Wilfoside C1N, cynanchum otophyllum glycoside A, separation method thereof and application of wilfoside C1N and cynanchum otophyllum glycoside A in preparation of medicine for treating constipation

Abstract

The invention provides wilfoside C1N, cynanchum otophyllum glycoside A, a separation method of the wilfoside C1N and the cynanchum otophyllum glycoside A, and application of the wilfoside C1N and the cynanchum otophyllum glycoside A in preparation of a medicine for treating constipation, and belongs to the technical field The preparation method comprises the following steps: crushing the root tuber part of cynanchum auriculatum, carrying out percolation extraction with ethanol, carrying out vacuum concentration to obtain an extract, dissolving, and carrying out repeated column chromatography separation and purification to obtain the wilfoside C1N and cynanchum otophyllum glycoside A. The wilfoside C1N and cynanchum otophyllum glycoside A have application values in preparation of the medicines for treating constipation, and are beneficial to saving raw material resources, reducing the production cost and improving the medication safety.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to wilfoside C1N, ginsenoside A, their separation method, and their application in the preparation of drugs for treating constipation. Background Technology

[0002] Constipation, especially chronic functional constipation, is one of the most common functional disorders of the digestive system. Its pathophysiological mechanisms are complex, mainly involving slow colonic transit, impaired pelvic floor muscle coordination during defecation, and abnormal intestinal sensory function. These changes lead to prolonged stool retention in the colon and excessive water absorption, resulting in hard, dry stools, accompanied by a series of clinical symptoms such as straining during defecation, a feeling of incomplete evacuation, and reduced defecation frequency. Specifically, it manifests as fewer than three spontaneous bowel movements per week, excessive straining during defecation, a feeling of anorectal obstruction or blockage, and the need for manual assistance during defecation, severely impacting patients' quality of life and mental and physical health. With changes in modern dietary structures (such as insufficient dietary fiber intake), faster pace of life, an aging population, and increased influence from psychological factors, the prevalence of constipation is showing a significant upward trend globally. Epidemiological surveys show that the prevalence of chronic constipation in Chinese adults is approximately 4%-10%, increasing with age, reaching as high as 15%-20% in people over 60 years of age. Chronic constipation not only causes discomfort such as bloating, abdominal pain, and loss of appetite, but also can induce cardiovascular and cerebrovascular accidents due to long-term straining during defecation. It is also closely related to colorectal diseases, anxiety and depression, and has become an important public health problem, placing a continuous burden on individual, family and social medical resources. The key pathophysiological basis of constipation lies in intestinal neuromuscular dysfunction and an imbalance in intestinal water metabolism. Normal intestinal propulsion depends on the dynamic balance between excitatory (e.g., acetylcholine, serotonin) and inhibitory (e.g., nitric oxide) neurotransmitters in the enteric nervous system. Experimental administration of opioid receptor agonists (e.g., loperamide) significantly inhibited intestinal peristalsis and intestinal fluid secretion, leading to decreased intestinal propulsion and hardened stool, successfully establishing a functional constipation model and confirming that intestinal motility is highly dependent on excitatory neural regulation. The concentration of key neurotransmitters (e.g., 5-HT) and their receptor expression levels in colonic tissue, as well as the activity of inhibitory mediators (e.g., nitric oxide), are closely related to intestinal transport function and stool characteristics. When excitatory neurotransmitters are relatively insufficient or inhibitory signals are too strong, it can lead to weak colonic smooth muscle contraction, delayed transport, and accompanying damage to the intestinal mucosal barrier and local inflammatory response, thus forming a vicious cycle of constipation. Therefore, assessing intestinal propulsion rate and stool water content is of great significance for determining the severity of constipation and selecting treatment targets. Summary of the Invention

[0003] The purpose of this invention is to provide wilfoside C1N, ginsenoside A, their separation method, and their application in the preparation of drugs for treating constipation, which helps to save raw material resources, reduce production costs, and improve drug safety. The technical solution of this invention is implemented as follows: This invention provides a method for separating wilfoside C1N and ginsenoside A, comprising the following steps: S1. The tuberous root of *Gnaphalium affine* was crushed, extracted by percolation with ethanol, concentrated under reduced pressure to obtain an extract, dissolved, and separated by silica gel column chromatography with gradient elution of chloroform-methanol solution to obtain 7 fractions, recorded as Fr. 1-Fr. 7; S2. Selected fraction Fr. 2 was further separated by silica gel column chromatography with gradient elution of petroleum ether-acetone solution to obtain 3 fractions, denoted as Fr. 2.1-Fr. 2.3. Selected fraction Fr. 2.2 was separated by ODS column chromatography with gradient elution of methanol-water solution to obtain 8 fractions, denoted as Fr. 2.2.1-Fr. 2.2.8. Among them, fraction Fr. 2.2.4 was purified to obtain ginsenoside A. S3. Selected fraction Fr. 3 was further separated by silica gel column chromatography, eluted with chloroform-methanol solution, to obtain 4 fractions, denoted as Fr. 3.1-Fr. 3.4. Selected fraction Fr. 3.3 was separated by ODS column chromatography, eluted with methanol-water gradient, to obtain 2 fractions, denoted as Fr. 3.3.1-Fr. 3.3.2. Among them, fraction Fr. 3.3.2 was purified to obtain wilfoside C1N. As a further improvement of the present invention, the volume ratio of the chloroform-methanol solution gradient elution in step S1 is 20:1 to 1:1. As a further improvement of the present invention, the volume ratio of the petroleum ether-acetone solution gradient elution in step S2 is 3:1 to 3:3; the volume ratio of the methanol-water solution gradient elution is 40:60 to 100:0. As a further improvement of the present invention, the volume ratio of the chloroform-methanol solution in step S3 is 20-30:1; the volume ratio of the methanol-water gradient elution is 50:50 to 100:0. This invention further protects wilfoside C1N and ginsenoside A obtained by the above separation method. This invention further protects the use of the aforementioned wilfoside C1N and ginsenoside A in the preparation of a medicament for treating constipation. As a further improvement of the present invention, the wilfoside C1N and ginsenoside A are formulated into tablets, capsules, granules, oral liquid preparations, subcutaneous preparations, or suppositories. The present invention has the following beneficial effects: The purpose of this invention is to provide the application of wilfoside C1N, sennaoside A, and their monomeric compounds isolated from the tuberous root of *Gnaphalium affine* (family Asclepiadaceae) in the preparation of a treatment for constipation. wilfoside C1N and sennaoside A showed good therapeutic effects in high, medium, and low dose groups, with the high-dose group exhibiting superior efficacy. Furthermore, the overall therapeutic effect of all treatment groups was better than the positive control, demonstrating excellent pharmacological and efficacy effects. This approach helps save raw material resources, reduce production costs, and improve medication safety, possessing promising development prospects and application value. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 The hydrogen spectrum of wilfoside C1N; Figure 2 The carbon spectrum and DEPT plot of wilfoside C1N; Figure 3 The hydrogen spectrum of ginsenoside A; Figure 4 The carbon spectrum and DEPT plot of ginsenoside A; Figure 5 This is a histopathological image of mouse colon tissue. Detailed Implementation The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1 This embodiment provides a method for separating wilfoside C1N and ginsenoside A. S1. 15 kg of dried and pulverized tuberous root parts of *Gnaphalium affine* were extracted by percolation with 70% ethanol and concentrated under reduced pressure to obtain 686 g of extract. The extract was dissolved in an appropriate amount of methanol and subjected to silica gel (200-300 mesh) column chromatography with chloroform-methanol (20:1-1:1) gradient elution. After TLC examination, the fractions were combined to obtain 7 fractions (Fr. 1-Fr. 7). S2. Fr. 2 was purified by silica gel column chromatography with a petroleum ether-acetone (3:1-3:3) gradient elution to obtain Fr. 2.1-Fr. 2.3. Fr. 2.2 was separated by ODS column chromatography with a methanol-water (40:60-100:0) gradient elution to obtain Fr. 2.2.1-Fr. 2.2.8. Fr. 2.2.4 was purified by repeated silica gel column chromatography (chloroform-methanol 40:1 and petroleum ether-acetone 3:1), followed by ODS column separation with a methanol-water (60:40-70:30) gradient elution and Sephadex LH-20 column chromatography (chloroform-methanol 1:1) to obtain ginsenoside A (36 mg). The structure is as follows: ; Figure 3 , 4 The figure shows the characterization spectrum of ginsenoside A. As can be seen from the figure, this compound is ginsenoside A. S3. Fr. 3 was purified by silica gel column chromatography with chloroform-methanol (25:1) elution to obtain Fr. 3.1-Fr. 3.4. Fr. 3.3 was separated by ODS column chromatography with methanol-water (50:50-100:0) gradient elution to obtain Fr. 3.3.1-Fr. 3.3.2. Fr. 3.2 was purified by repeated silica gel column chromatography (chloroform-methanol 25:1, chloroform-acetone 4:1, and petroleum ether-acetone 3:2), followed by ODS column separation with methanol-water (70:30-80:20) gradient elution to obtain wilfoside C1N (18 g). The structural formula is as follows: . Figure 1 , 2 The figure shows the characterization spectrum of wilfoside C1N. As can be seen from the figure, this compound is wilfoside C1N. Example 2 1. Experimental Methods 1.1. Model Establishment and Drug Administration Sixty mice were selected to establish a functional constipation mouse model using loperamide hydrochloride suspension: mice were fasted for 12 hours but allowed free access to water, and were administered loperamide hydrochloride suspension at 10 ml / kg via gavage once daily for 14 days. Modeling was considered successful when decreased appetite, dry stools, lethargy, and pellet-like stools were observed. Ten normal control rats were administered an equal volume of physiological saline via gavage once daily for 14 days. Sixty male mice were selected for modeling and randomly divided into the following groups after acclimatization: model group, positive control group (mosapride), low-dose wilfoside C1N group (50 mg / kg), medium-dose wilfoside C1N group (100 mg / kg), and high-dose wilfoside C1N group (200 mg / kg), as well as low-dose sinomenine A group (50 mg / kg), medium-dose sinomenine A group (100 mg / kg), and high-dose sinomenine A group (200 mg / kg). After preliminary testing to determine the appropriate dosage and volume, mice were fasted for 12 hours but allowed free access to water before administration of the prescribed dosage, once daily for 14 consecutive days via gavage. Body weight was recorded before administration and on day 7 after administration. Changes in mouse activity, food intake, and body weight were also observed. 1.2. Indicator Testing Colon tissue was fixed in paraformaldehyde for HE staining and observation. 1.3. Data Statistics Experimental data are expressed as x±s; one-way ANOVA with LSD multiple comparison analysis was used to analyze differences between groups, and GraphPad Prism was used for statistical analysis and plotting. 2. Experimental Results 2.1. Mouse organ index The results of the statistical analysis of organ indices are shown in Table 1. Compared with the normal group, there were no significant differences in the various organ indices of mice caused by the test drugs wilfoside C1N and ginsenoside A. P >0.05), indicating that the test drugs wilfoside C1N and ginsenoside A did not affect the weight of the organs in mice.

[0004] Table 1. Organ indices of mice (x±s, n=8)

[0005] 2.2. Intestinal propulsion rate and fecal water content As shown in Table 2, compared with the normal group, both indicators in the model group were significantly reduced ( P<0.001 indicates that the constipation model was successfully established: fecal water content decreased by approximately 57%, and intestinal propulsion rate decreased by approximately 42%. Compared with the model group, both indicators in the positive drug, wilfoside C1N, and ginsenoside A groups significantly rebounded. P <0.001).

[0006] Table 2. Intestinal propulsion rate and fecal water content (x±s, n=8)

[0007] Note: Compared with the normal group, ### P <0.001; compared with the model group, **P <0.01, * **P <0.001 2.3. Pathological changes in mouse colon tissue like Figure 5 As shown, in the normal group, the colonic mucosa was intact, with continuous epithelium, abundant goblet cells, regular glands, no inflammatory cell infiltration, and normal muscular layer thickness. In the model group, however, there were mucosal defects, epithelial shedding, a sharp decrease in goblet cells, disordered glandular atrophy, extensive inflammatory cell infiltration in the lamina propria, and a thinned or unevenly thickened muscular layer. The mucosal structure in the model group was basically restored, with each layer approaching normal. In the wilfoside C1N group, with increasing dosage, mucosal defects gradually repaired, the epithelium remained intact, goblet cells increased, glandular arrangement recovered, inflammatory cells significantly decreased, and muscular layer thickness approached normal. In the ginsenoside A group, the mucosal structure was significantly repaired, the epithelium was neat, goblet cells were dense, glands were regular, inflammatory cells almost disappeared, and muscular layer thickness returned to normal; the overall effect was comparable to the normal group. In summary, wilfoside C1N and ginsenoside A alleviate constipation by increasing intestinal propulsion and fecal water content, with all dosage groups showing superior pharmacological activity. This invention used loperamide hydrochloride suspension to construct a functional constipation mouse model. After 14 days of continuous administration, the results showed that compared to the normal group, the fecal water content in the model group decreased by approximately 57%, and the intestinal propulsion rate decreased by approximately 42%, both indicators being highly significant (P<0.001), indicating successful establishment of the constipation model. Compared to the model group, both indicators in the positive control group, wilfoside C1N, and ginsenoside A groups significantly improved. Furthermore, in the wilfoside C1N group, with increasing dosage, mucosal defects gradually repaired, the epithelium remained intact, goblet cells increased, glandular arrangement recovered, inflammatory cells significantly decreased, and muscle layer thickness approached normal. In the ginsenoside A group, the mucosal structure was significantly repaired, the epithelium was neat, goblet cells were dense, glands were regular, inflammatory cells almost disappeared, and muscle layer thickness returned to normal. The overall effect was comparable to the normal group. These results indicate that wilfoside C1N and ginsenoside A have a superior effect on improving constipation. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for separating wilfoside C1N and ginsenoside A, characterized in that, Includes the following steps: S1. The tuberous root of *Gnaphalium affine* was crushed, extracted by percolation with ethanol, concentrated under reduced pressure to obtain an extract, dissolved, and separated by silica gel column chromatography with gradient elution of chloroform-methanol solution to obtain 7 fractions, recorded as Fr. 1-Fr. 7; S2. Selected fraction Fr. 2 was further separated by silica gel column chromatography with gradient elution of petroleum ether-acetone solution to obtain 3 fractions, denoted as Fr. 2.1-Fr. 2.

3. Selected fraction Fr. 2.2 was separated by ODS column chromatography with gradient elution of methanol-water solution to obtain 8 fractions, denoted as Fr. 2.2.1-Fr. 2.2.

8. Among them, fraction Fr. 2.2.4 was purified to obtain ginsenoside A. S3. Selected fraction Fr. 3 was further separated by silica gel column chromatography, eluted with chloroform-methanol solution, to obtain 4 fractions, denoted as Fr. 3.1-Fr. 3.

4. Selected fraction Fr. 3.3 was separated by ODS column chromatography, eluted with methanol-water gradient, to obtain 2 fractions, denoted as Fr. 3.3.1-Fr. 3.3.

2. Among them, fraction Fr. 3.3.2 was purified to obtain wilfoside C1N.

2. The separation method according to claim 1, characterized in that, The volume ratio of the chloroform-methanol solution gradient elution in step S1 is 20:1 to 1:

1.

3. The separation method according to claim 1, characterized in that, In step S2, the volume ratio of the petroleum ether-acetone gradient elution is 3:1 to 3:3; the volume ratio of the methanol-water gradient elution is 40:60 to 100:

0.

4. The separation method according to claim 1, characterized in that, The volume ratio of the chloroform-methanol solution in step S3 is 20-30:1; the volume ratio of the methanol-water gradient elution is 50:50 to 100:

0.

5. A method for separating wilfoside C1N and ginsenoside A as described in any one of claims 1-4.

6. The use of wilfoside C1N and ginsenoside A as described in claim 5 in the preparation of a medicament for treating constipation.

7. The application according to claim 6, characterized in that, The wilfoside C1N and ginsenoside A are formulated into tablets, capsules, granules, oral liquid preparations, subcutaneous preparations, or suppositories.

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