阅读说明：本技术 一种金盏银盘活性成分的外用制剂及应用 (External preparation of active ingredients of calendula officinalis and application ) 是由 孟庆廷 宗剑飞 厉保秋 于 2021-02-01 设计创作，主要内容包括：本发明涉及中药及天然药物领域,具体涉及一种以鬼针聚炔苷为首的金盏银盘活性成分的外用制剂及其应用。其中金盏银盘活性成分选自鬼针聚炔苷或进一步加入金丝桃苷、大黄素、槲皮素中至少一种。作为外用制剂使用,鬼针聚炔苷或其与金丝桃苷、大黄素、槲皮素任一种、任两种或三种组合使用均具有一定的镇痛作用。本发明的外用制剂对热刺激疼痛、机械刺激疼痛、酸性物质刺激腹膜所致腹痛、炎症性疼痛、缺血性内脏痛等具有良好的镇痛效果。所述镇痛药物为减轻热刺激疼痛、机械刺激疼痛、酸性物质刺激腹膜所致腹痛、炎症性疼痛、缺血性内脏痛,且外用给药更为安全,可避免胃肠道给药的胃肠道刺激作用和静脉注射给药的潜在不良作用。(The invention relates to the field of traditional Chinese medicines and natural medicines, in particular to an external preparation containing active ingredients of a calendula silver disc and taking bidens parviflora as a first component and application thereof. Wherein the active component of herba Sidae Rhombifoliae is selected from bidens Podophyllum alkyne glycoside or further comprises at least one of hyperoside, emodin, and quercetin. The composition can be used as external preparation, and the podophyllum polyyne glycoside or its combination with one, two or three of hyperoside, emodin and quercetin has certain analgesic effect. The external preparation of the present invention has a good analgesic effect on thermal stimulation pain, mechanical stimulation pain, abdominal pain due to stimulation of the peritoneum by an acidic substance, inflammatory pain, ischemic visceral pain, and the like. The analgesic drug can relieve thermal stimulation pain, mechanical stimulation pain, abdominal pain caused by stimulation of acid substances to peritoneum, inflammatory pain and ischemic visceral pain, is safer in external administration, and can avoid gastrointestinal tract stimulation effect of gastrointestinal tract administration and potential adverse effect of intravenous injection administration.)

1. An external preparation of an active ingredient of a calendula, which is characterized in that the active ingredient of the external preparation is bidens bipinnata polyacetylene or a combination of the bidens bipinnata polyacetylene and at least one of hyperoside, emodin and quercetin.

2. The external preparation according to claim 1, wherein the active ingredient of the external preparation is a combination of bidens bipinnata polyacetylene and one of hyperoside, emodin and quercetin, and the weight part ratio of the bidens bipinnata polyacetylene to any one of the hyperoside, the emodin and the quercetin is 1 (4-12).

3. The external preparation according to claim 2, wherein the active ingredient of the external preparation is a combination of bidens bipinnata polyacetylene and one of hyperoside, emodin and quercetin, wherein the weight part ratio of bidens bipinnata polyacetylene to any one of hyperoside, emodin and quercetin is 1: 8.

4. The external preparation according to claim 1, wherein the active ingredients of the external preparation are a combination of bidens bipinnata polyacetylene glycoside and any two of hyperoside, emodin and quercetin, and the weight ratio of any two of hyperoside, emodin and quercetin is 1 (1-4).

5. The external preparation according to claim 4, wherein the weight ratio of any two of the hyperoside, emodin and quercetin in the external preparation is 1:1, and the weight ratio of the bidens bipinnata polyacetylene to any one of the hyperoside, emodin and quercetin in the external preparation is 1: 6.

6. The external preparation according to claim 1, wherein the active ingredients of the external preparation are a combination of bidens bipinnata polyacetylene glycoside and three ingredients of hyperoside, emodin and quercetin, wherein the weight ratio of the hyperoside, the emodin and the quercetin is 1:2: 1; the weight ratio of the podophyllum polyacetylene glycoside to the hyperin is 1: 4.

7. The external preparation according to any one of claims 1 to 6, wherein the external preparation is any one selected from a topical ointment, a topical cream, a topical gel, a topical emulsion, and a topical suspension.

8. The external preparation according to claim 7, wherein the external preparation is a gel, and the gel matrix of the gel is at least one selected from carbomer, sodium carboxymethylcellulose and lecithin.

9. The external preparation according to claim 7, further comprising at least one percutaneous absorption enhancer selected from the group consisting of propylene glycol, glycerin, polyethylene glycol and dimethyl sulfoxide.

10. Use of the external preparation according to claim 7 for the preparation of an analgesic drug, wherein the analgesic drug is an analgesic drug for relieving any one of thermal stimulation pain, mechanical stimulation pain, abdominal pain due to stimulation of the peritoneum with an acidic substance, inflammatory pain, and ischemic visceral pain.

Technical Field

The invention belongs to the field of traditional Chinese medicines and natural medicines, and particularly relates to an external preparation containing active ingredients of calendula and application thereof.

Background

The Marigold pan is whole plant of Bidens pilosa Linn. or Bidens biternata (Lour.) Merr. Et Sherff, also named Xianshan broom, Qianlingzhen, Bidens marigold, Bidens bipinnata, and Veronica officinalis. The product has effects of dispelling superficial evils, clearing away heat, removing toxic substance, and removing blood stasis, and can be used for treating influenza, encephalitis B, sore throat, enteritis, dysentery, jaundice, intestinal abscess, infantile convulsion, malnutritional stagnation, scabies, hemorrhoid, etc. Subsequent modern researches find that the bidens plant mainly contains compounds such as hyperoside, iso-ocannin-7-O-glucoside, ocannin, emodin, echinacoside, quercetin, polyacetylene and polyacetylene glycoside (plum courage, Jianhai Qiang, Zhang Qinghua. separation and identification of chemical components of the bidens disks. food and medicine, 2012, 14 th volume, 07: 270 page; plum, Liu, Xiong, Lizhong, Wang Shaojun, Liubo, Chengjie, Zhang faith. separation and identification of chemical components of the bidens disks. Jiangxi medicine, 2011, 10 th volume, 42 th volume, 346 page 51), has antibacterial, cardiovascular protecting, antiinflammatory, analgesic, xerophthalmia resisting, and antiaging effects, and can be used for preventing and treating common cold, appendicitis, dysentery, gastralgia, and hyperinsulinemia.

In the active ingredients of the calendula, the hyperoside, the emodin, the quercetin and the like have wide pharmacological activities, for example, the hyperoside has the effects of resisting inflammation, easing pain, resisting oxidation, protecting cardiac muscle and liver, resisting tumors and the like (Wanglimin, Wangsheng, Zhuqishuang, poplar, snow white, Liuming Yuan. the hyperoside has the effect of inhibiting tumors of tumor-bearing mice, the Heilongjiang medical science, No. 2 in 2010: 62-63 pages); emodin has antiinflammatory, antibacterial, liver protecting, anticancer, kidney protecting, purgative, pancreas protecting, diuresis, cholagogue, endotoxin reducing effects (study summary of pharmacological action of emodin at tensor. Chinese medicine declaration: 2006, 23 rd period: 12-14 pages); quercetin has various pharmacological actions such as anti-tumor, anti-oxidation, anti-infection, immunosuppression, cardiovascular protection, anti-inflammation and blood sugar regulation (Luomingxu, rhodamine, Zhao Wanhong. research on pharmacological actions of Quercetin progress. Chinese national folk medicine journal.No. 17: page 12-14 in 2014). Wherein, the ocaining has the function of resisting platelet aggregation (Wangjingwei, Zhang Yongwei, Lixinxia, Lilinlin, Wang Ye, Luxin, Wanli Feng, Mao Xinmin, research on the extraction separation and the function of resisting platelet aggregation of the ocaining, academic journal of Xinjiang medical university, 12 th stage of 2018: 1535. page 1538.).

The polyacetylene glycoside compound in the calendula silver disk is called Bipinnatolyacetylenicoside (BPC) with the chemical name of 2-beta-D-glucopyranosyl-1, 13-dihydroxy-3 (trans), 11 (trans) -tridecadiene-5, 7, 9-triyne, and belongs to the polyacetylene compound (Liujun, Chenfeihu, xuhui, Wangxiangyu, Linmeiying, Thangjian, Zhanglin, Bipino polyacetylene glycoside pair CCl₄Protective effects of induced acute liver injury in mice). The active ingredient is firstly discovered and confirmed by Wangjianpin, a university of traditional Chinese medicine, Shandong, and the like. Published in 1992 in the fast report of Chinese chemistry. In 2011, li bin and the like, which is a national academy of traditional Chinese medicine in Jiangxi, the podophyllum polyyne glycosides (bin, Liu Xin, Xiongjie, Laiyang Wen, Wang Shaojun, Liu Bo, Chenjie, Zhang faith.) are reported to be extracted and separated from the calendula silver disc, and the separation of chemical components of the calendula silver disc is carried out in 10 th stage of 2011, 51-53 pages in Jiangxi traditional Chinese medicine. 1997. The research of Wangjianpin and Liujianjin in 2012 respectively reports the in vitro inhibition effect of the podophyllum on leukemia cells and the CCl₄Protective action of induced acute liver injury in mice (the inhibitory action of Bidens pilosa extract on leukemia cells in vitro, Chinese medicinal materials 1997 5 th phase 247 and 249 page; Liujian Jun, Chengfei Huo, xu Hui, Wang Xiao Yu, Linmei English, Tangwen Jian, Zhang Tu Ling, Bidens pilosa Polyacetylenic glycoside on CCl)₄Protective effects of induced acute liver injury in mice). The preparation process for extracting and separating the podophyllum polyacetylene glycoside from the calendula silver disc generally adopts ethanol extraction (bin, Liu Xin, bear Jie, Leizian, Wang Shaojun, Liubo, Chenjie, Zhang faithful. separation of chemical components of the calendula silver disc. Jiangxi traditional Chinese medicine, No. 10: 51-53, CN201110294966.7 in 2011).

So far, a great deal of researches on the active ingredients of the bidens biternata in the industry have been reported, but the researches on different pharmacological effects of different active ingredients are not comprehensive, the researches on the external preparation of the bidens biternata and the preparation of the refined ingredients of the external preparation are less reported, and the researches on the analgesic effect are less.

Disclosure of Invention

In view of the above prior art, the objects of the present invention include:

firstly, a preparation of the active ingredients of the calendula officinalis, in particular to an external preparation of the active ingredients of the calendula officinalis, so as to reduce side effects caused by unnecessary ingredients.

The external preparation of the active ingredient of the calendula officinalis, the active ingredient is the bidens bipinnata polyacetylene or the combination of the bidens bipinnata polyacetylene and any one or two or three of hyperin, emodin and quercetin, namely the active ingredient comprises: a podophyllum polyacetylene glycoside; combination of polyrhizamide and hyperoside; a combination of podophyllum polyacetylene glycoside and emodin; combining the podophyllum polyyne glycoside and the quercetin; a combination of podophyllum polyacetylene glycoside, hyperin and emodin; combining the polygypenoside, hyperoside and quercetin; combining the podophyllum polyyne glycoside, emodin and quercetin; the composition comprises polyvidone, hyperoside, emodin, and quercetin.

Wherein when the active ingredient of the external preparation is the combination of the bidens bipinnata polyacetylene and any one of the hyperin, the emodin and the quercetin, the weight part ratio of the bidens bipinnata polyacetylene to any one of the hyperin, the emodin and the quercetin in the external preparation is 1 (4-12), and the preferable ratio is 1: 8.

When the active ingredients of the external preparation are the combination of the bidens bipinnata polyacetylene and any two of the three ingredients of the hyperoside, the emodin and the quercetin, the weight part ratio of any two of the three ingredients of the hyperoside, the emodin and the quercetin in the external preparation is 1 (1-4), and the preferable ratio is 1: 1. When the weight part ratio of any two of the three components of the hyperoside, the emodin and the quercetin in the external preparation is 1:1, the preferred weight part ratio of the bidens bipinnata polyacetylene to any one of the three components of the hyperoside, the emodin and the quercetin in the preparation is 1: 6.

Wherein, when the active ingredients of the external preparation are the combination of the bidens bipinnata polyacetylene and three ingredients of hyperoside, emodin and quercetin, the weight ratio of the hyperoside, the emodin and the quercetin in the external preparation is preferably 1:2: 1. The weight ratio of the podophyllum polyacetylene glycoside to the hyperin in the preparation is preferably 1: 4.

The active ingredients in the external preparation have analgesic effect. Therefore, the weight parts of the active ingredients in the external preparation are only preferred proportions, and the external preparation can be used in combination according to any proportion on the premise of ensuring that different active ingredients reach analgesic doses.

The external preparation of the active ingredients of the calendula officinalis can be selected from ointment, cream, gel, external emulsion, external suspension and the like, wherein the external preparation of the active ingredients of the calendula officinalis contains necessary auxiliary materials of the external preparation besides the active ingredients, and comprises at least one transdermal absorption enhancer, such as azone transdermal absorption enhancers, such as laurocapram; such as alcohol percutaneous absorption enhancer, such as propylene glycol, glycerol, polyethylene glycol, etc., such as dimethyl sulfoxide and its homologues. When the external preparation is a gel preparation, the necessary auxiliary materials also comprise a gel matrix, such as one or two of carbomer, sodium carboxymethylcellulose and lecithin. When the gel base is lecithin, the skilled person will appreciate that it should be prepared by dissolving it in an organic solvent and then adding water to make it a low viscosity liquid. When the external preparation is ointment or cream, the above necessary adjuvants further comprise at least one of glyceryl monostearate, white vaseline, stearic acid, Tween 80, sodium laurylsulfate, and ethylparaben.

The invention also aims to provide a new application of the active ingredients of the calendula officinalis, in particular to an application of the active ingredients of the calendula officinalis in preparing external analgesic drugs. Wherein the analgesic drug includes pain due to thermal stimulation, pain due to stimulation of the peritoneum by acidic substances, inflammatory pain, mechanical stimulation pain, and ischemic visceral pain. Wherein the active component of the calendula officinalis is at least selected from the group consisting of bidens bipinnata polyacetylen, and can further comprise any one or two or three of hyperin, emodin and quercetin.

Wherein, when the active component is the combination of the bidens bipinnata polyacetylene and any one of the hyperin, the emodin and the quercetin, the weight part ratio of the bidens bipinnata polyacetylene to any one of the hyperin, the emodin and the quercetin in the external preparation is 1 (4-12), and the optimal ratio is 1: 8.

Wherein, when the active ingredient is the combination of the bidens pilosa polyacetylene glycoside and any two of the hyperin, the emodin and the quercetin, the weight part ratio of any two of the hyperin, the emodin and the quercetin in the external preparation is 1 (1-4), and the optimal ratio is 1: 1. When the weight ratio of any two of the three components of the hyperoside, the emodin and the quercetin in the external preparation is 1:1, the preferred weight ratio of the bidens bipinnata polyacetylene to any one of the three components of the hyperoside, the emodin and the quercetin in the preparation is 1: 6.

Wherein, when the active ingredients are the bidens pilosa polyacetylene glycoside and three ingredients of hyperoside, emodin and quercetin, the weight ratio of the hyperoside, the emodin and the quercetin in the external preparation is 1:2: 1. The weight ratio of the podophyllum polyacetylene glycoside to the hyperin in the preparation is 1: 4.

The active ingredients in the external preparation have analgesic effect, and the weight ratio of the active ingredients is related to the dosage level range of different active ingredients. Therefore, the weight ratio of the active ingredients in the external preparation is only the preferable ratio, and the external preparation can be used in combination according to any ratio on the premise of ensuring that different active ingredients achieve analgesic dose.

The inventor finds that the bidens parviflora pall, one of the active ingredients of the bidens parviflora, has certain analgesic effect on various pain models including inflammatory pain, thermal stimulation pain, mechanical stimulation pain, cancer pain and the like when being externally applied. As the hyperoside, the emodin and the quercetin also have analgesic effect, the inventor further researches show that the combination of the bidens bipinnata polyacetylene with the hyperoside, the emodin and the quercetin has better analgesic effect. The invention also provides an external preparation of the active ingredients of the calendula officinalis, which has the advantages of convenient administration and high safety compared with oral administration or injection administration; in another aspect, the invention provides novel activity of the active ingredient bidens bipinnata propargyl in the bidens biternata, thereby providing a novel medicine for treating pain.

Detailed Description

The technical solution of the present invention will be described below with specific examples. The following specific examples are only for explaining and illustrating the technical solutions of the present invention, and do not limit the scope of the present invention.

EXAMPLE 1 preparation of an external preparation of Polypopyrin

The formula is as follows:

10g of bidens bipinnata polyacetylene;

14 g of glycerin monostearate;

20g of white vaseline;

20g of stearic acid;

20g of liquid paraffin;

28 g of glycerol;

2g of sodium dodecyl sulfate;

0.2g of ethylparaben;

96 g of distilled water.

Weighing raw and auxiliary materials according to the proportion of the formula, mixing the glyceryl monostearate, the white vaseline, the stearic acid and the liquid paraffin according to the formula amount, heating and melting to obtain an oil phase, adding the bidens bipinnata polyacetylene glycoside according to the formula amount, uniformly stirring, and keeping the temperature at 85 ℃. Mixing glycerol, sodium dodecyl sulfate, ethylparaben and distilled water, heating to dissolve to obtain water phase, and maintaining at 85 deg.C. Slowly adding the water phase into the oil phase under stirring, and stirring and cooling to obtain ointment.

EXAMPLE 2 preparation of Polyacetylenic glycoside and Hyperoside external preparations

The formula is as follows:

2g of bidens bipinnata polyacetylene;

hyperoside 16g

14 g of glycerin monostearate;

20g of white vaseline;

20g of stearic acid;

20g of liquid paraffin;

28 g of glycerol;

2g of sodium dodecyl sulfate;

0.2g of ethylparaben;

96 g of distilled water.

Weighing raw and auxiliary materials according to the proportion of the formula, mixing the glyceryl monostearate, the white vaseline, the stearic acid and the liquid paraffin according to the formula amount, heating and melting to obtain an oil phase, adding the bidens bipinnata polyacetylene glycoside and the hyperin according to the formula amount, uniformly stirring, and keeping the temperature at 85 ℃. Mixing glycerol, sodium dodecyl sulfate, ethylparaben and distilled water, heating to dissolve to obtain water phase, and maintaining at 85 deg.C. Slowly adding the water phase into the oil phase under stirring, and cooling to obtain a compound ointment of polyrhizaside/hyperoside.

EXAMPLE 3 preparation of an external preparation of Coprinus japonicus Polyacetylenic glycoside and emodin

The formula is as follows:

2g of bidens bipinnata polyacetylene;

emodin 16g

14 g of glycerin monostearate;

20g of white vaseline;

20g of stearic acid;

20g of liquid paraffin;

28 g of glycerol;

2g of sodium dodecyl sulfate;

0.2g of ethylparaben;

96 g of distilled water.

Weighing raw materials and auxiliary materials according to the proportion of the formula, mixing the glyceryl monostearate, the white vaseline, the stearic acid and the liquid paraffin according to the formula amount, heating and melting to obtain an oil phase, adding the phalloidin and the emodin according to the formula amount, uniformly stirring, and keeping the temperature at 85 ℃. Mixing glycerol, sodium dodecyl sulfate, ethylparaben and distilled water, heating to dissolve to obtain water phase, and maintaining at 85 deg.C. Slowly adding the water phase into the oil phase under stirring, and cooling to obtain a compound ointment.

Example 4 preparation of an external preparation of Polypopyridoside and Quercetin

The formula is as follows:

1g of bidens bipinnata polyacetylene;

quercetin 9 g

14 g of glycerin monostearate;

20g of white vaseline;

20g of stearic acid;

20g of liquid paraffin;

28 g of glycerol;

2g of sodium dodecyl sulfate;

0.2g of ethylparaben;

96 g of distilled water.

Weighing raw and auxiliary materials according to the proportion of the formula, mixing the glyceryl monostearate, the white vaseline, the stearic acid and the liquid paraffin according to the formula amount, heating and melting to obtain an oil phase, adding the bidens bipinnata polyacetylene glycoside and the quercetin according to the formula amount, uniformly stirring, and keeping the temperature at 85 ℃. Mixing glycerol, sodium dodecyl sulfate, ethylparaben and distilled water, heating to dissolve to obtain water phase, and maintaining at 85 deg.C. Slowly adding the water phase into the oil phase under stirring, mixing, and cooling to obtain a compound ointment of polyglycoside/quercetin.

Example 5 preparation of an external preparation of Polyacetylenic glycoside of Bidens bipinnata, Hyperoside and Quercetin

2g of bidens bipinnata polyacetylene;

12g of hyperin;

12g of quercetin;

10g of glycerin monostearate;

20g of white vaseline;

20g of stearic acid;

10g of liquid paraffin;

8g of triethanolamine;

6g of laurocapram;

0.2 of ethylparaben;

99.8 g of distilled water.

Weighing raw and auxiliary materials according to the proportion of the formula, mixing glyceryl monostearate, white vaseline, stearic acid and liquid paraffin according to the formula amount, heating and melting to obtain an oil phase, adding bidens bipinnata polyacetylene glycoside, hyperoside and quercetin according to the formula amount, uniformly stirring, and keeping the temperature at 80 ℃; mixing triethanolamine, laurocapram and ethylparaben, stirring, and keeping the temperature at 80 deg.C to obtain water phase; adding the water phase into the oil phase while stirring, and adding distilled water; stirring to completely emulsify, and cooling to obtain compound cream containing polydiyne-glycoside/hyperoside/quercetin.

EXAMPLE 6 preparation of an external preparation of Polyacetylenic glycoside, Hyperoside and emodin

2g of bidens bipinnata polyacetylene;

12g of hyperin;

12g of emodin;

10g of glycerin monostearate;

20g of white vaseline;

20g of stearic acid;

10g of liquid paraffin;

8g of triethanolamine;

6g of laurocapram;

0.2 of ethylparaben;

99.8 g of distilled water.

Weighing raw and auxiliary materials according to the proportion of the formula, mixing glyceryl monostearate, white vaseline, stearic acid and liquid paraffin according to the formula, heating and melting to obtain an oil phase, adding the polyyne-glycoside, hyperoside and emodin according to the formula, uniformly stirring, and keeping the temperature at 80 ℃; mixing triethanolamine, laurocapram and ethylparaben, stirring, and keeping the temperature at 80 deg.C to obtain water phase; adding the water phase into the oil phase while stirring, and adding distilled water; stirring to completely emulsify, and cooling to obtain compound cream containing polyynidine glycoside/hyperoside/emodin.

Example 7 preparation of an external preparation of Coprinus japonicus Polyacetylenic glycoside, emodin, Quercetin

2g of bidens bipinnata polyacetylene;

12g of emodin;

12g of quercetin;

10g of glycerin monostearate;

20g of white vaseline;

20g of stearic acid;

10g of liquid paraffin;

8g of triethanolamine;

6g of laurocapram;

0.2 of ethylparaben;

99.8 g of distilled water.

Weighing raw and auxiliary materials according to the proportion of the formula, mixing glyceryl monostearate, white vaseline, stearic acid and liquid paraffin according to the formula amount, heating and melting to obtain an oil phase, adding the podophyllum polyacetylene glycoside, emodin and quercetin according to the formula amount, uniformly stirring, and keeping the temperature at 80 ℃; mixing triethanolamine, laurocapram and ethylparaben, stirring, and keeping the temperature at 80 deg.C to obtain water phase; adding the water phase into the oil phase while stirring, and adding distilled water; stirring to completely emulsify, and cooling to obtain compound cream containing polyyne-glycoside/emodin/quercetin.

Example 7 preparation of an external preparation of Polyacetylenic glycoside of Bidens bipinnata, Hyperoside, emodin, Quercetin

2g of bidens bipinnata polyacetylene;

8g of hyperin;

emodin 16 g;

8g of quercetin;

94010 g of carbomer, namely carbomer,

50g of glycerin is added into the mixture,

tween 802 g;

70g of ethanol;

0.5g of ethylparaben;

1000g of distilled water.

Weighing raw and auxiliary materials according to the formula proportion, mixing carbomer 940, glycerol and tween 80 in the formula amount with 250ml of distilled water, uniformly stirring, and standing overnight to fully swell; mixing ethylparaben, ethanol, hyperoside, emodin and quercetin according to the prescription amount, stirring uniformly, adding into gel matrix, adding the balance of distilled water, and stirring uniformly to obtain the compound gel of bidens bipinnata polyacetylene glycoside/hyperoside/emodin/quercetin.

Example 8 Studies of analgesic Effect of Coprinus japonicus Polyacetylenic glycoside on mouse Hot plate pain model

Purpose of the experiment

Research on analgesic effect of synephrine on pain caused by simple heat stimulation (without other inflammation stimulation) by using mouse hot plate pain model

2 method

2.1 Experimental animals: healthy female Kunming mice weigh 18-22 g.

2.2 animal models and group administration

Adjusting the temperature of the water bath to 55 +/-0.5 ℃, and placing the hot plate on the water bath until the temperature reaches 55 +/-0.5 ℃. The mice were placed on a hot plate to determine the latency of the response required for the mice to contact the hot plate until they lick or jump their feet. 24 mice with response latencies above 5s and below 30s were selected according to the standard and recorded as basal response latencies.

The mice qualified for model making are randomly divided into 4 groups, namely a model group, a low (0.25mg/d) and medium (0.5mg/d) Bidens bipinnata polyacetylene glycoside group, a high dose group (1.0 mg/d) and 6 mice per group, and skin preparation is carried out on the back. Dimethyl sulfoxide (DMSO) is smeared on the skin preparation part of the model group, and the DMSO mixed solution of the etoposide is smeared on the etoposide group. Each group was administered 1 time daily for 7 consecutive days.

2.3 index and investigation

The reaction latency was determined again according to the method under item 2.2 for each group at 2h from the last administration, and recorded as post-administration reaction latency. Thermal response threshold growth value(s) = post-drug response latency-basal response latency.

2.4 statistical analysis

Group comparisons were considered significantly different using ANOVA analysis with p < 0.05.

3 results

The statistical results of the basal response latency, post-drug response latency and thermal response threshold growth values of the mice in each group are shown in Table 1. Compared with the multiple groups of ANOVA, the differences of the basal reaction latency of the mice in each group are not significant (p is more than 0.05). The reaction latency and the thermal reaction threshold value increase value of each dosage group of the bidens polyyne glucoside are obviously higher than those of a DMSO group (p is less than 0.01). Wherein the increase values of the reaction latency and the thermal reaction threshold of the Podophyllum bipinnatum polyacetylene glycoside group drug are increased in a dose-dependent manner. The reaction latency period of the high and medium dose groups of the podophyllum polyyne-glycoside is obviously higher than that of the low dose group (p is less than 0.01). The thermal response threshold value of the high and medium dose groups of the podophyllum polyyne-glycoside is obviously increased higher than that of the low dose group (p <0.05 or p < 0.01).

TABLE 1 Demosponin external use experiment the thermal response threshold(s) of mice in each group

Example 9 study of analgesic Effect of Coprinus japonicus Polyacetylenic glycoside for external use on mouse acetic acid writhing model

Purpose of the experiment

The analgesic effect of the podophyllotoxin for external use on the pain caused by the stimulation of the peritoneum by the common abdominal pain model, namely the acidic chemical substance, is researched by a mouse acetic acid writhing model. Exploring the dosage of the podophyllum polyyne glucoside with the inhibition rate of acetic acid writhing more than 50 percent.

2 method

2.1 Experimental animals: healthy Kunming mice are female and male, and have the weight of 18-22 g.

2.2 animal models and group administration

24 mice were randomly divided into 4 groups, 6 mice/group, and male and female halves, namely model group, low (0.25mg/d), medium (0.5mg/d), high dose group (1.0 mg/d), and dorsal skin preparation. Dimethyl sulfoxide (DMSO) is smeared on the skin preparation part of the model group, and the DMSO mixed solution of the etoposide is smeared on the etoposide group. Each group was administered 1 time daily for 7 consecutive days. 2h after the last administration, each group of mice was injected with 0.7% glacial acetic acid, 0.2 ml/mouse.

2.3 index and investigation

The number of writhing of each group of mice within 15min after intraperitoneal injection of glacial acetic acid was examined, and the writhing inhibition rate (%) = (number of writhing of DMSO group-number of writhing of administered group)/number of writhing of DMSO group x 100%.

2.4 statistical analysis

Group comparisons were considered significantly different using ANOVA analysis with p < 0.05.

3 results

The results of the number of writhing times and the writhing inhibition rate of each group of mice are shown in Table 2. Compared with multiple groups of ANOVA, the twisting times of each dose group of the bidens bipinnata polyacetylene glycoside are obviously lower than those of a DMSO group (p is less than 0.01), wherein the twisting times of a middle dose group are lower than those of a low dose group, the twisting inhibition rate is higher than that of the low dose group, and the difference of the two groups is not obvious (p =0.091, p = 0.065); the number of writhing was significantly lower in the high dose group than in the low dose group (p < 0.01); the twisting inhibition rate of the high-dose group reaches more than 50 percent, and is obviously higher than that of the medium (p <0.05) and low-dose groups (p < 0.01).

TABLE 2 external application of Coprinus comatus for testing the number of writhing and the inhibition rate of writhing in each group of mice

Example 10 Studies of analgesic Effect of Bidens bipinnata polyacetylene glycoside for external use on mouse formalin inflammatory pain model

Purpose of the experiment

Formalin is injected into the soles of mice to induce inflammation, and the analgesic effect of the bidens polyacetylene glycoside externally applied to the pain behaviors of the mice suffering from the plantar inflammation is researched.

2 method

2.1 Experimental animals: healthy Kunming mice, male, with a weight of 18-22 g.

2.2 animal models and group administration

24 mice were randomly divided into 4 groups, 6 mice/group, i.e., model group, low (0.25mg/d), medium (0.5mg/d), high dose (1.0 mg/d) spongylon, and dorsal skin preparation. Dimethyl sulfoxide (DMSO) is smeared on the skin preparation part of the model group, and the DMSO mixed solution of the etoposide is smeared on the etoposide group. Each group was administered 1 time daily for 7 consecutive days. 2h after the last dose, the right hind paw of each group of mice was injected subcutaneously with formalin (5% concentration, 10 μ L injection volume).

2.3 index and investigation

The time for licking or biting the right hind paw in two phases, 0-10min (first phase) and 10-45min (second phase) after formalin injection of each group of mice, namely the licking time, is examined.

2.4 statistical analysis

Group comparisons were considered significantly different using ANOVA analysis with p < 0.05.

3 results

The time results of licking or biting the right hind paw for the first and second phase time periods for each group of mice are shown in table 3. Compared with multiple groups of ANOVA, the first phase of the middle and high dose groups of the bidens bipinnata polyacetylene is remarkably shorter than that of a DMSO group (p is less than 0.05); the first phase among the three groups of the phalloidin has no significant difference in licking time (p > 0.05). The licking time of the second phase of the middle and high dose group of the phalloidin is significantly lower than that of the DMSO group (p <0.01), wherein the licking time of the second phase of the high dose group of the phalloidin is significantly lower than that of the middle and low dose group (p < 0.01).

TABLE 3 external experiments with Coprinus japonicus Polyacetylenic glycosides the first and second phase licking time(s) of mice in each group

Example 11 Studies of analgesic Effect of Coprinus japonicus polyacetylene glycoside on mouse model of mechanical stimulation pain

Purpose of the experiment

The analgesic effect of the synephrine externally applied to the simple mechanical stimulation pain (without inflammation stimulation) of the mice is researched by a mouse tail root tenderness model.

2 method

2.1 Experimental animals: healthy Kunming mice, male, with a weight of 18-22 g.

2.2 animal models and group administration

Fixing the body and tail of the mouse, and stimulating with pressure at a position 1cm away from the tail root, wherein the corresponding pressure is the threshold of mechanical stimulation pain when the mouse is pressed and painful at the tail root and is hoarse. 24 mice with a mechanical stimulation pain threshold value lower than 300g are screened and randomly divided into 4 groups and 6 groups, namely a model group, a low (0.25mg/d) and medium (0.5mg/d) picropodophyllin, a high dose group (1.0 mg/d) and back skin preparation. Dimethyl sulfoxide (DMSO) is smeared on the skin preparation part of the model group, and the DMSO mixed solution of the etoposide is smeared on the etoposide group. Each group was administered 1 time daily for 7 consecutive days. The mechanical irritation pain threshold was measured by re-fixing the body and tail 2h after the last dose.

2.3 index and investigation

Comparing the post-dose (M1) and pre-dose (M0) and post-dose (M1) mechanical pain threshold Differences (DM) for each group

2.4 statistical analysis

Group comparisons were considered significantly different using ANOVA analysis with p < 0.05.

3 results

The measurement results and statistical results of the mechanical stimulation pain threshold M0 before administration, the mechanical stimulation pain threshold M1 after administration, and the mechanical stimulation pain threshold difference DM before and after administration for each group of mice are shown in Table 4. The difference between M0 groups was not significant (p > 0.05). The etoposide high dose group M1 was significantly higher than the DMSO group and the etoposide low dose group (p < 0.01). DM in the middle and high dose groups of the podophyllum polyacetylene is obviously higher than that in the DMSO group (p < 0.01).

TABLE 4 Demosponin external application experiment mechanical stimulation pain threshold (g) of mice in each group

Example 12 Studies of analgesic Effect of Coprinus cinereus polyacetylene on rat ischemic visceral pain model

Purpose of the experiment

Ischemic visceral pain is induced by ligation of mesenteric vessels of rats, and the analgesic effect of the podophyllum-ethidium-poly-alkyne glycoside on the ischemic visceral pain is researched.

2 method

2.1 Experimental animals: healthy SD mice, males, weighing 180-200 g.

2.2 animal models and group administration

24 rats were randomly divided into 4 groups and 6 rats per group, i.e., model group, low (0.25mg/d), medium (0.5mg/d), high dose (1.0 mg/d) and dorsal skin preparation. Dimethyl sulfoxide (DMSO) is smeared on the skin preparation part of the model group, and the DMSO mixed solution of the etoposide is smeared on the etoposide group. Each group was administered 1 time daily for 7 consecutive days. 6h after the last administration of the drug, the mesentery is exposed by operation after anesthesia, 3 mesentery blood vessels are ligated to each rat, the abdominal cavity is closed, and antibiotic is injected prophylactically to avoid postoperative infection.

2.3 index and investigation

The number of times of licking the abdomen or vulva, contraction of the abdomen or stretching of the body and contraction of the whole body or flank was observed within 1h after surgery for each group of rats as a score for ischemic visceral pain.

2.4 statistical analysis

Group comparisons were considered significantly different using ANOVA analysis with p < 0.05.

3 results

The results of the scoring of ischemic visceral pain in the rats of each group are shown in Table 5. The visceral pain score in the high dose group was significantly lower than that in the DMSO group (p <0.01), with the visceral pain score in the high dose group being significantly lower than that in the low dose group (p < 0.01).

TABLE 5 external experiments with Coprin cinereus Polyacetylenic glycosides the visceral pain scores in 1h after ligation of the rats in each group

Example 13 study of analgesic Effect of Bidens bipinnata polyacetylene glycoside Compound on mouse acetic acid writhing model

Purpose of the experiment

The analgesic effect of the podophyllum polyyne-glycoside compound externally used on the common abdominal pain model, namely pain caused by the stimulation of acid chemical substances to peritoneum, is researched by using a mouse acetic acid writhing model.

2 method

2.1 Experimental animals: healthy Kunming mice are female and male, and have the weight of 18-22 g.

2.2 animal models and group administration

30 mice were randomly divided into 5 groups, 6 mice/group, and male and female halves, namely, a model group, a phalloidin/hyperoside complex group, a phalloidin/emodin complex group, and a phalloidin/quercetin complex group. The mice in each group were prepared for skin preparation on the back. Dimethyl sulfoxide (DMSO) is smeared on the skin preparation part of the model group, a DMSO mixed solution is smeared on the bidens polyacetylene glycoside group (the dosage is 0.25mg/d according to the bidens polyacetylene glycoside), a DMSO mixed solution is smeared on the bidens polyacetylene glycoside/hyperin compound group (the dosage is 0.25mg/d according to the bidens polyacetylene glycoside, the dosage is 2.00 mg/d according to the hyperin), a DMSO mixed solution is smeared on the bidens polyacetylene glycoside/emodin compound group (the dosage is 0.25mg/d according to the bidens polyacetylene glycoside, the dosage is 2.00 mg/d according to the emodin), and a DMSO mixed solution is smeared on the bidens polyacetylene glycoside/quercetin compound group (the dosage is 0.25mg/d according to the bidens polyacetylene glycoside, and the dosage is 2.00 mg/d according to the quercetin). Each group was administered 1 time daily for 7 consecutive days. 2h after the last administration, each group of mice was injected with 0.7% glacial acetic acid, 0.2 ml/mouse.

2.3 index and investigation

The number of writhing of each group of mice within 15min after intraperitoneal injection of glacial acetic acid was examined, and the writhing inhibition rate (%) = (number of writhing of DMSO group-number of writhing of administered group)/number of writhing of DMSO group x 100%.

2.4 statistical analysis

Group comparisons were considered significantly different using ANOVA analysis with p < 0.05.

3 results

The results of statistics of the number of writhing times and the writhing inhibition rate of each group of mice are shown in Table 6. The comparison among groups shows that the twisting times of the administration group are all obviously lower than those of a DMSO group (p is less than 0.01), wherein the twisting times of the bidens polyacetylene glycoside/hyperin compound group and the bidens polyacetylene glycoside/quercetin compound group are all obviously lower than those of the bidens polyacetylene glycoside group (p is less than 0.01). The twisting inhibition rate of the bidens polyacetylene glycoside/hyperin compound group and the bidens polyacetylene glycoside/quercetin compound group is higher than 50%.

TABLE 6 Compound external application experiment of Coprinus comatus alkyne glycoside for various mice writhing times and writhing inhibiting rate

Example 14 study of analgesic Effect of Bidens bipinnata polyacetylene compound on mouse Hot plate pain model

Purpose of the experiment

Research on analgesic effect of Bidens bipinnata polyacetylene compound externally used on pain (without other inflammation stimulation) caused by simple heat stimulation by using mouse hot plate pain model

2 method

2.1 Experimental animals: healthy female Kunming mice weigh 18-22 g.

2.2 animal models and group administration

Adjusting the temperature of the water bath to 55 +/-0.5 ℃, and placing the hot plate on the water bath until the temperature reaches 55 +/-0.5 ℃. The mice were placed on a hot plate to determine the latency of the response required for the mice to contact the hot plate until they lick or jump their feet. 12 mice with response latencies above 5s and below 30s were selected according to the standard and recorded as basal response latencies.

Randomly dividing the mice qualified by modeling into 2 groups, 6 mice per group, namely a model group, a phalloidin compound group (0.25mg/d of phalloidin, 1 mg/d of hyperoside, 2 mg/d of emodin and 1 mg/d of quercetin), and preparing skin on the back. The model group skin preparation part is coated with dimethyl sulfoxide (DMSO), and the bidens polyacetylene glycoside compound groups are coated with DMSO mixed solution of bidens polyacetylene glycoside compound active ingredients. Each group was administered 1 time daily for 7 consecutive days.

2.3 index and investigation

The reaction latency was determined again according to the method under item 2.2 for each group at 2h from the last administration, and recorded as post-administration reaction latency. Thermal response threshold growth value(s) = post-drug response latency-basal response latency.

2.4 statistical analysis

Group comparisons were considered significantly different using the t-test with p < 0.05.

3 results

The statistical results of the basal response latency, post-drug response latency and thermal response threshold growth values of the mice in each group are shown in Table 7. The difference of the two groups of basic reaction latencies is not significant (p is more than 0.05), and the increase values of the reaction latencies and the thermal reaction threshold values of the etoposide compound group after the drug is taken are both significantly higher than those of a DMSO group (p is less than 0.01).

TABLE 7 Deplostes-Podophyllotoxin external use experiment for mice thermal response threshold(s) of each group