阅读说明：本技术 二至方在制备防治便秘的药物中的用途 (Application of two formulas in preparation of medicine for preventing and treating constipation ) 是由 高秀梅 毛浩萍 刘二伟 吕彬 赵鑫 周巍 陈璐 蔡明琪 于 2021-02-09 设计创作，主要内容包括：本发明属于中医药领域,涉及二至方在制备防治便秘的药物中的用途。具体地,本发明涉及选自如下的(1)-(3)项中的任一项在制备治疗或预防便秘的药物中的用途：(1)中药组合物,包括女贞子和墨旱莲；(2)提取物,其为第(1)项中的中药组合物的水提物和/或乙醇溶液提取物；(3)药物制剂,其包含第(2)项中的提取物,以及一种或多种药学上可接受的辅料；优选地,所述药物制剂为二至丸。二至方或其提取物能够有效地治疗和/或预防便秘的药物；有效地抑制慢传输型便秘或阿片类药物引起的便秘。(The invention belongs to the field of traditional Chinese medicines, and relates to application of two formulas in preparation of a medicine for preventing and treating constipation. Specifically, the present invention relates to the use of any one of the following items (1) to (3) for the preparation of a medicament for treating or preventing constipation: (1) a Chinese medicinal composition comprising fructus Ligustri Lucidi and Ecliptae herba; (2) an extract which is an aqueous extract and/or an ethanol solution extract of the Chinese medicinal composition of item (1); (3) a pharmaceutical formulation comprising the extract of item (2), and one or more pharmaceutically acceptable excipients; preferably, the pharmaceutical formulation is a two to one pill. A drug capable of effectively treating and/or preventing constipation; effectively inhibit slow transit constipation or opioid-induced constipation.)

1. Use of any one of items (1) to (3) selected from the following in the preparation of a medicament for treating or preventing constipation:

(1) a Chinese medicinal composition comprising fructus Ligustri Lucidi and Ecliptae herba;

(2) an extract which is an aqueous extract and/or an ethanol solution extract of the Chinese medicinal composition of item (1);

(3) a pharmaceutical formulation comprising the extract of item (2), and one or more pharmaceutically acceptable excipients; preferably, the pharmaceutical formulation is a pill (e.g., a pellet), a granule, an oral liquid, a tablet or a capsule.

2. Use according to claim 1, wherein the constipation is slow transit constipation, such as opioid induced constipation.

3. The use according to any one of claims 1 to 2, wherein,

the traditional Chinese medicine composition comprises: 0.5 to 1.5 weight portions of glossy privet fruit and 0.5 to 1.5 weight portions of yerbadetajo herb;

preferably, the Chinese medicinal composition comprises: 0.8 to 1.2 weight portions of glossy privet fruit and 0.8 to 1.2 weight portions of yerbadetajo herb;

preferably, the Chinese medicinal composition comprises: 1 part of glossy privet fruit and 1 part of yerbadetajo herb;

preferably, the traditional Chinese medicine composition consists of glossy privet fruit and yerbadetajo herb;

preferably, the traditional Chinese medicine composition is prepared from two formulas;

preferably, the glossy privet fruit and the eclipta are medicinal materials or medicinal powder; preferably, the average particle size of the medicinal powder is 100-250 meshes.

4. The use according to any one of claims 1 to 3, wherein, in item (2), the extract is produced by a production method comprising the steps of:

1) reflux-extracting the Chinese medicinal composition of item (1) with 50-95% (v/v) ethanol solution for 1 or more times to obtain Chinese medicinal composition extractive solution; or reflux-extracting the raw materials in the Chinese medicinal composition of item (1) with 50-95% (v/v) ethanol solution for 1 or more times, and mixing extractive solutions of the raw materials to obtain mixed extractive solution;

2) concentrating the Chinese medicinal composition extractive solution or mixed extractive solutions to obtain concentrate;

3) the concentrate is lyophilized to obtain a lyophilized product, i.e., an extract.

5. Use according to claim 4, characterized by any one or any more of items A-C as follows:

A. in step 1), the concentration of the ethanol solution is independently 50% -85% (v/v), 55% -80% (v/v), 55% -75% (v/v), 55% -65% (v/v), or 60% (v/v);

B. in step 1), the amount of the ethanol solution is independently 3-15 times, 5-12 times or 5-10 times of the volume;

C. in step 1), the extraction time is independently 0.5-5 hours, 0.5-3 hours, or 0.5-2 hours.

6. The use according to any one of claims 1 to 5, wherein the unit dose of item (1), item (2) or item (3) is 3 to 15g, preferably 6 to 12g, particularly preferably 8 to 10g, such as 9g, calculated on the weight of the Chinese medicinal composition.

7. The use according to any one of claims 1 to 6, wherein the amount of the drug to be administered in accordance with item (1), item (2) or item (3) is 3 to 15 g/human, preferably 6 to 12 g/human, particularly preferably 8 to 10 g/human, such as 9 g/human, calculated on the weight of the Chinese medicinal composition.

8. The use according to any one of claims 1 to 7, wherein the route of administration according to item (1), item (2) or item (3) is oral administration.

9. The use according to any one of claims 1 to 8, wherein in item (3), the pharmaceutical formulation is an oral formulation.

10. The use according to any one of claims 1 to 9, wherein in item (3), the pharmaceutical formulation is a tablet, capsule, pill or granule.

Technical Field

The invention belongs to the field of traditional Chinese medicines, and relates to application of two formulas in preparation of a medicine for preventing and treating constipation. The invention also relates to a second-component extract.

Background

The main manifestations of constipation are a decrease in the number of bowel movements and difficulty in defecation, with many patients having bowel movements less than 3 times a week, and in the severe cases, up to 2-4 weeks. Some patients may be marked by difficulty in defecation, which may be as long as 30 minutes or more, or several times a day, but difficult to defecate and in small numbers. In addition, there are cases of abdominal distension, anorexia, and abdominal pain before defecation due to improper administration of laxatives.

Slow Transit Constipation (STC) is mainly manifested by decreased defecation frequency, difficulty in defecation and decreased or eliminated defecation tendency, and in recent years, the disease is highly developed and is more ill in China. At present, the pathogenesis of STC is not clear, and the study on the pathophysiology of STC in academic circles is not deep enough, so that the quantification and treatment of constipation are not unified, and laxatives and gastrointestinal motility promoting medicines are mainly adopted for clinical treatment.

According to the WHO three-step treatment principle of cancer pain, opioid analgesics are the first choice for treating moderate and severe cancer pain. Domestic reports suggest that opioid analgesics induce constipation with a rate of up to 90% -100% and persist throughout the course of analgesic treatment. With the prolonged taking of opioid, constipation is aggravated and gastrointestinal side effects such as nausea, vomiting, abdominal distention and pain are accompanied, so many patients stop the drug therapy once opioid constipation occurs. Opioid-induced constipation (OIC) is a slow transit constipation; the gastrointestinal side effects such as constipation caused by opioid drugs have become an important problem to be solved urgently in the process of treating cancer.

The second prescription is prepared by mixing glossy privet fruit and yerbadetajo herb in quality, wherein the glossy privet fruit is used as the medicine and collected in winter solstice, and the yerbadetajo herb is used as the medicine and collected in summer solstice; made into pills called Erzhi pills. Erzhi pill is a domestic medicine produced by enterprises such as the Leren Tang pharmaceutical factory, a member of Tianjin pharmaceutical industry group. It is used for tonifying liver and kidney, nourishing yin, and stopping bleeding. Can be used for treating deficiency of liver-yin and kidney-yin, dizziness, tinnitus, dry throat and nose, soreness of waist and knees, and menorrhagia. The pharmacological actions of the two formulas mainly comprise an immunoregulation action, an antioxidation and anti-aging action, a liver protection action, an anti-osteoporosis action, an anti-inflammatory action, an anti-tumor action, a hormone-like action and the like, and the application in the aspect of constipation is not seen.

At present, there is still a need to develop new drugs for preventing and treating constipation, particularly drugs suitable for slow-transit constipation or opioid-induced constipation.

Disclosure of Invention

Through intensive research and creative work, the inventor surprisingly finds that the combination of the glossy privet fruit and the eclipta can effectively prevent and treat constipation, particularly slow-transit constipation. The following invention is thus provided:

one aspect of the present invention relates to use of any one selected from the following items (1) to (3) for the preparation of a medicament for treating or preventing constipation:

(1) a Chinese medicinal composition comprising fructus Ligustri Lucidi and Ecliptae herba;

(2) an extract which is an aqueous extract and/or an ethanol solution extract of the Chinese medicinal composition of item (1);

(3) a pharmaceutical formulation comprising the extract of item (2), and one or more pharmaceutically acceptable excipients; preferably, the pharmaceutical formulation is a pill (e.g., a pellet), a granule, an oral liquid, a tablet or a capsule.

In some embodiments of the invention, the use, wherein the constipation is slow transit constipation, such as opioid-induced constipation.

In some embodiments of the invention, the use, wherein,

the traditional Chinese medicine composition comprises: 0.5 to 1.5 weight portions of glossy privet fruit and 0.5 to 1.5 weight portions of yerbadetajo herb;

preferably, the Chinese medicinal composition comprises: 0.8 to 1.2 weight portions of glossy privet fruit and 0.8 to 1.2 weight portions of yerbadetajo herb;

preferably, the Chinese medicinal composition comprises: 1 part of glossy privet fruit and 1 part of yerbadetajo herb;

preferably, the traditional Chinese medicine composition consists of glossy privet fruit and yerbadetajo herb;

preferably, the traditional Chinese medicine composition is prepared from two formulas;

preferably, the glossy privet fruit and the eclipta are medicinal materials or medicinal powder; preferably, the average particle size of the medicinal powder is 100-250 meshes. The medicinal material can be crude drugs or decoction pieces.

In some embodiments of the invention, the use, wherein, in item (2), the extract is prepared by a preparation method comprising the following steps:

1) reflux-extracting the Chinese medicinal composition of item (1) with 50-95% (v/v) ethanol solution for 1 or more times to obtain Chinese medicinal composition extractive solution; or reflux-extracting the raw materials in the Chinese medicinal composition of item (1) with 50-95% (v/v) ethanol solution for 1 or more times, and mixing extractive solutions of the raw materials to obtain mixed extractive solution;

2) concentrating the Chinese medicinal composition extractive solution or mixed extractive solutions to obtain concentrate;

3) the concentrate is lyophilized to obtain a lyophilized product, i.e., an extract.

In some embodiments of the invention, the use is characterized by any one or more of items a-C as follows:

A. in step 1), the concentration of the ethanol solution is independently 50% -85% (v/v), 55% -80% (v/v), 55% -75% (v/v), 55% -65% (v/v), or 60% (v/v);

B. in step 1), the amount of the ethanol solution is independently 3-15 times, 5-12 times or 5-10 times of the volume;

C. in step 1), the extraction time is independently 0.5-5 hours, 0.5-3 hours, or 0.5-2 hours.

A, B and/or C, wherein the term "independently" refers to the case where the composition of item (1) is subjected to reflux extraction as a whole or the case where each of the ingredients in the composition of item (1) is subjected to reflux extraction separately, and the two cases are independent of each other.

In some embodiments of the invention, the use is described, wherein the unit dose of item (1), item (2) or item (3) is 3 to 15g, preferably 6 to 12g, particularly preferably 8 to 10g, such as 9g, calculated on the weight of the Chinese medicinal composition.

In some embodiments of the invention, the use is described, wherein the administration dose of item (1), item (2) or item (3) is 3 to 15 g/human, preferably 6 to 12 g/human, particularly preferably 8 to 10 g/human, such as 9 g/human, calculated on the weight of the Chinese medicinal composition;

preferably, the route of administration according to item (1), item (2) or item (3) is oral administration.

In some embodiments of the invention, the use, wherein, in item (3), the pharmaceutical preparation is an oral preparation, such as a tablet, a capsule, a pill or a granule.

In the present invention, the term "two-to formula" means a formulation composed of fructus ligustri lucidi and eclipta (commonly known as eclipta alba), and the term "two-to pill" means a formulation obtained by industrial production, if not otherwise specified.

Generally, the pharmaceutical preparation of the present invention contains 0.1 to 90% by weight of the Chinese medicinal composition or the extract thereof as a main ingredient. The pharmaceutical formulations may be prepared according to methods known in the art. For this purpose, the principal agent may, if desired, be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants, in a suitable administration form or dosage form for human use.

The term "excipient" as used herein refers to an excipient or vehicle for administering a principal agent, including, but not limited to, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, coating materials, and the like. Adjuvants are generally described in "Remington's Pharmaceutical Sciences" by e.w. martin. Examples of adjuvants include, but are not limited to, aluminum monostearate, aluminum stearate, carboxymethylcellulose, sodium carboxymethylcellulose, crospovidone, glyceryl isostearate, glyceryl monostearate, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxystearyl hydroxystearate, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose monohydrate, magnesium stearate, mannitol, microcrystalline cellulose, and the like.

The second-party or second-party extract may be formulated into pharmaceutical preparations, including dosage forms suitable for oral administration, dosage forms suitable for parenteral injection (e.g., intravenous injection, subcutaneous injection) (e.g., as solutions), dosage forms suitable for topical administration (e.g., as ointments, patches or creams), and dosage forms suitable for rectal administration (e.g., as suppositories), and the like.

The term "effective amount" refers to a dose that achieves treatment, prevention, alleviation and/or alleviation of a disease or disorder described herein in a subject.

The term "continuous administration" means that daily administration is effected over a period of time, and the number of times per day of administration is not particularly limited, for example, 1 time, 2 times, 3 times or more.

In the present invention, the concentration of the ethanol solution is a volume percent concentration (v/v%) if not specifically stated.

In the present invention, the weight of the drug administered to human or mouse is the total weight of the glossy privet fruit and the eclipta, unless otherwise specified. In the case where the dose administered is per person (/ human), this is meant for adults, preferably adults having a body weight of 60 to 70 kg.

Advantageous effects of the invention

A drug capable of effectively treating and/or preventing constipation; effectively inhibit slow transit constipation or opioid-induced constipation.

Drawings

FIG. 1: two-fold effect on fecal pellet count after constipation in mice caused by loperamide hydrochloride.

FIG. 2: two-fold effect on wet stool weight of mice after constipation caused by loperamide hydrochloride

FIG. 3: two-fold effect on stool dry weight after constipation in mice caused by loperamide hydrochloride

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: preparation of the two-component extract (1)

Preparing a glossy privet fruit extract: taking 1kg of fructus Ligustri Lucidi (provided by Hebei Anshun Quanlong medicinal company), adding 10 times of 80% ethanol, heating to boil, reflux extracting for 2 times (2 hr for each time), mixing extractive solutions, concentrating to concentrated solution, and vacuum drying at 50 deg.C to obtain fructus Ligustri Lucidi extract 254g, with the paste yield calculated to be 25.4%.

Preparing the eclipta extract: taking 1kg of herba Ecliptae (provided by Hebei Anshun Quanlong medicinal materials Co., Ltd.), adding 10 times of drinking water, heating to boil, reflux-extracting for 2 times (2 h each time), mixing the extractive solutions, concentrating and drying the extractive solution to obtain 232g of herba Ecliptae extract, and calculating the extract yield to be 23.2%.

Mixing the glossy privet fruit extract and the eclipta alba extract according to a crude drug mass ratio of 1:1, mixing to obtain the extract of the second formula.

Example 2: preparation of the second best extract (2)

The crude drugs of the glossy privet fruit and the yerbadetajo herb used in the second and third formulas are purchased from Beijing Tongrentang Tianjin south-Kaihai pharmacy and are identified to meet the standard of Chinese pharmacopoeia.

Preparing a glossy privet fruit extract: soaking 500g fructus Ligustri Lucidi in 5L 60% (volume percentage concentration, the same below) ethanol for 0.5h, heating and reflux extracting for 2h, and filtering to obtain decoction; adding 5L 60% ethanol, extracting for 2 hr, filtering, and mixing the decoctions; concentrating decoction by using a rotary evaporator, and then performing freeze-drying for 12h to obtain 120.0g of glossy privet fruit extract, wherein the extraction rate is 24% by calculation.

Preparing the eclipta extract: soaking 500g Ecliptae herba in 5L 60% (volume percentage concentration, the same below) ethanol for 0.5h, heating and reflux extracting for 2h, and filtering to obtain decoction; adding 5L 60% ethanol, extracting for 2 hr, filtering, and mixing the decoctions; concentrating the decoction by using a rotary evaporator, and then drying by cooling and freeze-drying for 12h to obtain 118.5g of eclipta extract, wherein the extraction rate is calculated to be 23.7%.

Mixing the glossy privet fruit extract and the eclipta alba extract according to a crude drug mass ratio of 1:1, mixing to obtain the extract of the second formula.

Example 3: research on effect of extract of second-generation formula on promoting intestinal tissue contraction

1. Test sample and test Material

Sample preparation: example 2 a ligustrum lucidum extract and an eclipta extract are prepared as a two-component extract to formulate a two-component suspension. The specific configuration steps are as follows:

first, a 28mg/mL dose group suspension was prepared: adding 336mg fructus Ligustri Lucidi extract and 331.8mg Ecliptae herba extract into 10ml pure water, mixing well, storing at 4 deg.C, shaking well before use, adding 1ml medicinal liquid into 9ml k-H liquid to make the concentration of the medicinal liquid in bath be 28 mg/ml. Then diluting the mixture according to the ratio of 1:1 in sequence to obtain other concentrations.

Preparation of isolated intestinal muscle: healthy male guinea pigs are taken, 180 g and 200g are taken, fasting is carried out for 24h before the experiment, water is freely drunk in the period, the abdominal cavity is immediately cut open after the cervical dislocation, the stomach and the whole intestine (the part from the cardia of the stomach to the anus of the rectum) are taken out, and the mesentery and the connected abdominal fat are preliminarily separated. Taking small intestine of guinea pig 8-10cm from esophagus downwards, taking colon of guinea pig about 8-10cm from anus upwards, separating surface blood vessel and mesentery, and cutting into 1-2cm intestine for use. Using a mixture of 95% O₂+5％CO₂Introducing mixed gas into low temperature K-H solution (Kreb-Henseleit solution) for 0.5 hr to flush intestinal cavity contents and intestinal fluid, and storing in K-H solution.

2. Experimental methods

The two ends of the intestinal canal are knotted by silk threads, one end is tied on a fixed hook, and the other end is tied on a tension transducer connected with a signal acquisition system. Keeping the temperature in the water bath at 37 + -0.5 deg.C, immersing the intestinal segment in the bath containing 10ml of K-H solution, and continuing to aerate properly to make the bubbles in continuous linear shape or fisheye shape.

Setting the initial tension of the isolated intestinal segment to be 1g, balancing for 0.5h, recording the normal activity curve of the colon (or small intestine) for 5min, adding experimental medicines, and observing and recording the change condition of the colon (or small intestine) contraction curve.

2.1 Effect of the two-fold extract on Ex vivo intestinal muscle (small intestine and colon, respectively) Activity

When the guinea pig isolated intestine section is stable for 0.5H in a tissue bath tank, after regular contraction is generated, recording a 5min normal activity curve, then respectively adding two-to-one suspension with different dosages into each bath tank to make the final concentration of the drug in the bath tank respectively be 3.5mg/mL, 7mg/mL, 14mg/mL and 28mg/mL (3.5mg/mL is that the extract in each mL of K-H liquid in the bath tank is 1.75mg/mL of glossy privet fruit crude drug and 1.75mg/mL of eclipta crude drug according to the mass of the crude drug, and the other concentrations are analogized), observing for 5min after adding the drug, and recording the change condition of the average contractility after the drug acts.

2.2 Effect of the two-fold extract on the Activity of atropine-relaxed guinea pig Ex vivo intestinal muscles (small intestine and colon, respectively)

Atropine is an M-cholinergic receptor antagonist, has the effects of remarkably relaxing intestinal muscles and inhibiting contraction, and can be used for simulating the intestinal muscle movement state under the constipation condition. So that atropine is added into the bath tank of each group except the normal control group which is added with pure water with the same volume, so that the final concentration of the atropine in the bath tank is 10^-4M, when the intestinal contraction is remarkably inhibited and stable for 3-5min, adding two-component suspensions with different doses into each component except for an atropine component, adding pure water with the same volume into each component, so that the final concentration of the drug in a tank is 3.5mg/mL, 7.0mg/mL, 14.0mg/mL and 28.0 mg/mL (the final concentration of the extract in K-H liquid per mL in the bath tank is 1.75mg/mL of glossy privet fruit crude drug and 1.75mg/mL of eclipta crude drug according to the mass of the crude drugs, and the like) and observing for 5min after adding the drug, and recording the change condition of the average contractility after the drug acts.

3. Results of the experiment

3.1.1 Effect of the two-fold extract on the Ex vivo intestinal motility of Guinea pigs

As shown in table 1 below.

Table 1: effect of the two-component extract on the Ex vivo Small intestine Activity of Guinea pig (n ═ 4)

P <0.01 compared to before administration of the two-way dose.

Table 1 shows that the two-way extract has obvious contractile promotion effect on the guinea pig ex vivo small intestine and has certain dose correlation. The intestinal contractility increases in a short time after administration, but the drug action time is short, and the contractility gradually decreases with time. Compared with the extract before administration, the intestinal contractility of the two-component extract is remarkably increased after administration, and the contractility is highest (P <0.01 or P <0.05) 1min after administration, and then gradually decreased within 3min and 5min after administration with time.

3.1.2 Effect of the two-fold extract on the Ex vivo Colon Activity in Guinea pigs

As shown in table 2 below.

Table 2: effect of the two-component extract on the Ex vivo Colon Activity in Guinea pigs (n ═ 8)

P <0.01 compared to before administration of the two-way dose.

Table 2 shows that the two-component extract has a significant promoting effect on spontaneous shrinkage of isolated colon of guinea pig, which is represented by a significant increase in contractility after administration, and the increase in contractility is more significant with the increase in the amount of the two-component formulation. Compared with the extract before administration, the intestinal contractility of the two-component extract is increased significantly (P <0.01 or P <0.05) after administration, and the change of the contractility is most significant (P <0.01 or P <0.05) at 1min after administration, and then the contractility is gradually decreased with the time.

The results in tables 1 and 2 demonstrate that the two-way formulation has an effect of directly promoting the contraction of the small intestine and colon, suggesting that the anti-constipation effect of the two-way formulation is broad-spectrum in nature.

3.2.1 Effect of the two-fold extract on the Exotol Small bowel Activity of atropine-dosed Guinea pigs

As shown in table 3 below.

Table 3: effect of the two-part extract on the Exotoxin-administered guinea pig in vitro intestinal motility (n ═ 6)

P <0.01, compared to before administration of the two-way formulation; compared to after atropine administration, # p < 0.01.

Table 3 shows that atropine (10)^-4M) can obviously inhibit the spontaneous activity of the detached small intestine of the guinea pig (P is less than 0.01 or P is less than 0.05), and compared with the atropine before administration, the atropine can reduce the intestinal contractility by about 1/2; compared with the atropine and pure water group, the two-component extract group with different dosages can obviously reverse the relaxation of the atropine to the isolated small intestine of the guinea pig. Significant recovery of intestinal contractility (P <0.01 or P <0.05) was observed at 1min, 3min, and 5min after administration, and was higher than the initial contractility before administration.

3.2.2 Effect of two-fold extract on Activity of atropine-relaxing guinea pig in vitro Colon

As shown in table 4 below.

Table 4: effect of the two-part extract on Ex vivo Colon movements in atropine-dosed guinea pigs (n ═ 8)

P <0.01, compared to before administration of the two-way formulation; compared to after atropine administration, # p < 0.01.

Table 4 shows that atropine (10)^-4M) can obviously inhibit the spontaneous activity of the isolated colon of the guinea pig (P is less than 0.01 or P is less than 0.05) after the administration, compared with the control group and before the administration, the atropine can obviously reduce the intestinal tension, which is about 50 percent of the normal state; compared with the atropine model group, the two-component extract group with different dosages can obviously reverse the relaxation of the atropine to the isolated colon of the guinea pig, and the intestinal contractility can be obviously recovered 1min, 3min and 5min after the administration (P is less than 0.01). And the antagonism of the extract of the second-component formula on atropine has certain dose correlation.

The results in tables 3 and 4 demonstrate that the two-way formulation has antagonistic action on atropine-induced intestinal muscle relaxation, suggesting that the two-way formulation is associated with M receptors.

Example 4: clinical study of Erzhi pill for constipation

1. Test sample and test object

Second, pill: orchan hall pharmaceutical factory, a new pharmaceutical industry group member of Tianjin (specification of 1.7g per 10 capsules), comprises the following components: glossy privet fruit and eclipta alba (the mass ratio of the two medicinal materials is 1: 1). The preparation process is as follows (standard number: WS 3-B-2457-97):

prescription: 500g of glossy privet fruit (steamed) and 500g of yerbadetajo herb.

The preparation method comprises the following steps: taking 300g of glossy privet fruit, and crushing into fine powder; decocting the rest fructus Ligustri Lucidi and Ecliptae herba in water twice, each for 2 hr, mixing decoctions, filtering, concentrating the filtrate to obtain soft extract with relative density of 1.30, adding fructus Ligustri Lucidi powder, mixing, drying, pulverizing into fine powder, sieving (leaving appropriate amount of concentrated extract powder as coating), making into pill with water, drying, coating with the concentrated extract powder, polishing, and drying.

Dispersing tablets of mosapride citrate: chengdu Kanghong pharmaceutical industry group GmbH (5 mg standard).

A subject: patients with slow-conducting constipation.

Subjects were screened according to the following criteria:

the diagnosis standard conforms to the diagnosis standard of slow-conductive constipation in the Roman III standard: defecation is hard or blocky when exceeding 1/4, defecation is difficult when exceeding 1/4, defecation is not clean when exceeding 1/4, anorectal blockage is caused when exceeding 1/4, defecation exceeding 1/4 needs manual assistance, fewer than 3 times of defecation are needed every week, and more than 2 items are met; symptoms appear 6 months before diagnosis, and the diagnosis standard is satisfied in the last 3 months, so that the basis for diagnosing irritable bowel syndrome is insufficient. The traditional Chinese medicine diagnosis standard refers to the standard of 'traditional Chinese medicine disease diagnosis curative effect standard': the defecation period is prolonged, or the period is not long but the feces are dry and hard to remove, or the feces are not hard and the defecation is not smooth although the feces are satisfactory; syndrome differentiation refers to qi secret, syndrome: the main symptoms are dry stool, difficulty in defecation and abdominal distention, and the secondary symptoms are chest and hypochondrium fullness, belching, hiccup, inappetence, bowel sound, flatus, difficulty in defecation, thin white or thin yellow or thin greasy tongue coating, and wiry or wiry, slow or wiry, rapid or wiry and tense pulse.

Exclusion criteria: (ii) those aged > 75 or < 18 years old; colonoscope, anorectal pressure measurement and other tests show that organic lesions exist in the intestinal tract; ③ patients with serious liver, kidney and cardiovascular diseases; fourthly, serious complication of digestive tract; patients in gestation and lactation period; sixthly, allergic constitution patients; seventhly, the abnormal spirit can not be matched with researchers; and the people who voluntarily participate in the research and do not sign an informed consent.

2. Experimental methods

2.1 methods of administration

Control group (42 persons): the mosapride citrate dispersible tablet is taken half an hour before meals, 10mg is taken each time, 3 times a day, and the treatment is continuously carried out for 2 weeks.

Treatment group (38 humans): and (4) administering the Erzhiwan pill for treatment. 20g (specification: 1.7g of the weight of each 10 grains, equivalent to 4.6g of crude drug, namely 2.3g of glossy privet fruit and 2.3g of eclipta alba; equivalent to 1g of the extract of the second or third formula in the example 2) are orally taken after meal, 2 times a day, and the patient is properly administrated for 2-4 weeks to order light and easily digestible diet, avoid pungent and thick taste, regulate emotion, pay attention to defecation habits, regularly defecate every day and drink more clear water in the early morning.

2.2 curative effect standard: the evaluation is carried out by referring to the clinical research guidelines (trial) of new Chinese medicines, and the nimodipine method is used for calculation. And (3) healing: clinical symptoms and physical signs disappear or basically disappear, the tongue picture is completely normal, and the curative effect index is more than or equal to 95 percent; the effect is shown: the clinical symptoms and physical signs are obviously improved, the tongue picture is close to normal, and the curative effect index is 70-95 percent; the method has the following advantages: the clinical symptoms and physical signs are all better, the tongue picture is basically normal, and the curative effect index is 30-70%; and (4) invalidation: the clinical symptoms, physical signs and tongue symptoms are not improved or even aggravated, and the curative effect index is less than 30 percent. The total effective rate is calculated according to the healing and significant proportion.

3. Results of the experiment

As shown in table 5 below.

Table 5: comparison of clinical effects of two groups

Group of	n	Recovery method	Show effect	Is effective	Invalidation	Total effective rate
							Treatment group	38	20	14	4	0	89.1％
Control group	42	15	14	8	5	72.3％

The results show that the total effective rate of the treatment group is 89.1 percent, and the total effective rate of the control group is 72.3 percent. The difference was statistically significant (P <0.05) for both groups of comparisons.

Example 5: in-vivo study on effect of two-component extract on chronic constipation mice

1. Experimental Material

1.1 animals

SPF male ICR (CD-1) mice, with a body mass of 18-20g, were purchased from Beijing Wittinglima laboratory animal technology Co., Ltd, license number SCXK (Jing) 2016-. Is raised in the animal experiment center of Tianjin Chinese medicine university.

1.2 chemical reagents

Glossy privet fruit and yerbadetajo herb: the second-generation extract was prepared as in example 2.

Loperamide hydrochloride: SIGMA-ALDRICH, CAS: 34552-83-5.

Gum arabic powder: beijing Solaibao science and technology, CAS: 9000-01-5.

Activated carbon powder: fochen (Tianjin) Chemicals, Inc., CAS: no: 7440-44-0.

2. Preparation of test solutions

2.1.1 preparation of loperamide suspension

Loperamide hydrochloride is dissolved in pure water according to the administration concentration and is stored in a dark place at the temperature of-4 ℃.

2.1.2 preparation of the two-part extract suspension

The suspension of the second or third party extract is prepared by mixing fructus Ligustri Lucidi and Ecliptae herba at a ratio of 1:1, and storing at-4 deg.C with pure water.

2.1.3 preparation of carbon colloidal suspensions

Mixing Arabic gum with pure water at a ratio of 10g:80ml, heating and boiling with electromagnetic oven until the solution becomes transparent, adding 5g of activated carbon powder, boiling for 3 times, cooling, diluting to 100ml, storing at 4 deg.C, and shaking before use (not suitable for long storage, and mixing as required).

3. Experimental methods

3.1 establishment of mouse model with Chronic Constipation

30 ICR (CD-1) mice were acclimatized for 1 week during which they were free to drink water. Randomly divided into 5 groups, which were:

blank group, model group, low dose group of two-way formula extract, medium dose group of two-way formula extract and high dose group of two-way formula extract.

The other 4 groups except the blank group were administered with a dose of 10mg/kg by gavage at 9 am to establish a constipation model in mice. Preparing loperamide suspension of 1mg/ml by using pure water, wherein the administration volume is 10ml/kg, 1 time/day and 8 days; the blank group is filled with pure water, and the administration volume is 10ml/kg, 1 time/day, and 8 days.

3.2 two-way extract dosing regimen

The remaining 3 groups, except the blank group, model group, were given different doses of the two-way extract suspension treatment at 12 o' clock at noon. In 2020 < pharmacopoeia of the people's republic of China >, the recommended dosage of each adult of glossy privet fruit and yerbadetajo herb is 6-12g, the equivalent dosage of a mouse is calculated according to a body surface area formula, and then the dosage of extracts of two or more different dosage groups is formulated, namely a low dosage group: 1.56g/kg, middle dose group: 3.12g/kg, high dose group: 6.24g/kg, the administration volume is 10ml/kg, 1 time/day, 8 days; the blank group and the model group are subjected to pure water intragastric administration, the administration volume is 10ml/kg, 1 time/day, and the administration lasts 8 days.

3.36 h determination of the number of particles/wet weight and dry weight of the feces

After the last administration, mice were raised in a single cage, during which free food and water intake was ensured, and feces were collected for 6 hours, and the number of pellets and wet weight were recorded. And (3) setting the temperature of the constant-temperature drying oven to be 60 ℃, drying for 4h, taking out dried excrement, weighing for the 1 st time, then continuously drying for 10min, weighing for the 2 nd time, and recording the measured weight as the dry weight of the excrement if the weight of the excrement in two continuous times is the same.

4. Results of the experiment

As shown in fig. 1-3.

The results show that the number of particles of the excrement, the wet weight and the dry weight of the excrement of the mice in the model group are obviously reduced compared with those in the control group (p < 0.01). The two-way 3.12g/kg dose group significantly increased the number of fecal pellets and the fecal weight (including wet and dry weight) of the mice compared to the model group, indicating that the two-way was effective in increasing the total fecal amount of the constipated mice.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that the invention is not limited thereto. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such modifications are intended to be within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.