Application of venenum bufonis, venenum bufonis active ingredient and venenum bufonis active ingredient composition in relieving inflammatory pain and bone cancer pain

Abstract

The invention discloses venenum bufonis, venenum bufonis active ingredients and application of a venenum bufonis active ingredient composition in relieving inflammatory pain and bone cancer pain, and belongs to the technical field of venenum bufonis analgesic active ingredient research. It is found that the venenum bufonis medicinal material can relieve inflammatory pain and bone cancer pain, and it is clear that the main active ingredients of the venenum bufonis medicinal material are cinobufagin and resibufogenin; the cinobufagin and the resibufogenin have a good analgesic effect in a certain ratio range, inflammation pain can be relieved when the mass ratio of the cinobufagin to the resibufogenin is 4: 5-5: 1, and bone cancer pain can be relieved when the mass ratio of the cinobufagin to the resibufogenin is 1: 10-5: 1. The active ingredients cinobufagin and resibufogenin are enlightenment that cinobufagin and resibufogenin can become substitutes of endangered medicinal material venenum bufonis for relieving inflammation pain and bone cancer pain; in addition, it is found for the first time that the analgesic effect of resibufogenin is similar to that of morphine, no addiction exists, and it is prompted that resibufogenin is expected to be developed into a novel analgesic drug.

Description

Technical Field

[0001] The invention relates to the technical field of research on toad venom analgesic active ingredients, and in particular to application 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 plays an increasingly important role in the prevention and treatment of malignant tumors and cancer pain. A number of Chinese medicines with significant anti-cancer and analgesic effects have been discovered, such as toad venom, matrine, and cinobucoids, among which toad venom is more effective. Toad venom is the secretion of the parotid gland of the Chinese giant toad (Bufo bufo gargarizans Cantor) and the black-rimmed toad (Bufo melanostictus Schneider) of the Toad family. It is sweet, spicy, warm, and toxic. It has the effects of detoxification, swelling, refreshing, opening the mind, strengthening the heart, and relieving pain. Toad venom, which is listed in the Chinese Pharmacopoeia, has been used for centuries as a drug for the treatment of cardiovascular diseases, inflammatory diseases, cancer, and chronic pain. However, the compound toad venom paste made from toad venom is mainly used for pain caused by various cancers such as lung, liver, and stomach, and there are few reports on the treatment of bone cancer pain.

[0003] At present, toads are facing the problem of resource shortage and high commodity prices, and the contradiction between supply and demand is huge. Therefore, in order to protect the ecological environment, reduce the cost of toad venom analgesics, and lower the price of toad venom analgesics, it is of great significance to study the analgesic active ingredients of toad venom to replace toad venom.

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

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

[0006] Nonsteroidal anti-inflammatory drugs have a "ceiling" effect, and long-term use can cause liver damage. Opioids are highly addictive, and overdose can be fatal. Patients who take opioids to treat chronic pain quickly develop analgesic tolerance, and often fail to achieve the same pain relief effect over time. In addition, opioids can slow or prevent bone remodeling and even increase bone brittleness, adding an additional burden to patients who are being treated for bone cancer pain and hindering bone cancer treatment.

[0007] In summary, it is urgent to develop safe, analgesic and non-addictive drugs to treat bone cancer pain. Literature reports that toad venom can be used to relieve cancer pain, but there are few studies on the treatment of bone cancer pain, and there is no screening of its effective ingredients and addiction research. Therefore, the present invention intends to study the effect of toad venom in relieving bone cancer pain and clarify its active ingredients that exert their efficacy to replace toad venom. Summary of the invention

[0008] To solve the above problems, the present invention provides a use of toad venom, toad venom active ingredients and toad venom active ingredient composition in relieving inflammatory pain and bone cancer pain. While studying the analgesic mechanism of toad venom, it is clearly found that the main active ingredients in toad venom that exert their medicinal effects are cinobufogenin and sebufogenin, indicating that the active ingredients can replace the endangered medicinal material toad venom to relieve inflammatory pain and bone cancer pain.

[0009] To achieve the above-mentioned purpose, the present invention provides the use of toad venom in relieving inflammatory pain and bone cancer pain. The active ingredients of toad venom for relieving inflammatory pain include cinobucini, resebucin and bufotoxin; the active ingredients of toad venom for relieving bone cancer pain include cinobucini and resebucin.

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

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

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

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

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

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

[0016] (1) It was first discovered that the active ingredients of toad venom that relieve inflammatory pain are cinobucoid, sebucoid and tofu taridine, and the analgesic effect of cinobucoid and sebucoid is comparable to that of toad venom, suggesting that the combination of cinobucoid and sebucoid may replace toad venom to treat inflammatory pain.

[0017] (2) It was discovered for the first time that toad venom can relieve bone cancer pain, providing a candidate drug for the treatment of bone cancer pain; it was further discovered that esterbufagin and its combination with cinonabufagin can relieve bone cancer pain, suggesting that esterbufagin and its combination with cinonabufagin may replace toad venom to relieve bone cancer pain;

[0018] (3) The present invention discovered for the first time that the analgesic effect of seboflavin is similar to that of morphine and is non-addictive, suggesting that it has the potential to be developed into 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 on the inflammatory pain model mice in Example 1;

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

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

[0023] Figure 4 The results of the analgesic effects of other active molecules in toad venom in Example 4 on mice with inflammatory pain model;

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

[0025] Figure 6 The analgesic effect results of the combination of Chinese toad venom and bisbufogenin on the inflammatory pain model mice in Example 6;

[0026] Figure 7 The results of the effect of single oral administration of toad venom on mechanical allodynia in rats with bone cancer pain model 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] Fig. 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] Fig.10 The results of the effect of single oral administration of cinobucoid venom and sebucoid venom on mechanical allodynia in rats with bone cancer pain model in Example 10; among them, ###, P<0.001 model group vs sham operation group; *, P<0.05, **, P<0.05, ***, P<0.001 sebucoid venom 3 mg / kg group vs model group; &, P<0.05 sebucoid venom 1.5 mg / kg group vs model group; $, P<0.05 cinobucoid venom 6 mg / kg group vs model group;

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

[0031] Fig.12 The results of the effect of continuous oral administration of cinobucoid venom and sebucoid venom on the spontaneous pain response of rats with bone cancer pain model in Example 12; among them, ##, P<0.01, ###, P<0.001 model group vs sham operation group; *, P<0.05, ***, P<0.001 sebucoid venom 3 mg / kg group vs model group; &&, P<0.01 sebucoid venom 1.5 mg / kg group vs model group; $$, P<0.01 cinobucoid venom 6 mg / kg group vs model group;

[0032] Fig.13 The results of Example 13 are the effects of oral administration of cinobucoid venom and esterbufagin 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] Fig.14 The results of the effect of continuous oral administration of cinobucoid venom and esterbufagin on mechanical allodynia in rats with bone cancer pain model in Example 14; 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] Fig.15 The results of the effect of continuous oral administration of cinobucoid venom and esterbufagin on the spontaneous pain response of rats with bone cancer pain model in Example 15; 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] Fig.16 The results of the effects of cetirizine and morphine hydrochloride on mechanical allodynia in rats with bone cancer pain model in Example 1 are shown; among them, ###, P<0.001 model group vs sham operation group; ***, P<0.001 cetirizine group vs model group; &, P<0.05, &&, P<0.01 morphine ip group vs model group;

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

[0037] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention more clearly understood, the embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings and embodiments. 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 embodiments and preparation of required solutions:

[0039] Clean-grade 18-22g Kunming female mice and 180-200g 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 instrument was purchased from Shanghai Yuyan Scientific Instrument Co., Ltd.; toad venom medicinal materials were provided by Shanghai Hutchison Pharmaceuticals Co., Ltd.; acupuncture plantar pain tester was purchased from Shenzhen Ruiwode Life Science 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.; 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 saline in sequence to prepare 1 mL of matrix solution.

[0043] Embodiment 1

[0044] Analgesic effect of toad venom 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, including 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] The inflammatory pain model was established by injecting 10 μL of 2% carrageenan solution into the hind limbs of the mice in the model group. The normal group and the model group were given 0.5% sodium carboxymethyl cellulose solution by intragastric administration, and the drug group was given the corresponding drug (dissolved in 0.5% sodium carboxymethyl cellulose). The thermal paw withdrawal latency (s) of the mice was tested at 0.5, 1, 2, 4 and 6 h after administration.

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

[0049] The heat withdrawal latency of mice was tested by an intelligent hot plate instrument, and the reaction endpoint was the appearance of licking the paw of mice. The pain threshold of each group of mice before administration was tested by a hot plate instrument (temperature set to 55°C) (the average value was taken after two tests, and the interval between the two measurements was >15min), and unqualified mice (latency less than 5s or greater than 30s) were discarded. Qualified mice were randomly divided into groups for the experiment, and the heat withdrawal latency at 0.5, 1, 2, 4, and 6h after oral administration was tested and recorded.

[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 among different groups were tested by Two-way test. P < 0.05 was considered to be statistically significant.

[0052] The analgesic effect of toad venom on inflammatory pain model mice Figure 1 As shown. The latency of thermal paw withdrawal in mice with carrageenan-induced inflammatory pain was significantly reduced after modeling. Compared with the normal group, the latency of the model group was significantly reduced at 0.5-6h (P<0.01), proving that the model was successfully established. Compared with the model group, the latency of toad venom 150mg / kg increased significantly at 0.5, 2, 4 and 6h after administration (P<0.05), the latency of toad venom 175mg / kg increased significantly at 0.5-6h after administration (P<0.01), and the latency of toad venom 200mg / kg increased significantly at 1-6h after administration (P<0.05). The positive control ibuprofen 100mg / kg group also increased significantly at 0.5-6h after administration (P<0.05). The above results show that toad venom has a good analgesic effect in the carrageenan-induced inflammatory pain model.

[0053] Embodiment 2

[0054] Screening of analgesic activity of five lactone active molecules from toad venom 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 + cinobucina toxin base 3 mg / kg group, model + bufotoxin genin 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] The inflammatory pain model was established by injecting 10 μL of 2% carrageenan solution into the hind limbs of the mice in the model group. The normal group and the model group were given the matrix solution, and the drug group was given the corresponding drug (dissolved in the matrix solution). The thermal paw 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.

[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 among 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 venom on inflammatory pain model mice are shown in the following table. Figure 2 As shown. The latency of thermal paw withdrawal in mice with carrageenan-induced inflammatory pain was significantly reduced after modeling. Compared with the normal group, the latency of the model group was significantly reduced at 0.5-6h (P<0.05), proving that the model was successfully established. Compared with the model group, the latency of thermal paw withdrawal in the bufaline group was significantly increased at 0.5h after administration (P<0.05), the latency of bufaline was significantly increased at 2h after administration (P<0.001), and the latency of bufaline was significantly increased at 2, 4, and 6h after administration (P<0.01), while the latency of cinobucini and esterbufagin was significantly increased at 0.5-6h after administration (P<0.05), and the latency of the positive control ibuprofen 100mg / kg group was also significantly increased at 0.5-6h after administration (P<0.05).

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

[0061] Embodiment 3

[0062] Analgesic effect of cinobufogenin, bufalin and bufalin 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 + cinobucini 3 mg / kg group, model + bufalin 3 mg / kg group, model + composition 1 (the mass ratio of cinobucini to bufalin was 12:5) 3 mg / kg group, model + composition 2 (the mass ratio of cinobucini, bufalin and bufalin was 20:8:9) 3 mg / kg group and positive control ibuprofen 100 mg / kg group.

[0065] The inflammatory pain model was established by injecting 10 μL of 2% carrageenan solution into the hind limbs of the mice in the model group. The normal group and the model group were given the matrix solution, and the drug group was given the corresponding drug (dissolved in the matrix solution). The thermal paw 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.

[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 among different groups were tested by Two-way test. P < 0.05 was considered to be statistically significant.

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

[0069] By further screening the three active molecules according to the ratio in the toad venom, it was found that the analgesic effect of composition 1 was enhanced, while composition 2 (i.e., adding bufotoxin) did not further enhance the analgesic effect. The above results show that the combination of cinobucoid venom and esterbufogenin has an analgesic effect in the carrageenan-induced inflammatory pain model, which is better than single administration.

[0070] Embodiment 4

[0071] Analgesic effects of other active molecules in toad venom on inflammatory pain model mice

[0072] (1) Grouping and administration: 50 female Kunming mice were randomly divided into 10 groups, 5 mice in each group, including normal group, model group, model + telocinobufagin 3 mg / kg group, model + cinobufolin 3 mg / kg group, model + toad taridin 3 mg / kg group, model + sabbufagin 3 mg / kg group, model + 5-hydroxytryptamine 3 mg / kg group, model + N-methyl-5-hydroxytryptamine 3 mg / kg group, model + toad tryptamine 3 mg / kg group and model + toad ternidine 3 mg / kg group. The inflammatory pain model was established by injecting 10 μL of 2% carrageenan solution into the plantar hind limbs of mice in the model group, and the drug administration method was adopted. The normal group and the model group were given the matrix solution, and the drug administration group was given the corresponding drug (dissolved in the matrix solution). The thermal paw withdrawal latency (s) of the mice was tested 0.5, 1, 2, 4 and 6 hours after administration, and 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 by Two-way test. P < 0.05 was considered to be statistically significant.

[0074] The analgesic effects of other active molecules in toad venom on inflammatory pain model mice are shown in the following table: 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 had significantly reduced latency at 0.5-6h (P<0.05), proving that the model was successfully established. Compared with the model group, only toad taridin had a significant increase in thermal paw withdrawal latency at 0.5-6h after administration (P<0.05), and the latency of the other 7 molecules did not change significantly after administration (P>0.05). The above results show that toad taridin has an analgesic effect in the carrageenan-induced inflammatory pain model.

[0075] Embodiment 5

[0076] Analgesic effect of cinobucoid venom, esterbufogenin and bufotoxin combined with toad venom of the same content on inflammatory pain model mice

[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 cinobucina toxin base and sebucina toxin genin is 12:5) 3 mg / kg group, model + composition 3 (mass ratio of cinobucina toxin base, sebucina toxin genin and toad tardin is 25:10:4) 3 mg / kg group, model + toad venom 43 mg / kg group and positive control ibuprofen 100 mg / kg group. The inflammatory pain model was established by injecting 10 μL 2% carrageenan solution into the plantar of the hind limbs of the mice in the model group, and the administration method was gavage. The normal group and the model group were given the matrix solution, and the drug administration group was given the corresponding drug (dissolved in the matrix solution). The thermal paw withdrawal latency (s) of the mice was tested 0.5, 1, 2, 4 and 6 hours after administration, and 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 by Two-way test. P < 0.05 was considered to be statistically significant.

[0079] The analgesic effects of the combination of cinobucoid venom, esterbufogenin and bufotoxin and the same content of toad venom on mice with inflammatory pain model are shown in the following table. Figure 5 As shown. The heat paw withdrawal latency of mice with inflammatory pain induced by carrageenan was significantly reduced after modeling. Compared with the normal group, the model group had significant differences at 0.5-6h (P<0.01), proving that the model was successfully established. Compared with the model group, the heat paw withdrawal latency of the composition 1 group increased significantly at 0.5-6h after administration (P<0.05), the heat paw withdrawal latency of the composition 3 group increased significantly at 1-6h after administration (P<0.05), the heat paw withdrawal latency of the toad venom 43mg / kg group increased significantly at 0.5-6h after administration (P<0.05), and the latency of the positive control ibuprofen 100mg / kg group increased significantly at 1-6h after administration (P<0.05). Compared with the composition 1 group, the composition 3 group did not significantly increase the latency (P>0.05), and there was no significant difference compared with the toad venom medicinal material group (ns: P>0.05). The above results show that when the mass ratio of cinona tofu venom to ester tofu venom is 12:5, it has an analgesic effect in the carrageenan-induced inflammatory pain model, and its analgesic effect is equivalent to that of toad venom.

[0080] Embodiment 6

[0081] Analgesic effect of cinobufogenin and bisbufogenin on inflammatory pain model mice

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

[0083] The inflammatory pain model was established by injecting 10 μL of 2% carrageenan solution into the plantar hind limbs of mice in the model group. The normal group and the model group were given the matrix solution, and the drug group was given the corresponding drug (dissolved in the matrix solution). The thermal paw 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 that 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 by Two-way test. P < 0.05 was considered to be statistically significant.

[0085] The analgesic effect of cinobucoid venom and esterbufagin on inflammatory pain model mice Figure 6 As shown. The latency of thermal paw withdrawal in mice with carrageenan-induced inflammatory pain was significantly reduced after modeling. Compared with the normal group, the model group had significant differences at 0.5-6h (P<0.05), proving that the model was successfully established. Compared with the model group, the latency of composition group 6 increased significantly at 6h after administration (P<0.001), the latency of composition group 7 increased significantly at 2h after administration (P<0.01), the latency of composition group 1 increased significantly at 1-6h after administration (P<0.05), the latency of composition group 8 increased significantly at 2 and 6h after administration (P<0.05), and the latency of composition groups 4, 5, 9 and 10 did not change significantly (P>0.05). The above results show that the mass ratio of cinobucini to esterbufagin is 4:5-5:1, which has analgesic effect in the carrageenan-induced inflammatory pain model.

[0086] Embodiment 7

[0087] Effect of single intragastric 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 meloxicam (2.5 mg / kg) and lidocaine hydrochloride (2 mg / kg) were used for analgesia. 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 micro-injector was used to inject tumor cells into the tibial bone marrow cavity. The micro-injectors of the model group and the drug-treated group in turn inhaled 2 μL of gelatin sponge aqueous solution, 1 μL of air, and 5 μL of tumor cells (5×10 5 The sham operation group was treated with 5 μL HBSS without tumor cells as a control.

[0089] (2) Grouping and administration: 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 rats in the model group. The drug administration was started on the 11th day after the establishment of the model. The normal group and the model group were given 0.5% CMC-Na solution, and the drug administration group was given the corresponding drug (dissolved in 0.5% CMC-Na solution). The mechanical paw withdrawal force (g) of the rats was tested 1, 2, 4 and 6 hours after drug administration.

[0090] (3) Determination of mechanical withdrawal force: Place the rat on the plantar acupuncture pain tester and allow it to move freely. When the rat stops exploring and becomes relatively quiet, move the stimulation unit and use the mirror to aim the stimulation needle at the stimulation area on the surface of the paw. Then press the start button on the side of the stimulation unit handle. The avoidance force value measured when the rat lifts its paw due to pain is used as an observation index. The mechanical withdrawal force of the rat is tested 1, 2, 4, and 6 hours after drug administration. To avoid errors, each hind limb is measured 3 times and the average value is taken.

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

[0092] Effect of single oral administration of toad venom on mechanical allodynia in rats with bone cancer pain model Figure 7As 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 the 60mg / kg toad venom group increased significantly at 1, 2 and 4h after administration (P<0.05), and there was no significant difference in the avoidance force of the 30mg / kg toad venom group (P>0.05). The positive control zoledronic acid 30μg / kg group had a significant increase in avoidance force at 0-6h after administration (P<0.01). The above results show that a single oral administration of 60mg / kg toad venom can relieve bone cancer pain in rats.

[0093] Embodiment 8

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

[0095] (1) The establishment method of the model group rats and the sham operation group rats was the same as that in Example 7, but the administration method was different: 30 SD female rats were randomly divided into 5 groups, 6 rats in each group, 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, and the administration began on the 11th day after the model was established, and continued for 11 days, using the method of gavage administration. The normal group and the model group were given 0.5% CMC-Na solution, and the administration group was given the corresponding drug (dissolved in 0.5% CMC-Na solution). The mechanical paw withdrawal force (g) of the rats was tested 1 day before modeling, 4, 7, 11, 14, 17 and 21 days after modeling. The mechanical paw withdrawal force determination method 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 among different groups were tested by Two-way test. P < 0.05 was considered to be statistically significant.

[0097] Effect of continuous oral administration of toad venom on mechanical allodynia in rats with bone cancer pain model Figure 8 As 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<0.05), proving that the model was successfully established. Compared with the model group, the avoidance force of the 60 mg / kg toad venom group increased significantly on days 11, 14, 17 and 21 after administration (P<0.05), and there was no significant difference in the avoidance force of the 30 mg / kg toad venom group (P>0.05). The positive control zoledronic acid group had a significant increase in avoidance force on days 11, 14, 17 and 21 after administration (P<0.05). The above results show that continuous oral administration of 60 mg / kg toad venom can significantly increase the mechanical avoidance force of rats with bone cancer pain and relieve bone cancer pain.

[0098] Embodiment 9

[0099] Effect of continuous intragastric 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, the grouping and the 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 min. After the rats were quiet, the number of spontaneous pain responses of the rats' hind limbs on the operated side was recorded within 5 min. The behaviors were 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 among different groups were tested by Two-way test. P < 0.05 was considered statistically significant.

[0103] Effect of continuous oral administration of toad venom on spontaneous pain response in rats with bone cancer pain model Fig. 9 As shown. The number of spontaneous pain reactions in rats increased significantly after inoculation of tumor cells. Compared with the sham operation group, the number of spontaneous pain reactions in the model group increased significantly from 11 to 21 days (P<0.05), proving that the model was successfully established. Compared with the model group, the number of spontaneous pain reactions in the 60 mg / kg toad venom group was significantly reduced on days 14, 17 and 21 after administration (P<0.05), and there was no significant difference in the number of spontaneous pain reactions in the 30 mg / kg toad venom group (P>0.05). The number of spontaneous pain reactions in the positive control zoledronic acid group was significantly reduced on days 11, 14, 17 and 21 after administration (P<0.01). The above results show that continuous intragastric administration of 60 mg / kg toad venom can significantly reduce the number of spontaneous pain reactions in rats with bone cancer pain and relieve bone cancer pain.

[0104] Embodiment 10

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

[0106] (1) Grouping and administration:

[0107] 36 SD female mice were randomly divided into 6 groups, 6 in each group, sham operation group, model group, model + cinobucini venom 3 mg / kg group, model + cinobucini venom 6 mg / kg group, model + sepibucini 1.5 mg / kg group and model + sepibucini venom 3 mg / kg group. The establishment method of the model rats and sham operation rats was the same as that of Example 7. The drugs were administered on the 11th day of model establishment by 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 paw withdrawal force (g) of the rats was tested 1, 2, 4 and 6 hours after administration. The test method was the same as that of 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 among different groups were tested by Two-way test. P < 0.05 was considered to be statistically significant.

[0109] Effects of single oral administration of cinobucoid venom and bisbufogenin on mechanical allodynia in rats with bone cancer pain model Fig.10 As shown. The mechanical avoidance force of rats was significantly reduced after tumor cell inoculation. 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, there was no significant difference in the avoidance force of the 3mg / kg group of cinobucoid venom after administration (P>0.05), the avoidance force of the 6mg / kg group of cinobucoid venom was significantly increased at 2 and 4h after administration (P<0.05), and the avoidance force of the 1.5 and 3mg / kg groups of esterbufagin was significantly increased at 1-6h after administration (P<0.05). The above results show that a single oral administration of 6mg / kg cinobucoid venom and esterbufagin can significantly increase the mechanical avoidance force of rats with bone cancer pain and relieve bone cancer pain.

[0110] Embodiment 11

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

[0112] (1) Grouping and administration: 36 SD female mice were randomly divided into 6 groups, 6 mice in each group, sham operation group, model group, model + cinobucini venom 3 mg / kg group, model + cinobucini venom 6 mg / kg group, model + sepibucini 1.5 mg / kg group and model + sepibucini venom 3 mg / kg group. The establishment method of the model rats and sham operation rats was the same as that of Example 7. The administration began on the 11th day of model establishment and continued for 11 days. The administration method was gavage. The normal group and the model group were given the matrix solution, and the drug administration group was given the corresponding drug (dissolved in the matrix solution). The mechanical paw 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 test method was the same as that of 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 among different groups were tested by Two-way test. P < 0.05 was considered to be statistically significant.

[0114] Effects of continuous oral administration of cinobucoid venom and esterbufagin on mechanical allodynia in rats with bone cancer pain model Fig.11 As shown. The mechanical avoidance force of rats was significantly reduced after tumor cell inoculation. Compared with the sham operation group, the avoidance force of the model group was significantly reduced from 11 to 21 days (P<0.001), which proved that the model was successfully established. Compared with the model group, there was no significant difference in the avoidance force of the 3 mg / kg group of cinobucini toxin after administration (P>0.05), the avoidance force of the 6 mg / kg group of cinobucini toxin was significantly increased on days 17 and 21 after administration (P<0.05), the avoidance force of the 1.5 mg / kg group of esterbufagin toxin was significantly increased on days 11 and 14 after administration (P<0.05), and the avoidance force of the 3 mg / kg group of esterbufagin toxin was significantly increased on days 11, 14, 17 and 21 after administration (P<0.05). The above results show that continuous oral administration of 6 mg / kg cinobucini toxin and esterbufagin toxin can significantly increase the mechanical avoidance force of rats with bone cancer pain and relieve bone cancer pain.

[0115] Embodiment 12

[0116] Effects of continuous intragastric administration of cinobufogenin and sebufogenin 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, the grouping and the 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 among different groups were tested by Two-way test. P < 0.05 was considered statistically significant.

[0119] The results of the continuous oral administration of cinobufogenin and bisbufogenin on the spontaneous pain response of rats with bone cancer pain model are as follows Fig.12As shown. The number of spontaneous pain reactions in rats increased significantly after inoculation of tumor cells. Compared with the sham operation group, the number of spontaneous pain reactions in the model group increased significantly from 14 to 21 days (P<0.01), proving that the model was successfully established. Compared with the model group, there was no significant difference in the number of spontaneous pain reactions in the 3 mg / kg group of cinobucini toxin after administration (P>0.05), the number of spontaneous pain reactions in the 6 mg / kg group of cinobucini toxin after administration was significantly reduced 21 days after administration (P<0.01), the number of spontaneous pain reactions in the 1.5 mg / kg group of esterbufagin toxin after administration was significantly reduced 21 days after administration (P<0.01), and the number of spontaneous pain reactions in the 3 mg / kg group of esterbufagin toxin after administration was significantly reduced 14, 17 and 21 days after administration (P<0.05). The above results show that continuous oral administration of the 6 mg / kg group of cinobucini toxin and the esterbufagin toxin group can significantly reduce the number of spontaneous pain reactions in rats with bone cancer pain, thereby relieving bone cancer pain.

[0120] Embodiment 13

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

[0122] (1) Grouping and administration: Forty-two female SD mice were randomly divided into 7 groups, with 6 mice in each group, namely, sham operation group, model group, model + composition 4 (the mass ratio of cinobucina toxin base to esterobacillus genin was 1:10) 3 mg / kg group, model + composition 11 (the mass ratio of cinobucina toxin base to esterobacillus genin was 1:2) 3 mg / kg group, model + composition 7 (the mass ratio of cinobucina toxin base to esterobacillus genin was 1:1) 3 mg / kg group, model + composition 8 (the mass ratio of cinobucina toxin base to esterobacillus genin was 5:1) 3 mg / kg group and model + composition 9 (the mass ratio of cinobucina to esterobacillus genin was 10:1) 3 mg / kg group. The establishment method of the model group rats and the sham operation group rat models was the same as that in Example 7. The drugs were administered on the 11th day after the model establishment by 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, 2, 4 and 6 h after administration. The test method was the same as that 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 among different groups were tested by Two-way test. P < 0.05 was considered to be statistically significant.

[0124] Effects of oral administration of cinobucoid venom and esterbufagin on mechanical allodynia in rats with bone cancer pain model Fig.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 group 4 was significantly increased 1-6h after administration (P<0.05), the avoidance force of composition groups 11, 7 and 8 was significantly increased 2h after administration (P<0.05), and there was no significant difference in avoidance force after administration of composition group 9 (P>0.05). The above results show that when the mass ratio of cinobucoid venom toxin base to ester toxin ligin is 0.1-5, it can significantly increase the mechanical avoidance force of rats with bone cancer pain and relieve bone cancer pain.

[0125] Embodiment 14

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

[0127] (1) Grouping and administration: Forty-two female SD mice were randomly divided into 7 groups, with 6 mice in each group, namely, sham operation group, model group, model + composition 4 (the mass ratio of cinobucina toxin base to esterobacillus genin was 1:10) 3 mg / kg group, model + composition 11 (the mass ratio of cinobucina toxin base to esterobacillus genin was 1:2) 3 mg / kg group, model + composition 7 (the mass ratio of cinobucina toxin base to esterobacillus genin was 1:1) 3 mg / kg group, model + composition 8 (the mass ratio of cinobucina toxin base to esterobacillus genin was 5:1) 3 mg / kg group and model + composition 9 (the mass ratio of cinobucina to esterobacillus genin was 10:1) 3 mg / kg group. The establishment method of the model group rats and the sham operation group rat models is the same as that in Example 7. The drug administration started on the 11th day after the model establishment and continued for 11 days. The drug administration method was gavage. The normal group and the model group were given the matrix solution, and the drug administration group was given the corresponding drug (dissolved in the matrix 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 testing method was the same as that 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 among different groups were tested by Two-way test. P < 0.05 was considered to be statistically significant.

[0129] Effects of continuous oral administration of cinobufogenin and bisbufogenin on mechanical allodynia in rats with bone cancer pain model Fig.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 composition 4 group increased significantly from 11 to 21 days after administration (P<0.001), the avoidance force of composition 11 group increased significantly from 11 to 14 days after administration (P<0.05), the avoidance force of composition 7 group increased significantly from 11 to 14 days after administration (P<0.05), the avoidance force of composition 8 group increased significantly from 11 to 14 days after administration (P<0.05), and there was no significant difference in avoidance force after administration of composition 9 group (P>0.05). The above results show that when the mass ratio of cinobucini to ester tofu venom is 0.1-5, it can significantly increase the mechanical avoidance force of rats with bone cancer pain and relieve bone cancer pain.

[0130] Embodiment 15

[0131] Effects of continuous intragastric administration of cinobufogenin 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, the grouping and the 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 among different groups were tested by Two-way test. P < 0.05 was considered statistically significant.

[0134] The results of the continuous oral administration of cinobufogenin and bisbufogenin on the spontaneous pain response of rats with bone cancer pain model are as follows Fig.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 cinobucini to ester tofu venom 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] Embodiment 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 meloxicam (2.5 mg / kg) and lidocaine (2 mg / kg) were used for analgesia. 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 micro-injector was used to inject tumor cells into the tibial bone marrow cavity. The micro-injectors of the model group and the drug-treated group in turn inhaled 2 μL of gelatin sponge aqueous solution, 1 μL of air, and 5 μL of tumor cells (5×10 5 The sham operation group was treated with 5 μL HBSS without tumor cells as a control.

[0138] (2) Grouping and administration: 40 SD female rats were randomly divided into 5 groups, 8 rats in each group, sham operation group, model group, model + bispyribacin 3 mg / kg (ig) group, model + morphine hydrochloride 3 mg / kg (ig) group and model + morphine hydrochloride 3 mg / kg (ip) group. The bone cancer pain model was established in the model group rats, and the drug was 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 administration group was administered with the corresponding drug (dissolved in the matrix solution). The mechanical paw withdrawal force (g) of the rats was tested 1, 2, 4 and 6 hours after the administration. The method for determining the mechanical paw withdrawal force was the same as that 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 by Two-way test. P < 0.05 was considered to be statistically significant.

[0140] Effects of bispyribacin and morphine hydrochloride on mechanical allodynia in rats with bone cancer pain model Fig.16 As shown. The mechanical avoidance force of rats was significantly reduced after tumor cell inoculation. 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 esterbufagin genin was significantly increased 1-6h after intragastric administration (P<0.001), and the avoidance force of the morphine group was significantly increased 1-2h after intraperitoneal administration (P<0.05), but there was no significant difference in avoidance force after intragastric administration (P>0.05). The above results show that under the same administration method and the same dosage, esterbufagin genin has a stronger analgesic effect than morphine hydrochloride.

[0141] Embodiment 17

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

[0143] (1) Pre-test period (Day 1): the preliminary test phase of the basic value.

[0144] The mice were placed in the middle of 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 not between 40% and 60%) and mice with less than 20 shuttle times were eliminated according to the baseline value.

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

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

[0147] Every morning, the saline group was injected with saline intraperitoneally and placed in the medicine-accompanying box, while the drug group was injected with the corresponding drug (dissolved in the matrix solution) and placed in the non-drug-accompanying box. Every afternoon, the two groups exchanged boxes for training, that is, the saline group was injected with saline intraperitoneally and placed in the non-drug-accompanying box, while the drug group was injected with the corresponding drug (dissolved in the matrix solution) and placed in the medicine-accompanying box. After the injection, they were placed in the box and stayed for 45 minutes. The training lasted for 5 consecutive days.

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

[0149] Open the partition, place the mouse in the middle and allow it to freely enter the two chambers, and record the time the mouse stays in each chamber within 15 minutes. The result score is expressed as the preference rate of the companion medicine box, that is, the preference rate of the companion medicine box = the companion medicine box / (companion medicine box + raw salt box) activity time.

[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 Fig.17 As shown. Compared with the pre-test stage, the time that mice in the positive control morphine dependence group stayed on the drug-accompanying box in the test stage was significantly prolonged (P<0.001), and the time that mice in the 10mg / kg and 5mg / kg groups of retibufogenin stayed on the drug-accompanying box did not change significantly (P<0.05); the time that mice in the normal group stayed on the drug-accompanying box also did not differ significantly (P<0.05). The above 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 solution of the present invention rather than to limit it. 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 solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. The use of toad venom in relieving inflammatory pain and bone cancer pain, characterized in that: The active ingredients of toad venom for relieving inflammatory pain include cinobucini, sebuffogenin and bufotoxin; The active ingredients of toad venom for relieving bone cancer pain include cinobucini and bisbufagin.

2. A toad venom active ingredient composition for relieving inflammatory pain, characterized in that: The invention relates to a composition comprising the cinobucini and bisbufagin as described in claim 1.

3. A toad venom active ingredient composition for relieving inflammatory pain according to claim 2, characterized in that: The mass ratio of cinobucini to bufotoxin is 4:5 to 5:

1.

4. A composition of toad venom active ingredients for relieving bone cancer pain, characterized in that: The invention relates to a composition comprising the cinobucini and bisbufagin as described in claim 1.

5. The composition of toad venom active ingredients for relieving bone cancer pain according to claim 4, characterized in that: The mass ratio of cinobucini to bufotoxin is 1:10 to 5:

1.

6. A use of the resibufogenin as claimed in claim 1 in the preparation of analgesic drugs, characterized in that: Resifugin is non-addictive.

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