Huangliang Lian Cucurbitan-type Saponin Extract and Its Use in Preparing Medicament for Treating Constipation

Abstract

The present invention belongs to the research and development field of new drugs in traditional Chinese medicine, specifically discloses the Huanglian cucurbitan-type saponin extract and its use in the preparation of medicine for treating constipation. Based on the active orientation, the present invention discovers that cucurbitan-type saponin is the subcuticular effective site of Huanglian, in which 10 kinds of cucurbitan-type tetracyclic triterpenoid saponins with 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanosta as the parent structure are included, the total content is >50%, and no iridoid components are included. Here, the compound 2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,25-dien-22-one is a newly discovered cucurbitan-type tetracyclic triterpenoid saponin. Pharmacological research shows that the cucurbitan-type saponin extract from Huanglian can promote defecation and increase the water content of feces in normal and constipated model mice. It can be used to prepare medicines for treating constipation.

Description

[Technical field]

[0001] The present invention belongs to the field of research and development of new drugs in traditional Chinese medicine, specifically to an extract of Euonymus humilis cucurbitan-type saponins and its use in preparing medicine for treating constipation. [Background technology]

[0002] Huanglian was introduced as a "Hu medicine" in the Tang Dynasty and has gradually been absorbed into the theoretical system of traditional Chinese medicine. It is an important medicine for clearing deficiency and heat in the clinical practice of traditional Chinese medicine. Its properties are cold, its taste is bitter, it belongs to the liver, stomach and large intestine meridians, and it has the effects of clearing heat and cooling blood, drying and eliminating dampness, and it is used for childhood fever, bone steaming fever, damp heat diarrhea, jaundice hematuria, swelling and pain caused by hemorrhoids, etc. Modern research has proven that Huanglian contains compounds such as iridoids, cucurbitane-type triterpenoids, phenylethanoid glycosides, and phenolic glycosides, as well as phenolic acids such as vanillic acid, cinnamic acid, and ferulic acid, and the 2020 edition of the Chinese Pharmacopoeia stipulates that the total amount of picroside I and picroside II is 9.0% or more. Modern pharmacology shows that Huanglian has the effects of hepatoprotection and choleresis, blood lipid regulation, antiasthma, anti-inflammatory and hypoglycemic effects, etc. General research suggests that these are related to the compounds of iridoids, phenylethanoid glycosides and phenolic glycosides contained in it.

[0003] Huanglian is a commonly used medicine in pediatrics. Some scholars have found in the course of long-term clinical practice that Huanglian preparations that do not need to be decoctioned have a hygroscopic effect that has not been recorded in traditional materia medica works ("Huanglian preparations that do not need to be decoctioned" Shanxi Traditional Chinese Medicine, Vol. 32, No. 2, 2016). Animal experiments have further demonstrated that the decoction of the herb Huanglian also has a hygroscopic effect ("New Hygroscopic Efficacy of Huanglian Based on the Analysis of Spectral-Effect Relationship and Basic Research on Its Substances", Master's Thesis, Shanxi University of Traditional Chinese Medicine, 2017). Through the research and analysis of spectral effects, vanillic acid has been confirmed to be one of the hygroscopic active substances of Huanglian "New Uses of Vanillic Acid in Huanglian Extract", CN106983737B). Vanillic acid is a phenolic acid compound produced by secondary metabolism in plants. It is used in the food industry as a spice, food additive, preservative, etc. and is widely found in plants such as rice, strawberry, sugar cane, mango, and wheat. Pharmacological activity tests have demonstrated that vanillic acid has antioxidant, anti-inflammatory, anti-apoptotic, and antiplatelet aggregation effects. This suggests that in addition to vanillic acid, the active ingredients of Huanglian are still needed to be further confirmed.

[0004] It has been reported in the literature that Huanglian Rhizome can increase gastrointestinal motility, protect the gastrointestinal mucosa, and relieve constipation symptoms by regulating the intestinal flora structure and metabolic product synthesis ("Study on the mechanism of action of Huanglian Rhizome in treating constipated mice based on 16S rDNA and GC-MS technology", Chinese Journal of Traditional Chinese Medicine), but the specific components of Huanglian Rhizome that act as laxatives are still unknown.

[0005] Chronic constipation is a common gastrointestinal disease or symptom in clinical practice, manifesting as decreased frequency of bowel movements, dry and hard stools, and / or difficult defecation, severely affecting the quality of life of patients. In the Rome IV diagnostic criteria published in 2016, diseases related to chronic constipation include functional constipation, opioid-induced constipation, constipation-predominant irritable bowel syndrome, and functional defecation disorder, etc. Currently, the use of laxatives to improve various symptoms of chronic constipation is the main means of treatment, mainly: (1) bulking laxatives such as methylcellulose increase the water content and volume of the feces to promote defecation; (2) osmotic laxatives such as lactulose form a hypertonic state in the intestine, absorb water, and stimulate intestinal peristalsis; (3) stimulant laxatives such as anthraquinone-containing vegetable laxatives act on the enteric nervous system to increase intestinal motility; and (4) demulcent laxatives such as glycerin lubricate the intestinal lining and soften the stool. There are many types of laxatives, and short-term use can obviously improve bowel movements, but long-term use is unsatisfactory or has the risk of carcinogenicity. For example, long-term use of anthraquinone-containing laxatives can worsen constipation and induce melanosis coli, and long-term use of phenolphthalein poses the risk of carcinogenicity. In 2021, the National Medical Products Administration suspended the manufacture, sale and use of phenolphthalein tablets and phenolphthalein lozenges in China.

[0006] With the deepening of research into the pathogenesis of chronic constipation, research into innovative drugs has been progressing with the strategy of promoting intestinal secretion and regulating the absorption and metabolism of bile acids. The former is represented by drugs such as linaclotide (trade name Ling Zeshu), a cellular guanylate cyclase C receptor (GC-C) agonist, which increases intracellular cGMP by activating intestinal epithelial cell GC-C, and further opens the cystic fibrosis transmembrane conductance regulator (CFTR) to promote the secretion of chloride ions and bicarbonate, increase the water content of the intestinal contents, and promote defecation. The latter is an apical sodium-dependent bile acid transporter (ASBT) inhibitor such as elobixibat (trade name GOOFICE), approved in Japan, which increases the flow of bile acids to the colon by inhibiting the reabsorption of bile acids by ileal ASBT, and further promotes the intestinal secretion of more water, promotes intestinal peristalsis, and improves the patient's natural defecation. Currently, China lacks innovative medicines for treating chronic constipation with independent intellectual property rights, and medicines from foreign manufacturers approved in China are expensive, increasing the burden on the country's public health and medical system. Summary of the Invention

[0007] In order to solve the problems raised in the above background art, the present invention adsorbs Huangliang Rhizome extract with macroporous resin, then separates it into various eluted components according to different polarities, and uses animal models to trace the active components in the sublaminar region based on the active orientation. Then, by combining with techniques such as chemical color identification reaction, spectroscopy, chromatography, mass spectrometry and nuclear magnetic resonance, it proves that cucurbitan-type saponins are the effective site of Huangliang Rhizome in the sublaminar region, and further proves through pharmacological experiments that Huangliang Rhizome cucurbitan-type saponin extract has the effect of treating constipation.

[0008] The present invention relates to a white powder, molecular formula C 36 H 58 O 10The present invention provides a tetracyclic triterpenoid saponin of Euonymus humilis cucurbitan type having a molecular weight of 650, a chemical name of 2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,25-dien-22-one (compound IX), which is named Euonymus humilis cucurbitan-type saponin A and has the following structural formula: [ka]

[0009] The present invention further provides an extract of cucurbitan-type saponins from Huangliang L., which contains 10 kinds of cucurbitan-type tetracyclic triterpenoid saponins with 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanosta-5-en-22-one as the parent nucleus, specifically, compound I, 25-acetoxy-2β-glucoxy-3,16-dihydroxy-9-methyl-19-norlanosta-5-en-22-one, compound II, 2β- glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanosta-5-en-22-one, compound III, 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one, compound IV, 2β-glucoxy-3,16,20,22-tetrahydroxy-9-methyl-19-norlanosta-5,24-diene, compound V, 25-acetoxy-2β-glucoxy- 3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one, compound VI, 2β-glucoxy-3,16-dihydroxy-4,4,9,14-tetramethyl-19-norlanosta-5-en-20-one, compound VII, 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5-en-22-one, compound VIII, 2β-glucoxy-3,16,20 -trihydroxy-9-methyl-19-norlanosta-5,24-dien-22-one, compound IX 2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,25-dien-22-one, and compound X 25-acetoxy-2β-glucoxy-3,16-dihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one, with the total content of the 10 compounds being >50%, and no iridoid components being contained.

[0010] The method for preparing the Euonymus chinensis cucurbitan-type saponin extract comprises: (1) the herb Huangliang Rhizome is decoctioned with water or reflux-extracted with organic solvent, the aqueous extract is directly adsorbed by macroporous adsorption resin, and the organic solvent extract is first recovered, dispersed in water, and then adsorbed by macroporous adsorption resin; (2) washing with water until colorless; (3) stripping with an organic solvent; (4) concentrating the eluate to obtain a crude extract of Euonymus chinensis cucurbitan-type saponins; (5) purifying the crude extract of Euonymus chinensis cucurbitan-type saponins by column chromatography to obtain an extract of Euonymus chinensis cucurbitan-type saponins; The herbal medicine Picrorrhiza kurroa is the rhizome of the Indian Picrorrhiza kurroa Royle ex Benth or the Tibetan Picrorrhiza scrophulariiflora Pennell, both of which belong to the Scrophulariaceae family.

[0011] The macroporous adsorption resin is any one of AB-8, D4020, D101, D860021, and HP20, the column chromatography is any one of silica gel, alumina, and ODS column chromatography, and the organic solvent is methanol or ethanol.

[0012] Through the study of extraction methods, the present invention determines the Huangliang cucurbitan-type saponin extract and its preparation process, which are mainly composed of compounds I to X. The total content of 10 compounds is >50%, and does not contain iridoid components. This preparation process can obtain the Huangliang cucurbitan-type saponin extract, the total content of the above cucurbitan-type triterpenoid saponin compounds is between 50% and 100%. The present invention establishes an HPLC content measurement method for compounds I to X.

[0013] Another object of the present invention is to provide the use of Euonymus humilis cucurbital saponin extract in the preparation of medicine for treating constipation.Pharmacological experiments show that Euonymus humilis cucurbitan saponin extract can promote defecation in normal mice and mice with constipation model, significantly increase fecal water content, and have obvious laxative effect. [Brief description of the drawings]

[0014] [Figure 1] Figure 1 shows the HPLC spectra of the ethanol extract of Coptis Rhizome and each eluate, where A is 275 nm, B is 230 nm, and C is 200 nm. [Diagram 2] FIG. 2 is an infrared spectrum of compound IX. [Diagram 3] FIG. 3 is a high-resolution mass spectrum of compound IX. [Figure 4] FIG. 4 is a 1H-NMR spectrum of compound IX. [Diagram 5] FIG. 5 is a 13C-NMR spectrum of compound IX. [Figure 6] FIG. 6 shows the major 1H, 1H-COSY and HMBC signals of compound IX. [Figure 7] FIG. 7 is a base peak chromatogram of the Euonymus humilis cucurbitan-type saponin extract, in which A is the negative ion mode and B is the positive ion mode. [Figure 8] Figure 8 is a liquid chromatogram of the Euonymus rhizoma cucurbitan-type saponin extract. [Figure 9] FIG. 9 shows the ultraviolet absorption spectra of Euonymus rhizoma cucurbitan-type tetracyclic triterpenoid saponins I to X. [Figure 10] FIG. 10 shows the structural formulas of Euonymus humilis cucurbitan-type tetracyclic triterpenoid saponins I to X. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described below clearly and completely, obviously, the described embodiments are some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without paying creative labor, fall within the protection scope of the present invention.

[0016] The instruments and equipment used in the following examples are all routine laboratory instruments and equipment unless otherwise specified, and the experimental materials used in the following examples are all routinely purchased from biochemical reagent stores unless otherwise specified.

[0017] Example 1: 2 kilograms of Tibetan Huangliang Coptis Rhizome are taken, boiled with water, and extracted three times, with 20 liters of water added each time, boiled for 1 hour, filtered, mixed with the extract, adsorbed with D101 macroporous adsorption resin, and successively eluted with water and 30% ethanol by volume, until 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (V) is no longer detected by thin-layer chromatography, and 60% ethanol eluate is collected. The eluate is removed by ethanol recovery under reduced pressure, and 151 grams of Huangliang Coptis Rhizome cucurbitan-type saponin crude extract is obtained. HPLC detection revealed that 25-acetoxy-2β-glucoxy-3,16-dihydroxy-9-methyl-19-norlanost-5-en-22-one (I), 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanost-5-en-22-one (II), 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl -19-norlanosta-5,23-(E)-dien-22-one (III), 2β-glucoxy-3,16,20,22-tetrahydroxy-9-methyl-19-norlanosta-5,24-diene (IV), 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (V), 2β- Glucoxyl-3,16-dihydroxy-4,4,9,14-tetramethyl-19-norlanost-5-en-20-one (VI), 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanost-5-en-22-one (VII), 2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanost-5, The total content of 24-dien-22-one (VIII), 2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,25-dien-22-one (IX) and 25-acetoxy-2β-glucoxy-3,16-dihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (X) was found to be 9.5%.This crude cucurbitan-type saponin extract was added to ethyl acetate for purification, and the ethyl acetate was recovered under reduced pressure to obtain 31 grams of Huanglian cucurbitan-type saponin extract, of which HPLC detection showed that the total content of cucurbitan-type tetracyclic triterpenoid saponins I to X was 51%. This saponin extract was subjected to column chromatography on silica gel, gradient elution with ethyl acetate as the mobile phase and TLC detection, the saponin portion was collected, the solvent was recovered, and 18 grams of purified Huanglian cucurbitan-type saponin extract was obtained, in which the total content of cucurbitan-type tetracyclic triterpenoid saponins I-X was 72% by HPLC detection, in which the content of 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (V) was 53%, and no iridoid glycoside components were detected by liquid chromatography-mass spectrometry.

[0018] Example 2: 2 kilograms of Tibetan Coptis Rhizome was taken, added with anhydrous ethanol, heated, extracted three times, 10 liters each time, reflux time 1 hour, filtered, mixed extracts, concentrated under reduced pressure until the smell of ethanol disappeared, diluted with water, adsorbed on AB-8 macroporous adsorption resin column, eluted with water and 20% ethanol by volume, collected 50% ethanol eluate until 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (V) was not detected by thin layer chromatography. Ethanol was collected from the eluate under reduced pressure, and 168 grams of Coptis Rhizome cucurbitan-type saponin crude extract was obtained. This crude cucurbitan-type saponin extract was added to ethyl acetate for purification, and the ethyl acetate was recovered under reduced pressure to obtain 36 grams of Huanglian cucurbitan-type saponin extract, of which HPLC detection showed that the total content of cucurbitan-type tetracyclic triterpenoid saponins I to X was 56%. This Euonymus humilis cucurbitan-type saponin extract was taken and subjected to alumina column chromatography, and a mixed solution of ethyl acetate and ethanol was used as the mobile phase for gradient elution and TLC detection. The saponin portion was collected, and the solvent was recovered to obtain 23 grams of purified Euonymus humilis cucurbitan-type tetracyclic triterpenoid saponin extract. HPLC detection showed that the total content of cucurbitan-type tetracyclic triterpenoid saponins I-X was 74%, where the content of 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (V) was 57%, and liquid chromatography-mass spectrometry showed that no iridoid glycoside components were detected.

[0019] Example 3: 2 kilograms of Tibetan Coptis Rhizome was taken, added with 95% ethanol, heated, extracted three times, 10 liters each time, reflux time 1 hour, filtered, mixed extracts, concentrated under reduced pressure until the smell of ethanol disappeared, diluted with water, adsorbed on HP-20 macroporous adsorption resin column, eluted with water and 30% ethanol by volume, collected 60% ethanol eluate until 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (V) was not detected by thin layer chromatography. Ethanol was collected from the eluate under reduced pressure, and 157 grams of Coptis Rhizome cucurbitan-type saponin crude extract was obtained. This crude cucurbitan-type saponin extract was added to ethyl acetate for purification, and the ethyl acetate was recovered under reduced pressure to obtain 31 grams of Huanglian cucurbitan-type saponin extract, of which HPLC detection showed that the total content of cucurbitan-type tetracyclic triterpenoid saponins I to X was 62%. This Euonymus humilis cucurbitan-type saponin extract was taken and subjected to ODS column chromatography, and a mixed solution of methanol and water was used as the mobile phase for gradient elution and TLC detection, the saponin portion was collected, and the solvent was recovered to obtain 18 grams of purified Euonymus humilis cucurbitan-type tetracyclic triterpenoid saponin extract, in which the total content of cucurbitan-type tetracyclic triterpenoid saponins I-X was 84% ​​by HPLC detection, in which the content of 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (V) was 68%, and no iridoid glycoside components were detected by liquid chromatography-mass spectrometry.

[0020] Example 4: 2 kilograms of Indian Coptis Rhizome was taken, boiled with water, and extracted three times, with 20 liters of water added each time, boiled for 1 hour, filtered, mixed with the extract, adsorbed with D4020 macroporous adsorption resin, and successively eluted with water and 30% ethanol by volume concentration, and 60% ethanol eluate was collected until 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (V) was not detected by thin-layer chromatography. The eluate was removed by ethanol recovery under reduced pressure, and 155 grams of Coptis Rhizome cucurbitan-type saponin crude extract was obtained. This crude cucurbitan-type saponin extract was added to ethyl acetate for purification, and the ethyl acetate was recovered under reduced pressure to obtain 33 grams of Huanglian cucurbitan-type saponin extract, of which HPLC detection showed that the total content of cucurbitan-type tetracyclic triterpenoid saponins I to X was 53%. This saponin extract was subjected to column chromatography on silica gel, gradient elution with ethyl acetate as the mobile phase and TLC detection, the saponin portion was collected, the solvent was recovered, and 21 grams of purified Huanglian cucurbitan-type saponin extract was obtained, in which the total content of cucurbitan-type tetracyclic triterpenoid saponins I-X was 77% by HPLC detection, in which the content of 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (V) was 51%, and no iridoid glycoside components were detected by liquid chromatography-mass spectrometry.

[0021] Example 5, take 2 kilograms of Indian Coptis Rhizome, add anhydrous ethanol, heat, extract 3 times, 10 liters each time, reflux time 1 hour, filter, mix the extract, concentrate under reduced pressure until the smell of ethanol disappears, dilute with water, adsorb on D860021 macroporous adsorption resin column, sequentially eluted with water and 30% ethanol by volume, collect 60% ethanol eluate until 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (V) is not detected by thin layer chromatography. Ethanol is collected from the eluate under reduced pressure, and 161 grams of Coptis Rhizome cucurbitan-type saponin crude extract is obtained. This crude cucurbitan-type saponin extract was added to ethyl acetate for purification, and the ethyl acetate was recovered under reduced pressure to obtain 34 grams of Huanglian cucurbitan-type saponin extract, of which HPLC detection showed that the total content of cucurbitan-type tetracyclic triterpenoid saponins I to X was 52%. This Euonymus humilis cucurbitan-type saponin extract was taken and subjected to alumina column chromatography, and a mixed solution of ethyl acetate and ethanol was used as the mobile phase for gradient elution and TLC detection. The saponin portion was collected, and the solvent was recovered to obtain 19 grams of purified Euonymus humilis cucurbitan-type tetracyclic triterpenoid saponin extract. HPLC detection showed that the total content of cucurbitan-type tetracyclic triterpenoid saponins I-X was 81%, where the content of 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one (V) was 60%, and liquid chromatography-mass spectrometry showed that no iridoid glycoside components were detected.

[0022] Experimental example 1: Screening of subcutaneous active ingredients of Phellodendron amplexicaule 1. Experimental materials: Medicines and reagents: Huanglian tablets, Shanxi Provincial Traditional Chinese Medicine Hospital Herbal Medicine Room (Batch No.: 21010129), D101 macroporous adsorption resin was purchased from Tianjin Bohong Resin Technology Co., Ltd., and ethanol was purchased from Beijing Dima Outai Science and Technology Development Center.

[0023] Experimental animals: SPF KM mice, half female and half male, a total of 120 mice, weighing 20±2g, production qualification certificate number: SCXK(Kyoto)2014-0013, purchased from Beijing Huafukang Biological Technology Co., Ltd.

[0024] 2. Experimental method: Preparation of Ethylhexylglycerin ethanol extract: 200g of Ethylhexylglycerin pieces were weighed, crushed and passed through a 10 mesh sieve, 1500ml of ethanol was added, and the mixture was heated under reflux for 1.5 hours. The mixture was filtered and the supernatant was removed. The remaining residue was extracted twice more, and the filtrate was mixed three times. The ethanol was concentrated and recovered under reduced pressure using a rotary evaporator, and then evaporated in a water bath to obtain 83.2g of dry paste.

[0025] Preparation of macroporous resin eluate: 500 g of D101 macroporous adsorption resin was placed in a saturated salt solution and soaked for 18-20 h, washed with distilled water until it became colorless, soaked in 5% hydrochloric acid by mass for 2-4 h, and after the acid was depleted, washed with distilled water until it became neutral, and then placed in a glass column with a diameter of 5 cm for use.

[0026] 80g of dried paste of Euonymus rhizoma ethanol extract was taken, 500ml of distilled water was added to dissolve and remove insoluble substances, and the mixture was passed through a pretreated macroporous resin column to fully adsorb the resin. The mixture was eluted with pure water until it was almost colorless, and the Molish reaction was negative. The mixture was then successively replaced with 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol and 90% ethanol to elute the macroporous resin. Each eluate was collected and evaporated in a water bath to obtain 42.31g, 6.63g, 11.82g, 9.14g, 4.53g, 3.21g, 0.83g, 0.42g, 0.33g and 0.24g, respectively.

[0027] Preparation of test drug: 4 g of the ethanol extract of Euonymus persica was weighed out and added with 10 ml of distilled water to prepare a 0.1 g / ml suspension. In addition, each of the dissolved components equivalent to 4 g of the extract was weighed out and used in the same manner to prepare a suspension.

[0028] Mouse lagoon experiment: Grouping and medication: 120 mice were acclimated for 3 days and randomly divided into blank group, Euonymus persicae ethanol extract group, water elution group, 10% ethanol elution group, 20% ethanol elution group, 30% ethanol elution group, 40% ethanol elution group, 50% ethanol elution group, 60% ethanol elution group, 70% ethanol elution group, 80% ethanol elution group and 90% ethanol elution group, with 10 mice in each group, half of which were female and half were male. Except for the blank group, which was given distilled water by gavage, the remaining groups were given the corresponding medication, with the gavage dose being 20ml / kg.

[0029] Observation index: After forced oral administration or drug administration, the mice were placed in separate cages with filter paper laid on the bottom and observed continuously for 6 hours. The number of mice with loose stools in each group was recorded, and the defecation rate of each group was calculated based on the following formula: defecation rate = number of defecation animals / total number of animals in the group x 100%.

[0030] 3. Experimental results: This experiment used 4g / kg ethanol extract as a control, and systematically compared the effects of each extract at a dose equivalent to 4g / kg ethanol extract on the defecation and fecal characteristics of normal mice. After forced oral administration or administration, the mice were continuously observed for 6 hours. As shown in Table 1, all 10 mice in the ethanol extract group had loose stools, with a 100% rate of defecation. At the same time, mice in the 50% and 60% ethanol extract groups had defecation, among which, all 10 mice forced oral administration of 0.16g / kg 50% extract had loose stools, with a 100% rate of defecation.

[0031] [Table 1]

[0032] Experimental example 2: Study on the separation, purification and identification of chemical substances in the active ingredients of Phellodendron amplexicaule 1. Experimental materials: Drugs and reagents: Androsin, vanillic acid, picroside I and picroside II control samples were all purchased from Shanghai Jiji Biochemical Technology Co., Ltd., the ethanol extract of Huanglian and its ethanol eluates with different volume fractions of macroporous resin, and chromatography-grade acetonitrile were purchased from Beijing Dimaoutai Science and Technology Development Center.

[0033] Experimental equipment: Bruker AVANCE NEO 500 MHz nuclear magnetic resonance spectrometer (Bruker, Switzerland), ACQUITY UPLC I-CLASS / Xevo G2-XS QTOF liquid chromatography-mass spectrometry chromatograph (Waters, USA), Nicolet iN10 MX infrared imaging microscope (ThermoFisher, USA), Waters preparative high-performance liquid chromatograph, binary gradient infusion pump (1525 type) with autosampler (2707 type), UV-Vis absorbance detector (2489 type), Diamonsil ODS chromatography column (150mmX21.2mm, 10μm), Milli-Q ultrapure water system (Millipore, USA), EYELASB-1000 rotary evaporator (Eyela, Japan), XS105 electronic balance (Mettler, Switzerland). Toledo), D101 macroporous adsorption resin (Tianjin Haiguang Chemical Co., Ltd.), column chromatography silica (200-300 mesh, Qingdao Marine Processing Factory), pure acetonitrile for chromatography (ThermoFisher, USA), and all other reagents were analytical reagents (Tianjin Zhiyuan Chemical Reagents Co., Ltd.).

[0034] 2. Experimental method: Preparation of standard solution: 5 mg each of androsin, vanillic acid, picroside I and picroside II were weighed out and placed in a volumetric flask, and an appropriate amount of methanol was added. The solution was ultrasonicated for 30 minutes, cooled, and then the solution was diluted to 50 ml and passed through a 0.22 μm microporous membrane for use.

[0035] Preparation of test sample solutions: Appropriate amounts of the ethanol extract of Phellodendron amplexicaule and each extract of the macroporous resin were precisely weighed and placed in a 50 ml volumetric flask, and methanol was added. The mixture was ultrasonically treated for 30 minutes to prepare a 10 mg / ml solution, which was then passed through a 0.22 μm microporous membrane for use.

[0036] High-performance liquid chromatography (HPLC-UV) analysis conditions: Chromatography column: Shim-pack C18-ODS chromatography column (250 mm x 4.6 mm, 5 μm), mobile phase: A is 0.1% phosphoric acid aqueous solution, B is acetonitrile, flow rate: 1.0 ml min -1 Injection volume: 10 μl, column temperature: 25° C., detection wavelengths: 200 nm, 230 nm and 275 nm. Mobile phase gradient elution conditions: 0-2 min, 15% B, 2-32 min, 15-25% B, 32-70 min, 25%-50% B, 70-90 min, 50-80% B.

[0037] Semi-preparative reversed-phase chromatography conditions: Chromatography column: Diamonsil C18-ODS chromatography column (150 mm x 21.2 mm, 10 μm), mobile phase: pure water / acetonitrile 70 / 30 (v / v), flow rate: 20.0 ml min -1 , injection volume: 1000 μl, detection wavelength: 200 nm.

[0038] UPLC-ESI-QTOF / MS analysis conditions: Chromatographic conditions: ACQUITY UPLC HSS T3 C18 chromatographic column (2.1 × 100 mm, 1.8 μm), mobile phase: 0.1% formic acid in acetonitrile (A) - 0.1% formic acid in water (B), gradient elution (0-1.2 min, 15% A, 1.2-6 min, 15% → 25% A, 6-36 min, 25% → 55% A), flow rate 0.2 mL / min, column temperature 40 °C, injection volume 2 μL.

[0039] Mass spectrum conditions: XEVO G2-XS ESI ion source, positive / negative ion mode, cone voltage 40 V, ion source temperature 100 °C, desolvation temperature 400 °C, cone gas flow 50 L / h, desolvation gas flow 700 L / h. Scan range m / z 100-1500, calibration solution: leucine-enkephalin, [M+H] 556.2771, [MH] 554.2615. MS was analyzed using Masslynx 4.1 software. E Mass spectral data were collected in Continuum mode with a scan rate of 0.2 / s and collision energy of 20V–35V.

[0040] 3. Experimental results: HPLC-UV spectrum: As shown in Figure 1, the maximum absorption wavelength of iridoid glycosides, phenolic glycosides and phenolic acids is about 280 nm, and the maximum absorption wavelength of cucurbitane-type tetracyclic triterpenoids is about 230 nm, or terminal absorption. The present invention uses high performance liquid chromatography gradient elution combined with multi-wavelength analysis to analyze the chemical substances in each component of Euonymus persicae. The concentrations of all reagents are 10 mg / ml, and 10 μl are injected. Compared with the standard, at 275 nm wavelength (Figure 1A), the ethanol extract of Huanglian contains androsin (peak 1), vanillic acid (peak 2), picroside II (peak 3), and picroside I (peak 4); the 10% ethanol eluate is rich in androsin, the 20% and 30% ethanol eluates are both rich in vanillic acid and picroside II, the 40% ethanol eluate is rich in picroside I, and the eluate with 50% or more does not contain more compounds; at 230 nm wavelength (Figure 1B), the 50% ethanol eluate contains the main peak 8; and at 200 nm wavelength (Figure 1C), the 50% ethanol eluate contains unknown compounds 5-11. The above analysis results suggest that peaks 5-11 may be the antibacterial active compounds in the 50% ethanol eluate, and further separation and identification studies are needed.

[0041] Compound identification results: The 50% ethanol eluate was further purified with ethyl acetate, and then repeatedly separated and purified using a reversed-phase high-performance liquid fractionation column. A total of 10 types of cucurbitane-type triterpenoid saponins were identified, and the identification results are as follows:

[0042] Compound I: White powder, soluble in chloroform and ethyl acetate, UV spectrum shows terminal absorption, 1 H-NMR(500MHz,CD3OD)δ:0.88(3H,s),1.04(3H,s),1.13(3H,s),1.22(3H,s),1 .28(3H,s),2.22(3H,s),1.42(1H,d,H-15a),1.75(1H,ddd,H-1a),1.86(1H,br d,H-8),1.89(1H,dd,H-15b),1.92(1H,dd,H-7a),2.53(1H,ddd,H-7b),2.58(1H,H-10),2.59(1H,ddd,H-1b),3.09(1H,d,H-17),3.66(1 H,d,H-3),3.76(1H,dd,H-6b'),3.99(1H,dd,H-6a'),4.29(1H,ddd,H-2),4.52(1H,d,H-1'),4.93(1H,br.t,H-16),5.68(1H,br.d,H-6), 13 C-NMR(125MHz,CD3OD)δ:28.34(C-1),77.57(C-2),77.13(C-3),42.26(C-4),141.73(C-5),121.31(C-6),25.31 (C-7),44.60(C-8),35.74(C-9),38.04(C-10),32.83(C-11),30.58(C-12),49.85(C-13),49.56(C-14),46.60( C-15),72.75(C-16),69.64(C-17),19.24(C-18),29.00(C-19),211.83(C-20),31.86(C-21),27.41(C-28),26. 19(C-29), 18.65(C-30), 102.06(C-1'), 75.23(C-2'), 78.12(C-3'), 71.79(C-4'), 77.92(C-5'), 62.89(C-6'). ESI-MS / MS(-)m / z:597.43[M+HCOO] -, 551.41[MH] - , molecular weight 552, molecular formula C 30 H 48 O9 is presented. Compared with the literature, compound I is 2β-glucoxy-3,16-dihydroxy-4,4,9,14-tetramethyl-19-norlanost-5-en-20-one. The structural formula is as follows: [ka]

[0043] Compound II: White powder, soluble in chloroform and ethyl acetate, melting point: 166-168°C, UV spectrum shows terminal absorption, 1 H-NMR(500MHz,CD3OD)δ:1.03(3H,s),1.11(3H,s),1.14(3H,s),1.20(3H,s),1.27(3H,s),1.37 (3H,s),1.37(3H,s),1.47(3H,s), 1.88(1H,dd,H-7a),2.63(1H,d,H-17),2.48(1H,ddd,H-7b), 3.66(1H,d,H-3),3.75(1H,dd,H-6b'),3.98(1H,dd,H-6a'),4.28(1H,br.d,H-2),4.52(1H,d,H -1'),4.46(1H,br.t,H-16),5.68(1H,br.d,H-6),2.78(1H,ddd,H-23a),2.94(1H,ddd,H-23b), 13C-NMR(125MHz,CD3OD)δ:28.42(C-1),77.55(C-2),77.06(C-3),42.22(C-4),141.70(C-5),121.51(C-6),25.45(C-7),44.05(C-8),3 5.41(C-9),38.12(C-10),31.73(C-11),32.77(C-12),49.81(C-13),49.84(C-14),47.08(C-15),72.01(C-16),60.09(C-17),18.45( C-18), 29.39 (C-19), 81.36 (C-20), 25.25 (C-21), 217.43 (C-22), 32.95 (C-23), 38.23 (C-24), 70.80 (C-25), 29.19 (C-26), 28.90 (C-2) 7),27.42(C-28),26.20(C-29),19.23(C-30),101.93(C-1'),75.17(C-2'),78.06(C-3'),71.73(C-4'),77.87(C-5'),62.87(C-6'). ESI-MS / MS(-)m / z:713.48[M+HCOO] - , 667.47[MH] - , molecular weight 668.9, molecular formula C 36 H 60 O 11 is presented. Compared with the literature, compound II is 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanost-5-en-22-one. The structural formula is as follows: [ka]

[0044] Compound III: White powder, soluble in chloroform and ethyl acetate, melting point: 156-160°C, UV λ max (nm): 230, 11H-NMR (500 MHz, CD3OD) δ: 1.03 (3H, s), 1.11 (3H, s), 1.14 (3H, s), 1.20 (3H, s), 1.27 (3H, s), 1.40 (3H, s), 1.40 (3H, s), 1.47 (3H, s), 1.30 (1H, d, H-15a), 1.75 (1H, d, H-15b), 1.75 (1H, ddd, H-1a), 1.88 (1H, ddd, H-7a), 2.45 (1H, d, H-17), 2.48 (1H, ddd, H-7b), 2.50 (1H, ddd, H-1b), 3.68 (1H, d, H-3), 3.74 (1H, dd, H-6b’), 3.96 (1H, dd, H-6a’), 4.28 (1H, br.d, H-2), 4.51 (1H, d, H-1’), 4.51 (1H, br.t, H-16), 5.67 (1H, br.d, H-6), 6.87 (1H, d, H-24), 7.04 (1H, d, H-23). 13 13C-NMR (125 MHz, CD3OD) δ: 28.40 (C-1), 77.61 (C-2), 77.12 (C-3), 42.25 (C-4), 141.72 (C-5), 121.42 (C-6), 25.27 (C-7), 44.12 (C-8), 35.45 (C-9), 38.14 (C-10), 31.63 (C-11), 32.72 (C-12), 49.11 (C-13), 49.82 (C-14), 47.07 (C-15), 72.37 (C-16), 60.04 (C-17), 18.63 (C-18), 29.25 (C-19), 81.46 (C-20), 25.15 (C-21), 205.23 (C-22), 121.55 (C-23), 155.14 (C-24), 71.48 (C-25), 29.29 (C-26), 28.93 (C-27), 27.42 (C-28), 26.17 (C-29), 19.24 (C-30), 102.03 (C-1’), 75.27 (C-2’), 78.16 (C-3’), 71.74 (C-4’), 77.97 (C-5’), 62.87 (C-6’). ESI-QTOF-MS / MS (-) m / z: 711.47 [M+HCOO] - , 665.46 [M-H] - , molecular weight 666.8, molecular formula C 36 H 58 O 11is presented. Compared with the literature, compound III is 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one. The structural formula is as follows: [ka]

[0045] Compound IV: White powder, soluble in chloroform and ethyl acetate, melting point: 175-179°C, UV spectrum shows terminal absorption, 1 H-NMR(500MHz,CD3OD)δ:0.94(3H,s),1.03(3H,s),1.08(3H,s),1.11(3H,s),1.18(3H,s),1.24(3H ,s),1.62(3H,s),1.77(3H,s),1.88(1H,dd,H-7b),2.21(1H,dd,J=6.6,14.0Hz,H-23b),2.30(1H,br dd,J=6.2,13.6Hz,H-23a),2.39(1H,d,H-17),2.42(1H,br d,H-7a),3.37(1H,tr,H-22),3.60(1H,br,H-3),3.67(1H,dd,J=5.4,11.8Hz,H-6b'),3.90(1H,br d,J=11.8Hz,H-6a'),4.28(1H,br d,J=11.3Hz,H-2),4.30(1H,d,J=7.8Hz,H-1'),4.56(1H,tr,J=7.4Hz,H-16),5.23(1H,tr,J=6.9Hz,H-24),5.59(1H,br d,J=4.9Hz,H-6), 13C-NMR(125MHz,CD3OD)δ:28.5(C-1),76.6(C-2),75.7(C-3),41.6(C-4),141.6(C-5),120.0(C-6),24.7(C-7),42.9(C-8), 34.5(C-9),36.9(C-10),31.3(C-11),32.1(C-12),48.8(C-13),49.4(C-14),46.1(C-15),71.7(C-16),56.9(C-17),18.5( C-18),28.0(C-19),76.6(C-20),24.5(C-21),81.1(C-22),31.5(C-23),124.3(C-24),131.8(C-25),25.9(C-26),18.0(C- 27), 27.2 (C-28), 26.2 (C-29), 18.1 (C-30), 101.3 (C-1'), 75.7 (C-2'), 78.6 (C-3'), 71.7 (C-4'), 78.7 (C-5'), 62.7 (C-6'). ESI-MS / MS(-)m / z:651.47[MH] - ,697.48[M+HCOO] - , molecular weight 652, molecular formula C 36 H 60 O 10 .Compared with the literature, it is determined that compound IV is 2β-glucoxy-3,16,20,22-tetrahydroxy-9-methyl-19-norlanosta-5,24-diene. The structural formula is as follows: [ka]

[0046] Compound V: White powder, soluble in chloroform and ethyl acetate, melting point 125-128°C, UV λ max (nm): 230, 11H-NMR (500 MHz, CD3OD) δ: 0.94 (3H, s), 1.02 (3H, s), 1.03 (3H, s), 1.10 (3H, s), 1.18 (3H, s), 1.39 (3H, s), 1.54 (3H, s), 1.56 (3H, s), 2.00 (3H, s, OAc), 2.34 (1H, d, J = 7.2 Hz, H-17), 2.43 (2H, br.d, J = 15.3 Hz, H-7), 3.68 (1H, dd, J = 10.7, 5.2 Hz, H-6b’), 3.89 (1H, d, J = 10.7 Hz, H-6a’), 4.29 (1H, br.d, J = 10.6 Hz, H-2), 4.43 (1H, d, J = 7.7 Hz, H-1’), 4.47 (1H, br.t, J = 7.8 Hz, H-16), 5.58 (1H, d, J = 5.2 Hz, H-6), 6.77 (1H, d, J = 15.8 Hz, H-23), 6.95 (1H, d, J = 15.8 Hz, H-24). 13 13C-NMR (125 MHz, CD3OD) δ: 28.0 (C-1), 76.7 (C-2), 75.7 (C-3), 41.6 (C-4), 141.6 (C-5), 120.0 (C-6), 24.7 (C-7), 42.7 (C-8), 34.9 (C-9), 37.0 (C-10), 30.9 (C-11), 32.0 (C-12), 49.0 (C-13), 49.0 (C-14), 46.8 (C-15), 71.7 (C-16), 60.2 (C-17), 18.6 (C-18), 20.0 (C-19), 80.2 (C-20), 25.4 (C-21), 204.6 (C-22), 122.8 (C-23), 149.6 (C-24), 79.9 (C-25), 26.3 (C-26), 26.7 (C-27), 27.2 (C-28), 26.2 (C-29), 18.7 (C-30), 101.7 (C-1’), 75.5 (C-2’), 78.8 (C-3’), 71.3 (C-4’), 78.6 (C-5’), 62.7 (C-6’), 21.8 (OAC, C-1), 169.8 (OAC, C-2). ESI-MS / MS (-) m / z: 707 [M-H] - , 753 [M+HCOO] - , molecular weight 708, molecular formula C 38 H 60 O 12. Comparing with the literature, it is determined that compound V is 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one. The structural formula is as follows: [ka]

[0047] Compound VI: White powder, soluble in chloroform and ethyl acetate, UV λ max (nm): 279, 1H-NMR(500MHz,CD3OD)δ:0.93(3H,s,H-18),0.94(3H,s,H-19),1.03(3H,s,H-28),1.08(3H,s,H-30),1.18(3H,s,H-29),1.43(6H,s,H-26,H-27),1.94(3H,s,OCOMe)、1.12(3H,d,J=6.7Hz,H-21),1.26(1H,d,J=12.8Hz,H-15b),1.47(1H,m,H-12b),1.50(1H,m,H-11b),1.65(1H,d,J=12.4Hz,H-1b),1.72(1H,m,H-15a),1.73(1H,m,H-11a),1.76(1H,m,H-8),1.77(1H,m,H-1a),1.84(1H,m,H-7b),1.94(1H,m,H-12a),1.98(2H,m,H-24a,H-24b),2.03(1H,dd,J=10.6,6.4Hz,H-17),2.40(1H,m,H-7a),2.45(1H,m,H-10),2.56(1H,m,H-23b),2.68(1H,m,H-23a),2.71(1H,m,H-20),3.18(1H,dd,J=9.2,7.8Hz,H-2’),3.27(1H,d,J=8.7Hz,H-4’),3.30(1H,m,H-5’),3.37(1H,dd,J=9.2,8.7Hz,H-3’),3.60(1H,br s,H-3),3.66(1H,dd,J=11.9,6.0Hz,H-6’b),3.89(1H,dd,J=11.9,2.2Hz,H-6’a),3.99(1H,t,J=7.8Hz,H-16),4.20(1H,m,H-2),4.42(1H,d,J=7.8Hz,H-10),5.58(1H,d,J=5.9Hz,H-6)、 13C-NMR(125MHz,CD3OD)δ:29.0(C-1),77.5(C-2),77.1(C-3),42.2(C-4),141.7(C-5),121.5(C-6),25.4(C-7),44.9(C-8),35.6(C- 9),38.0(C-10),32.8(C-11),31.5(C-12),48.8(C-13),49.5(C-14),47.5(C-15),77.5(C-16),58.0(C-17),17.4(C-18),28.4(C-1) 9),50.3(C-20),17.1(C-21),217.6(C-22),37.0(C-23),35.6(C-24),83.1(C-25),26.2(C-26),26.2(C-27),27.4(C-28),26.2(C- 29),19.0(C-30),22.3(OAC,C-1),172.5(OAC,C-2),102.0(C-1'),75.2(C-2'),77.9(C-3'),71.7(C-4'),78.1(C-5'),62.8(C-6'). ESI-MS / MS(-)m / z:693.46[MH] - , molecular weight 694, molecular formula C 38 H 62 O 11 is presented. Comparing with the literature, compound VI is 25-acetoxy-2β-glucoxy-3,16-dihydroxy-9-methyl-19-norlanost-5-en-22-one. The structural formula is as follows: [ka]

[0048] Compound VII: White powder, soluble in chloroform and ethyl acetate, UV λ max (nm): 230, 275, 1H-NMR(500MHz, CD3OD) δ: 1.03(3H, s), 1.12(3H, s), 1.14(3H, s), 1.20(3H, s), 1.27(3H, s), 1.46(3H, s), 1.52(3H, s), 1.52(3H, s), 1.31(1H, d, H-15a), 1.75(1H, ddd, H-1a), 1.78(1H, d, H-15b), 1.85(H, ddd, H-7a), 1.87(1H, br.d, H-8), 2.03(3H, s, OAc), 2.42(1H, d, H-17), 2.46(H, ddd, H-7b), 2.53(1H, ddd, H-1b), 2.75(1H, ddd, H-23a), 2.92(1H, ddd, H-23b), 3.69(1H, d, H-3), 3.77(1H, dd, H-6b’), 3.99(1H, dd, H-6a’), 4.29(1H, ddd, H-2), 4.48(1H, br.t, H-16), 4.53(1H, d, H-1’), 5.67(1H, br.d, H-6). 13 C-NMR(125MHz, CD3OD) δ: 28.93(C-1), 77.48(C-2), 77.07(C-3), 42.23(C-4), 141.69(C-5), 121.53(C-6), 25.24(C-7), 44.02(C-8), 35.40(C-9), 38.06(C-10), 32.72(C-11 / C-23), 31.71(C-12), 49.74(C-13), 49.60(C-14), 47.08(C-15), 71.95(C-16), 60.29(C-17), 19.18(C-18), 28.42(C-19), 81.40(C-20), 25.50(C-21), 216.89(C-22), 32.72(C-23), 35.89(C-24), 83.07(C-25), 26.29(C-26), 26.18(C-27), 27.42(C-28), 26.20(C-29), 18.50(C-30), 101.97(C-1’), 75.17(C-2’), 78.09(C-3’), 71.67(C-4’), 77.81(C-5’), 62.78(C-6’), 22.32(OAC, C-1), 172.40(OAC, C-2). ESI-MS / MS(-) m / z: 709.47[M-H] - , molecular weight 710, molecular formula C 38 H 62 O12 .Compared with the literature, it is determined that compound VII is 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanost-5-en-22-one. The structural formula is as follows: [ka]

[0049] Compound VIII: White amorphous powder, soluble in chloroform and ethyl acetate, UV spectrum shows terminal absorption. 1 H-NMR(500MHz,CD3OD)δ:1.03(3H,s),1.12(3H,s),1.14(3H,s),1.20(3H,s),1.27(3H,s),1.45(3H,s),1.70(3H, s),1.80(3H,s),1.32(1H,d,H-15a),1.74(1H,ddd,H-1a),1.86(H,ddd,H-7a),1.87(1H,br.d,H-8),2.44(1H,dd,H -17),2.47(H,br.dd,H-7b),2.52(1H,ddd,H-1b),2.52(1H,H-10),3.69(1H,d,H-3),3.76(1H,dd,H-6b'),3.98(1H ,dd,H-6a'),4.28(1H,ddd,H-2),4.50(1H,br.t,H-16),4.52(1H,d,H-1'),5.35(1H,H-24a),5.66(1H,br.d,H-6). 13C-NMR(125MHz,CD3OD)δ:29.0(C-1),76.1(C-2),75.7(C-3),42.2(C-4),140.3(C-5),120.1(C-6),25.3(C-7),44.1(C-8), 35.5(C-9),38.1(C-10),32.8(C-11),31.7(C-12),49.8(C-13),49.0(C-14),47.1(C-15),70.6(C-16),58.8(C-17),17.8(C -18),28.4(C-19),80.0(C-20),25.5(C-21),214.7(C-22),37.1(C-23),116.9(C-24),134.0(C-25),16.8(C-26),25.8(C- 27), 27.5 (C-28), 26.2 (C-29), 17.1 (C-30), 100.6 (C-1'), 73.8 (C-2'), 76.7 (C-3'), 70.3 (C-4'), 76.4 (C-5'), 61.4 (C-6'). ESI-QTOF-MS / MS(-)m / z:649.44[MH] - , molecular weight 650.8, molecular formula C 36 H 58 O 10 is presented. Comparing with the literature, it is determined that compound VIII is 2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,24-dien-22-one. The structural formula is as follows: [ka]

[0050] Compound IX: White powder, soluble in chloroform and ethyl acetate. UV spectrum shows terminal absorption. Vanillin-concentrated sulfuric acid reaction is brownish yellow, and both Liebermann-Burchard and Molish reactions are positive. (-)ESI-MS shows adduct ion peak m / z: 695.4019 [M+HCOOH] - (Calculated molecular weight: 650.4030), molecular formula C 36 H 58 O 10 This compound is presumed to belong to the triterpenoid glycoside class of compounds. As shown in Table 2,1 Two alkene hydrogen signals δ from H-NMR spectrum H 5.58 (1H, br d, J = 5.3 Hz) and δ H 4.69 (2H, d, J = 7.1 Hz), 10 oxymethylene or methine group hydrogen signals δ H 4.43(1H,d,J=7.8Hz),δ H 4.39(1H,t,J=7.5Hz),δ H 4.21(1H,ddd,J=12.1,4.3,2.3Hz),δ H 3.93(1H,dd,J=11.9,2.0Hz),δ H 3.70(1H,dd,J=11.9,5.6Hz),δ H 3.63(1H,d,J=2.3Hz),δ H 3.38(1H,m),δ H 3.31, 3.24 (2H,m) and δ H 3.19 (1H, dd, J = 9.3, 7.8 Hz), 7 unimodal methyl signals δ H 1.73, 1.36, 1.18, 1.10, 1.06, 1.03 and 0.94 can be observed, 13 In combination with the 36 carbon signals displayed in the C-NMR and DEPT spectra, a carbonyl signal δ C 216.7, two pairs of alkene carbon signals δ C 141.7 and δ C 121.5 and δ C 146.3 and δ C 110.5 is visible. In addition, nine oxycarbon signals and seven methylcarbon signals are also displayed, suggesting that compound VII contains a cucurbitane-type tetracyclic triterpenoid structure. δ H 4.43 (1H, d, J = 7.8 Hz) is the proton signal of the glucose end group. From the coupling constant of 7.8 Hz, glucose is assumed to be in the β configuration. Similarly, the six carbon signals on glucose are δ C 102.0, 75.2, 78.1, 71.7, 77.8 and 62.8 are also seen in the carbon spectrum, where δ C102.0 is the carbon signal of the terminal group. The IR spectrum of FIG. 2 shows that the compound has an OH absorption peak at 3444 cm -1 ) and CH bonds (2937cm -1 ), C=O bond (1700cm -1 ), C=C bond (1647cm -1 From the above data, it can be inferred that compound VII is a cucurbitan-type glucoside. 1 H, 1 Through H-COSY and HSQC, the planar structure of compound VII was determined, which was very similar to that of compound VI. The only difference was that the double bond position of the 17-bit side chain was Δ 25 The primary HMBC information also supports the above conclusion, i.e., H3-27 and C-24 / C-25 / C-26, H2-26 and C-24 / C-27, and H2-24 and C-22 / C-23 / C-25 / C-26 / C-27 are related.

[0051] Based on the above information, the structure of compound IX is determined to be 2β-d-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,25-dien-22-one, which is a new unreported cucurbitan glycoside compound and is named Huanglian cucurbitan saponin A. The structural formula is as follows: [ka]

[0052] [Table 2(1)] [Table 2(2)]

[0053] a,b Signal peak assignments are interchangeable.

[0054] Compound X: White powder, soluble in chloroform and ethyl acetate, melting point 132-135°C, UV λ max (nm): 200, 230, 1 H-NMR(500MHz,CD3OD)δ:1.22(3H,s),1.04(3H,s),1.64(3H,s),1.63(3H,s),1.11(3H,s),1.27(3H,s) ,1.18(3H,s),2.09(3H,s),1.38(1H,d,H-15a),1.75(1H,ddd,H-1a),1.79(1H,dd,H-15b),1.86(1H,br d,H-8),1.89(1H,dd,H-7a),2.17(1H,d,H-17),2.48(1H,ddd,H-7b),2.51(1H ,ddd,H-1b),2.52(1H,H-10),3.13(1H,dd,H-20),3.68(1H,d,H-3),3.76(1H, dd,H-6b'),3.99(1H,dd,H-6a'),4.11(1H,br.t,H-16),4.28(1H,ddd,H-2),4 .52(1H,d,H-1'),5.67(1H,br.d,H-6),6.39(1H,d,H-23),7.07(1H,d,H-24), 13C-NMR(125MHz,CD3OD)δ:28.97(C-1),77.57(C-2),77.13(C-3),42.26(C-4),141.68(C-5),121.50(C-6),25.37(C-7),44.97(C-8),35. 59(C-9),38.03(C-10),32.84(C-11),31.44(C-12),47.05(C-15),77.40(C-16),58.48(C-17),17.36(C-18),28.43(C-19),47.02(C-20) ),17.80(C-21),207.46(C-22),127.58(C-23),151.06(C-24),80.92(C-25),26.73(C-26),26.60(C-27),27.44(C-28),26.19(C-29),1 8.87(C-30),102.03(C-1'),75.25(C-2'),78.12(C-3'),71.78(C-4'),77.92(C-5'),62.85(C-6'),21.84(OAC,C-1),172.00(OAC,C-2). ESI-MS / MS(-)m / z:737.52[M+HCOO] - , 691.51[MH] - , molecular weight 692, molecular formula C 38 H 60 O 11 is presented. Comparing with the literature, compound X is 25-acetoxy-2β-glucoxy-3,16-dihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one. The structural formula is as follows: [ka]

[0055] Experimental example 3: Study on the preparation process of Phellodendron amplexicaule cucurbitan-type saponin extract In order to obtain a cucurbitan-type saponin extract with few impurities and high content from the herbal medicine Euonymus persicae, the present invention has conducted extensive research and finally completed the following preparation process.

[0056] Huangliang Rhizome is either decoctioned with water or reflux extracted with an organic solvent. The cucurbitane-type triterpenoid saponin compounds have relatively low polarity, and decoction with water is not very efficient, requiring 4 to 6 times to achieve complete extraction. Therefore, it is best to use an organic solvent such as methanol or ethanol for reflux extraction. Ethanol is safe and inexpensive, so reflux extraction with ethanol is preferred.

[0057] Research has shown that the compounds of cucurbitan-type triterpenoid saponins from Huangliang Coptis can be adsorbed by macroporous adsorption resin, so that the compounds of cucurbitan-type triterpenoid saponins can be preliminarily separated from impurities such as polysaccharides, iridoids, and phenolic glycosides by using the adsorption method with macroporous adsorption resin. The water decoction can be directly adsorbed by macroporous adsorption resin, while the organic solvent extract can be collected to a certain volume, and then dispersed by adding water, and then adsorbed by macroporous adsorption resin. First, some impurities are eluted with a low concentration of organic solvent, and then the components of cucurbitan-type triterpenoid saponins are eluted with a high concentration of organic solvent. The eluate is concentrated under reduced pressure or atmospheric pressure to obtain the crude extract of cucurbitan-type saponins from Huangliang Coptis, which also contains polysaccharides and phenolic glycosides, and the content of cucurbitan-type saponins is less than 50%. The macroporous adsorption resin described in the above preparation process may be one of AB-8, D4020, D101, 860021, HP20, or other manufacturers' adsorption resins with the same or similar functions. The organic solvent used for removing and eluting impurities is generally a mixed solution of an organic solvent such as methanol and ethanol and water, and an aqueous ethanol solution is preferred. When using an aqueous solution of a low concentration of an organic solvent to elute impurities, the so-called low concentration is generally not more than 30%, with the upper limit being that the saponin compounds are not eluted.

[0058] The crude extract of Euonymus rhizoma cucurbitan-type saponins obtained by the above method is dispersed in water, and then extracted with an organic solvent, and the extract is concentrated under reduced pressure and dried to obtain Euonymus rhizoma cucurbitan-type saponins extract, in which the content of saponins is increased. The organic solvent used for extraction can be a water-immiscible solvent such as chloroform or ethyl acetate, and ethyl acetate is preferred. Finally, the Euonymus rhizoma cucurbitan-type saponins extract obtained by the above method can be further purified by column chromatography to obtain a higher purity Euonymus rhizoma cucurbitan-type saponins extract, and the column chromatography can be selected from silica gel, alumina or ODS column chromatography.Through high performance liquid chromatography detection, the main components were identified as compound I, 25-acetoxy-2β-glucoxy-3,16-dihydroxy-9-methyl-19-norlanost-5-en-22-one; compound II, 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanost-5-en-22-one; compound III, 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanost-5-en-22-one; tetrahydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one, compound IV 2β-glucoxy-3,16,20,22-tetrahydroxy-9-methyl-19-norlanosta-5,24-diene, compound V 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one, compound VI 2β-glucoxy-3,16-dihydroxy-4,4,9,14-tetramethyl-19-norlanost-5-en-20-one, VII 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanost-5-en-22-one, VIII 2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanost-5, It was found that the total content of the extract was between 50% and 100%, and that it contained 24-dien-22-one, compound IX 2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,25-dien-22-one, and compound X 25-acetoxy-2β-glucoxy-3,16-dihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one. At the same time, it did not contain any iridoid glycosides.

[0059] Experimental Example 4: Content Analysis Method of Euonymus chinensis Cucurbitan-type Saponin Extract and Mass Spectral Detection Spectrophotometric methods can be used to measure the total content of cucurbitan-type saponin compounds, but on the one hand, this method is not highly accurate, and on the other hand, it cannot specifically detect the saponin components comprehensively. Therefore, a liquid chromatography-mass spectrometry and HPLC content measurement method for Euonymus humilis cucurbitan-type saponin extract is established.

[0060] Medicines and reagents: Huangliang Huangliang cucurbitan-type saponin extract prepared in this laboratory, with a purity of 99.2% by liquid chromatography analysis; cucurbitan-type tetracyclic triterpenoid saponin compounds I-X isolated and purified in this experiment; mass spectrum grade acetonitrile purchased from Beijing DMM Outlet Science and Technology Development Center.

[0061] Experimental equipment: High-performance liquid chromatograph: Agilent Techologies 1200 series, Shim-pack C18-ODS chromatography column (250mmX4.6mm, 5μm), quaternary pump: G1311A, autosampler: G1329A, column heater: G1316A, DAD diode array detector: G1315D, HP Chemstation chromatography workstation liquid chromatography-mass spectrometry equipment: ACQUITY UPLC I CLASS-G2-XS QTOF (Waters, USA), MassLynx V4.1 mass spectrum workstation (Waters, USA), ESI ion source.

[0062] Preparation of standard solution: 5 mg of each of compounds I to X was accurately weighed and placed in a volumetric flask, an appropriate amount of methanol was added, ultrasonicated for 30 minutes, cooled, and the volume was adjusted to 50 ml, and the solution was passed through a 0.22 μm microporous membrane for use.

[0063] Preparation of Euonymus humilis cucurbitan-type saponin extract solution: 50 mg of Euonymus humilis cucurbitan-type saponin extract was accurately weighed and placed in a volumetric flask, an appropriate amount of methanol was added, ultrasonicated for 30 minutes, cooled, and the volume was adjusted to 50 ml, and the solution was passed through a 0.22 μm microporous membrane for preparation for use.

[0064] UPLC-ESI-QTOF / MS analysis conditions: Chromatographic conditions: ACQUITY UPLC HSS T3 C18 chromatography column (2.1 × 100 mm, 1.8 μm), mobile phase: 0.1% formic acid in acetonitrile (A) - 0.1% formic acid in water (B), gradient elution (0-6 min, 25% → 31% A, 6-36 min, 31% → 50% A, 36-38 min, 50% → 95% A), flow rate 0.2 mL / min, column temperature 40 °C, injection volume 2 μL.

[0065] Mass spectrum conditions: XEVO G2-XS ESI ion source, positive / negative ion mode, cone voltage 40 V, ion source temperature 100 °C, desolvation temperature 400 °C, cone gas flow 50 L / h, desolvation gas flow 700 L / h. Scan range m / z 100-1500, calibration solution: leucine-enkephalin, [M+H] 556.2771, [MH] 554.2615. MS was analyzed using Masslynx 4.1 software. E Mass spectral data were collected in Continuum mode with a scan rate of 0.2 / s and collision energy of 20V–35V.

[0066] High-performance liquid chromatography conditions: Chromatography column: Shim-pack C18-ODS chromatography column (250 mm x 4.6 mm, 5 μm), mobile phase: A is 0.1% phosphoric acid water, B is acetonitrile, flow rate: 1.0 ml min -1 Injection volume: 10 μl, column temperature: 20° C., detection wavelength: 200 nm. Gradient elution: 0-15 min, 15% → 25% B, 15-40 min, 25% → 30% B, 40-50 min, 30% → 30% B, 50-65 min, 30% → 40% B.

[0067] Liquid chromatography-mass spectrometry results: The Huanglian cucurbitan-type saponin extract was passed through ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry to obtain base peak intensity chromatograms in positive and negative ion modes, respectively. As shown in Figure 7, when compared with standard substances, cucurbitan-type triterpenoid saponins I to X were found, while iridoid glycosides such as picroside I and picroside II were not detected.

[0068] Liquid chromatographic analysis results: 10 μl of the mixed control solution and the test solution were accurately transferred with a pipette and injected into the liquid chromatograph. As shown in FIG. 8, vanillic acid and cinnamic acid were observed, peak 1 is compound I, peak 2 is compound II, peak 3 is compound III, peak 4 is compound IV, peak 5 is compound V, peak 6 is compound VI, peak 7 is compound VII, peak 8 is compound VIII, peak 9 is compound IX, and peak 10 is compound X. The area of ​​each peak was recorded and calculated, which showed that the total content of compounds I to X was as high as 68.1%. Liquid chromatography also did not detect any iridoid glycosides such as picroside I and picroside II.

[0069] Experimental example 5: Study on the effect of Euonymus humilis cucurbitan-type saponin extract on defecation in mice Drugs and reagents: Huanglian cucurbitan-type saponin extract (CTTS) prepared in our laboratory, vanillic acid and cinnamic acid purchased from Shanghai Jiji Biochemical Technology Co., Ltd., linaclotide capsules (Ling Zeshu), Almac Pharma Services Limited, batch number W054121, specification 290 μg / capsule.

[0070] Experimental animals: SPF KM mice, half female and half male, weighing 20±2g, production qualification certificate number: SCXK(Kyoto)2014-0013, purchased from Beijing Huafukang Biological Technology Co., Ltd.

[0071] Preparation of test drugs: 100mg each of vanillic acid and cinnamic acid were weighed out, 5ml of distilled water was added, and ultrasonic treatment was performed for 10min to prepare a 20mg / ml suspension; an appropriate amount of Huanglian cucurbitan-type saponin extract was weighed out, and 5mg / ml, 10mg / ml and 20mg / ml suspensions were prepared in the same manner; the contents of linaclotide capsules were taken, and crushed with distilled water to prepare a 5μ / ml suspension; the contents were left at 4℃ for use.

[0072] Preparation of carbon suspension: 80 ml of distilled water was weighed out, 10 g of gum arabic was added, boiled, and after dissolving, 5 g of activated carbon was added, boiled three times, cooled, and then the volume was adjusted to 100 ml, and left at 4°C for use.

[0073] Study on the effect on defecation of normal mice: After three days of adaptive feeding, the mice were divided into blank group, linaclotide group (50μg / kg), vanillic acid group (200mg / kg), cinnamic acid group (200mg / kg), Huanglian cucurbitan-type saponin extract low dose group (50mg / kg), medium dose group (100mg / kg) and high dose group (200mg / kg), with 10 mice in each group, half female and half male. On the day of the test, all mice in each group were given carbon suspension (10ml / kg) by gavage, and after 30 minutes, except for the blank group, which was given distilled water by gavage, the remaining groups were given the corresponding medicine, and the gavage intake was 10ml / kg. Each mouse was placed in a separate cage and observed continuously for 12 h. The time of the first melena of each mouse was recorded, feces were collected and the amount of feces excreted was weighed and recorded. At the same time, the moisture content of the feces was calculated after the feces had completely dried.

[0074] Study on the effect of dehydration and drying on defecation in dehydrated constipation model mice: In order to make dehydrated constipation model, mice were fed adaptively, then deprived of water for 72 hours without fasting. On the next day, the model mice were randomly divided into model control group, linaclotide group (50μg / kg), vanillic acid group (200mg / kg), cinnamic acid group (200mg / kg), Huangliang Coptis cucurbitan-type saponin extract low dose group (50mg / kg), medium dose group (100mg / kg) and high dose group (200mg / kg). At the same time, mice that were not deprived of water were set as blank group. Each group had 10 mice, half of which were female and half were male, and all the mice in each group were forced to take carbon suspension (10ml / kg) by oral force. After 30 minutes, except for the blank group that was forced to take distilled water by oral force, the remaining groups were given the corresponding medicine, and the forced oral intake was 10ml / kg. Each mouse was placed in a separate cage and observed continuously for 12 h. The time of the first melena of each mouse was recorded, and feces were collected and the amount of feces excreted was weighed and recorded. At the same time, the moisture content of the feces was calculated after the feces had completely dried.

[0075] Experimental results: Study on the effect of Euonymus humilis cucurbitan-type saponin extract on defecation in normal mice: In this experiment, the laxative effect of Euonymus humilis cucurbitan-type saponin extract was evaluated in normal mice, with linaclotide as the positive control, and vanillic acid and cinnamic acid control groups set up at the same time. Mice in each group were forcefully orally administered water or test solution, and then continuously observed for 12 hours. As shown in Table 3, the time to first melena in the mice in the linaclotide group was significantly shorter than that in the blank group (P<0.05), and the number of defecations and the water content of feces were significantly increased (P<0.05, P<0.05). The time to first melena, the number of defecations and the water content of feces in the mice in the vanillic acid and cinnamic acid groups did not change significantly compared to the blank group. The time to first melena in the three dose groups of Euonymus persicae cucurbitan-type saponin extract was dose-dependently decreased, and the amount of defecation and the water content of feces were increased compared to the blank group. The amount of defecation and the water content of feces in the medium dose group (100mg / kg) were all significantly increased compared to the blank group (P<0.05, P<0.05).

[0076] [Table 3]

[0077] Note: # P<0.05, ## P<0.01 compared with blank group.

[0078] Study on the effect of Euonymus humilis cucurbitan-type saponin extract on defecation in dehydrated and dried constipated model mice: In this experiment, the laxative effect of Euonymus humilis cucurbitan-type saponin extract was evaluated in a replicated dehydrated and dried constipated model mouse that was deprived of water and not fasted for 72 hours, with linaclotide as the positive control, and vanillic acid and cinnamic acid control groups also set up. After the modeling was completed, the mice in each group were forced to orally administer water or test liquid, and then continuously observed for 12 hours. As shown in Table 4, the time of first melena in the mice in the model group was significantly prolonged compared with the blank group (P<0.01), and the defecation volume and fecal moisture content were significantly decreased compared with the blank group (P<0.01, P<0.001). Compared with the model group, the mice in the linaclotide, vanillic acid and cinnamic acid control groups did not show any significant improvement in all three indicators. Compared with the model group, the three dose groups of Euonymus persicae cucurbitan-type saponin extract showed a dose-dependent shortening of the time of first melena, and an increase in defecation volume and fecal moisture content. The defecation volume and fecal moisture content of the medium dose group (100mg / kg) were all significantly increased compared with the model group (P<0.05, P<0.05).

[0079] [Table 4]

[0080] Note: ## P<0.01, ### P<0.001 compared with blank group; * P<0.05, ** P<0.01, *** P<0.001 compared with the model group.

[0081] It was shown that 100 mg / kg of Euonymus persicae cucurbitan-type saponin extract could promote defecation in normal mice, shortening the time to the first melena and increasing the amount of stool excreted. Furthermore, using dehydrated constipated model mice, it was shown that 100 mg / kg of Euonymus persicae cucurbitan-type saponin extract could similarly shorten the time to the first melena and increase the amount of stool excreted and the water content of feces, while vanillic acid (200 mg / kg) and cinnamic acid (200 mg / kg) had no obvious effect on defecation in the two model mice.

[0082] Experimental Example 6: Effect of Phellodendron rhizoma cucurbitan-type tetracyclic triterpenoid saponin compounds on defecation in dehydrated constipated model mice Medicines and reagents: Huanglian cucurbitan-type saponin extract (CTTS) and compounds I-X prepared in this laboratory, linaclotide capsules (Linclotide), Almac Pharma Services Limited, batch number W054121, specification 290 μg / capsule.

[0083] Experimental animals: SPF KM mice, half female and half male, weighing 20±2g, production qualification certificate number: SCXK(Kyoto)2014-0013, purchased from Beijing Huafukang Biological Technology Co., Ltd.

[0084] Preparation of test drugs: Weigh out an appropriate amount of cucurbitane-type tetracyclic triterpenoid saponin I-X compounds, add 5ml of distilled water, and ultrasonicate for 10 minutes to prepare 5mg / ml and 10mg / ml suspensions, weigh out an appropriate amount of Phellodendron amplexicaule cucurbitane-type saponin extract, and prepare a 10mg / ml suspension in the same manner, take the contents of linaclotide capsules, add distilled water and crush to prepare a 5μ / ml suspension, and leave at 4℃ for use.

[0085] Grouping and medication: To create a dehydrated constipation model, the mice were fed adaptively, and then deprived of water without fasting for 72 h continuously. The next day, the model mice were randomly divided into a model control group, a linaclotide group (50 μg / kg), a Phellodendron candida cucurbitan-type saponin extract group (100 mg / kg), and a low-dose group (50 mg / kg) and a high-dose group (100 mg / kg) of compounds I-X. At the same time, the mice that were not deprived of water were set as a blank group, with 10 mice in each group, half of which were female and half were male. Except for the blank group, which was given distilled water by force orally, the remaining groups were given the corresponding medicine, and the amount of forced oral intake was 10 ml / kg. The mice were placed in separate cages and observed continuously for 12 h. The feces of each mouse were collected and weighed, and the moisture content of the feces was calculated after it was completely dried.

[0086] Experimental results: Mice in each group were forced to be orally administered water or test liquid, and then continuously observed for 12 hours. As shown in Table 5, the amount of defecation and the water content of feces in the model group were significantly decreased compared with the blank group (P<0.001, P<0.001); the mice in the linaclotide 50μg / kg treatment group did not show any significant improvement compared with the model group; the mice in the Euonymus persicae cucurbitan-type saponin extract 100mg / kg treatment group all showed significant increases compared with the model group (P<0.001, P<0.001). The amount of defecation and the water content of feces in the high dose group (100mg / kg) of Euonymus persicae cucurbitan-type tetracyclic triterpenoid saponin compounds I-X all showed significant increases compared with the model group.

[0087] [Table 5]

[0088] Note: ### P<0.001 compared with blank group; * P<0.05, ** P<0.01, *** P<0.001 compared with the model group.

Claims

1. White powder, molecular formula C ３６ H ５８ O １０ A tetracyclic triterpenoid saponin of Euonymus humilis cucurbitan type, characterized in that it has a molecular weight of 650 and the chemical name is 2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,25-dien-22-one, i.e., compound IX, which is named Euonymus humilis cucurbitan-type saponin A and has the structural formula as follows: 【Chemistry 1】

2. The extract of Euonymus rhizomeliad cucurbitan-type saponin contains 10 kinds of cucurbitan-type tetracyclic triterpenoid saponins having 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanosta-5-en-22-one as the parent structure, specifically, 25-acetoxy-2β-glucoxy-3,16-dihydroxy-9-methyl-19-norlanosta-5-en-22-one as compound I, 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanosta-5-en-22-one as compound II, Compound III, 2β-glucoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one; Compound IV, 2β-glucoxy-3,16,20,22-tetrahydroxy-9-methyl-19-norlanosta-5,24-diene; Compound V, 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,25-trien-22-one; lulanosta-5,23-(E)-dien-22-one, compound VI 2β-glucoxy-3,16-dihydroxy-4,4,9,14-tetramethyl-19-norlanosta-5-en-20-one, compound VII 25-acetoxy-2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5-en-22-one, compound VIII 2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5 , 24-dien-22-one, compound IX 2β-glucoxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,25-dien-22-one, and compound X 25-acetoxy-2β-glucoxy-3,16-dihydroxy-9-methyl-19-norlanosta-5,23-(E)-dien-22-one, the total content of the 10 compounds being >50%, and no iridoid components are contained.

3. (1) the herb Huangliang Rhizome is decoctioned with water or reflux-extracted with an organic solvent, the aqueous extract is directly adsorbed by a macroporous adsorption resin, and the organic solvent extract is first recovered, dispersed in water, and then adsorbed by a macroporous adsorption resin; (2) eluting the macroporous adsorption resin with water until it becomes almost colorless; (3) stripping the resin with an organic solvent and recovering the eluate; The method for preparing the Huanglian cucurbitan-type saponin extract according to claim 2, characterized in that it comprises: (4) concentrating to obtain a crude Huanglian cucurbitan-type saponin extract; and (5) purifying the crude Huanglian cucurbitan-type saponin extract by column chromatography to obtain a cucurbitan-type saponin extract.

4. The method for preparing the Huanglian cucurbitan-type saponin extract according to claim 3, characterized in that the herbal medicine Huanglian is the rhizome of the Scrophulariaceae plant, Picrorhiza kurroa Royle ex Benth, or the Tibetan Huanglian Picrorhiza scrophulariiflora Pennell.

5. The method for preparing a Coptis chinensis cucurbitan-type saponin extract according to claim 3, characterized in that the macroporous adsorption resin is any one of AB-8, D4020, D101, D860021 and HP20, the column chromatography is any one of silica gel, alumina and ODS column chromatography, and the organic solvent is methanol or ethanol.

6. Use of the Huanglian cucurbitan-type saponin extract of claim 2 in the preparation of a medicine for treating constipation.

7. The use according to claim 6, characterized in that the extract of Euonymus persicae cucurbitan-type saponins and a pharmacologically acceptable excipient form a pharmaceutical composition.

8. The use according to claim 7, characterized in that the pharmaceutical composition is formed into a dosage form of a tablet, a capsule, a granule, a patch or a patch.

Images