Application of toad venom, toad venom active ingredients and toad venom active ingredient combination in relieving inflammatory pain and bone cancer pain

Abstract

The present invention discloses the use of toad venom, its active ingredients, and a combination of its active ingredients in relieving inflammatory pain and bone cancer pain, belonging to the technical field of research on analgesic active ingredients of toad venom. The present invention discovers that toad venom can relieve inflammatory pain and bone cancer pain and clarifies that its main active ingredients are cinobufagin and resefobufogenin. Cinobufagin and resefobufogenin have a good analgesic effect within a certain ratio range. A mass ratio of cinobufagin to resefobufogenin of 4:5 to 5:1 can relieve inflammatory pain, and a mass ratio of cinobufagin to resefobufogenin of 1:10 to 5:1 can relieve bone cancer pain. The present invention also provides the suggestion that the active ingredients cinobufagin and resefobufogenin can become substitutes for the endangered medicinal material toad venom in relieving inflammatory pain and bone cancer pain. In addition, the present invention discovers for the first time that resefobufogenin has an analgesic effect similar to that of morphine and is non-addictive, suggesting that it has the potential to be developed into a new analgesic drug.

Description

Technical Field

[0001] The present invention relates to the technical field of research on analgesic active ingredients of toad venom, and in particular to applications of toad venom, toad venom active ingredients and toad venom active ingredient compositions in relieving inflammatory pain and bone cancer pain. Background Art

[0002] Traditional Chinese medicine (TCM) plays an increasingly important role in the prevention and treatment of malignant tumors and cancer pain. A number of Chinese herbs with significant anticancer and analgesic effects have been discovered, including toad venom, matrine, and cinobucoids, with toad venom being particularly effective. Toad venom is a secretion from the parotid glands of the Chinese giant toad (Bufo bufo gargarizans Cantor) and the black-orbiting toad (Bufo melanostictus Schneider), both members of the Toad family. It is sweet, pungent, warm, and toxic, and has detoxifying, swelling-reducing, mind-stimulating, heart-strengthening, and analgesic properties. Listed in the Chinese Pharmacopoeia, toad venom has been used for centuries as a treatment for cardiovascular disease, inflammatory diseases, cancer, and chronic pain. However, compound toad venom pastes made with toad venom are primarily used for pain caused by various cancers, including those of the lungs, liver, and stomach, with few reports on its use in treating bone cancer pain.

[0003] Currently, toads face resource constraints and high commodity prices, creating a significant supply-demand imbalance. Therefore, to protect the ecological environment and reduce the cost and price of toad venom analgesics, research into the analgesic active ingredients of toad venom as a potential alternative to toad venom is of great significance.

[0004] Cancer-induced bone pain (CIBP) refers to severe and common pain caused by bone metastases or primary bone tumors. 70% of patients with advanced cancer experience cancer pain, and over 80% of pain is attributed to bone pain caused by metastatic cancer.

[0005] Bone cancer pain is currently the most severe and persistent type of cancer pain. Currently, cancer pain treatment follows the international three-step analgesic strategy, which is based on a three-step analgesic ladder from non-opioids to weak opioids to strong opioids. Non-opioid analgesics, including acetaminophen and nonsteroidal anti-inflammatory drugs, are used in the first step for mild pain; weak opioids, including tramadol, hydrocodone, and codeine, are used in the second step for mild to moderate pain; and strong opioids, including morphine, fentanyl, buprenorphine, and tapentol, are used in the third step for moderate to severe pain.

[0006] Nonsteroidal anti-inflammatory drugs (NSAIDs) have a "ceiling" effect, and long-term use can lead to liver damage. Opioids are highly addictive, and overdose can be fatal. Patients taking opioids for chronic pain quickly develop analgesic tolerance, and over time, they often fail to achieve the same pain relief. Furthermore, opioids can slow or prevent bone remodeling and even increase bone brittleness, placing an additional burden on patients undergoing bone cancer pain treatment and hindering bone cancer treatment.

[0007] In summary, there is an urgent need to develop safe, highly effective, and non-addictive drugs for treating bone cancer pain. Literature reports suggest that toad venom can alleviate cancer pain, but there is little research on its use in treating bone cancer pain, and there is no research on its active ingredients or addictive properties. Therefore, this study aims to investigate the effects of toad venom in alleviating bone cancer pain and identify the active ingredients that exert its efficacy as an alternative to toad venom. Summary of the Invention

[0008] To solve the above problems, the present invention provides a method for using toad venom, its active ingredients, and a composition of its active ingredients in alleviating inflammatory pain and bone cancer pain. While studying the analgesic mechanism of toad venom, the present invention clearly shows that the main active ingredients in toad venom that exert their efficacy are cinobufagin and esterbufagin, suggesting that its active ingredients can replace the endangered medicinal material toad venom to relieve inflammatory pain and bone cancer pain.

[0009] To achieve the above objectives, the present invention provides the use of toad venom for relieving inflammatory pain and bone cancer pain, wherein the active ingredients of toad venom for relieving inflammatory pain include cinobufagin, resebufugin and bufadin; and the active ingredients of toad venom for relieving bone cancer pain include cinobufagin and resebufugin.

[0010] A toad venom active ingredient composition for relieving inflammatory pain is a composition comprising the above-mentioned cinobufagin and bisbufogenin.

[0011] Preferably, the mass ratio of cinobufagin to bisulphofugin is 4:5 to 5:1.

[0012] A composition of toad venom active ingredients for relieving bone cancer pain is a composition comprising the above-mentioned cinobufagin and bisbufogenin.

[0013] Preferably, the mass ratio of cinobufagin to bisulphofugin is 1:10 to 5:1.

[0014] A use of the above-mentioned resebufugin in the preparation of analgesic drugs, wherein the resebufugin is non-addictive.

[0015] The use of the toad venom, toad venom active ingredients, and toad venom active ingredient compositions of the present invention in relieving inflammatory pain and bone cancer pain has the following technical effects:

[0016] (1) It was discovered for the first time that the active ingredients of toad venom that relieve inflammatory pain are cinobufagin, esterbufogenin, and bufotoxin. Moreover, the analgesic effect of cinobufagin and esterbufogenin combined is comparable to that of toad venom, suggesting that the combination of cinobufagin and esterbufogenin may replace toad venom in the treatment of inflammatory pain.

[0017] (2) For the first time, it was discovered that toad venom can relieve bone cancer pain, providing a candidate drug for the treatment of bone cancer pain. It was further discovered that esterbufogenin and its combination with cinobufogenin can relieve bone cancer pain, suggesting that esterbufogenin and its combination with cinobufogenin may replace toad venom in relieving bone cancer pain.

[0018] (3) The present invention discovered for the first time that the analgesic effect of retiboflavin is similar to that of morphine and is non-addictive, suggesting that it is promising for development as a new analgesic drug.

[0019] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The analgesic effect of the toad venom medicinal material in Example 1 on the inflammatory pain model mice;

[0021] Figure 2 The analgesic effect of the five lactone active molecules of Bufon venom in Example 2 on mice with inflammatory pain model;

[0022] Figure 3 The results of Example 3 are the analgesic effect of the combination of sinobufagin, seribugenin and bufalin on the inflammatory pain model mice;

[0023] Figure 4 The analgesic effect of other active molecules in Toad Venenum in Example 4 on mice with inflammatory pain model;

[0024] Figure 5 The analgesic effect of the combination of Chinese toad venom toxin base, esterbufogenin and bufadin and the same amount of toad venom medicinal materials on the inflammatory pain model mice in Example 5;

[0025] Figure 6 This is the analgesic effect result of the combination of sinobufagin and bisbufogenin on the inflammatory pain model mice in Example 6;

[0026] Figure 7 The results of the effect of a single oral administration of toad venom on mechanical allodynia in the rat model of bone cancer pain in Example 7; among them, ###, P < 0.001 model group vs. sham operation group; *, P < 0.05, ***, P < 0.001 toad venom 60 mg / kg group vs. model group; &&, P < 0.01 zoledronic acid group vs. model group;

[0027] Figure 8 The results of the effect of continuous oral administration of toad venom on mechanical allodynia in rats with bone cancer pain model in Example 8; among them, #, P < 0.05, ###, P < 0.001 model group vs sham operation group; *, P < 0.05, **, P < 0.01, ***, P < 0.001 zoledronic acid group vs model group; &, P < 0.05, &&, P < 0.01, &&&, P < 0.001 toad venom 60 mg / kg group vs model group;

[0028] Figure 9 The results of the effect of continuous oral administration of toad venom on the spontaneous pain response of rats with bone cancer pain model in Example 9; among them, #, P < 0.05, ###, P < 0.001 model group vs sham operation group; **, P < 0.01, ***, P < 0.001 zoledronic acid group vs model group; &, P < 0.05, &&, P < 0.01, &&&, P < 0.001 toad venom 60 mg / kg group vs model group;

[0029] Figure 10 The results of Example 10 are the effects of a single oral administration of cinobufogenin and sebufogenin on mechanical allodynia in rats with bone cancer pain model; among them, ###, P < 0.001 model group vs. sham operation group; *, P < 0.05, **, P < 0.05, ***, P < 0.001 sebufogenin 3 mg / kg group vs. model group; &, P < 0.05 sebufogenin 1.5 mg / kg group vs. model group; $, P < 0.05 cinobufogenin 6 mg / kg group vs. model group;

[0030] Figure 11 The results of Example 11 are the effects of continuous oral administration of cinobufogenin and sebufogenin on mechanical allodynia in rats with bone cancer pain model; among them, ###, P < 0.001 model group vs. sham operation group; *, P < 0.05, **, P < 0.01, ***, P < 0.001 sebufogenin 3 mg / kg group vs. model group; &, P < 0.05 sebufogenin 1.5 mg / kg group vs. model group; $, P < 0.05, $$, P < 0.01 cinobufogenin 6 mg / kg group vs. model group;

[0031] Figure 12 The results of Example 12 are the effects of continuous oral administration of cinobufogenin and sebufogenin on the spontaneous pain response of rats with bone cancer pain model; among them, ##, P < 0.01, ###, P < 0.001 model group vs sham operation group; *, P < 0.05, ***, P < 0.001 sebufogenin 3 mg / kg group vs model group; &&, P < 0.01 sebufogenin 1.5 mg / kg group vs model group; $$, P < 0.01 cinobufogenin 6 mg / kg group vs model group;

[0032] Figure 13 The results of Example 13 are the effects of oral administration of a combination of cinobufogenin and esterbufogenin on mechanical allodynia in rats with bone cancer pain model; among them, ###, P < 0.001 model group vs. sham operation group; *, P < 0.05, **, P < 0.01 combination group 4 vs. model group; &, P < 0.05 combination group 11 vs. model group; $, P < 0.05 combination group 7 vs. model group; Δ, P < 0.05 combination group 8 vs. model group;

[0033] Figure 14 The results of Example 14 are the effects of continuous oral administration of cinobufogenin and bisbufogenin on mechanical allodynia in rats with bone cancer pain model; among them, ##, P < 0.01, ###, P < 0.001 model group vs sham operation group; ***, P < 0.001 combination group 4 vs model group; &, P < 0.05, &&, P < 0.01 combination group 11 vs model group; $, P < 0.005 combination group 7 vs model group; Δ, P < 0.05, ΔΔ, P < 0.01 combination group 8 vs model group;

[0034] Figure 15 The results of the effects of continuous oral administration of cinobufogenin and bisbufogenin on the spontaneous pain response of rats with bone cancer pain model in Example 15 are shown; among them, ##, P < 0.01, ###, P < 0.001 model group vs. sham operation group; **, P < 0.01***, P < 0.001 combination group 4 vs. model group; &&, P < 0.01 combination group 11 vs. model group; $$, P < 0.01 combination group 7 vs. model group; Δ, P < 0.05 combination group 8 vs. model group;

[0035] Figure 16 The results of the effects of cetirifugrin and morphine hydrochloride on mechanical allodynia in rats with bone cancer pain model are shown in Example 1; among them, ###, P < 0.001 model group vs. sham operation group; ***, P < 0.001 cetirifugrin group vs. model group; &, P < 0.05, &&, P < 0.01 morphine ip group vs. model group;

[0036] Figure 17 These are the results of investigating the addictive effects of continuous intraperitoneal injection of resebufugin on mice in Example 17; ***, P<0.001 Day 7 vs Day 1. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages disclosed in the embodiments of the present invention more clear, the embodiments of the present invention are further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0038] Sources of instruments and drugs in the examples and preparation of required solutions:

[0039] Clean-grade 18-22 g Kunming female mice and 180-200 g SD female mice were purchased from Beijing Weitong Lihua Company; carrageenan was purchased from Sigma; MRMT-1 rat breast cancer cell line was purchased from Guangzhou Jinio Biotechnology Co., Ltd.; sodium carboxymethyl cellulose was purchased from Adamas; dimethyl sulfoxide (DMSO), anhydrous ethanol, and castor oil were purchased from Adamas; YLS-6B intelligent hot plate apparatus was purchased from Shanghai Yuyan Scientific Instrument Co., Ltd.; toad venom was provided by Shanghai Hutchison Pharmaceuticals Co., Ltd.; a plantar acupuncture pain tester was purchased from Shenzhen Ruiwode Life Science Technology Co., Ltd.; Shutai 50 was purchased from Virbac; thiazine hydrochloride injection and meloxicam were purchased from Qilu Animal Health Products Co., Ltd.; lidocaine hydrochloride was purchased from Shanghai Myrel Biochemical Technology Co., Ltd.; and Hanks' balanced salt solution (HBSS) was purchased from Sigma.

[0040] Preparation of 2% carrageenan solution: Weigh 20 mg of carrageenan powder and dissolve it in 1 mL of normal saline to prepare a 2% carrageenan solution.

[0041] Preparation of 0.5% sodium carboxymethylcellulose (CMC-Na) solution: Weigh 50 mg of sodium carboxymethylcellulose powder and dissolve it in 10 mL of normal saline to prepare 0.5% sodium carboxymethylcellulose solution.

[0042] Preparation of matrix solution: Take 10 μL of DMSO solution, then add 40 μL of ethanol, 500 μL of castor oil and 900 μL of normal saline in sequence to prepare 1 mL of matrix solution.

[0043] Example 1

[0044] Analgesic effect of Toad Venenum on inflammatory pain model mice

[0045] (1) Grouping and administration:

[0046] Thirty female Kunming mice were randomly divided into 6 groups, with 5 mice in each group, namely normal group, model group, model + toad venom 150 mg / kg group, model + toad venom 175 mg / kg group, model + toad venom 200 mg / kg group and positive control ibuprofen 100 mg / kg group.

[0047] In the model group, mice were injected intraplantar with 10 μL of a 2% carrageenan solution to establish an inflammatory pain model. The normal and model groups were administered a 0.5% sodium carboxymethylcellulose solution by gavage, while the drug-treated group received the corresponding drug (dissolved in 0.5% sodium carboxymethylcellulose). The thermal withdrawal latency (s) of the mice was measured 0.5, 1, 2, 4, and 6 hours after drug administration.

[0048] (2) Determination of thermal paw withdrawal latency:

[0049] The thermal withdrawal latency of mice was measured using an intelligent hot plate apparatus, with paw licking as the endpoint. The pain threshold of each group of mice was measured using a hot plate apparatus (set to 55°C) before administration (two tests were performed and the average was taken, with an interval of >15 minutes between the two measurements). Unqualified mice (with a latency less than 5 seconds or greater than 30 seconds) were discarded. Qualified mice were randomly divided into groups for the experiment, and the thermal withdrawal latency was measured and recorded 0.5, 1, 2, 4, and 6 hours after oral administration.

[0050] (3) Statistical analysis:

[0051] The mean and SD values ​​of the thermal withdrawal latency of mice in the same group were calculated, and then the statistical differences between different groups were tested by Two-way test. P < 0.05 was considered to be statistically significant.

[0052] The analgesic effect of Toad Venenum on inflammatory pain model mice Figure 1 As shown. The thermal paw withdrawal latency of mice with carrageenan-induced inflammatory pain was significantly reduced after modeling. Compared with the normal group, the model group had significantly lower latencies at 0.5-6 hours (P < 0.01), demonstrating successful model establishment. Compared with the model group, the 150 mg / kg toad venom significantly increased the latencies at 0.5, 2, 4, and 6 hours after administration (P < 0.05), the 175 mg / kg toad venom significantly increased the latencies at 0.5-6 hours after administration (P < 0.01), and the 200 mg / kg toad venom significantly increased the latencies at 1-6 hours after administration (P < 0.05). The positive control, the 100 mg / kg ibuprofen group, also showed a significant increase in the latencies at 0.5-6 hours after administration (P < 0.05). These results demonstrate that toad venom has a potent analgesic effect in the carrageenan-induced inflammatory pain model.

[0053] Example 2

[0054] Screening of analgesic activity of five lactone active molecules from Venenum Bufonis on inflammatory pain model mice

[0055] (1) Grouping and administration: Forty female Kunming mice were randomly divided into 8 groups, with 5 mice in each group, namely, normal group, model group, model + cinobufoxin 3 mg / kg group, model + bufotoxin 3 mg / kg group, model + bufotoxin 3 mg / kg group, model + bufotoxin 3 mg / kg group, model + bufotoxin 3 mg / kg group, and positive control ibuprofen 100 mg / kg group.

[0056] Inflammatory pain models were established in the model group mice by intraplantar injection of 10 μL of a 2% carrageenan solution into the hind paws. The normal and model groups were administered the matrix solution, while the drug-treated group received the corresponding drug (dissolved in the matrix solution). The thermal withdrawal latency (s) of the mice was measured 0.5, 1, 2, 4, and 6 h after drug administration, using the same testing method as in Example 1.

[0057] (2) Statistical analysis:

[0058] The mean and SD values ​​of the thermal withdrawal latency of mice in the same group were calculated, and then the statistical differences between different groups were tested by Two-way test. P < 0.05 was considered to be statistically significant.

[0059] The analgesic effects of five lactone active molecules of Toad Venenum on inflammatory pain model mice are shown in the following results: Figure 2 As shown. The thermal paw withdrawal latency of mice with carrageenan-induced inflammatory pain was significantly reduced after modeling. Compared with the normal group, the model group had significantly lower latencies at 0.5-6 hours (P < 0.05), demonstrating successful model establishment. Compared with the model group, the thermal paw withdrawal latency in the bufalin group was significantly increased at 0.5 hours after administration (P < 0.05), the latency was significantly increased at 2 hours after administration (P < 0.001), and the latency was significantly increased at 2, 4, and 6 hours after administration (P < 0.01). Both cinobufogenin and sefobufogenin significantly increased the latency at 0.5-6 hours after administration (P < 0.05). The positive control, ibuprofen 100 mg / kg, also had a significant increase in latency at 0.5-6 hours after administration (P < 0.05).

[0060] The above results indicate that cinobufagin and esterbufagin have analgesic effects in the carrageenan-induced inflammatory pain model, and are superior to bufalin, bufatoxin and bufatoxin.

[0061] Example 3

[0062] Analgesic effects of cinobufagin, esterbufogenin and bufalin combined on inflammatory pain model mice

[0063] (1) Grouping and administration:

[0064] Forty female Kunming mice were randomly divided into 8 groups, with 5 mice in each group, including normal group, model group, model + cinobufogenin 3 mg / kg group, model + bufalin 3 mg / kg group, model + composition 1 (the mass ratio of cinobufogenin to bufalin is 12:5) 3 mg / kg group, model + composition 2 (the mass ratio of cinobufogenin, bufalin and bufalin is 20:8:9) 3 mg / kg group and positive control ibuprofen 100 mg / kg group.

[0065] Inflammatory pain models were established in the model group mice by intraplantar injection of 10 μL of a 2% carrageenan solution into the hind paws. The normal and model groups were administered the matrix solution, while the drug-treated group received the corresponding drug (dissolved in the matrix solution). The thermal withdrawal latency (s) of the mice was measured 0.5, 1, 2, 4, and 6 h after drug administration, using the same testing method as in Example 1.

[0066] (2) Statistical analysis:

[0067] The mean and SD values ​​of the thermal withdrawal latency of mice in the same group were calculated, and then the statistical differences between different groups were tested by Two-way test. P < 0.05 was considered to be statistically significant.

[0068] The analgesic effect of cinobufagin, esterbufogenin and bufalin on inflammatory pain model mice was shown in the following results. Figure 3 As shown. The thermal paw withdrawal latency of mice with carrageenan-induced inflammatory pain was significantly reduced after modeling. Compared with the normal group, the model group had significantly reduced latency at 0.5-6 hours (P < 0.01), proving that the model was successfully established. Compared with the model group, the latency of cinobufogenin significantly increased at 2, 4, and 6 hours after administration (P < 0.05), the latency of bufalin significantly increased at 0.5, 1, 4, and 6 hours after administration (P < 0.05), the latency of bufalin significantly increased at 4 and 6 hours after administration (P < 0.05), the latency of composition 1 significantly increased at 1-6 hours after administration (P < 0.001), and the latency of composition 2 significantly increased at 4 and 6 hours after administration (P < 0.01). The positive control ibuprofen 100 mg / kg group had significantly increased latency at 1-6 hours after administration (P < 0.01).

[0069] Further screening of the three active molecules by combining them according to the ratios used in Toad Venenum revealed that Composition 1 enhanced the analgesic effect, while Composition 2 (with the addition of bufalin) did not further enhance the analgesic effect. These results demonstrate that the combination of cinobufogenin and sefobufogenin exhibits analgesic activity in the carrageenan-induced inflammatory pain model, superior to either agent alone.

[0070] Example 4

[0071] Analgesic effects of other active molecules in Toad Venenum on inflammatory pain model mice

[0072] (1) Grouping and administration: 50 female Kunming mice were randomly divided into 10 groups, with 5 mice in each group, namely normal group, model group, model + telocinobufagin 3 mg / kg group, model + cinobufolin 3 mg / kg group, model + bufotoxin 3 mg / kg group, model + bufotoxin 3 mg / kg group, model + 5-hydroxytryptamine 3 mg / kg group, model + N-methyl-5-hydroxytryptamine 3 mg / kg group, model + bufotoxin 3 mg / kg group and model + bufotoxin 3 mg / kg group. Inflammatory pain model was established by intraplantar injection of 10 μL 2% carrageenan solution in the hind limbs of mice in the model group. The drug administration method was gavage. The normal group and model group were given the matrix solution, and the drug administration group was given the corresponding drug (dissolved in the matrix solution). The thermal withdrawal latency (s) of the mice was tested 0.5, 1, 2, 4 and 6 hours after administration. The test method was the same as that in Example 1.

[0073] (2) Statistical analysis: The mean and SD values ​​of the thermal withdrawal latency of mice in the same group were calculated, and then the statistical differences between different groups were tested using a two-way test. P < 0.05 was considered to be statistically significant.

[0074] The analgesic effects of other active molecules in Toad Venenum on inflammatory pain model mice are as follows Figure 4 As shown. The thermal paw withdrawal latency of mice with carrageenan-induced inflammatory pain was significantly reduced after modeling. Compared with the normal group, the model group showed significantly lower latencies at 0.5-6 hours (P<0.05), confirming the successful establishment of the model. Compared with the model group, only toad taridone showed a significant increase in thermal paw withdrawal latency at 0.5-6 hours after administration (P<0.05). The other seven molecules showed no significant changes in latency after administration (P>0.05). These results demonstrate that toad taridone has an analgesic effect in the carrageenan-induced inflammatory pain model.

[0075] Example 5

[0076] Analgesic effects of cinobufagin, esterbufogenin and bufadin combined with the same amount of toad venom in mice with inflammatory pain model

[0077] (1) Grouping and administration: 30 female Kunming mice were randomly divided into 6 groups, 5 mice in each group, including normal group, model group, model + composition 1 (mass ratio of cinobufogenin to sirolimus is 12:5) 3 mg / kg group, model + composition 3 (mass ratio of cinobufogenin, sirolimus to bufotoxin is 25:10:4) 3 mg / kg group, model + venom of toad 43 mg / kg group and positive control ibuprofen 100 mg / kg group. Inflammatory pain model was established by intraplantar injection of 10 μL 2% carrageenan solution into the hind limbs of mice in the model group. The normal group and model group were given matrix solution, and the drug group was given the corresponding drug (dissolved in matrix solution). The thermal withdrawal latency (s) of the mice was tested 0.5, 1, 2, 4 and 6 h after administration. The test method was the same as that in Example 1.

[0078] (2) Statistical analysis: The mean and SD values ​​of the thermal withdrawal latency of mice in the same group were calculated, and then the statistical differences between different groups were tested using a two-way test. P < 0.05 was considered to be statistically significant.

[0079] The analgesic effects of the combination of cinobufogenin, esterbufogenin and bufadin and the same amount of toad venom on mice with inflammatory pain model are shown in the following table. Figure 5 As shown. The thermal paw withdrawal latency of mice with carrageenan-induced inflammatory pain was significantly reduced after modeling. Compared with the normal group, the model group showed significant differences at 0.5-6 hours (P < 0.01), demonstrating successful model establishment. Compared with the model group, the thermal paw withdrawal latency of Composition 1 group was significantly increased 0.5-6 hours after administration (P < 0.05), and the thermal paw withdrawal latency of Composition 3 group was significantly increased 1-6 hours after administration (P < 0.05). The thermal paw withdrawal latency of the Toad Venenum 43 mg / kg group was significantly increased 0.5-6 hours after administration (P < 0.05), and the latency of the positive control ibuprofen 100 mg / kg group was significantly increased 1-6 hours after administration (P < 0.05). Compared with Composition 1 group, Composition 3 group did not significantly increase the latency (P > 0.05), and there was no significant difference compared with the Toad Venenum 43 mg / kg group (ns: P > 0.05). The above results show that the mass ratio of cinobufogenin to esterbufogenin is 12:5, which has an analgesic effect in the carrageenan-induced inflammatory pain model, and its analgesic effect is comparable to that of toad venom.

[0080] Example 6

[0081] Analgesic effect of cinobufagin and bisbufogenin combined in inflammatory pain model mice

[0082] (1) Grouping and administration: 50 female Kunming mice were randomly divided into 10 groups, with 5 mice in each group, including the normal group, the model group, the model + composition 4 (the mass ratio of cinobufogenin to esterbufogenin was 1:10) 3 mg / kg group, the model + composition 5 (the mass ratio of cinobufogenin to esterbufogenin was 2:5) 3 mg / kg group, the model + composition 6 (the mass ratio of cinobufogenin to esterbufogenin was 4:5) 3 mg / kg group, the model + composition 7 (the mass ratio of cinobufogenin to esterbufogenin was 1:10) 3 mg / kg group The groups were divided into model + composition 1 (the mass ratio of cinobucoid venom to esterobucoid venom is 1:1) 3 mg / kg group, model + composition 1 (the mass ratio of cinobucoid venom to esterobucoid venom is 12:5) 3 mg / kg group, model + composition 8 (the mass ratio of cinobucoid venom to esterobucoid venom is 5:1) 3 mg / kg group, model + composition 9 (the mass ratio of cinobucoid venom to esterobucoid venom is 10:1) 3 mg / kg group and model + composition 10 (the mass ratio of cinobucoid venom to esterobucoid venom is 50:1) 3 mg / kg group.

[0083] The inflammatory pain model was established by intraplantar injection of 10 μL of 2% carrageenan solution into the hind paws of mice in the model group. The drug was administered by gavage. The normal group and the model group were given the matrix solution, and the drug-treated group was given the corresponding drug (dissolved in the matrix solution). The thermal withdrawal latency (s) of the mice was tested 0.5, 1, 2, 4, and 6 h after administration. The testing method was the same as in Example 1.

[0084] (2) Statistical analysis: The mean and SD values ​​of the thermal withdrawal latency of mice in the same group were calculated, and then the statistical differences between different groups were tested using a two-way test. P < 0.05 was considered to be statistically significant.

[0085] The analgesic effect of cinobufogenin and esterbufogenin on inflammatory pain model mice was shown in the following results. Figure 6 As shown. The thermal paw withdrawal latency of mice with carrageenan-induced inflammatory pain was significantly reduced after modeling. Compared with the normal group, the model group showed significant differences at 0.5-6 hours (P<0.05), proving that the model was successfully established. Compared with the model group, the latency of Composition 6 group was significantly increased 6 hours after administration (P<0.001), the latency of Composition 7 group was significantly increased 2 hours after administration (P<0.01), the latency of Composition 1 group was significantly increased 1-6 hours after administration (P<0.05), and the latency of Composition 8 group was significantly increased 2 and 6 hours after administration (P<0.05). However, there was no significant change in the latency of Compositions 4, 5, 9, and 10 groups (P>0.05). These results indicate that a mass ratio of cinobufagin to sefobagin of 4:5 to 5:1 has an analgesic effect in the carrageenan-induced inflammatory pain model.

[0086] Example 7

[0087] Effects of single oral administration of toad venom on mechanical allodynia in rats with bone cancer pain model

[0088] (1) Establishment of bone cancer pain model: SD rats were anesthetized with Shutai 50 (50 mg / kg) combined with thiazine hydrochloride (5 mg / kg), and analgesic was given with meloxicam (2.5 mg / kg) and lidocaine hydrochloride (2 mg / kg). The left knee joint was shaved and disinfected. The knee joint was fixed with the left finger, and a No. 7 needle was used to drill a hole at the knee joint (edge ​​of the patellar ligament) along the longitudinal axis of the tibia toward the distal end of the tibia, with a depth of about 1.5 cm. Then, a microinjector was used to inject tumor cells into the tibial bone marrow cavity. The microinjectors of the model group and the drug-treated group successively aspirated 2 μL of gelatin sponge aqueous solution, 1 μL of air, and 5 μL of tumor cells (5×10 5 The sham-operated group received 5 μL HBSS without tumor cells as a control.

[0089] (2) Grouping and administration: 30 female SD rats were randomly divided into 5 groups, with 6 rats in each group, namely, sham operation group, model group, model + toad venom 30 mg / kg group, model + toad venom 60 mg / kg group, and model + positive control zoledronic acid 30 μg / kg group. The bone cancer pain model was established in the model group rats. Drug administration was started on the 11th day after the model establishment by gavage. The normal group and model group were given 0.5% CMC-Na solution, and the drug group was given the corresponding drug (dissolved in 0.5% CMC-Na solution). The mechanical withdrawal force (g) of the rats was tested 1, 2, 4, and 6 h after drug administration.

[0090] (3) Mechanical withdrawal force measurement: The rats were placed on the plantar acupuncture pain tester and allowed to move freely. When the rats stopped exploring and became relatively quiet, the stimulation unit was moved and the stimulation needle was aimed at the stimulation area on the surface of the paw with the help of a mirror. Then, the start button on the side of the stimulation unit handle was pressed. The withdrawal force value measured when the rats lifted their paws due to pain was used as an observation index. The mechanical withdrawal force of the rats was tested 1, 2, 4, and 6 hours after drug administration. To avoid errors, each hind limb was measured 3 times and the average value was taken.

[0091] (4) Statistical analysis: The mean and SD values ​​of the mechanical withdrawal force of rats in the same group were calculated, and then the statistical differences between different groups were tested by two-way test. P < 0.05 was considered to be statistically significant.

[0092] Effects of single oral administration of toad venom on mechanical allodynia in rats with bone cancer pain model Figure 7As shown. After tumor cell inoculation, rats' mechanical avoidance force was significantly reduced. Compared with the sham-operated group, the model group showed significantly lower avoidance force from 0 to 6 hours (P < 0.001), confirming the successful establishment of the model. Compared with the model group, the 60 mg / kg venom of Toad group showed a significant increase in avoidance force at 1, 2, and 4 hours after administration (P < 0.05). There was no significant difference in avoidance force in the 30 mg / kg venom of Toad group (P > 0.05). The positive control, zoledronic acid 30 μg / kg, showed a significant increase in avoidance force from 0 to 6 hours after administration (P < 0.01). These results indicate that a single oral administration of 60 mg / kg venom of Toad can alleviate pain in rats with bone cancer.

[0093] Example 8

[0094] Effects of continuous oral administration of toad venom on mechanical allodynia in rats with bone cancer pain model

[0095] (1) The establishment method of the model rats and sham operation group rat models is the same as that in Example 7, except for the different administration methods: 30 SD female rats were randomly divided into 5 groups, 6 rats in each group, namely, sham operation group, model group, model + toad venom 30 mg / kg group, model + toad venom 60 mg / kg group and model + positive control zoledronic acid 30 μg / kg group. The bone cancer pain model was established in the model rats. The drugs were administered starting on the 11th day after the model was established and continued for 11 days. The drugs were administered by gavage. The normal group and the model group were administered with 0.5% CMC-Na solution, and the drug administration group was administered with the corresponding drugs (dissolved in 0.5% CMC-Na solution). The mechanical paw withdrawal force (g) of the rats was tested 1 day before modeling and 4, 7, 11, 14, 17 and 21 days after modeling. The method for determining the mechanical paw withdrawal force was the same as that in Example 7.

[0096] (2) Statistical analysis: The mean and SD values ​​of the mechanical withdrawal force of rats in the same group were calculated, and then the statistical differences between different groups were tested by two-way test. P < 0.05 was considered to be statistically significant.

[0097] The results of the study on the effect of continuous oral administration of Toad Venenum on mechanical allodynia in rats with bone cancer pain model are as follows: Figure 8 As shown. After tumor cell inoculation, rats' mechanical avoidance force was significantly reduced. Compared with the sham-operated group, the model group showed significantly lower avoidance force from 7 to 21 days (P < 0.05), confirming the successful establishment of the model. Compared with the model group, the 60 mg / kg Toad Venenum group showed a significant increase in avoidance force on days 11, 14, 17, and 21 after administration (P < 0.05). There was no significant difference in avoidance force in the 30 mg / kg Toad Venenum group (P > 0.05). The positive control, zoledronic acid, showed a significant increase in avoidance force on days 11, 14, 17, and 21 after administration (P < 0.05). These results indicate that continuous oral administration of 60 mg / kg Toad Venenum can significantly increase the mechanical avoidance force of rats with bone cancer pain and alleviate bone cancer pain.

[0098] Embodiment 9

[0099] Effects of continuous oral administration of toad venom on the number of spontaneous pain responses in rats with bone cancer pain model

[0100] (1) The method for establishing the bone cancer pain model, grouping and administration method were the same as those in Example 8, except for the test content: the number of spontaneous pain responses of rats was tested 1 day before modeling and 4, 7, 11, 14, 17 and 21 days after modeling.

[0101] (2) Spontaneous pain response test: The rats were placed in a transparent plexiglass box to adapt to the environment for about 20 minutes. After the rats were quiet, the number of spontaneous pain responses of the rats' hind limbs on the operated side was recorded within 5 minutes. The behavior was defined as follows: ① spontaneous withdrawal (i.e., lifting the affected limb); ② spontaneous protection (i.e., raising the affected limb); ③ jumping or lameness (i.e., intermittent jumping without using the affected limb during exercise).

[0102] Statistical analysis: The mean and SD values ​​of the spontaneous pain response times of rats in the same group were calculated, and then the statistical differences between different groups were tested by Two-way test. P < 0.05 was considered to be statistically significant.

[0103] The results of the effects of continuous oral administration of toad venom on the spontaneous pain response of rats with bone cancer pain model are as follows Figure 9 As shown. The number of spontaneous pain responses in rats after tumor cell inoculation increased significantly. Compared with the sham-operated group, the model group had a significant increase in spontaneous pain responses on days 11-21 (P < 0.05), demonstrating successful model establishment. Compared with the model group, the 60 mg / kg Toad Venenum group had a significant decrease in spontaneous pain responses on days 14, 17, and 21 after administration (P < 0.05). There was no significant difference in the number of spontaneous pain responses in the 30 mg / kg Toad Venenum group (P > 0.05). The positive control, zoledronic acid, group had a significant decrease in spontaneous pain responses on days 11, 14, 17, and 21 after administration (P < 0.01). These results demonstrate that continuous oral administration of 60 mg / kg Toad Venenum can significantly reduce the number of spontaneous pain responses in rats with bone cancer pain and alleviate bone cancer pain.

[0104] Example 10

[0105] Effects of single oral administration of cinobufagin and bisbufogenin on mechanical allodynia in rats with bone cancer pain model

[0106] (1) Grouping and administration:

[0107] Thirty-six female SD rats were randomly divided into six groups, each consisting of six rats: a sham-operated group, a model group, a model + cinobufogenin 3 mg / kg group, a model + cinobufogenin 6 mg / kg group, a model + sefobufogenin 1.5 mg / kg group, and a model + sefobufogenin 3 mg / kg group. The model and sham-operated rat models were established in the same manner as in Example 7. Drugs were administered on day 11 of model establishment by gavage. The normal and model groups were administered with the matrix solution, and the drug-treated groups were administered with the corresponding drug (dissolved in the matrix solution). Mechanical withdrawal force (g) was measured in the rats 1, 2, 4, and 6 h after administration, using the same testing method as in Example 7.

[0108] (2) Statistical analysis: The mean and SD values ​​of the mechanical withdrawal force of rats in the same group were calculated, and then the statistical differences between different groups were tested by two-way test. P < 0.05 was considered to be statistically significant.

[0109] The results of the single oral administration of cinobufogenin and esterbufogenin on mechanical allodynia in rats with bone cancer pain model are as follows Figure 10 As shown. After tumor cell inoculation, the rats' mechanical avoidance force was significantly reduced. Compared with the sham-operated group, the model group showed significantly lower avoidance force from 0 to 6 hours (P < 0.001), confirming the successful establishment of the model. Compared with the model group, the 3 mg / kg cinobufogenin group showed no significant difference in avoidance force after administration (P > 0.05). The 6 mg / kg cinobufogenin group showed a significant increase in avoidance force 2 and 4 hours after administration (P < 0.05). The 1.5 and 3 mg / kg cinobufogenin groups also showed a significant increase in avoidance force from 1 to 6 hours after administration (P < 0.05). These results indicate that a single oral administration of 6 mg / kg cinobufogenin and cinobufogenin significantly increases the mechanical avoidance force of rats with bone cancer pain and alleviates bone cancer pain.

[0110] Example 11

[0111] Effects of continuous oral administration of cinobufagin and bisbufogenin on mechanical allodynia in rats with bone cancer pain model

[0112] (1) Grouping and administration: 36 SD female rats were randomly divided into 6 groups, 6 rats in each group, namely, sham operation group, model group, model + cinobufogenin 3 mg / kg group, model + cinobufogenin 6 mg / kg group, model + retibufogenin 1.5 mg / kg group, and model + retibufogenin 3 mg / kg group. The model establishment method of the rats in the model group and the sham operation group was the same as that in Example 7. The administration started on the 11th day after the model establishment and continued for 11 days. The administration method was gavage. The normal group and the model group were administered with the matrix solution, and the drug administration group was administered with the corresponding drug (dissolved in the matrix solution). The mechanical withdrawal force (g) of the rats was tested 1 day before model establishment and 4, 7, 11, 14, 17, and 21 days after model establishment. The testing method was the same as that in Example 7.

[0113] (2) Statistical analysis: The mean and SD values ​​of the mechanical withdrawal force of rats in the same group were calculated, and then the statistical differences between different groups were tested by two-way test. P < 0.05 was considered to be statistically significant.

[0114] The results of the effects of continuous oral administration of cinobufogenin and esterbufogenin on mechanical allodynia in rats with bone cancer pain model are as follows Figure 11 As shown in the results, mechanical avoidance force in rats was significantly reduced after tumor cell inoculation. Compared with the sham-operated group, the model group showed significantly lower avoidance force on days 11-21 (P < 0.001), confirming the successful establishment of the model. Compared with the model group, the 3 mg / kg cinobufogenin group showed no significant difference in avoidance force after administration (P > 0.05). However, the 6 mg / kg cinobufogenin group showed a significant increase in avoidance force on days 17 and 21 after administration (P < 0.05). The 1.5 mg / kg cinobufogenin group showed a significant increase in avoidance force on days 11 and 14 after administration (P < 0.05), and the 3 mg / kg cinobufogenin group showed a significant increase in avoidance force on days 11, 14, 17, and 21 after administration (P < 0.05). These results indicate that continuous oral administration of 6 mg / kg cinobufogenin and cinobufogenin significantly increases mechanical avoidance force in rats with bone cancer pain and alleviates bone cancer pain.

[0115] Example 12

[0116] Effects of continuous oral administration of cinobufagin and bisbufogenin on the number of spontaneous pain responses in rats with bone cancer pain model

[0117] (1) The method for establishing the bone cancer pain model, grouping, and administration method were the same as those in Example 11, except for the test content: the number of spontaneous pain responses of rats was tested 1 day before modeling and 4, 7, 11, 14, 17, and 21 days after modeling. The test method was the same as that in Example 9.

[0118] (2) Statistical analysis: The mean and SD values ​​of the spontaneous pain response times of rats in the same group were calculated, and then the statistical differences between different groups were tested by two-way test. P < 0.05 was considered to be statistically significant.

[0119] The results of the effects of continuous oral administration of cinobufogenin and esterbufogenin on the spontaneous pain response of rats with bone cancer pain model are as follows Figure 12As shown. The number of spontaneous pain responses in rats significantly increased after tumor cell inoculation. Compared with the sham-operated group, the model group had a significant increase in spontaneous pain responses from day 14 to 21 (P < 0.01), demonstrating successful model establishment. Compared with the model group, the number of spontaneous pain responses in the 3 mg / kg cinobufogenin group did not differ significantly (P > 0.05). However, the number of spontaneous pain responses in the 6 mg / kg cinobufogenin group was significantly reduced 21 days after administration (P < 0.01). The number of spontaneous pain responses in the 1.5 mg / kg cinobufogenin group was significantly reduced 21 days after administration (P < 0.01). The number of spontaneous pain responses in the 3 mg / kg cinobufogenin group was significantly reduced 14, 17, and 21 days after administration (P < 0.05). These results indicate that continuous oral administration of the 6 mg / kg cinobufogenin group and the 3 mg / kg cinobufogenin group significantly reduces the number of spontaneous pain responses in rats with bone cancer pain, thereby alleviating bone cancer pain.

[0120] Example 13

[0121] Effects of single oral administration of cinobufagin and bisbufogenin on mechanical allodynia in rats with bone cancer pain model

[0122] (1) Grouping and administration: Forty-two SD female mice were randomly divided into seven groups, with six mice in each group, namely, the sham operation group, the model group, the model + composition 4 (the mass ratio of cinobufogenin to esterobafignin was 1:10) 3 mg / kg group, the model + composition 11 (the mass ratio of cinobufogenin to esterobafignin was 1:2) 3 mg / kg group, the model + composition 7 (the mass ratio of cinobufogenin to esterobafignin was 1:1) 3 mg / kg group, the model + composition 8 (the mass ratio of cinobufogenin to esterobafignin was 5:1) 3 mg / kg group, and the model + composition 9 (the mass ratio of cinobufogenin to esterobafignin was 10:1) 3 mg / kg group. The model establishment method for rats in the model group and the sham-operated group was the same as in Example 7. Drugs were administered on the 11th day after model establishment by gavage. The normal group and the model group were administered with the matrix solution, and the drug-treated group was administered with the corresponding drug (dissolved in the matrix solution). The mechanical withdrawal force (g) of the rats was tested 1, 2, 4, and 6 h after administration. The testing method was the same as in Example 7.

[0123] (2) Statistical analysis: The mean and SD values ​​of the mechanical withdrawal force of rats in the same group were calculated, and then the statistical differences between different groups were tested by two-way test. P < 0.05 was considered to be statistically significant.

[0124] The results of oral administration of cinobufogenin and esterbufogenin on mechanical allodynia in rats with bone cancer pain model are as follows Figure 13As shown. The mechanical avoidance force of rats was significantly reduced after inoculation of tumor cells. Compared with the sham operation group, the avoidance force of the model group was significantly reduced at 0-6h (P<0.001), proving that the model was successfully established. Compared with the model group, the avoidance force of composition 4 group was significantly increased 1-6h after administration (P<0.05), the avoidance force of composition 11, 7 and 8 groups was significantly increased 2h after administration (P<0.05), and there was no significant difference in the avoidance force of composition 9 group after administration (P>0.05). The above results show that when the mass ratio of cinobucoid venom toxin base is 0.1-5, it can significantly increase the mechanical avoidance force of rats with bone cancer pain and relieve bone cancer pain.

[0125] Example 14

[0126] Effects of continuous oral administration of cinobufagin and bisbufogenin on mechanical allodynia in rats with bone cancer pain model

[0127] (1) Grouping and administration: Forty-two SD female mice were randomly divided into seven groups, with six mice in each group, namely, the sham operation group, the model group, the model + composition 4 (the mass ratio of cinobufogenin to esterobafignin was 1:10) 3 mg / kg group, the model + composition 11 (the mass ratio of cinobufogenin to esterobafignin was 1:2) 3 mg / kg group, the model + composition 7 (the mass ratio of cinobufogenin to esterobafignin was 1:1) 3 mg / kg group, the model + composition 8 (the mass ratio of cinobufogenin to esterobafignin was 5:1) 3 mg / kg group, and the model + composition 9 (the mass ratio of cinobufogenin to esterobafignin was 10:1) 3 mg / kg group. The model establishment method for rats in the model group and the sham-operated group was the same as in Example 7. Drug administration began on the 11th day after model establishment and continued for 11 consecutive days. The drug administration method was gavage. The normal group and the model group were administered with the matrix solution, and the drug-treated group was administered with the corresponding drug (dissolved in the matrix solution). The mechanical withdrawal force (g) of the rats was tested 1 day before model establishment and 4, 7, 11, 14, 17, and 21 days after model establishment. The testing method was the same as in Example 7.

[0128] (2) Statistical analysis: The mean and SD values ​​of the mechanical withdrawal force of rats in the same group were calculated, and then the statistical differences between different groups were tested by two-way test. P < 0.05 was considered to be statistically significant.

[0129] The results of the combined effects of cinobufogenin and esterbufogenin on mechanical allodynia in rats with bone cancer pain model were as follows: Figure 14As shown. The mechanical avoidance force of rats was significantly reduced after inoculation of tumor cells. Compared with the sham operation group, the avoidance force of the model group was significantly reduced from 7 to 21 days (P<0.01), proving that the model was successfully established. Compared with the model group, the avoidance force of the composition 4 group increased significantly from 11 to 21 days after administration (P<0.001), the avoidance force of the composition 11 group increased significantly 11 and 14 days after administration (P<0.05), the avoidance force of the composition 7 group increased significantly 11, 14 and 21 days after administration (P<0.05), the avoidance force of the composition 8 group increased significantly 11 and 14 days after administration (P<0.05), and there was no significant difference in the avoidance force of the composition 9 group after administration (P>0.05). The above results show that when the mass ratio of cinobucoid venom to ester tofu genin is 0.1-5, it can significantly increase the mechanical avoidance force of rats with bone cancer pain and relieve bone cancer pain.

[0130] Example 15

[0131] Effects of continuous oral administration of cinobufagin and bisbufogenin on the number of spontaneous pain responses in rats with bone cancer pain model

[0132] (1) The method for establishing the bone cancer pain model, grouping, and administration method were the same as those in Example 14, except for the test content: the number of spontaneous pain responses of rats was tested 1 day before modeling and 4, 7, 11, 14, 17, and 21 days after modeling. The test method was the same as that in Example 9.

[0133] (2) Statistical analysis: The mean and SD values ​​of the spontaneous pain response times of rats in the same group were calculated, and then the statistical differences between different groups were tested by two-way test. P < 0.05 was considered to be statistically significant.

[0134] The results of the effects of continuous oral administration of cinobufogenin and esterbufogenin on the spontaneous pain response of rats with bone cancer pain model are as follows Figure 15 As shown. The number of spontaneous pain reactions in rats increased significantly after inoculation of tumor cells. Compared with the normal group, the number of spontaneous pain reactions in the model group increased significantly from 11 to 21 days (P<0.01), proving that the model was successfully established. Compared with the model group, the number of spontaneous pain reactions in the composition 4 group was significantly reduced from 11 to 21 days after administration (P<0.01), the number of spontaneous pain reactions in the composition 11 group was significantly reduced from 14 to 17 days after administration (P<0.01), the number of spontaneous pain reactions in the composition 7 group was significantly reduced from 17 to 21 days after administration (P<0.01), the number of spontaneous pain reactions in the composition 8 group was significantly reduced from 17 days after administration (P<0.05), and there was no significant difference in the number of spontaneous pain reactions in the composition 9 group after administration (P>0.05). The above results show that when the mass ratio of cinobucoid venom to esterbufagin is 0.1-5, it can significantly reduce the number of spontaneous pain reactions in rats with bone cancer pain, thereby relieving bone cancer pain.

[0135] Example 16

[0136] Effects of bisbufotoxin and morphine hydrochloride on mechanical allodynia in a rat model of bone cancer pain

[0137] (1) Establishment of bone cancer pain model: SD rats were anesthetized with Shutai 50 (50 mg / kg) combined with thiazine (5 mg / kg), and analgesic was given with meloxicam (2.5 mg / kg) and lidocaine (2 mg / kg). The left knee joint was shaved and disinfected. The knee joint was fixed with the left finger, and a No. 7 needle was used to drill a hole at the knee joint (edge ​​of the patellar ligament) along the longitudinal axis of the tibia toward the distal end of the tibia, with a depth of about 1.5 cm. Then, a microinjector was used to inject tumor cells into the tibial bone marrow cavity. The microinjectors of the model group and the drug-treated group successively aspirated 2 μL of gelatin sponge aqueous solution, 1 μL of air, and 5 μL of tumor cells (5×10 5 The sham-operated group received 5 μL HBSS without tumor cells as a control.

[0138] (2) Grouping and Administration: Forty female SD rats were randomly divided into five groups, each with eight rats: a sham operation group, a model group, a model group plus 3 mg / kg (ig) of bispyribacin, a model group plus 3 mg / kg (ig) of morphine hydrochloride, and a model group plus 3 mg / kg (ip) of morphine hydrochloride. A bone cancer pain model was established in the model group rats, and the drugs were administered on the 11th day after the model was established. The normal group and the model group were administered with the matrix solution, and the drug-administered group was administered with the corresponding drug (dissolved in the matrix solution). The mechanical withdrawal force (g) of the rats was measured 1, 2, 4, and 6 h after administration. The method for determining the mechanical withdrawal force was the same as in Example 7.

[0139] (3) Statistical analysis: The mean and SD values ​​of the mechanical withdrawal force of rats in the same group were calculated, and then the statistical differences between different groups were tested using a two-way test. P < 0.05 was considered to be statistically significant.

[0140] The results of the effects of bismuthine and morphine hydrochloride on mechanical allodynia in rats with bone cancer pain model are as follows Figure 16 As shown in the figure. After tumor cell inoculation, the rats' mechanical avoidance force was significantly reduced. Compared with the sham-operated group, the model group showed significantly lower avoidance force from 0 to 6 hours (P < 0.001), confirming the successful establishment of the model. Compared with the model group, the avoidance force of retibufogenin significantly increased from 1 to 6 hours after oral administration (P < 0.001). The avoidance force of the morphine group increased significantly from 1 to 2 hours after intraperitoneal administration (P < 0.05), but there was no significant difference in avoidance force after oral administration (P > 0.05). These results indicate that, under the same administration method and dosage, retibufogenin has a stronger analgesic effect than morphine hydrochloride.

[0141] Embodiment 17

[0142] The addictiveness of bufotoxin was evaluated using the Conditioned Place Preference (CPP) test.

[0143] (1) Pre-test period (Day 1): the stage of pre-testing the basic value.

[0144] The mice were placed in the middle of the two boxes, and the time they stayed in the two boxes was recorded within 15 minutes. Mice with a natural preference for a certain box (preference rate was not between 40% and 60%) and mice with less than 20 shuttle times were eliminated based on the baseline value.

[0145] (2) Training period (Day 2-6): training phase.

[0146] 24 qualified mice were divided into normal saline group, 10 mg / kg and 5 mg / kg thiazolin groups and positive control morphine 10 mg / kg group, with 6 mice in each group. According to the basal value, the non-preferred box of mice was used as the companion box, and the CPP box was closed with a partition.

[0147] Each morning, the saline group received an intraperitoneal injection of saline and was placed in a medicine-attached box. The drug group received an intraperitoneal injection of the corresponding drug (dissolved in a matrix solution) and was placed in a non-medicine-attached box. Each afternoon, the two groups exchanged training boxes. The saline group received an intraperitoneal injection of saline and was placed in a non-medicine-attached box. The drug group received an intraperitoneal injection of the corresponding drug (dissolved in a matrix solution) and was placed in a medicine-attached box. After the injection, the mice were placed in the box and remained there for 45 minutes. This training continued for five consecutive days.

[0148] (3) Testing period (Day 7): Testing phase.

[0149] The partition was opened, and the mouse was placed in the center, allowing it to freely enter both chambers. The time the mouse spent in each chamber was recorded over a 15-minute period. The results were scored as the preference rate for the drug-companion chamber, i.e., the preference rate for the drug-companion chamber = the activity time in the drug-companion chamber / (activity time in the drug-companion chamber + the raw salt chamber).

[0150] (4) Statistical analysis: The mean and SD values ​​of the drug box preference rate of rats in the same group were calculated, and then the statistical differences between different groups were tested by two-way test. P < 0.05 was considered to be statistically significant.

[0151] The results of the study on the addictive effect of continuous intraperitoneal injection of bufotoxin on mice are as follows Figure 17 As shown in the figure, compared with the pre-test phase, mice in the positive control morphine-dependent group spent significantly longer on the drug-companion side during the test phase (P<0.001). There was no significant change in the time spent on the drug-companion side in the 10 mg / kg and 5 mg / kg groups of retibufogenin (P<0.05). There was also no significant difference in the time spent on the drug-companion side in the normal group (P<0.05). These results indicate that retibufogenin has no addictive effect.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of an active ingredient of toad venom in the preparation of a drug for relieving inflammatory pain and bone cancer pain, characterized in that: The active ingredients of toad venom in drugs that relieve inflammatory pain include bufadin; The active ingredients of toad venom in the drug for relieving bone cancer pain include sebufugin or a combination of cinobufugin and sebufugin; The mass ratio of the composition of cinobufagin and resebufugin is 1:10-5:1.

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