray to several bacteria
Figure BDA0003138902140000042
(2) Tissue repair capability detection
Selecting 10 SD rats with weight of about 230g and age of 8 weeks, and dividing the SD rats into a medicinal group and a control group at random, wherein the SD rats are healthy and have normal eating, drinking, defecation, urination, body temperature and respiration through clinical detection, and each group comprises 5 rats. Anesthetizing rat, and removing skin hair of 2 × 2cm on both sides of rat spine with shaver2A circular wound of 10mm diameter, up to the fascia, was created at the skin of the shaved area using a skin sampler, and all rats were modeled at the same location on the back, with repeated shaves every 5 days. Each group of rats is smeared with the medicine for 3 times every day, 1mL of the medicine is smeared every time, and sterile dressing is replaced after each smearing, so that the rats are naturally healed; the control group was not dosed. On days 1, 4, 7, 10, and 14 after modeling, 1 rat was randomly selected from each group at these 5 time points, and normal skin up to the fascia including within 1cm around the wound was excised, and the wound healing rate was calculated (see table 3). The wound healing rate is (area of original wound surface-area of residual wound surface)/area of original wound surface × 100%. The results show that the compound radix sophorae flavescentis spray group and the control group have obvious difference (P)<0.05), the wound healing rate of the rat in the compound sophora flavescens spray group can reach 100% on the 14 th day after modeling.
Table 3 rat wound healing rate (%)
Figure BDA0003138902140000043
Note: the differences were significant, as compared to the control group.
3. Detection of safety of compound sophora flavescens spray to skin
(1) Detection of acute toxicity to skin
The number of male and female parts of 25 adult healthy rats is close, and the weight of the adult healthy rats is about 200 g. The groups were randomly divided into a 70% ethanol solvent control group, an intact skin low dose group, an intact skin high dose group, a damaged skin low dose group, and a damaged skin high dose group, with 5 per group. Depilating rat dorsal spine at both sides for 10cm 24h before administration2. 1mL of the drug is evenly sprayed on the depilating area on the back of the rat, the broken skin group is applied after a # -shaped wound is scratched on the depilating area on the back of the rat by a disposable syringe, and the broken skin group is fixed by medical gauze and adhesive tape. After spraying the drug for 24h, the residue on the back of the rat was gently removed with warm water and observed daily for 7 consecutive days. Attention is paid to systemic toxic manifestations and death conditions of rats after administration, including changes in animal body weight, skin, hair, eyes, mucous membranes and secretions, as well as changes in respiratory system, circulatory system, central nervous system, limb activities, and the like. The test results are as follows:
after the skin of the rat is removed from the test object, the weight, the skin, the hair, the eyes, the mucous membrane and the secretion of the rat are normal and no obvious change exists in the respiratory system, the circulatory system, the central nervous system, the activities of four limbs and the like after continuous observation for 7 days. Comparing the body weight of each test group with that of the control group on days 1, 4 and 7, no significant difference (P >0.05) is observed, see Table 4.
TABLE 4 weight comparison (g) of rat in compound radix Sophorae Flavescentis spray for acute toxicity test
Figure BDA0003138902140000051
(2) Long term toxicity test on skin
The number of male and female parts of 25 adult healthy rats is close, and the weight of the adult healthy rats is about 200 g. Randomly dividing into blank control group, compound radix Sophorae Flavescentis spray low dose group (0.5 mL), compound radix Sophorae Flavescentis spray medium dose group (1 mL), compound radix Sophorae Flavescentis spray high dose group (2 mL), 70% ethanol control group (1 mL), and 5 pieces per group. Depilating rat dorsal spine at both sides for 10cm 24h before administration2。
The medicine is applied on the depilated area of the back of rat, and fixed with medical gauze and adhesive tape, 1 time daily, at least 6h contact, and continuously administered for 14 d. In addition to daily clinical symptoms, general indices, and pathological examination, hematology (red blood cells (RBC), White Blood Cells (WBC), Hemoglobin (HB), Platelets (PLT), monocyte count (Mon), lymphocyte count (Lym), neutrophil count (Neu)), hematology-chemistry indices (alanine transaminase (ALT), glutamic-oxaloacetic transaminase (AST), hematurin (UREA), blood Glucose (GLU), Creatinine (CREA), Creatine Kinase (CK), total Cholesterol (CHOL), etc.) was performed when the animals were killed, organ indices (heart, liver, spleen, lung, kidney) were weighed and calculated at the time of killing, and organ and skin tissues were made into paraffin sections and observed by HE staining. The results are as follows:
firstly, detecting the influence on the body weight
The experimental results show that the skin, hair, eyes, mucous membrane and secretion of each group of rats are normal, and the respiratory system, the circulatory system, the central nervous system, the activities of four limbs and the like have no obvious changes. There was no significant difference (P >0.05) between each experimental group and the blank control group, and the results are shown in table 5.
Table 5 long term toxicity test rat body weight comparison (n ═ 5)
Figure BDA0003138902140000061
② blood routine index influence detection
The hematology index of the rats in each experimental group has no significant difference (P is more than 0.05) compared with that of the blank control group, and the experimental result is shown in the table 6.
Table 6 long term toxicity test results (n ═ 5) from hematology in rats
Figure BDA0003138902140000062
Third, the influence of blood biochemical index is detected
The blood biochemical indexes of the rats in each experimental group have no significant difference (P is more than 0.05) compared with those of a blank control group, and the experimental results are shown in table 7.
Table 7 long-term toxicity test results of biochemical tests of rat blood (n ═ 5)
Figure BDA0003138902140000071
4. Skin irritation detection of compound radix sophorae flavescentis spray
One skin irritation experiment: 10 adult healthy rats, half male and half female, weigh about 200 g. Rats were randomly divided into 2 groups of 5 rats each, a complete skin group and a damaged skin group, respectively, and the experiment employed a self-control. Rats were depilated from both sides of the dorsal spine 24h before administration, approximately 10cm per side2. The hairs were shaved again every 5 days. In the depilation region, 0.5mL of the drug is administered to the left side one time or more, and 70% ethanol solvent with the same amount is smeared to the right side, and fixed by medical gauze and adhesive tape. After 24h, the residue was removed, and the application site was observed for erythema and edema at 1h, 24h, 48h and 72h, and the recovery of the above changes and the time.
Multiple skin irritation experiments: the administration method of multiple experiments is consistent with that of one experiment, and the administration method is applied once a day for 1 week continuously. And (5) judging a result: a skin irritation response score; evaluation of skin irritation intensity. The experimental results are as follows:
after the rat is subjected to the compound radix sophorae flavescentis spray and the 70% ethanol solution in equal amount once, the complete skin group and the control group are continuously observed for 3 days, no obvious erythema and edema are seen in the complete skin group and the control group, slight redness and swelling appear in the damaged skin group, new hairs can be seen in the damaged skin and the complete skin, the wound of the damaged skin is basically healed in the 4 th day, no obvious bleeding is seen, and the healing conditions of the damaged skin on the two sides of the rat are consistent.
After multiple times of medicine stimulation, scoring and strength evaluation are carried out according to the research guidelines of new traditional Chinese medicines. In the intact skin group, 1 rat skin showed slight erythema, in the damaged skin group, 3 rats showed slight erythema, and in the control group, there was no abnormality. After the administration is finished, erythema gradually disappears in each group, part of hairs fall off in the damaged skin group, no erythema and edema are seen, the skin is smooth and normal in color, normal new hairs can be seen in each group, and the damaged skin on two sides of the rat is not obviously abnormal in recovery condition. The results are shown in Table 8.
Table 8 multiple skin irritation test evaluations (n ═ 5)
Figure BDA0003138902140000072
Figure BDA0003138902140000081
5. Skin sensitization detection of compound sophora flavescens spray
15 adult healthy guinea pigs, with a similar number of males and females, weigh approximately 300 g. The medicine is divided into 3 groups at random, each group comprises 5 medicines, a positive control group and a negative control group. Depilating the dorsal spine of guinea pig 24h before administration to about 9cm2. The positive sensitizer is prepared from 2, 4-dinitrochlorobenzene to 1% sensitization concentration and 0.1% excitation concentration.
Sensitization and contact: the test article was applied to the left area and fixed with medical gauze and tape for 6 hours. 7d and 14d, repeated in the same manner. The other compositions have the same method.
And (3) exciting contact: the test article was smeared on the right side area 14d after the last sensitization with the test article, removed after 6h, observed immediately, and then observed again at 24/48/72 h.
And (5) judging a result: skin hypersensitivity scoring criteria; and (5) classifying sensitization rate. The results are as follows:
according to the research on skin sensitization of guinea pigs, the sensitization response score and the sensitization degree evaluation are carried out according to the research guidelines of new traditional Chinese medicines. The drug was washed with warm water 6h after the challenge in 3 test groups, and the skin of each guinea pig in each group was continuously observed and recorded after 1h, 24h, 48h, and 72h after the drug administration for the presence or absence of erythema, edema, and the like, and the death of the animals. In the experimental process, the skins of guinea pigs of the medicine group and the negative control group do not have erythema and edema, and do not have systemic anaphylactic reaction such as asthma, dyspnea, shock and the like, 4 skin of guinea pigs can be obviously seen after the positive control group is stimulated to contact with the medicine group, 1 mild erythema of guinea pigs, 2 skin of guinea pigs have obvious edema, hair has slight yellow stain, erythema of 3 guinea pigs is obviously aggravated after 24h, hair yellow stain is aggravated, 48h severe erythema is regressed, edema is relieved, erythema is regressed gradually for 72h, edema disappears, animals of the positive control group also do not have anaphylactic symptoms such as unstable standing, dyspnea, shock and the like, and do not die without animals. The sensitization incidence is the total number of cases with allergic reaction on the skin/total number of test animals x 100%, and the results are shown in table 9.
Table 9 evaluation of skin sensitization test (n ═ 5)
Figure BDA0003138902140000082
6. Compound radix sophorae flavescentis spray for treating cow foot rot
In the clinical examination of a certain cow farm, 20 adult cows with definite foot rot diagnosis are selected as test animals, and the species, the weight, the group combination and the like of the animals are consistent. Test cattle are randomly divided into a compound sophora flavescens spray test group and a 2% iodophor control group, wherein 10 cows in the test group and 10 cows in the control group are respectively numbered.
Cleaning hoof of affected cow, treating with compound radix Sophorae Flavescentis spray 2 times per day for 7 days in experimental group, spraying affected part with 2% iodophor 2 times per day in control group, and continuously applying for 7 days. The spray covers the whole affected area, and the physiological activities and clinical reactions of the cows before and after administration are observed. The results of the clinical treatment experiments were as follows:
clinical observation shows that no clinical drug reaction is seen when the test group and the control group are used. The basal physiological indexes of the sick dairy cows, such as body temperature, pulse and respiration, do not change obviously, the spirit is good, the appetite and the milk yield are increased, and the lameness is greatly improved.
The compound sophora flavescens spray for treating the cow foot rot is compared with iodophor for treating the cow foot rot: the clinical symptoms such as red and swollen hoof, ulceration, lameness and the like are better than those of the iodophor treatment group, and the milk yield increasing effect is also better than that of the iodophor group. The symptom improving time and the curing time are obviously shortened compared with the iodophor group. The results are shown in Table 10.
TABLE 10 Experimental results of treating cow foot rot with compound radix Sophorae Flavescentis spray
Figure BDA0003138902140000091

Claims (6)

1. A preparation method of a compound sophora flavescens spray for treating cow foot rot is characterized by comprising the following steps:
1) weighing radix Sophorae Flavescentis, cortex Phellodendri and rhizoma Smilacis Glabrae according to weight parts, mixing, and making into Chinese medicinal micropowder; the proportion of the radix sophorae flavescentis, the golden cypress and the rhizoma smilacis glabrae is as follows: 2-3 parts of radix sophorae flavescentis, 1-1.5 parts of golden cypress and 2-3 parts of rhizoma smilacis glabrae;
2) according to the weight ratio of 1 kg: 2L-1 kg: adding a material-liquid ratio of 2.5L into the traditional Chinese medicine micro powder obtained in the step 1), leaching for 20-28 h by using 70% ethanol solution, filtering, centrifuging, and taking supernatant to obtain a leaching solution;
3) adding boric acid and salicylic acid into the leaching liquor obtained in the step 2), and fully stirring; the addition amount of the boric acid and the salicylic acid is 10 g/L;
4) adding a color developing agent brilliant blue into the leaching liquor obtained in the step 3), fully stirring uniformly, subpackaging, sealing and storing; the addition amount of the developer brilliant blue is 0.15-0.20 g/L.
2. The preparation method according to claim 1, wherein the ratio of radix Sophorae Flavescentis, cortex Phellodendri and rhizoma Smilacis Glabrae in step 1) is as follows: 2 parts of radix sophorae flavescentis, 1 part of cortex phellodendri and 2 parts of rhizoma smilacis glabrae.
3. The preparation method according to claim 1, wherein the particle size of the Chinese medicinal micropowder obtained in the step 1) is 300-400 mesh.
4. The preparation method according to claim 1, wherein the feed-liquid ratio in step 2) is 1 kg: 2.5L, and the leaching time is 24 h.
5. The use of the spray of compound radix Sophorae Flavescentis obtained by the preparation method of any one of claims 1-4 in the preparation of a medicine for treating cow foot rot.
6. The use of claim 5, wherein the compound spray is sprayed on affected parts 2 times a day for 5-7 days.
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