CN117482116A - Use of toad skin based extract for preparing pharmaceutical composition for treating bone pain - Google Patents

Abstract

The invention relates to application of a toad skin extract in preparing a pharmaceutical composition for treating bone pain, a pharmaceutical composition containing the toad skin extract and a preparation method for preparing the toad skin extract. The pharmaceutical composition of the invention comprises the toad skin extract of the invention and optionally one or more pharmaceutically acceptable excipients and/or carriers and may also comprise one or more other pharmaceutically active ingredients. The invention also particularly discloses various external pharmaceutical preparations containing the toad skin extract for treating bone pain.

Description

Use of toad skin based extract for preparing pharmaceutical composition for treating bone pain

Technical Field

The invention belongs to the technical field of medicine preparation, relates to application of a toad skin extract in preparing a pharmaceutical composition for treating bone pain diseases, a pharmaceutical composition containing the toad skin extract and a method for preparing the toad skin extract, and particularly discloses various external pharmaceutical preparations containing the toad skin extract for treating various bone pain diseases.

Background

Bone pain refers generally to various rheumatic bone pain, joint pain, lumbago, cervical spondylosis, gout, bone pain caused by traumatic injury and sprain, bone tumor, etc.

Traumatic injury is one of common diseases of human beings, and the patients have sprains and tendons, bruises, muscle swelling, blood stasis, meridian bone pain and the like, so that the travel, work, life and the like of the patients are influenced; the rheumatic bone pain is caused by wind, cold and damp mixed qi, is a arthralgia formed by mixing various pathogenic factors, and is retained in joints to block meridians, so that the joints are painful, numb, swollen, stiff and even paralyzed, and great pain is brought to patients; for various reasons, the incidence of arthralgia, sciatica, rheumatism, rheumatoid disease and the like is on the rise, and the disease becomes a frequently occurring disease. Because rheumatalgia is a systemic disease mainly affecting joints, bones, muscles, blood vessels and related soft tissues or connective tissues, which is mainly manifested by chronic and non-suppurative inflammation of a plurality of joints, the rheumatalgia has a great deal of damage to the body. If the patients are not treated in time, a series of complications such as pneumonia, urinary tract infection and heart diseases are easily caused, and the patients with serious illness can have malformed bone rigidity, even joint dislocation and the like; gout is a group of diseases of long-term purine metabolic disorder and tissue injury caused by blood uric acid elevation, and has the clinical characteristics that: hyperuricemia, recurrence of acute arthritic response, tophus formation, chronic arthritis and joint deformity, uric acid stones and gouty kidney parenchymal lesions appear in the later stages of the disease. Gout has high morbidity, difficult healing and high repetition rate after healing, is accompanied with various complications, is listed as a remarkable difficult and complicated disease, and has an unsatisfactory common treatment effect; metastatic cancer bone pain (metastatic cancer-induced bont pain, CIBP) is caused by tumor bone metastasis, which is the most common metastasis of advanced malignant tumors, and can occur in any tumor, with breast, lung, and prostate cancers being particularly common. CIBP severely affects the quality of life of tumor patients, one of the most difficult types of pain to alleviate in the pain area. The general strategy of CIBP treatment is to relieve pain and improve comprehensive treatment with the quality of life as a main aim, and a treatment scheme of long-term combination of a three-step analgesic drug and bisphosphonate for periodic application is commonly adopted clinically at present to obtain a satisfactory result, but problems of dry mouth, constipation, nausea, hypodynamia and the like caused by expensive opioid analgesics and drug resistance and potential addiction risks caused by continuous application are difficult to eliminate.

At present, the means for treating the rheumatic bone pain mainly comprise drug treatment, physical treatment, massage acupuncture treatment, closed therapy, operation treatment and the like, and the simple use of the drugs for treating the rheumatism or the massage, acupuncture and massage can only relieve pain symptoms, so that the treatment purpose can not be achieved, the regular application of the drugs has a certain influence on viscera and organs, and the problem can not be fundamentally solved. The traditional Chinese medicine has the defects of complex prescription, high cost, long treatment course, large side effect, repeated attack after healing and the like at different degrees. The operation treatment has high cost, and the treatment itself can cause the pain of the patient, and the pain is hard to be accepted.

Therefore, there is still an urgent need in the art to provide a pharmaceutical product for treating bone pain, which has definite and good effects, preferably has the advantages of high safety, easy quality control, convenient and reliable sources, and the like.

The cutis Bufonis is dry whole body of Bufo siccus Bufo bufogargarizans cantor and Bufo siccus Bufo melanostictus Schneider of Bufo siccus belonging to Bufo siccus family. Collecting, processing, generally after capturing in spring and autumn, taking out or not taking out Bufonis venenum on Bufo siccus, removing viscera by laparotomy, and removing together with chin and abdomen. The body cavity is spread, dried in the sun, and then dried in an oven with charcoal fire, turned over at any time, and dried to be fully dried (the process needs to be turned over at any time to avoid burning), or the body cavity is dried by adopting an electric heating oven to control the temperature. It is sweet, pungent and cool; is toxic. It has effects in clearing away toxic materials, relieving swelling, tonifying heart, and relieving pain. Can be used for treating malignant boil, carbuncle, chronic tracheitis, sore throat, edema, etc.

The bufogenin is mainly bufogenin (Bufogenin), resibufogehin (resibugin), cinobufagin (cinobugin), bufogenin (Cinobufagin), bufalin (Bufalin), bufalin (Tenocinobugin) and the like. Also contains indole derivatives such as bufonin (bufotaline), bufotaline and 5-hydroxytryptamine, epinephrine, beta-sitosterol, cholesterol, etc. (Mei Quanxi, manual of modern Chinese medicine and clinical application, chinese medical Press, 1 st edition, 493 month 10 in 2008).

In clinical application, the toad has many researches on detoxification, anti-inflammation, acute and chronic suppurative infection, leukemia, anti-tumor, anticancer auxiliary drugs and the like, for example, the toad lipid extract has a certain clinical effect on various cancers, especially on lymphocytic leukemia (Jiang Faxian, the main code of clinical and research on venenum bufonis, traditional Chinese medicine literature, ocean publishing, beijing, 1994, page 58). The water-soluble extract of Bufo siccus has inhibiting effect on cells such as human neurocollagen tumor, liver cancer, leukemia and uterine cancer cultured in vitro (Zhang Yuyao, etc., experimental study of water-soluble total components of Bufonis venenum, nanjing university of Chinese medicine, 1998,7 (2): 33), (Yang Sujuan, clinical application and research progress of Bufo siccus and Bufonis venenum, journal of Chinese medicine, 1992,9 (5): 2). The domestic live oviductus Ranae is made into Momordica Charantia tissue liquid injection for relieving cough and asthma, and treating senile chronic tracheitis; boiling oviductus Ranae, making into paste, soaking in 95% ethanol, extracting twice, recovering ethanol, and extracting bufogenin injection for treating advanced schistosomiasis complicated with colon cancer (Cao Chunlin, chinese medicinal preparation, national health Press, 10 th edition 1983, pages 608-609). Also, 200g of toad and 1000 ml of 0.9% sodium chloride solution are added to prepare injection for treating psoriasis, dermatitis herpetiformis, furuncle, etc. (Cao Chunlin, chinese medicinal preparation, national health publishing company, 10 th edition 1983, page 1670-1671). The folk use of the toad powder has a certain effect on treating tumors, but has higher toxicity, and then the extract prepared by taking the toad skin (toad skin) as a raw material has lower toxicity. The composition is verified by delamination method to contain Bufotenine, bufotenidine, epinephrine, bufotoxin, bufonin essence, and Bufotoxin. Can be used for treating hepatocarcinoma, esophageal carcinoma, seminoma, sarcoma, psoriasis, behcet's disease, lupus erythematosus, scleroderma, etc. (general health department, pharmaceutical inspection, chinese herbal medicine data Association, pages 148-149).

Although traditional Chinese medicine has the record of analgesic effect on toad skin, the study and application of toad skin on analgesic are rarely reported in literature. The present literature has researches on the analgesic function of the toad venom, such as' the toad venom can detoxify and detumescence, has good analgesic effect, is clinically used for detumescence and analgesia of carbuncle, cellulite, pharyngitis and other symptoms, is also commonly used for pain caused by toothache, muscle strain, bone spur, arthritis and the like, and can be used as a surgical anesthetic. This effect of the product is related to its action on the central nervous system. The analgesic active ingredients of the toad venom mainly exist in chloroform extract, 6 active ingredients separated from the chloroform extract have analgesic effect, and have a certain dose-effect relationship, and the analgesic effect of 0.49% concentration of the active ingredients is stronger than that of ibuprofen with lower concentration (Shen Yingjun main edition, traditional Chinese medicine pharmacology, 2 nd edition, people health press, 10 th edition of 2000, page 775). However, the application of toad skin as pain relieving is disclosed in patent literature, for example, CN101195002A discloses that the pain relieving health care traditional Chinese medicine plaster is prepared from 18 traditional Chinese medicinal materials such as toad skin and the like, and has the total effective rate of 94.03% for treating pain symptoms caused by cervical spondylosis, scapulohumeral periarthritis, hyperosteogeny and intervertebral disc protrusion. CN104096178A discloses a "rehabilitation patch for treating visceral pain and bone pain" which is a black plaster prepared from 35 Chinese medicinal materials such as toad skin and the like. Is suitable for acute and chronic gastroenteritis, duodenal bulbar ulcer, diarrhea, abdominal distention and stomachache; menoxenia, dysmenorrhea, hyperplasia of mammary glands; traumatic injury, fracture, lumbar sprain, lumbar muscle strain, chest and back pain, old injury, swelling, pain and numbness of joints of hands and feet, rheumatoid arthritis, cervical and lumbar disc herniation, hyperosteogeny, sciatica and the like. CN105125887a discloses a traditional Chinese medicine for treating tumor neck, shoulder, waist and leg pain, which is prepared from 35 traditional Chinese medicines such as toad skin, etc., and is prepared into capsules or honeyed pills by pulverizing, mixing and filling, and can effectively treat scapulohumeral periarthritis, cervical spondylosis, lumbar disc herniation and rheumatism and rheumatoid disease. CN110664856a discloses "a Chinese herbal medicine composition for external application for treating lumbago and skelalgia" which is a plaster preparation containing toad ingredients, and is matched with magnetite and tourmaline to carry out magnetic therapy on the lumbago and skelalgia affected part, so that the curative effect is further improved. CN111643534a discloses a traditional Chinese medicine formula for treating pain, which is prepared by decocting toad, szechuan lovage rhizome, cinnabar and borneol into powder or adding honey for decoction, and treating lumbago and skelalgia and sciatica by oral administration. CN114191504a discloses a "Chinese medicinal composition for treating various pains", which is prepared from 12 medicinal materials such as toad skin and the like, and is prepared into patches, ointments and ointments for treating cervical hyperplasia, scapulohumeral periarthritis, compulsive spondylitis, lumbar intervertebral disc herniation and the like. Although the above preparations have certain effects, the curative effect and the safety of the preparations are still to be further improved and verified, in addition, the ingredients of the preparations are more and complex, the problems of easiness in obtaining raw material sources and difficulty in quality control possibly need to be considered, meanwhile, the compositions of the medicines are complex, the effects of the toad skin in the compositions are difficult to prove, the effects of the toad skin in the compositions are also not cheap and easily available, the administration cost is reduced, and the potential problem of complex interaction among unknown ingredients possibly exists. Therefore, there is still an urgent need in the art to provide a pharmaceutical product for treating bone pain that is exactly good in effect and preferably has the advantages of high safety, convenient and reliable sources, etc.

Disclosure of Invention

The Bufo siccus skin is dry skin of Bufo siccus Bufo bufogargarizans cantor and Bufo siccus Bufo melanostictus Schneider of Bufo siccus belonging to Bufo siccus family. Pungent and cool in nature; is toxic. It enters heart, spleen, lung and large intestine meridians. It has effects in clearing away heat and toxic materials, killing parasites, treating infantile malnutrition, inducing diuresis, and relieving edema. The chemical components of Bufo siccus are complex, mainly bufogenin glycosides, such as Bufonis venenum toxin, such as bufogenin, lipobufogenin, cinobufagin, hydroxy cinobufagin (cinobufagin), bufogenin, and far bufogenin obtained by hydrolyzing Bufonis venenum toxin. Also contains indole derivatives such as bufonin (bufotaline), bufotaline 5-hydroxytryptamine, epinephrine, beta-sitosterol, cholesterol, etc. Traditionally, they are mainly used as detumescence, detoxification, pain-relieving and diuretic. It is used for treating carbuncle, furuncle, sore, infantile malnutrition, chronic tracheitis, sore throat, ascites, scrofula, and cancer.

However, the present inventors have unexpectedly found in the study that toad skin extract has unexpected effects on bone pain caused by various causes. The prior art shows that the toad skin is respectively researched and applied in the treatment of diseases by extracts of chloroform, ethanol and water, but no practical application is reported for bone pain caused by various reasons. The inventor finds that the mixed extract obtained by extracting toad skin without extracting toad venom with diethyl ether and then carrying out alcohol and chloroform fractional progressive extraction has good curative effect on bone pain caused by various diseases. Based on the above findings, the present inventors have completed this invention.

In one aspect of the present invention, there is provided a toad skin extract, wherein the toad skin extract is prepared by drying and pulverizing toad skin without venenum Bufonis, and extracting with diethyl ether, alcohol and chloroform, respectively, wherein the diethyl ether is medicinal diethyl ether, and the alcohol comprises any one or a combination of methanol and ethanol. Preferably ethanol, which is an aqueous acidic ethanol solution, is typically present at a concentration of 45% to 95%, preferably 55% to 95%, more preferably 65% to 90%, most preferably 80%. The acidic ethanol solution is aqueous alcohol containing inorganic acid, and the inorganic acid comprises hydrochloric acid, nitric acid, sulfuric acid or phosphoric acid, wherein the preferable inorganic acid is hydrochloric acid or sulfuric acid. More preferably, an aqueous ethanol solution containing 0.1% to 1% hydrochloric acid or sulfuric acid is used as an extraction solvent, particularly preferably an aqueous 80% ethanol solution containing 0.1% to 1% hydrochloric acid or sulfuric acid is used as an extraction solvent, and still more preferably an aqueous 80% ethanol solution containing 0.5% hydrochloric acid or sulfuric acid is used as an extraction solvent. The alcohol solvent in the present invention may be a combination of ethanol and a methanol solvent, including a combination of alcohol and water, a combination of methanol and ethanol, a combination of ethanol, methanol and acid, and water, and the like.

In another aspect of the invention, there is provided the use of the toad skin extract in the manufacture of a pharmaceutical composition for the treatment of bone pain. Wherein the pharmaceutical composition can be in the form of a pharmaceutical formulation product or can be in the form of an intermediate for preparing a pharmaceutical formulation product. The pharmaceutical composition comprises the toad skin extract of the invention and optionally one or more pharmaceutically acceptable excipients or carriers. The excipient or carrier may be, but is not limited to, water, ethanol, propylene glycol, polyethylene glycol, vegetable oil, glycerin, liquid paraffin, tween 80, span 80, surfactant, soft soap, lanolin, vaseline, sodium carboxymethyl cellulose, methylcellulose, starch, tragacanth, acacia, agar, bletilla gum, sodium alginate, polyvinyl amines, polyvinylpyrrolidone, polyvinyl alcohol, talc, kaolin, vegetable oil, soft soap, paraffin, and the like. In addition, the pharmaceutical composition may also comprise one or more other active pharmaceutical ingredients. Examples of such other pharmaceutically active ingredients include, but are not limited to, red sage extract, chuanxiong rhizome extract, clove extract, safflower extract, angelica extract, pricklyash peel extract, asarum extract, rhizoma corydalis extract, olibanum, myrrh, methyl salicylate, camphor, borneol, dragon's blood, artificial bezoar, artificial musk, taurine, menthol, shikimic acid, eucalyptus oil, zaocys dhumnade extract, prepared aconite root extract, prepared radix aconiti kusnezoffii extract, combined spicebush root extract, and the like.

In another aspect of the present invention, there is provided an external preparation comprising the toad skin extract and the pharmaceutical composition as described above, which may be any pharmaceutical preparation, including but not limited to solutions, lotions, liniments, tinctures, sprays, aerosols, emulsions, gels, suspensions, films, ointments, creams, patches. Preferred are liniments, tinctures, sprays, emulsions, and patches.

In yet another aspect of the present invention, there is provided a method for preparing the toad skin extract, the method comprising at least the steps of:

(1) Baking and drying toad skin without Bufonis venenum, pulverizing into superfine powder, and extracting toad skin with diethyl ether as solvent to obtain filter residue and diethyl ether extractive solution respectively;

(2) Leaching the filter residue in the step (1) by using the ethanol as a solvent to obtain filter residue and an extracting solution;

(3) Extracting the filter residue in the step (2) further by using chloroform as a solvent to obtain an extracting solution;

(4) After the solvents in the steps (1), (2) and (3) are respectively recovered, mixing to obtain a mixed material;

(5) Optionally removing impurities from the extraction mixture by filtration, centrifugation or other suitable means, and optionally concentrating and/or drying to obtain liquid or solid extracts of different forms and concentrations.

The invention provides a pharmaceutical composition for preparing a medicine for treating bone pain, which comprises the toad skin extract and optionally one or more pharmaceutically acceptable excipients or carriers, and can further comprise one or more other active pharmaceutical ingredients. Examples of such other active pharmaceutical ingredients include, but are not limited to, the ingredients described above.

Terms and definitions described in the present invention:

the term "pharmaceutically acceptable excipient and/or carrier" as used in this specification means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation auxiliary. Remington's Pharmaceutical Sciences ed.by Gennaro, mackpubishing, easton, pa.,1995 discloses various carriers for formulating pharmaceutical compositions and known techniques for preparing such pharmaceutical compositions. The person skilled in the art can also determine them based on technical knowledge and other literature.

The term "treating or preventing" as used in this specification means having a cure, elimination, alleviation, amelioration, or delay of action of, or preventing or delaying the onset of, a disease or condition experienced by a subject (including a human or mammal).

In addition, it is also contemplated that the pharmaceutical formulations of the present invention each comprise an effective amount of a pharmaceutically active ingredient. The effective amount is selected by a physician in light of treating the patient. An "effective amount" refers to the amount necessary to produce the desired biological response. It will be appreciated by those of ordinary skill in the art that the effective amount of the toad skin extract of the present invention can vary depending on a variety of factors, including the severity of the disease state, the age, weight and sex of the patient being treated, the diet, the time and frequency of administration; drug combination; reaction sensitivity; and tolerance response to treatment, etc.

The other active ingredients of the medicine are most commercial products, and the unusual active ingredient materials can be prepared according to published literature materials or be provided by entrusted qualified enterprises according to the prior art. The person skilled in the art can realize homemaking according to the mastered basic technical common knowledge.

In the present invention, "%" ratio is referred to as weight "%" ratio unless otherwise specified.

In the invention, toad skin is crushed into superfine powder with the particle size of 200 meshes.

The invention has the beneficial effects that the toad skin is respectively extracted by diethyl ether, ethanol and chloroform to obtain an ether, alcohol and chloroform extraction mixture, which means that the toad skin extract is used as the main component for treating the bone pain and is applied to the preparation of the pharmaceutical composition, the pain relieving advantage of treating the bone pain disease is exerted, and the defect of the utilization of the toad skin in the prior art is overcome.

The invention dries and pulverizes toad skin without extracted toad venom into 200 mesh superfine powder, the surface area is increased, a large amount of cells are broken, and a plurality of insoluble polymer substances easily enter the leaching liquid. The invention adopts the progressive fractional extraction of diethyl ether, ethanol and chloroform, is favorable for realizing the maximum dissolution rate in different fat-soluble solvents, and can leach the active ingredients contained in the solvent as much as possible to leach more active ingredients.

The invention has the advantages that the repeated experiments show that the extraction is carried out for three times respectively through diethyl ether, ethanol and chloroform, and the extraction rate is higher than that of diethyl ether, ethanol and chloroform, so that the defect that the single diethyl ether extract, chloroform extract or ethanol extract has influence on the analgesic effect in the prior art is possibly overcome.

The comparative experiments were performed as follows:

1. three experiments with diethyl ether

The method comprises the steps of firstly adopting toad skin without venenum bufonis, baking and drying, crushing into 200-mesh superfine powder, weighing 100g of superfine powder, adding 1000g of diethyl ether, soaking for 7 days at room temperature, stirring for 2 times a day during soaking, filtering after soaking, evaporating the soaking liquid to remove diethyl ether, and drying to obtain 3.9g of diethyl ether extract; extracting the filter residues for the second time and the third time, wherein the method steps are the same as above, the extracts are respectively 1.8g and 0.8g, and the total extraction of 6.5g for three times is equivalent to 6.5% of the original medicine amount;

2. Three experiments with ethanol as solvent

Firstly, adopting toad skin without toad venom extraction, baking and drying, crushing into 200-mesh superfine powder, weighing 100g of superfine powder, adding 1000g of 80% ethanol aqueous solution, soaking for 72 hours at room temperature, filtering the leaching solution, evaporating to remove ethanol, and drying to obtain 5.3g of ethanol extract; extracting the filter residues for the second time and the third time, wherein the method steps are the same as above, and the extracts are respectively 2.6g and 1.2g, and the total extraction of 9.1g for three times is 9.1% of the original medicine amount;

3. three experiments with chloroform as solvent

Firstly, baking and drying toad skin without venenum bufonis, crushing into 200-mesh superfine powder, weighing 100g of superfine powder, adding 1000g of chloroform, soaking for 48 hours at room temperature, oscillating from time to time, filtering the leaching solution, recovering the chloroform, and drying to obtain 3.8g of chloroform extract; the filter residues are extracted for the second time and the third time, the method steps are the same as above, the extracts are respectively 2.2g and 1.3g, and the total extraction of 7.3g is carried out for three times, which is equivalent to 7.3 percent of the original medicine amount.

Extraction test I was carried out by the method of the present invention

Baking and drying toad skin without Bufonis venenum, pulverizing into 200 mesh superfine powder, weighing 100g of superfine powder, adding 1000g of diethyl ether, soaking at room temperature for 7 days, stirring for 2 times a day during soaking, and filtering to obtain filter residue and diethyl ether extract respectively;

Respectively adding 1000g of 80% ethanol into the filter residues, leaching twice at room temperature for 72 hours each time, filtering, and combining the secondary leaching solutions to obtain filter residues and ethanol extract respectively;

adding 1000g of chloroform into the residues after ethanol leaching, oscillating and leaching at room temperature for 48 hours at intervals, filtering, and discarding filter residues to obtain chloroform extract;

the solvent is recovered from the above extractive solutions, and the extractive solutions are combined, dried, and pulverized to obtain 22-26 g of Bufo siccus skin extract (powder) which is equivalent to 22-26% of the original dosage.

The extraction test II was carried out by the method of the invention

Baking and drying toad skin without Bufonis venenum, pulverizing into 200 mesh superfine powder, weighing 100g of superfine powder, adding 1000g of diethyl ether, soaking at room temperature for 7 days, stirring for 2 times a day during soaking, and filtering to obtain filter residue and diethyl ether extract respectively;

adding 1000g of 80% ethanol containing 0.5% hydrochloric acid into the filter residue, heating, refluxing and extracting for 2 hours, and filtering to obtain filtrate; extracting the residue with 1000g of 80% ethanol containing 0.5% hydrochloric acid under reflux for 1 hr, filtering, mixing the filtrates, and collecting ethanol extractive solution;

Adding 1000g of chloroform into the filter residue, oscillating at room temperature, leaching for 48 hours, filtering, and discarding the filter residue to obtain chloroform extract;

the extract is respectively recovered from the solvents, and then is combined to obtain a mixture, and the mixture is dried and crushed to obtain 27g-30g of toad skin extract (powder), which is equivalent to 27-30% of the original drug quantity.

According to the comparison experiment, the traditional method which takes diethyl ether, ethanol and chloroform as toad skin extracts respectively has less mass, and compared with the method which takes diethyl ether, ethanol and chloroform as the mixture for progressive extraction for the next time, the method can realize the maximum extraction of diethyl ether-soluble, ethanol-soluble and chloroform-soluble components in toad skin, thereby not only improving the utilization rate of toad skin, but also saving the cost of raw and auxiliary materials such as solvents, and the like, and particularly the preparation of the mixed extract of the invention has better pain relieving effect in the use of clinical bone pain cases, has great practical value in the aspect of clinical pain relieving for the toad skin extracts, and also has the research direction of expanding the application range. As can be seen from a comparison of experiments I and II of the method of the invention, the extraction by hot reflux extraction with acid ethanol has the significance of high extraction rate compared with the extraction at room temperature by using ethanol alone, the final extract of experiment I is 22g-26g, which is equivalent to 22-26% of the original dosage, and the final extract of experiment II is 27g-30g, which is equivalent to 27-30% of the original dosage. This is beneficial for improving the application of raw materials and reducing costs.

The toad skin extract of the invention can be any external preparation for preparing a pharmaceutical composition for treating osteodynia, and the preparation can be, but is not limited to, solvent, lotion, liniment, tincture, spray, aerosol, emulsion, gel, suspension, film coating agent, ointment, cream and patch. Preferably liniment, tincture, emulsion, spray, ointment, and patch. The preparations can be absorbed through skin to directly reach focus, has rapid effect, and can better realize the analgesic treatment effect on rheumatic osteodynia, arthralgia, lumbago, gout and osteodynia caused by cervical spondylosis and bone tumor.

Description of the embodiments

As the preparation of toad skin extract, diethyl ether is usually adopted as a solvent to prepare the extract, ethanol or methanol is adopted as the solvent to prepare the extract, chloroform is adopted as the solvent to prepare the extract, and the application of water extract is also useful, however, any single solvent extract has incomplete extraction and factors affecting the application effect, however, the invention adopts diethyl ether, ethanol and chloroform to sequentially extract in a progressive way, so that the aim of fully extracting the toad functional components is fulfilled, and the extraction rate and the higher utilization rate are improved. In the preparation of the toad skin extract, the toad skin without the toad venom is used as a raw material, and is sequentially extracted by diethyl ether, alcohol and chloroform, so that the toad venom component can be reserved and extracted in the extraction process, and the efficacy of the toad venom component is displayed. The method at least comprises the following steps: (1) Baking toad skin without Bufonis venenum, pulverizing into superfine powder, and extracting toad skin with diethyl ether to obtain diethyl ether extract; (2) Extracting the filter residue obtained in the step (1) again by using the alcohol or the aqueous alcohol as a solvent to obtain an alcohol extract; (3) Extracting the filter residue in the step (2) with chloroform as a solvent to obtain chloroform extract; (4) After the solvents in the steps (1), (2) and (3) are respectively recovered, the solvents are combined to obtain a mixed material; and (5) optionally removing impurities from the extraction mixture by filtration, centrifugation or other suitable means, and optionally concentrating and/or drying to obtain liquid or solid extracts of different forms and concentrations. For example, the extraction may be performed at room temperature, or may be performed at room temperature with the aid of ultrasonic treatment to facilitate the extraction. The extraction can be carried out once or twice according to actual needs, and the extraction conditions with or without heating can be selected according to the physical properties of the solvent. It is also possible to carry out the reaction under heating, for example at the reflux temperature of the solvent. The extraction may be carried out generally twice, for example, the first time may be generally 1.5 to 2.5 hours and the second time may be generally 1 to 1.5 hours, and the two extracts (solutions) may be combined, and the multiple extracts (solvents) of multiple solvents may be combined. Optionally, the toad skin medicinal material may be subjected to pretreatment before extraction, for example, selecting, removing impurities, drying, and the drying conditions may be selected from a proper method such as oven drying, charcoal fire baking or microwave drying. The amount of the extraction solvent can be 8-12 times of the weight of the medicinal materials on the premise that the medicinal materials can be fully extracted. In order to save the preparation cost, the solvent can be recycled by a conventional method, the extract materials can be concentrated to different degrees according to the need after being filtered or centrifuged, and the extract materials can be crushed, sieved and dried according to the need, so that the extracts in different forms such as suspension, fluid extract, powder or granule are obtained.

Specific embodiments of the present invention will be described in detail by way of examples. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes, modifications and improvements within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. For a better description of the present invention, the following examples are provided for reference.

Example 1

Preparation of toad skin extract

(1) Oven drying Bufo siccus skin without Bufonis venenum, pulverizing into 200 mesh superfine powder, weighing 1kg of superfine powder, placing into brown small-mouth bottle, adding 10kg of medicinal diethyl ether, shaking sufficiently to make the material uniform, sealing, soaking at room temperature for 7 days, stirring or shaking for 2 times each day during soaking, filtering, and collecting the residue to obtain diethyl ether extractive solution;

(2) Adding 10kg of 80% ethanol into the filter residue obtained in the step (1), placing in a round bottom flask, heating and reflux-extracting for 2 hours, filtering, retaining filtrate, reflux-extracting the filter residue with 8kg of 80% ethanol for 1.5 hours, filtering, retaining the filter residue, and combining the two filtrates to obtain ethanol extract;

(3) Placing the dregs in the step (2) in a small-mouth bottle, adding 8kg of chloroform, sealing the bottle mouth, oscillating at intervals at room temperature, leaching for 8 hours, filtering, and removing the filter residue to obtain a chloroform extract;

(4) And (3) respectively recovering the solvents from the extracting solutions in the steps (1), (2) and (3), merging to obtain a mixed material, further concentrating the mixed material under reduced pressure to be dry, and then carrying out vacuum drying and crushing to obtain 23.4g of toad skin extract A (powder), wherein the amount of the toad skin extract A is 23.4% of that of the crude medicinal material.

Example 2

Preparation of toad skin extract

(1) Oven drying Bufo siccus skin without Bufonis venenum, pulverizing into 200 mesh superfine powder, weighing 1kg of superfine powder, placing into brown small-mouth bottle, adding 10kg of medicinal diethyl ether, shaking sufficiently to make the material uniform, sealing, soaking at room temperature for 7 days, stirring or shaking for 2 times each day during soaking, filtering, and collecting the residue to obtain diethyl ether extractive solution;

(2) Placing the filter residue in the step (1) into a round-bottom flask, adding 10kg of 80% ethanol containing 0.5% hydrochloric acid, heating and reflux-extracting for 2.5 hours, filtering, retaining the filtrate, carrying out the second reflux-extracting on the filter residue by 8kg of the 80% ethanol containing 0.5% hydrochloric acid for 1 hour, filtering, retaining the filter residue, and combining the two filtrates to obtain ethanol extract;

(3) Placing the residues obtained in the step (2) in a small-mouth bottle, adding 8kg of chloroform, sealing the bottle mouth, oscillating at room temperature from time to time, leaching for 8 hours, filtering, and discarding residues to obtain chloroform extract;

(4) Recovering solvents from the extractive solutions obtained in steps (1), (2) and (3), mixing to obtain a mixed material, concentrating the mixed material under reduced pressure to dry, vacuum drying, and pulverizing to obtain Bufo siccus skin extract B (powder) 296g which is 29.6% of the original medicinal material.

Example 3

Preparation of toad skin extract

(1) Oven drying Bufo siccus skin without Bufonis venenum, pulverizing into 200 mesh superfine powder, weighing 1kg of superfine powder, placing into brown small-mouth bottle, adding 10kg of medicinal diethyl ether, shaking sufficiently to make the material uniform, sealing, soaking at room temperature for 7 days, stirring or shaking for 2 times each day during soaking, filtering, and collecting the residue to obtain diethyl ether extractive solution;

(2) Placing the filter residue obtained in the step (1) into a round-bottom flask, adding 10kg of 80% ethanol containing 0.5% hydrochloric acid, performing ultrasonic extraction at room temperature for 1.5 hours, filtering, retaining the filtrate, performing ultrasonic extraction on the filter residue for 1 hour again by using 8kg of 80% ethanol containing 0.5% hydrochloric acid, filtering, retaining the filter residue, and combining the two filtrates to obtain ethanol extract;

(3) Placing the residues obtained in the step (2) into a small-mouth bottle, adding 8kg of chloroform, sealing the bottle mouth, oscillating at room temperature, leaching for 8 hours, filtering, and discarding residues to obtain chloroform extract;

(4) Recovering solvents from the extractive solutions obtained in steps (1), (2) and (3), mixing to obtain a mixed material, concentrating the mixed material under reduced pressure to dry, vacuum drying, and pulverizing to obtain 273g of Bufo siccus skin extract C (powder) which is 27.3% of the original medicinal material.

Formulation examples

Formulation example 1

External solution of toad skin extract and preparation thereof

Prescription of prescription

Toad skin extract B10 g

Dimethyl sulfoxide 32ml

Polyethylene glycol 300 to 1000ml

The preparation method comprises the following steps: dissolving Bufo siccus skin extract B in dimethyl sulfoxide, adding polyethylene glycol 300 to a sufficient amount, filtering, stirring, and packaging.

The usage method is as follows: is applied to affected part with bone pain 2-3 times daily.

Formulation example 2

Toad skin extract tincture and preparation method thereof

Prescription of prescription

Toad skin extract B10 g

Camphor 15g

Menthol 10g

Glycerol 50ml

Laurocapram 20ml

Tween 80 20ml

60% ethanol to 1000ml

The preparation method comprises the following steps: sequentially adding cutis Bufonis extract and glycerol into 60% ethanol, stirring, adding Camphora and Mentholum into mortar, grinding to liquefy, adding into the above solution, stirring, adding laurocapram, stirring, adding 60% ethanol to 1000ml, filtering, and packaging.

The usage method is as follows: dipping the liquid medicine with cotton ball or gauze to wipe the pain parts of bones for 3-4 times a day.

Formulation example 3

Toad skin extract liniment and preparation thereof

Prescription of prescription

Toad skin extract B15 g

Ligusticum wallichii extract 10g

15g of safflower extract

Shikimic acid 10g

Borneol 12g

95% ethanol 300ml

Dimethyl sulfoxide 120ml

Propylene glycol is added to 1000ml

The preparation method comprises the following steps: dissolving Bufo siccus skin extract, rhizoma Ligustici Chuanxiong extract, carthami flos extract, shikimic acid and Borneolum Syntheticum in ethanol, and filtering; and adding dimethyl sulfoxide into the filtrate, adding propylene glycol to 1000ml, stirring, and packaging.

The usage method is as follows: is applied to the affected parts of bone 3-4 times daily.

Formulation example 4

Toad skin extract emulsion and preparation thereof

Prescription of prescription

Toad skin extract B15 g

Sodium carboxymethylcellulose 20g

Sorbic acid 2g

Tween 80 20g

Span 80 g

Distilled water is added to 1000ml

The preparation method comprises the following steps: mixing tween 80 and span 80, and standing at 70-80deg.C; dissolving sodium carboxymethylcellulose in new boiling distilled water, dissolving cutis Bufonis extract and sorbic acid in appropriate amount of water, stirring, mixing the two solutions, heating to 70-80deg.C, slowly adding into the above prepared liquid, stirring thoroughly, packaging in glass bottle with stopper, and sealing.

The finished product is fine, moderate in consistency, light yellow viscous liquid and good in fluidity.

The usage method is as follows: is applied to the painful parts of bones for 2 times a day.

Formulation example 5

Toad skin extract tincture and preparation method thereof

Prescription of prescription

Toad skin extract B10 g

Prepared Sichuan aconite root extract 5g

5g of radix aconiti kusnezoffii preparata extract

Angelica sinensis extract 7.5g

7.5g of safflower extract

Olibanum 5g

Myrrh 5g

3g of artificial musk

Glycerol 10g

60% ethanol to 1000ml

The preparation method comprises the following steps: placing the Bufo siccus skin extract, radix Aconiti Preparata extract, radix Aconiti Kusnezoffii Preparata extract, radix Angelicae sinensis extract, carthami flos extract, olibanum, myrrha, artificial Moschus and glycerol in a ceramic container, soaking for 7 days, stirring 1 time a day, filtering after soaking, packaging in glass bottle, and sealing.

The usage method is as follows: topical application can be applied to affected part 3-4 times daily, or applied by soaking with sterile gauze.

Formulation example 6

Toad skin extract ointment and preparation thereof

Prescription of prescription

Toad skin extract B20 g

10g of red sage root extract

Asarum extract 7.5g

7.5g of rhizoma corydalis extract

Glycerol 200g

150g of liquid paraffin

Borneol 10g

Menthol 10g

Camphor 5g

Lanolin 200g

Vaseline is added to 1000g

The preparation method comprises the following steps: heating lanolin and vaseline to bake, and cooling to 60deg.C; adding cutis Bufonis extract, saviae Miltiorrhizae radix extract, herba asari extract, and rhizoma corydalis extract into a mortar, grinding, adding lanolin and vaseline mixture melted and cooled to 60deg.C under grinding, cooling to 50deg.C, sequentially adding ground and liquefied Borneolum, menthol and Camphora mixture, and liquid paraffin, grinding, cooling, and packaging.

The usage method is as follows: topical application for treating bone pain is carried out 2 times daily.

Formulation example 7

Toad skin extract cream and preparation thereof

Prescription of prescription

Toad skin extract B15 g

Shikimic acid 5g

Borneol 10g

Menthol 10g

130g of white lead and vaseline

Stearyl alcohol 90g

Liquid paraffin 60g

Sodium dodecyl sulfate 10g

Tween 80 12g

Propylene glycol 130g

Sodium bisulphite 2g

Laurocapram 20g

Distilled water is added to 1000g

The preparation method comprises the following steps: heating white vaseline, stearyl alcohol, liquid paraffin and laurocapram according to the formula amount to be eutectic-melted in a container, and keeping the temperature at 80 ℃ to obtain an oil phase for later use; placing Bufo siccus skin extract, sodium dodecyl sulfate, tween 80, sodium bisulphite and distilled water in another container, heating to boil, cooling to 80deg.C, and keeping the temperature to obtain water phase; mixing Borneolum Syntheticum and Mentholum, grinding to obtain liquefied shikimic acid, dissolving in hot propylene glycol, and maintaining at 50deg.C; adding the oil phase into the water phase under stirring, emulsifying thoroughly, slowly adding the propylene glycol mixture when the temperature is reduced to 50 ℃, and continuing stirring until the mixture is coagulated.

The usage method is as follows: it can be applied to cheek bones and finger joint pain, 2 times daily.

Formulation example 8

Toad extract film coating agent and preparation thereof

Prescription of prescription

Toad skin extract B10 g

Rhizoma corydalis extract 10g

10g of safflower extract

5g of artificial musk

20g of frankincense

Myrrh 20g

Borneol 5g

Camphor 5g

Menthol 5g

Polyvinylpyrrolidone 80g

75% ethanol to 1000ml

The preparation method comprises the following steps: taking about 800ml of 75% ethanol as a solvent, adding the prescription amount of frankincense and myrrh, soaking for 12 days, filtering to obtain filtrate, sequentially adding the prescription amount of toad skin extract, rhizoma corydalis extract and safflower extract into the filtrate, stirring for dissolving, standing for 1 hour, filtering, adding artificial musk, borneol, camphor, menthol and polyvinylpyrrolidone into the filtrate, stirring for dissolving, adding 75% ethanol to 1000ml, filtering, and sub-packaging.

The usage method is as follows: can be used for treating traumatic injury, muscle strain, soreness of tendons and bones, sprain, strain, contusion, etc., and can be applied to affected part for 1-2 times to form a film 3-4 times daily after the medicinal liquid is volatilized.

Formulation example 9

Bufo siccus extract pellicle and its preparation method

Prescription of prescription

Toad skin extract B2 g

15g of polyvinyl alcohol 486

Sodium carboxymethylcellulose 5g

Glycerol 4g

Distilled water is added to 100ml

The preparation method comprises the following steps: firstly, adding a proper amount of distilled water into polyvinyl alcohol 486 and sodium carboxymethylcellulose to soak overnight, adding the mixture into glycerol after the mixture is fully dissolved, and heating the mixture on a water bath to prepare mucilage for standby; dissolving Bufo siccus skin extract in distilled water, stirring, adding into the above prepared mucilage slowly to get 100g, heating in water bath, stirring, taking out, defoaming, pouring into glass plate with edges at 25cm×4cm periphery, spreading to make it uniform in thickness, oven drying at 50deg.C, taking out, cutting into 2.0cm×2.5cm films, sterilizing with ultraviolet lamp, and bagging.

The usage method is as follows: can be used for treating ear, nose, cheek bone, phalangeal pain, and affected part, 2 times daily.

Preparation example 10

Toad Pi Ningjiao agent and preparation thereof

Prescription of prescription

Toad skin extract B10 g

Shikimic acid 5g

Ligusticum wallichii extract 5g

Carbomer 15g

Glycerol 100g

Tween 80 20g

Polyvinyl alcohol 12440g

Polyethylene glycol 400

Distilled water is added to 1000ml

The preparation method comprises the following steps: placing 100g of glycerol into a mortar, adding 15g of carbomer, fully grinding to moisten, and adding a proper amount of distilled water; and adding 10g of toad skin extract, 5g of shikimic acid and 5g of ligusticum wallichii extract, 20g of tween 80 and a proper amount of distilled water to dissolve, adding into the milk, adding the melted polyvinyl alcohol 124 and polyethylene glycol 400 under stirring, fully and uniformly stirring to form gel, adding distilled water to 1000ml, and uniformly grinding.

In this embodiment, the polyvinyl alcohol 124 is selected as one of the drug carrier components, the porous property of the polyvinyl alcohol 124 is used as an effective controlled release matrix, and the polyethylene glycol 400 has good plasticity and spreadability, and has the advantages of delayed effect and controlled release.

The usage method is as follows: is applied to the painful parts of bones 2-3 times daily.

Formulation example 11

Toad skin extract spray and preparation thereof

Prescription of prescription

Toad skin extract B10 g

Borneol 2g

Camphor 10g

Menthol 1.5g

Glycerol 50g

Olibanum 40g

Laurocapram 5g

60% ethanol to 1000ml

The preparation method comprises the following steps: extracting Olibanum and cutis Bufonis with 60% ethanol 200ml, soaking for 7 days, and decanting the supernatant; squeezing the residues to obtain squeezed liquid, and mixing the supernatant with the squeezed liquid to obtain extractive solution; sequentially adding Borneolum Syntheticum, camphora, and Mentholum into 60% ethanol 500ml to dissolve, adding glycerol, stirring, adding above extractive solution, stirring, filtering, adding 60% ethanol to 1000ml, and spraying on affected parts for 3-4 times per day.

In order to illustrate the effects of the present invention, a clinical experiment will be further described below.

Clinical data

General data: the inventor carries out clinical observation on 222 cases, wherein 172 cases are men and 50 cases are women, the age is 22 at the minimum and 72 at the maximum; 52 cases of rheumatic arthritis, 12 cases of rheumatoid arthritis, 16 cases of cervical spondylosis, 20 cases of lumbar disc herniation, 12 cases of sciatica, 8 cases of lumbosacral joint pain, 10 cases of scapulohumeral periarthritis, 12 cases of knee meniscus injury, 18 cases of elbow joint pain, 8 cases of wrist joint pain, 6 cases of ankle joint pain, 8 cases of toe joint pain, 10 cases of thumb joint pain, 1 case of nose bridge fracture pain, 17 cases of knee joint swelling and pain, and 12 cases of traumatic injury pain. 222 cases were diagnosed based on medical history, physical signs and clinical examination.

The treatment method comprises the following steps: the product of the preparation example 1 of the invention is used for local external application of the scapulohumeral periarthritis pain, the treatment is carried out 2 to 3 times a day, and 10 days is a treatment course; the product of preparation example 2 is used for external wiping of liquid medicine for treating rheumatic arthritis pain patients, 3 to 4 times daily, and 7 days is a treatment course; the preparation of the product of the preparation example 3 is used for treating lumbosacral joint pain, the product is coated on the bone pain part 3 to 4 times daily, and 7 days is a treatment course; treatment of knee meniscus injury pain with the formulation of example 4, topical application, 2 times daily, 7 days as a course of treatment; the preparation of the preparation example 5 is used for treating rheumatoid arthritis, lumbar disc herniation and cervical spondylosis pain, the pain is applied locally, 3-4 times a day, and 10 days is a treatment course; the preparation of the preparation example 7 is used for treating the pain of thumb joints and toes, the topical application is carried out 2 times a day, and 7 days is a treatment course; treating knee joint swelling and pain and traumatic injury pain with preparation example 8, and topically applying to form a film 3-4 times daily, 7 days as a treatment course; the product of the example 9 is used for treating the fracture pain of the nasal bridge, and is locally applied to the affected part, 2 times a day, and 7 days is a treatment course; ankle joint pain was treated with formulation example 10, topically applied 2-3 times daily, 7 days as a course of treatment; the preparation of example 11 is used for treating sciatica, elbow joint pain and wrist joint pain, local spraying and local kneading by hand is combined, 3-4 times daily, and 7 days is a treatment course.

Treatment efficacy judgment

And (3) curing: the clinical symptoms such as joint pain, joint swelling, tenderness, joint swelling and pain disappear, the uncomfortable feeling disappears, the joint movement is normal, and the normal life is restored.

The method is effective: the clinical symptoms are relieved, pain and discomfort are obviously eliminated, but slight symptoms can also appear, and the normal life is only partially restored.

Invalidation: the clinical symptoms are unchanged.

Treatment results: 52 cases of rheumatic arthritis pain are treated, 8 treatment courses are treated on average, 46 cases are cured, 4 cases are effective, and 2 cases are ineffective; 12 cases of rheumatoid arthritis pain are treated, 6 treatment courses are treated averagely, 6 cases are cured, 5 cases are effective, and 1 case is ineffective; 16 cases of cervical spondylosis pain are treated, 7 treatment courses are treated on average, 8 cases are cured, 4 cases are effective, and 4 cases are ineffective; 20 cases of lumbar disc herniation pain are treated, 5 treatment courses are treated on average, 10 cases are cured, 4 cases are effective, and 6 cases are ineffective; 12 cases of sciatica are treated, 6 treatment courses are treated averagely, 8 cases are cured, 3 cases are effective, and 1 case is ineffective; 8 cases of lumbosacral joint pain are treated, 10 treatment courses are treated averagely, 6 cases are cured, 1 case is effective, and 1 case is ineffective; 10 cases of scapulohumeral periarthritis pain are treated, 5 treatment courses are treated averagely, 7 cases are cured, 2 cases are effective, and 1 case is ineffective; 12 cases of knee meniscus injury pain are treated, 5 treatment courses are averagely treated, 6 cases are cured, 4 cases are effective, and 2 cases are ineffective; 18 cases of elbow joint pain are treated, 3 treatment courses are treated on average, 14 cases are cured, 3 cases are effective, and 1 case is ineffective; 8 cases of wrist joint pain are treated, the average treatment is carried out for 4 treatment courses, 6 cases are cured, 2 cases are effective, and 0 case is ineffective; 6 cases of ankle joint pain are treated, the average treatment is carried out for 4 treatment courses, 5 cases are cured, 1 case is effective, and 0 case is ineffective; 8 cases of toe joint pain are treated, 3 treatment courses are treated on average, 4 cases are cured, 2 cases are effective, and 2 cases are ineffective; 10 cases of joint pain of the thumb and the hand are treated, 3 treatment courses are treated averagely, 8 cases are cured, 1 case is effective, and 1 case is ineffective; 1 case of pain caused by fracture of the nose bridge, 1 treatment course and 1 case of healing; 17 cases of knee joint swelling and pain are treated, 6 treatment courses are treated on average, 12 cases are cured, 4 cases are effective, and 1 case is ineffective; 12 cases of traumatic injury pain are treated, the average treatment is carried out for 4 treatment courses, 10 cases are cured, 2 cases are effective, and 0 cases are ineffective. The statistics of the treatments are carried out, and 222 cases of the treatments are summed, wherein the total cure rate is 70.72%, the effective rate is 18.92%, the ineffective rate is 10.36%, and the total effective rate is 89.64%.

Typical cases

1. If a male is 22 years old, the fracture of the nasal bridge is caused by mistake, the fracture is not misplaced, the local part is red and swollen, and the pain is hard to be satisfied, the film agent prepared in the example 9 is applied to the fracture part, the film agent is changed once every night, the film agent is applied twice a day, the pain is basically disappeared in the third day, the film agent is continuously applied for 7 days, and the pain is disappeared.

2. Wei Mou A, female, 58 years old, left and right knee joint pain, walking pain aggravates, normal steps are influenced, walking difficulty, the coating agent prepared by adopting the product of the example 8 is coated on the knee joint pain position of a patient, after the liquid medicine is coated, the coating agent is coated for 1-2 times after the liquid medicine is volatilized, so that a film is formed, the periodic dressing change is carried out at intervals of 4 times each day, after 2 treatment periods (14 days) of the medicine, the pain is relieved, after the 6 th treatment period, the pain is basically disappeared, after 7 treatment periods of the medicine are continuously coated, the function is normal, the pain disappears, and no recurrence is seen in follow-up visit.

3. When a man is aged 40 years, the left scapulohumeral periarthritis causes shoulder joint pain for more than half a year, the solution product of the embodiment 1 of the invention is used for local smearing, and meanwhile, the solution product is rubbed by hands for 10 minutes, 3 times a day and 10 days are one course of treatment, the pain is obviously relieved after three courses of treatment, and the pain disappears after two courses of treatment, so that the patient can exercise independently along with the relief and disappearance of the pain, the shoulder joint movement is flexible, and no pain is seen in follow-up visit.

4. Some male, 62 years old, retired officers, 2 years ago, sometimes painful, sometimes light, careless, after 2 months, aggravated right knee pain, difficult walking, clinical examination and diagnosis of rheumatic arthritis, the tincture product of the invention is adopted, the topical application is carried out 4 times a day, 10 days is a course of treatment, symptoms are obviously relieved after five courses of treatment, pain is relieved, five courses of treatment are continuously treated, pain is eliminated, healing is continued, and recurrence is not seen for 2 years following the follow-up.

5. Jiang Mou for men, 28 years old and elbow joint pain for 2 months, the spray product of the embodiment 11 of the invention is used for local spraying and local rubbing by hands, 3 times a day, 7 days are a treatment course, the pain is obviously relieved after the treatment of the treatment course, the pain symptoms are completely disappeared after the treatment of the treatment course, and no recurrence is seen in follow-up visit.

While the invention has been described with reference to certain preferred embodiments, it is not intended that the invention be limited to those embodiments, and it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Modifications and variations may be made without departing from the spirit and scope of the invention, and it is intended that the invention include all such modifications and variations as fall within the scope of the invention.

Claims (11)

1. The use of toad skin extract in preparing a pharmaceutical composition for treating bone pain, wherein the toad skin extract is obtained by drying and crushing toad skin without venenum bufonis, and extracting with diethyl ether, alcohol or aqueous alcohol and chloroform respectively, wherein the alcohol is any one or combination of methanol and ethanol, and the diethyl ether is medicinal diethyl ether.

2. The use according to claim 1, wherein the alcohol is ethanol.

3. The use according to claim 1, wherein the aqueous alcohol is an acidic aqueous alcohol.

4. The use according to claim 3, wherein the acidic aqueous alcohol is 80% ethanol containing 0.5% hydrochloric acid or sulfuric acid.

5. The use according to claim 1, wherein the pharmaceutical composition preferably contains pharmaceutically acceptable excipients and/or carriers and optionally one or more other pharmaceutically active ingredients.

6. The use according to claim 5, wherein the pharmaceutically acceptable excipients and/or carriers comprise water, ethanol, propylene glycol, polyethylene glycol, vegetable oil, glycerol, liquid paraffin, tween 80, span 80, surfactants, soft soaps, lanolin, petrolatum, sodium carboxymethyl cellulose, methylcellulose, starch, tragacanth, acacia, agar, bletilla gum, sodium alginate, polyvinylamines, polyvinylpyrrolidone, polyvinyl alcohol, talc, kaolin, vegetable oil, soft soaps, paraffin.

7. The use according to claim 5, wherein the other pharmaceutically active ingredient comprises a substance selected from the group consisting of: the traditional Chinese medicine composition comprises a red sage root extract, a ligusticum wallichii extract, a clove extract, a safflower extract, a Chinese angelica extract, a pricklyash peel extract, an asarum extract, a rhizoma corydalis extract, frankincense, myrrh, methyl salicylate, camphor, borneol, dragon's blood, artificial bezoar, artificial musk, taurine, menthol, shikimic acid, eucalyptus oil, a black-tail snake extract, a prepared aconite root extract, a prepared radix aconiti kusnezoffii extract and a lindera root extract.

8. The use according to claim 1, wherein the pharmaceutical composition is a topical pharmaceutical formulation.

9. The use according to claim 1, wherein the pharmaceutical composition is a solution, lotion, liniment, tincture, spray, aerosol, emulsion, gel, suspension, film, ointment, cream, patch.

10. The use according to claim 9, wherein the pharmaceutical composition is a liniment, a tincture, a spray, an emulsion, a patch.

11. The use of claim 1, wherein the toad skin extract is prepared by a process comprising at least the steps of:

(1) Baking toad skin without Bufonis venenum, pulverizing into superfine powder, and extracting toad skin with diethyl ether to obtain diethyl ether extract;

(2) Extracting the filter residue obtained in the step (1) again by using the alcohol or the aqueous alcohol as a solvent to obtain an alcohol extract;

(3) Extracting the filter residue in the step (2) with chloroform as a solvent to obtain chloroform extract;

(4) After the solvents in the steps (1), (2) and (3) are respectively recovered, the solvents are combined to obtain a mixed material; and

(5) Optionally removing impurities from the extraction mixture by filtration, centrifugation or other suitable means, and optionally concentrating and/or drying to obtain liquid or solid extracts of different forms and concentrations.