Pharmaceutical composition for loosening bowels to relieve constipation, and preparation method therefor and use thereof

Abstract

A pharmaceutical composition for loosening bowels to relieve constipation, and a preparation method therefor and a use thereof. The pharmaceutical composition comprises 10-300 parts of fried Fructus Cannabis, 10-200 parts of blanched Semen Pruni, 10-200 parts of fried Semen Sesami Nigrum, and 10-180 parts of fried Semen Raphani. Fructus Cannabis is dried ripe seeds of Cannabis sativa L.; Semen Pruni is dried ripe seeds of Prunus humilis Bge., Prunus japonica Thunb., or Prunus pedunculata Maxim.; Semen Sesami Nigrum is dried ripe seeds of Sesamum indicum L.; Semen Raphani is dried ripe seeds of Raphanus sativus L. The pharmaceutical composition relieves constipation through multiple metabolic pathways, such as an amino acid metabolism pathway, a short-chain fatty acid metabolism pathway, a lipid metabolism pathway, and an energy metabolism pathway.

Description

A laxative medicinal composition and its preparation method and application Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine compositions, and particularly relates to a laxative medicinal composition and a preparation method and application thereof. Background Art

[0002] Constipation, as a chronic condition, not only affects quality of life but, in more severe cases, can lead to gastrointestinal disorders such as gastrointestinal disorders, colon polyps, and colon cancer. Constipation is a common condition characterized by fewer than three bowel movements per week, hard stools, and / or difficulty defecating. Defecation difficulty includes straining, difficulty evacuating, a feeling of incomplete defecation, prolonged defecation, and the need for manual defecation. In Traditional Chinese Medicine (TCM), constipation is categorized as "constipation," "spleen obstruction," and "constipation." The Yellow Emperor's Classic of Internal Medicine first described "difficult defecation" and "inconvenience," while other texts also refer to "dryness and constipation," "spleen obstruction," and "difficult bowel movements." While Western medicine can effectively alleviate symptoms, it can cause numerous long-term side effects and lead to drug dependence. In recent years, reports on the use of Chinese herbal medicine to treat chronic functional constipation have increased. Evidence-based research confirms that TCM is safe and effective in treating functional constipation, improving clinical symptoms and offering significant advantages in overall efficacy.

[0003] Summary of the Invention

[0004] Technical problem to be solved: In response to the above technical problems, the present invention provides a laxative pharmaceutical composition and a preparation method and application thereof.

[0005] Technical solution: A laxative medicinal composition comprises 10-300 parts of stir-fried hemp seeds, 10-200 parts of stewed prunes, 10-200 parts of stir-fried black sesame seeds and 10-180 parts of stir-fried radish seeds; hemp seeds are the dried mature seeds of the Moraceae plant hemp (Cannabis sativa L.); prunes are the dried mature seeds of the Rosaceae plant Prunus humilis Bge., Prunus japonica Thunb. or Prunus peduncμLata Maxim.; black sesame seeds are the dried mature seeds of the Sesamum indicum L. of the Sesamaceae plant; and radish seeds (Raphanus sativus L.) are the dried mature seeds of the Cruciferae plant radish.

[0006] Preferably, the above composition further comprises 10-200 parts of peach powder.

[0007] Preferably, the above composition further comprises 10-300 parts of compound honey powder.

[0008] Preferably, the above-mentioned laxative pharmaceutical composition includes 14.1 kg of fried hemp seeds, 9.4 kg of stewed prunes, 9.4 kg of fried black sesame seeds, 7.05 kg of fried radish seeds, 7.5 g of peach powder and 15 g of compound honey powder.

[0009] The preparation method of the above-mentioned laxative pharmaceutical composition is characterized in that, according to proportion, fried hemp seeds, stewed prunes, fried black sesame seeds and fried radish seeds are put into a frying bag, and water is added for frying; after extraction, the mixture is concentrated, and then maltodextrin (MD15) is added and stirred until dissolved; the concentrated solution is spray-dried to obtain dry powder, and then honey powder and peach powder are added and granulated.

[0010] Preferably, according to the proportion, 14.1kg of fried hemp seeds, 9.4kg of stewed lily seeds, 9.4kg of fried black sesame seeds and 7.05kg of fried radish seeds are weighed, and the medicinal materials are put into a frying bag for frying; the amount of water added for the first extraction is 12 times the volume of the medicinal materials, and the extraction time is 2 hours; the amount of water added for the second extraction is 12 times the volume of the medicinal materials, and the extraction time is 1 hour; after the extraction is completed, the concentration process is entered, the concentration time is 140min, the concentrated liquid is filtered with a mesh size of 200 meshes, and the concentrated liquid is filtered with a mesh size of 200 meshes. The measured specific gravity of the liquid at 49°C is 1.0332, and the weight of the concentrated liquid is 46.94kg. Then, 2.8kg of maltodextrin (MD15) is added and stirred until dissolved; the concentrated liquid is spray-dried, and the inlet air temperature is set to 175°C, the exhaust air temperature is set to 95°C, the air delivery temperature is set to 45.4°C, and the peristaltic pump speed is set to 50rpm. A total of 12.76kg of dry powder is obtained. 2.5g of dry powder, 15g of honey powder and 7.5g of peach powder are taken in proportion and mixed and granulated to obtain compound hemp seed granules.

[0011] The preparation method of the stewed Prunus mume kernel is as follows: take clean Prunus mume kernel, place it in 10 times the amount of boiling water, heat and cook for 5 minutes, until the seed coat is slightly swollen, take it out, soak it in cold water for a while, take it out, rub the seed coat and seed kernel apart, dry it, and sieve out the seed coat.

[0012] The application of the prepared composition in the preparation of laxative drugs.

[0013] Beneficial Effects: Hemp seed enters the spleen, stomach, and large intestine meridians and is primarily used to treat intestinal dryness and constipation. Its chemical components primarily include fatty acids, fatty acid esters, lignin amides, flavonoids, alkaloids, steroids, terpenes, cannabinoids, amino acids, and proteins. Hemp seed is rich in essential unsaturated fatty acids such as oleic acid, linoleic acid, and α-linolenic acid. The ratio of linoleic acid (ω-6) to α-linolenic acid (ω-3) is 2.48-2.93:1, close to the WHO-recommended dietary ratio. Recent studies have shown that the fatty acids in hemp seed have pharmacological effects such as lowering blood lipids, providing antioxidant and anti-atherosclerotic effects, improving the intestinal microenvironment, and alleviating intestinal inflammation. Prunus mume enters the liver and gallbladder meridians, and some studies have also documented the lung meridian, promoting diuresis, reducing swelling, and promoting laxative effects. Flavonoids in Prunus mume include afudamidin, kaempferol, prurigoside A, prurigoside B, and amygdalin. Prunus mume glycosides have a strong laxative effect, with a mechanism similar to sennosides, achieving this effect by promoting intestinal motility. Prunus mume seeds are rich in oily components, which are also key ingredients in treating dry bowel constipation. Black sesame seeds are regulated by the liver, kidney, and large intestine meridians and are primarily used to nourish the liver and kidneys and moisten the intestines. Their main bioactive components include lignans, amino acids, unsaturated fatty acids, melanin, and minerals. Sesame oil contains over 80% unsaturated fatty acids, primarily linoleic and oleic acids. The lignans in sesame seeds primarily include sesamin and sesamolin, with sesamin exhibiting strong antioxidant, hepatoprotective, and anticancer activities. Radish seeds are regulated by the lung, spleen, and stomach meridians. They are neutral in nature, with a pungent and sweet flavor. They have the effects of promoting digestion, relieving bloating, and reducing qi and resolving phlegm. They are used to treat food stagnation, abdominal distension and pain, constipation, stagnation, diarrhea, and cough and wheezing caused by phlegm. The main chemical components of radish seeds include volatile oils, fatty acids, alkaloids, flavonoids, sterols, polysaccharides, and proteins, with glucoraphanin being its primary characteristic chemical component. The decoction pieces, prepared according to the optimal preparation method for each herb, provide beneficial effects in a laxative medicinal composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Total ion chromatograms of Fufang Maren Granules in negative (A) and positive (B) UPLC-QTOF-MS / MS modes;

[0015] Figure 2 Venn diagram of component-disease intersection targets;

[0016] Figure 3 GO functional pathway enrichment analysis;

[0017] Figure 4 KEGG pathway enrichment analysis;

[0018] Figure 5 Schematic diagram of the component-core target network;

[0019] Figure 6. Heat map of key target protein expression;

[0020] Figure 7 shows the results of the first stool experiment of Fufang Ma Ren Granule in mice; Note: * indicates a significant difference compared with the model group, ** indicates an extremely significant difference compared with the model group, and ## indicates an extremely significant difference compared with the blank group. DETAILED DESCRIPTION

[0021] The present invention will be further described with reference to the following examples, which are intended only to illustrate the present invention and are not intended to limit the present invention in any way.

[0022] Preparation of laxative medicinal composition slices

[0023] 1. Preparation of Stir-fried Hemp Seed: Hemp Seed is the dried mature fruit of the Moraceae plant Cannabis sativa L., and after removing impurities and peel, the dried mature seeds are obtained. Take an appropriate amount of raw Hemp Seed slices and place them in a stir-frying machine. Set the stir-frying temperature to 200°C, the stir-frying speed to 30 r / min, and stir-fry for 3 minutes until the hemp seed is slightly yellow and fragrant. Then, grind the stir-fried Hemp Seed into a fine powder, pass it through a 20-mesh sieve, and set aside.

[0024] Hemp seed is mainly used to treat blood deficiency, fluid deficiency, dry intestines and constipation. Hemp seed oil is the main component of hemp seed slices (its content accounts for 40% to 50% of the total weight of the slices), and it is also its medicinal ingredient. Hemp seed oil has the functions of lubricating the intestinal wall, softening stool, mildly stimulating the intestinal tract, increasing peristalsis, and reducing water absorption by the large intestine. Hemp seed oil is composed of a series of triglyceride compounds. The content determination results show that the content of the medicinal ingredients trilinoleic acid glycerol and fenugreek in the fried hemp seed slices is increased, reflecting the "synergistic" characteristics of the preparation, and the texture is crisper and easier to fry.

[0025] 2. Preparation of stir-fried radish seeds: Radish seeds are the dried mature seeds of the cruciferous plant radish (Raphanus sativus L.). Take an appropriate amount of raw radish seed slices and place them in a stir-frying machine. Set the stir-frying temperature to 220°C, the stir-frying machine speed to 30 r / min, and the stir-frying time to 3 minutes. Pulverize, pass through a 20-mesh sieve, and set aside. Prepare stir-fried radish seed slices according to this processing conditions. The surface is reddish-brown, bulging, easily crushed, and the interior is yellow.

[0026] Radish seeds have the benefits of promoting digestion and relieving bloating, lowering qi and resolving phlegm. They are used to treat food stagnation, abdominal distension and pain, constipation, dysentery, and cough and wheezing caused by phlegm. Radish seeds are a classic example of the "raw versus cooked" principle in traditional Chinese medicine (TCM). Stir-frying them causes them to settle, promoting digestion and relieving bloating. They are a commonly used digestive remedy in clinical practice. Modern research has shown that radish seeds primarily contain glucosinolates and sulfur-containing derivatives, alkaloids, flavonoids, fatty acids, volatile oils, and polysaccharides. Glucosinolates are characteristic components of radish seeds both before and after processing, with radixanolide being the most abundant single glucosinolate. The content of radixanolide in the aqueous extract of stir-fried radish seeds is over eight times higher than that of the raw extract. However, over-processing can lead to the complete loss of radixanolide. Stir-frying radish seeds can inhibit the activity of myrosinase, preventing the degradation of the glucosinolate glycoside.

[0027] 3. Preparation of stewed Prunus mume kernels: Clean Prunus mume kernels and place them in 10 times the volume of boiling water. Heat and cook for 5 minutes until the seed coat slightly swells. Remove and soak briefly in cold water. Remove, rub the seed coat and kernel apart, dry, and sieve out the seed coat. The extraction rates of amygdalin in the decoctions of three batches of raw, stewed, and stir-fried Prunus mume kernels were 22.56%-24.27%, 33.18%-33.42%, and 23.85%-24.52%, respectively. Stewing facilitates the dissolution of amygdalin from Prunus mume kernels. Prunus mume kernels contain enzymes that can hydrolyze amygdalin, and stewing kills the enzymes while preserving the amygdalin. The extraction rates of amygdalin in the raw and stir-fried Prunus mume kernels were lower than those in the stewed Prunus mume kernels. Crushing significantly improves the extraction rate, facilitating the dissolution of the active ingredient and increasing the extraction rate.

[0028] 4. Preparation of Roasted Black Sesame: Black sesame is the dried mature seed of Sesamum indicum L., a plant of the Sesamaceae family. Also known as sesame, black sesame, and sesame, it is an annual herb and a long-standing human food crop. Select an appropriate amount of black, plump black sesame seeds. Take an appropriate amount of raw black sesame seeds and place them in a stir-frying machine. Set the stir-frying temperature to 210°C, the stir-frying speed to 30 r / min, and the stir-frying time to 2 minutes. Crush the seeds, pass them through a 20-mesh sieve, let them cool, and stir-fry them for later use. Black sesame contains many nutrients and has high nutritional and medicinal value. There are many ways to prepare black sesame, and different preparation methods have different effects and uses. Stir-fried black sesame seeds have a significantly better bowel-moistening and laxative effect than other processed products.

[0029] 5. Preparation of Compound Honey Powder: This preparation method uses a mixture of honey, maltodextrin, whole milk powder, and β-cyclodextrin (8:6:6:1) to produce a milky white compound honey powder with a relatively uniform color, a rich honey aroma, and a smooth, mellow taste. At room temperature, honey is a sweet, mellow, and nutritious liquid with a variety of components, primarily sugars, which account for over 75% of the total honey content. Honey is highly viscous and hygroscopic, making it extremely difficult to produce pure honey powder. Furthermore, honey, which is primarily sugar-rich, lacks a balanced nutritional profile. This preparation method involves adding milk powder, maltodextrin, and β-cyclodextrin to honey, stirring thoroughly, and then spray-drying to produce honey powder. This honey powder formula can reduce honey viscosity and improve honey processing properties; it can also enhance the quality of honey products, enriching and balancing their nutritional content; and it can increase the depth of honey processing and add value to the honey process.

[0030] Adding honey can improve the sensory quality of the powder, but due to its high viscosity and strong hygroscopicity, increasing its content will sharply reduce the yield of honey powder. Therefore, its addition should not be too large during the preparation of honey powder, and it should be prepared according to the proportion. The special spatial structure of β-cyclodextrin makes it play a significant role in maintaining the original flavor of honey, so it is advisable to add it appropriately when preparing honey powder. Adding maltodextrin can significantly improve the yield of honey powder, but excessive addition will affect the sensory quality of honey powder. The addition of whole milk powder can increase the dry powder yield while also improving the relatively simple nutritional composition of honey. The above preparation method of compound honey powder is of great significance for improving the sensory, taste and beneficial effects of this formula.

[0031] Example 1

[0032] 1. First Preparation Method

[0033] 1.1 Preparation of granules

[0034] The pharmaceutical composition is as follows

[0035] Table 1

[0036] 1.1.1 Extraction

[0037] Weigh 9.4kg of stewed Prunus mume seeds, 14.1kg of stir-fried hemp seeds, 9.4kg of stir-fried black sesame seeds, and 7.05kg of stir-fried radish seeds, totaling 39.95kg. Place the herbs in a decoction bag and decoct. For the first extraction, add 12 times the amount of water and extract for two hours. For the second extraction, add 12 times the amount of water and extract for one hour.

[0038] 1.1.2 Concentration

[0039] After the extraction is completed, the concentration process begins. The concentration time is 140 minutes. The filter mesh size of the concentrate is 200 mesh. The measured specific gravity of the concentrate is 1.0332 (49°C). The weight of the concentrate is 46.94 kg. Then, 2.8 kg of maltodextrin (MD15) is added while hot and stirred until dissolved.

[0040] 1.1.3 Spray Drying

[0041] The concentrate was spray dried with an inlet air temperature of 175°C, an exhaust air temperature of 95°C, a delivery air temperature of 45.4°C, and a peristaltic pump speed of 50 rpm. A total of 12.76 kg of dry powder was obtained.

[0042] 1.1.4 Flavor correction

[0043] Weigh 2.5g of dry powder and weigh 5 portions in total. Weigh 10g, 12.5g, 15g, and 17.5g of honey powder according to the ratio of 1:4, 1:5, 1:6, and 1:7 respectively.

[0044] ①2.5g dry powder + 10g honey powder

[0045] ②2.5g dry powder + 12.5g honey powder

[0046] ③2.5g dry powder + 15g honey powder

[0047] ④2.5g dry powder + 17.5g honey powder

[0048] ⑤2.5g dry powder

[0049] Add 100mL of water respectively and stir until dissolved. There should be no wall adhesion, precipitation or lumps.

[0050] Table 2 Taste evaluation of 20 tasters on different ratios of dry powder and honey powder

[0051] Table 3 Taste evaluation of dry powder with different ratios and some other fruit powders

[0052] Taking into account the amount of auxiliary materials, daily consumption and other factors, 4 times the amount of compound honey powder was selected as the auxiliary material.

[0053] 1.1.5 Granulation

[0054] The total weight of the drug powder obtained in the preliminary test was 12.76 kg, and the compound honey powder was 4 times the drug weight, or 51.04 kg. After thoroughly mixing the drug and compound honey powder, totaling 63.8 kg, the powder was granulated in three steps. Because granulation may result in ungranulated powder or excessive adhesion leading to agglomeration of particles, the resulting granules need to be sieved. The resulting granules were passed through 20-mesh and 80-mesh vibrating sieves, respectively. The particles between 20-80 mesh were the target particles, totaling 57.12 kg.

[0055] 1.2 Preparation of capsules

[0056] The pharmaceutical composition is as follows

[0057] Table 4

[0058] The extract is weighed according to the above prescription, and supercritical extraction or microwave extraction is performed to obtain the extract, and then the prescribed amount of microcrystalline cellulose and starch are added and mixed evenly, granulated, dried, granulated, and filled into capsules.

[0059] 1.3 Preparation of extract

[0060] The pharmaceutical composition is as follows

[0061] Table 5

[0062] The extract is weighed according to the above prescription, and supercritical extraction or microwave extraction is performed to obtain an extract, and the obtained extract is concentrated to a relative density of 1.20-1.30 to obtain an extract.

[0063] 2. Pharmacodynamic Evaluation and Component Analysis of Granules (Example 1)

[0064] 2.1 Pharmacodynamic evaluation

[0065] 2.1.1 Mouse small intestinal motility experiment

[0066] ① Experimental groups:

[0067] Sixty mice were randomly divided into a blank group, a model group, a Ma Ren Wan group, and high-, medium-, and low-dose Compound Ma Ren Granule groups, with 10 mice in each group. The high-dose group received 12.8 g / kg (10.6 times the human dose), the medium-dose group received 6.4 g / kg (5.3 times the human dose), and the low-dose group received 3.2 g / kg (2.65 times the human dose). The Ma Ren Wan group received 6.4 g / kg of Ma Ren Wan (10.6 times the human dose) by oral gavage.

[0068] ② Experimental methods

[0069] The mice were gavaged once daily for 14 days, with a volume of 0.2 mL / 10 g / body weight per gavage. The model control group and the blank group were gavaged with distilled water, while the other groups were gavaged with the corresponding doses of the test substance. During this period, mice were allowed to eat and drink freely, and their body weights were recorded weekly. After gavage on the 14th day, the mice in the six groups were fasted for 24 hours. On the 15th day, the blank control group was gavaged with an equal volume of distilled water, while the model group, the Ma Ren Wan group, and the low-, medium-, and high-dose Runchang Tongbian Fang groups were gavaged with compound diphenoxylate (4 mg / kg). 30 minutes later, the low-, medium-, and high-dose Fufang Ma Ren Granule groups and the positive control group were gavaged with Chinese ink containing the corresponding test substance. The model control group and the blank group were gavaged with an equal volume of Chinese ink. Twenty-five minutes later, the animals were immediately sacrificed by cervical dislocation. The abdominal cavity was opened and the mesentery was isolated. The intestinal loops were cut from the pylorus at the upper end to the ileocecal region at the lower end. The small intestine was placed on a tray and gently pulled into a straight line. The total length of the small intestine was measured, and the distance from the pylorus to the ink front was measured as the ink propagation length. Calculate the ink advancement rate.

[0070] Ink propulsion rate (%) = ink propulsion length (cm) / total length of small intestine (cm) × 100%

[0071] ③Experimental results

[0072] Table 6 Results of the charcoal-powder propulsion experiment on mice with Fufang Maren granules Note: * indicates significant difference compared with the model group

[0073] The ink propulsion rates of mice in the low-, medium- and high-dose Compound Ma Ren Granule and Ma Ren Pill groups were all higher than those in the constipation model group. Among them, there were significant differences between the positive control group, the high-dose Compound Ma Ren Granule group and the model control group (P < 0.05), indicating that Compound Ma Ren Granule has a certain effect in improving constipation.

[0074] 2.1.2 Time of first black stool excretion and stool characteristics of mice

[0075] ① Experimental grouping

[0076] Sixty mice were randomly divided into a blank group, a model group, a Ma Ren Wan group, and high-, medium-, and low-dose Compound Ma Ren Granule groups, with 10 mice in each group. The high-dose group received 12.8 g / kg (10.6 times the human dose), the medium-dose group received 6.4 g / kg (5.3 times the human dose), and the low-dose group received 3.2 g / kg (2.65 times the human dose). The Ma Ren Wan group received 6.4 g / kg of Ma Ren Wan (10.6 times the human dose) by oral gavage.

[0077] ② Experimental methods

[0078] The mice were gavaged once daily for 14 days, with a volume of 0.2 mL / 10 g·BW per gavage. The model control group and the blank group were gavaged with distilled water, while the other groups were gavaged with the corresponding dose of the test substance. During this period, mice were allowed to eat and drink freely, and their body weights were recorded weekly. After gavage on the 14th day, the mice in the six groups were fasted for 24 hours, but not water. On the 15th day, the blank group was gavaged with an equal volume of distilled water, while the model control group, the Ma Ren Wan group, and the low-, medium-, and high-dose Runchang Tongbian Fang groups were gavaged with compound diphenoxylate (4 mg / kg). Thirty minutes later, the low-, medium-, and high-dose Runchang Tongbian Fang groups and the positive control group were gavaged with Chinese ink containing the corresponding test substance, while the model control group and the blank group were gavaged with an equal volume of Chinese ink. The timing was started simultaneously. Each mouse was housed individually in a cage with a piece of white paper placed inside. Mice in each group resumed normal food and water intake. The time was counted from the time of ink injection, and the time of the first black stool of each mouse was recorded. The fecal characteristics including fecal volume, fecal weight, fecal water content and state were observed within 5 hours.

[0079] Experimental results: see Figure 7.

[0080] The time to first defecation and stool weight of mice in the model group were significantly different from those in the blank group (P < 0.01), indicating successful modeling. The time to first defecation of mice in the low-, medium-, and high-dose Fufang Maren Granule and Maren Pill groups was shorter than that in the constipation model group. There was a significant difference between the positive control group and the model control group (P < 0.01), and a significant difference between the medium- and high-dose groups and the model control group (P < 0.05). The stool weight of mice in the low-, medium-, and high-dose groups and the positive control group was higher than that in the constipation model group. There was a significant difference between the medium-, high-dose groups and the positive control group and the model group (P < 0.01). The stool quantity of mice in the low-, medium-, and high-dose groups and the positive control group was greater than that in the constipation model group. There was a significant difference between the positive control group and the high-dose laxative group and the model control group (P < 0.05). These experimental results also indicate that Fufang Maren Granule has a certain effect on improving constipation.

[0081] 2.1 Component analysis method

[0082] 2.1.1 Chemical composition identification

[0083] This application uses UPLC-QTOF-MS / MS technology to analyze the components of one dosage form of this pharmaceutical composition. UPLC-QTOF-MS / MS technology, short for ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry (UPLC-QTOF-MS), offers advantages such as high sensitivity, high resolution, high accuracy, and a wide scanning range. It can provide accurate fragment ion information for compounds, providing an excellent technical means for multi-index component analysis of compound preparations.

[0084] 2.1.1.1 Sample preparation method

[0085] Grind the compound hemp seed granules, accurately weigh 1 g, add 5 mL of 75% methanol, ultrasonicate for 30 minutes, let it stand for 24 hours, weigh the mass, and make up the lost mass with 75% methanol. Then centrifuge at 12000 r / min for 10 minutes at 4°C, take the supernatant and pass it through a 0.22 μm microporous filter membrane for later use.

[0086] 2.1.1.2 UPLC-QTOF-MS / MS detection conditions

[0087] ①Chromatographic conditions

[0088] An ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) was used; the mobile phase consisted of 0.05% formic acid in water (A) and acetonitrile (B); the elution gradient was as follows: 0–3 min, 2%–5% B; 3–15 min, 5%–15% B; 15–20 min, 15%–30% B; 20–24 min, 30%–30% B; 24–28 min, 30%–80% B; 28–31 min, 80%–90% B; 31–35 min, 90%–98% B; 35–35.1 min, 98% B–2%; 35.1–38 min, 2%–2% B; the flow rate was 0.2 mL min-1, the column temperature was 35°C, and the injection volume was 2 μL.

[0089] ②Mass spectrometry conditions

[0090] Data acquisition was performed in full-scan automatic mode using an electrospray ionization (ESI) source in both positive and negative ion modes over the m / z range of 50 to 1000. In positive ion mode, the spray gas (GS1) was 55 psi, the auxiliary heater gas (GS2) was 55 psi, the curtain gas (CUR) was 35 psi, the temperature (TEM) was 500°C, the ionization voltage (ISVF) was 4500 V, the declustering voltage (DP) was 60 eV, and the collision voltage (CE) was set to 10 eV in TOF-MS mode. In IDA-MS / MS, the CE was set to 35 eV, and the collision voltage difference (CES) was set to 15 eV. In the negative ion mode, the settings of GS1, GS2, CUR, and TEM were the same as those for the positive ion mode, with an ionization voltage (ISVF) of -4500 V, a declustering voltage (DP) of -60 eV, a collision voltage (CE) of -10 eV in TOF-MS mode, and a CE of -35 eV and a collision voltage difference (CES) of 15 eV under IDA-MS / MS conditions.

[0091] 2.1.1.3 Chemical composition identification

[0092] A database for Fufang Maren Granules was established using online databases such as the TCMSP database, the Chinese Academy of Sciences Chemical Database, and ChemSpider, combined with relevant domestic and international literature. After mass spectrometry data acquisition, data were processed using PeakView software from AB. The database, containing chemical names and molecular formulas, was imported into the XIC Manager form to screen for target compounds. Retention times, accurate relative molecular masses, adducted ion information, and secondary ion fragmentation data were obtained. Chemical components were identified and verified based on relevant literature reports.

[0093] 2.2 Network pharmacology prediction analysis

[0094] Network pharmacology integrates multiple disciplines, including systems biology, pharmacology, information networks, and computer science, to construct a visual "drug-target-gene-disease" network to study the relationship between drugs and diseases and predict their mechanisms of action. Therefore, this application uses UPLC-QTOF-MS / MS technology to identify the chemical components of Compound Ma Ren Granules and, combined with network pharmacology, provides an intuitive and systematic explanation of the effect of Compound Ma Ren Granules on constipation, hoping to provide a research foundation and research ideas for further in-depth research on the mechanism of action of Compound Ma Ren Granules in improving constipation.

[0095] 2.2.1 Acquisition of chemical component targets and constipation-related disease targets

[0096] The SMILES numbers of the 80 identified chemical components were imported into the Swiss target prediction database, resulting in 862 component targets. Using "constipation" as the disease keyword, the Disgenet and Genecards databases were searched, and the results were merged and duplicates removed to obtain 5,333 constipation disease targets.

[0097] 2.2.2 Protein-protein interaction (PPI) network construction

[0098] Using Venny software, the drug ingredient targets of Fufang Ma Ren Granule were intersected with constipation targets, resulting in 491 intersection targets. A Venn diagram was then constructed, as shown in Figure 2. The resulting intersection targets were entered into the STRING online database, with the species defined as "Homo sapiens" and the score set to the highest confidence level (0.900) to construct a PPI network and establish the interaction relationships between targets. The PPI network was analyzed using Cytoscape software, and its node degrees (Degree) were calculated. A higher degree indicates a greater contribution of the corresponding protein. Using a screening criterion of twice the median Degree value, the core target network was extracted, resulting in 117 core targets.

[0099] 2.2.3 Pathway enrichment analysis

[0100] The 117 core targets were entered into the DAVID database, with "Homo sapiens" selected for both "list" and "background" for GO and KEGG analyses. GO enrichment analysis primarily encompassed three components: biological process, cellular component, and molecular function. Using a P < 0.05 screening criterion, 705 entries were included for biological process, 90 entries for cellular component, and 148 entries for molecular function. These included protein phosphorylation, transmembrane receptor protein tyrosine kinase activity, intracellular signal transduction, positive regulation of MAPK cascades, positive regulation of ERK1 and ERK2 cascades, and protein serine / threonine kinase activity. The top 15 entries or pathways were selected and imported into the Microbiome Informatics Online platform for visualization analysis, as shown in Figure 3.

[0101] Using P < 0.05 as the screening criterion, KEGG pathway enrichment yielded a total of 168 pathways, primarily involving the MAPK signaling pathway, ErbB signaling pathway, PI3K-Akt signaling pathway, Ras signaling pathway, colorectal cancer, and human cytomegalovirus infection. The top 15 entries or pathways were selected and imported into the Microbiology Online platform to create a bubble plot, as shown in Figure 4. The vertical axis represents the enrichment name, and the horizontal axis represents -log10 (pvalue). Larger bubbles indicate more genes in the pathway, and darker bubbles indicate a more significant correlation between the pathway and the gene. These results suggest that Fufang Ma Ren Granules can improve constipation by regulating multiple biological processes and acting on multiple targets.

[0102] 2.2.4 Construction of component-core target network diagram and heat map of key target protein tissue distribution

[0103] Cytoscape software was used to associate the components and core targets, resulting in a component-core target network as shown in Figure 5. Figure 5 contains 185 nodes, including 63 chemical components and 121 targets. On average, each chemical component corresponds to 16.3 targets. Nineteen components had Dgree values ​​greater than three times the median Dgree value, including N-trans-p-coumaryltyramide, methyl 4-[[2-[3-[4-(2-methoxyphenyl)piperazin-1-yl]pyrazin-2-yl]sμLfanylacetyl]amino]benzoate, and N-((2S,3S,5R)-2-amino-6-(butylamino)-3-hydroxy-5-methyl-6-oxohexyl)-N-isopropyl-4-methoxy-3-(3-methoxypropoxy)benzam The chemical components included 1,4-dihydro-1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, 1,4-diol, The results of the ingredient-core target network diagram show that the mechanism of action of Compound Ma Ren Granules in improving constipation is through multiple ingredients and multiple targets, which coincides with the "holistic view" of traditional Chinese medicine in treating diseases.

[0104] The Human Protein Atlas database was used to search for seven targets, including EGFR, CDK1, TERT, NOS2, MMP2, ESR1, and TNF. Protein expression levels for each target in the colon, rectum, small intestine, and stomach were collected, and a heat map of key target protein expression was plotted, as shown in Figure 6. The figure shows that all seven targets are expressed in four tissues, with the highest expression in the small intestine. From a target perspective, MMP2 has the highest protein expression among the four tissues. This result indirectly suggests that the mechanism of action of Fufang Maren Granule in improving constipation may be through its effects on the small intestine.

[0105] 2.3 Rat metabolomics study

[0106] ① Experimental grouping

[0107] Thirty-nine male Sprague-Dawley rats, weighing 180-200 g, were provided by the Experimental Animal Center of Nantong University (SPF) under license number SCXK(Su)2019-0001. They were housed at a temperature of 22 ± 4°C and a relative humidity of 55 ± 15%. The animal experiments were approved by the Ethics Committee of Jiangsu Provincial Hospital of Traditional Chinese Medicine (approval number: 2021DW-13-01).

[0108] ② Experimental methods

[0109] Thirty-nine rats were divided into a blank group, a model group, a positive control group (Ma Ren soft capsule group), and a low-dose and high-dose group of Compound Ma Ren Granules. The blank group had 7 rats, and the other groups had 8 rats. After 3 days of adaptive feeding, the blank group received distilled water orally, while the other groups received compound diphenoxylate (8 mg / kg) orally. This process continued for 14 days. The rats' living conditions and hair coat were observed. After the 14th day of gavage, the rats were placed in metabolic cages to collect 24-hour feces, and the feces quantity and weight were recorded. A statistically significant difference in 24-hour feces quantity and weight between the blank and model groups indicated successful modeling. After successful modeling, the blank group continued to be gavaged with distilled water, the model group was first gavaged with compound diphenoxylate (8 mg / kg) 1 hour later and then with distilled water, the positive control group was first gavaged with compound diphenoxylate (8 mg / kg) 1 hour later and then with Ma Ren soft capsule suspension (0.36 g / kg), the high-dose compound Ma Ren granule group was first gavaged with compound diphenoxylate (8 mg / kg) 1 hour later and then with compound Ma Ren granule suspension (4.8 g / kg), and the low-dose group was first gavaged with compound diphenoxylate (8 mg / kg) 1 hour later and then with compound Ma Ren granule suspension (2.4 g / kg). During this period, the rats were free to eat and drink, and their body weights were recorded weekly.

[0110] 2.3.1 Rat serum metabolomics study

[0111] ①Biological sample collection and processing

[0112] The rats were given the drug by gavage for one week. After the last administration, orbital blood was collected from the rats. The blood samples were placed at room temperature for 1 hour and centrifuged at 3500 r / min for 10 minutes. The supernatant was centrifuged again at 14000 r / min for 10 minutes at 4°C. The supernatant was collected, packaged, and stored in a -80°C refrigerator for later use.

[0113] ② Serum sample preparation

[0114] Take the serum sample from -80℃ freezer and thaw it at room temperature. Take 300μL serum sample in 2mL centrifuge tube, add 900μL acetonitrile, vortex for 1min, and centrifuge at 12000r·min at 4℃. -1 Centrifuge for 10 minutes, take the supernatant, transfer it to a 2 mL centrifuge tube, blow it to dryness with nitrogen at room temperature. Add 100 μL of acetonitrile to the residue, vortex for 1 minute, and rotate at 12000 r / min at 4 °C. -1 Centrifuge for 10 minutes and collect the supernatant for testing. Take 20 μL of each serum sample and mix them to obtain a quality control (QC) sample, and prepare the sample according to the above method.

[0115] ③Chromatographic and mass spectrometry conditions

[0116] ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm); mobile phase: 0.05% formic acid aqueous solution (A)-acetonitrile (B); flow rate: 0.3 mL·min-1, column temperature: 35°C, injection volume: 2 μL; gradient: as shown in the table below.

[0117] Table 7 Chromatographic gradient table

[0118] Data acquisition was performed in full scan automatic mode, electrospray ionization source (ESI), positive and negative ion mode scanning, scanning range m / z 50-1000, and mass spectrometry conditions are shown in the following table.

[0119] Table 8 Mass spectrometry conditions

[0120] ④Data processing

[0121] UPLC-Q-TOF-MS / MS technology was used to collect data from the serum of each group of rats in positive and negative ion modes. After data preprocessing, the obtained data were imported into MetaboAnalyst 5.0 online software was used to compare the difference fold (FC) and P value between the model group and the blank group, and between the model group and the high-dose group. FC>1.2 and P<0.05 or FC<0.8333 and P<0.05 were used as screening conditions. A total of 16 differential metabolites were identified in the two ion modes, including proline, cyclo(L-proline-L-valine), propionylcarnitine, nonylarabinofuranosyl glucoside, succinyldisalicylic acid, 7a,12a-dihydroxy-3-oxo-4-cholic acid, 3a,6b,7b,12a-tetrahydroxy-5b-cholic acid, α-methyl-metatyrosine, 12alpha-hydroxy-13,18-dehydrodopamine, acetylpterin C, mannose L, LacCer (d18:1 / 12:0), 1-β-hydroxycholic acid, MG compounds, LysoPc compounds, etc. The specific information is shown in Table 3-3. Compared with the blank group, 6 differential metabolites were upregulated in the model group and downregulated after drug treatment; 10 differential metabolites were first downregulated in the model group and upregulated after drug treatment.

[0122] Table 9 Endogenous differential metabolites in serum Note: NC is the blank group; MOD is the model group; HD is the high-dose group; ↑ indicates upregulation; ↓ indicates downregulation

[0123] ⑤ Differential metabolite pathway enrichment analysis

[0124] The 17 differential metabolites were imported into MetaboAnalyst 5.0 online software for KEGG pathway analysis, and a total of 6 metabolic pathways were obtained, mainly involving biological pathways such as glycosphingolipid biosynthesis-globular and heteroglobular series, glycosphingolipid biosynthesis-ganglionic series, sphingomyelin metabolism, arginine and proline metabolism, glycosphingolipid biosynthesis-lactose and neolactone series, and aminoacyl-tRNA biosynthesis.

[0125] Serum metabolomics results showed that Fufang Ma Ren Granule has a regulatory effect on the metabolism of rats with STC constipation. The differential metabolites screened mainly include amino acids, carnitine, glyceride compounds, bile acid, and lysophosphatidylcholine (LysoPC). PC (phosphatidylcholine) and LysoPC (lysophosphatidylcholine) are both endogenous lipid molecules. Studies have shown that both are associated with inflammation. Decreased PC content in intestinal mucus can lead to a breakdown of the mucus barrier, bacterial invasion, and intestinal inflammation. PC can reduce the secretion of TNF-α and is accompanied by the inactivation of NF-κB, thereby inhibiting the upregulation of proinflammatory factors induced by TNF-α. Under inflammatory conditions, PC can be hydrolyzed to LysoPC by phospholipase A2, so LysoPC can be considered a biomarker.

[0126] Carnitine is an amino acid found widely in the liver and primarily in the mitochondria of muscle cells. It serves as a transporter for fatty acids and plays a crucial role in the oxidation of fatty acids into ATP. Carnitine deficiency can lead to gastrointestinal smooth muscle motility disorders, which in turn can cause constipation. Metabolic results also indicate that propionylcarnitine levels decreased in modeling rats. Carnitine deficiency can lead to gastrointestinal motility disorders and constipation, but propionylcarnitine levels recovered after treatment.

[0127] Serum metabolic pathway enrichment analysis primarily involved six metabolic pathways: glycosphingolipid biosynthesis - globular and heteroglobular series, glycosphingolipid biosynthesis - ganglionic series, sphingomyelin metabolism, arginine and proline metabolism, and glycosphingolipid biosynthesis - lactose and neolactone series. Amino acids are the basis for the synthesis of numerous proteins involved in rapid epithelial cell turnover and mucin production. They not only improve intestinal barrier function and the expression of anti-inflammatory cytokine-related proteins, but also reduce oxidative stress, intestinal cell apoptosis, and the expression of proinflammatory cytokines in intestinal inflammation. Arginine is the sole substrate in the L-Arg / NO (arginine / nitric oxide) pathway. Arginine utilizes molecular oxygen to synthesize NO under the action of endothelial nitric oxide synthase. NO is an inhibitory transmitter of non-adrenergic and non-cholinergic nerve endings in the gastrointestinal tract, hyperpolarizing smooth muscle cell membranes and generating inhibitory junction potentials, thereby inhibiting gastrointestinal motility.

[0128] 2.3.2 Rat urine metabolomics study

[0129] ①Biological sample collection and processing

[0130] The drug was administered by gavage for one week. After the last administration, the rats were placed in metabolic cages to collect urine and feces for 24 hours, which were then packaged and stored in a -80℃ refrigerator for later use.

[0131] ② Urine sample preparation

[0132] Take the urine sample from the -80℃ refrigerator and thaw it at room temperature. Take 300μL of urine sample in a 2mL centrifuge tube, add 900μL of acetonitrile, vortex for 1min, and centrifuge at 12000r·min at 4℃. -1 Centrifuge for 10 minutes, take the supernatant, transfer it to a 2 mL centrifuge tube, blow it to dryness with nitrogen at room temperature. Add 100 μL of acetonitrile to the residue and re-dissolve it. -1 Centrifuge for 10 minutes and collect the supernatant for testing. Take 50 μL of each urine sample and mix them to obtain quality control (QC) samples. Prepare QC samples according to the above method.

[0133] ③Chromatographic and mass spectrometry conditions

[0134] Chromatographic conditions were the same as those for serum metabolomics.

[0135] ④Data processing

[0136] UPLC-Q-TOF-MS / MS technology was used to collect data on the urine of rats in each group in positive and negative ion modes. After preprocessing, the data were imported into MetaboAnalyst 5.0 online software. The difference fold (FC) and P value between the model group and the blank group, and between the model group and the high-dose group were compared. FC>1.2 and P<0.05 or FC<0.8333 and P<0.05 were used as screening conditions. A total of 20 differential metabolites were identified in the two ion modes, mainly including L-valine, dihydromethylcystine, isophthalic acid, phosphatidylcholine (18:1-16:1), xanthine acid, methionine sulfone, N-carboxymethylaspartic acid, 2-acetaminophenol, pyrimidine, deoxyguanosine, glycine, lactitol, prostaglandin A2, hexadecanoic acid, sphingomyelin SM (18:1 / 18:00), palmitic acid, stearic acid, acetyl phosphate, etc. The specific information is shown in Tables 3-4. Compared with the blank group, 15 differential metabolites including L-valine, dihydromethylcystine, prostaglandin A2, sphingolipids (18:1 / 18:00), and xanthine acid were upregulated in the model group and downregulated after treatment; 5 differential metabolites including phosphatidylcholine (18:1 / 16:1), deoxyguanosine, N-carboxymethylaspartic acid, methionine sulfone, and stearic acid were first downregulated in the model group and then upregulated after treatment.

[0137] Table 10 Endogenous metabolites in urine Note: NC is the blank group; MOD is the model group; HD is the high-dose group; ↑ indicates upregulation; ↓ indicates downregulation

[0138] ⑤ Differential metabolite pathway enrichment analysis

[0139] The 20 differential metabolites were imported into MetaboAnalyst 5.0 online software for KEGG pathway analysis, and a total of 8 metabolic pathways were obtained. The metabolic pathways mainly involved valine, leucine and isoleucine biosynthesis, pantothenate and CoA biosynthesis, sphingomyelin metabolism, pyruvate metabolism, unsaturated fatty acid biosynthesis, valine, leucine and isoleucine degradation, aminoacyl tRNA biosynthesis and purine metabolism.

[0140] Analysis of urine metabolomics results showed that the 20 differential metabolites screened out mainly included amino acids, prostaglandin A2 (PGA2), fatty acids, phosphatidylcholine (PC), sphingomyelin (SM), acetyl phosphate and other compounds. PGA2, as an inflammatory factor, was upregulated in the model group. This may be because when constipation occurs, gastrointestinal motility slows down, water reabsorption increases, and stool accumulates in the intestine for a long time, which causes changes in the intestinal microbiota and leads to the occurrence of intestinal inflammation. At the same time, PG, as an unsaturated fatty acid, can cause smooth muscle contraction and protect the gastric mucosa, small intestine and colon, among other physiological effects. PC is an amphiphilic molecule and an important component of cell membranes and lung surfactants. It also plays an important role in signaling pathways. Studies have shown that PC can effectively treat ulcerative colitis. Experimental results showed that PGA2 was upregulated in rats undergoing intestinal inflammation, confirming the occurrence of intestinal inflammation. PC (18:1 / 16:1) can be hydrolyzed to LysoPC by phospholipase A2 under inflammatory conditions. PC (18:1 / 16:1) levels were restored after treatment. L-valine is a branched-chain amino acid. Elevated serum concentrations of branched-chain amino acids activate the NF-κB pathway, promoting oxidative stress and inflammation, and exacerbating constipation.

[0141] Metabolic pathways enriched by urine metabolomics include those involved in amino acid biosynthesis and degradation, pantothenate and CoA biosynthesis, sphingomyelin metabolism, pyruvate metabolism, and unsaturated fatty acid biosynthesis. Pyruvate, a three-carbon ketoacid produced in the body and the final product of glycolysis, can be reduced to lactate to provide energy or oxidized into acetyl-CoA within the mitochondria, entering the tricarboxylic acid cycle. Therefore, pyruvate plays a pivotal role in the metabolic connection between the three major nutrients: sugar, fat, and amino acids. Compared to the pathways enriched by serum metabolomics, the pathways in the urine metabolome also involve amino acid biosynthesis and sphingomyelin metabolism.

[0142] 2.3.3 Rat fecal metabolomics study

[0143] ①Biological sample collection and processing

[0144] The drug was administered orally for one week. After the last administration, the rats were placed in metabolic cages to collect feces for 24 hours, which were then packaged and stored in a -80°C refrigerator for later use.

[0145] ② Preparation of stool samples

[0146] Take a stool sample from a -80°C refrigerator, thaw it at room temperature, weigh about 0.25 g of stool, grind it in a 2 mL centrifuge tube, add 1.5 mL of acetonitrile, vortex and shake for 3 minutes, ultrasonicate it at room temperature for 30 minutes, and then spin it at 12000 r / min at 4°C. -1 Centrifuge for 15 minutes, take the supernatant, transfer it to a 2 mL centrifuge tube, blow it to dryness with nitrogen at room temperature. Add 200 μL of acetonitrile to the residue, vortex for 1 minute, and rotate at 12000 r / min at 4 °C. -1 Centrifuge for 15 minutes and collect the supernatant for testing. Take 50 μL of the supernatant from each stool sample and mix them to obtain the quality control (QC) sample.

[0147] ③Chromatographic and mass spectrometry conditions

[0148] An ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) was used; the mobile phase was 0.05% formic acid in water (A)-acetonitrile (B); the flow rate was 0.2 mL min-1, the column temperature was 35°C, and the injection volume was 2 μL. The gradient is shown in the table below.

[0149] Table 11 Chromatographic gradient table

[0150] Data acquisition was performed in full-scan automatic mode, using an electrospray ionization (ESI) source, scanning in positive and negative ion modes, with a scan range of m / z 50 to 1000. The mass spectrometry conditions were set as shown in the table below.

[0151] Table 12

[0152] ④Data processing

[0153] UPLC-Q-TOF-MS / MS technology was used to collect data on the urine of rats in each group under positive and negative ion modes. The data were imported into MetaboAnalyst 5.0 online software. The difference fold (FC) and P value between the model group and the blank group, and between the model group and the high-dose group were compared. FC>1.2 and P<0.05 or FC<0.8333 and P<0.05 were used as screening criteria. A total of 13 differential metabolites were identified under the two ion modes, mainly including D-xylose, oxypurinol, succinic acid, 5,8,12-trihydroxydodecanoic acid, isocitric acid, sebacic acid, SM (d18:1 / 18:0), MG (14:1 / 0:0), LysoPC (16:0 / 0:0), 12-HEPE, PG (18:0), glycyrrhetinic acid, and adrenic acid. The specific information is shown in the table below. Compared with the blank group, four differential metabolites were upregulated in the model group and downregulated after drug treatment, namely isocitric acid, SM (d18:1 / 18:0) and PG (18:0), and glycyrrhetinic acid; nine differential metabolites, including D-xylose, oxypurinol, succinic acid, 5,8,12-trihydroxydodecanoic acid, sebacic acid, MG (14:1 / 0:0), LysoPC (16:0 / 0:0), and 12-HEPE, were first downregulated in the model group and then upregulated after drug treatment.

[0154] Table 13 Differential metabolites in feces Note: NC is the blank group; MOD is the model group; HD is the high-dose group; ↑ indicates upregulation; ↓ indicates downregulation

[0155] ⑤ Differential metabolite pathway enrichment analysis

[0156] The 13 differential metabolites were imported into MetaboAnalyst 5.0 online software for KEGG pathway analysis, and a total of 6 metabolic pathways were obtained. The metabolic pathways mainly involved butyrate metabolic pathway, pentose and gluconic acid interconversion pathway, citrate cycle (TCA cycle), sphingomyelin metabolic pathway, propionate metabolic pathway and alanine, aspartate and glutamate metabolic pathways.

[0157] By performing metabolomics analysis on feces, using Metaboanalyst online software and the HMDB database, a total of 13 differential metabolites were screened, mainly involving endogenous metabolites such as D-xylose, oxypurinol, SM (d18:1 / 18:0), MG (14:1 / 0:0), LysoPC (16:0 / 0:0), PG (18:0), and adrenaline acid; pathway enrichment analysis obtained six metabolic pathways, including butyrate metabolic pathway, pentose and gluconic acid mutual conversion pathway, citrate cycle (TCA cycle), sphingomyelin metabolic pathway, propionate metabolic pathway, and alanine, aspartate and glutamate metabolic pathways.

[0158] SM (d18:1 / 18:0) was detected in both urine and feces and was upregulated in the model group compared with the blank group. Sphingomyelin (SM) is a type of sphingolipid compound with properties similar to phosphatidylcholine (PC), both of which are important components of cell membranes. Sphingomyelin metabolism is mainly carried out by sphingomyelinase, of which alkaline sphingomyelinase (alk-SMase) is mainly distributed in the intestine and bile and is only expressed in the intestinal mucosa and liver. Alk-SMase can not only hydrolyze sphingomyelin, but also PC, LysoPC and platelet-activating factor (PAF). SM is mainly derived from diet and desquamated intestinal mucosal cells. When constipation occurs, stool accumulates in the intestine, making defecation difficult. Therefore, the levels of SM (d18:1 / 18:0) in the urine and feces of rats in the model group were upregulated. The occurrence of constipation is often accompanied by intestinal inflammation. PG (18:0), as an inflammatory factor, is upregulated in the model group, which indirectly confirms this.

[0159] Short-chain fatty acids (SCFA), also known as volatile fatty acids, are metabolites produced by intestinal anaerobic bacteria acting on dietary fiber. They mainly include acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid. They play an important role in maintaining the normal function of the large intestine and the morphology and function of colonic epithelial cells. SCFA not only serve as the main energy source for intestinal mucosal cells in the colon lumen, but also reduce the production of pro-inflammatory factors and reduce the occurrence of intestinal inflammation. Among them, butyric acid can promote intestinal ion absorption and is of great significance for maintaining intestinal mucosal homeostasis. In addition, L-glutamate can act in the colon as a precursor of butyrate and acetate in intestinal microorganisms, and affect the expression of amino acid receptors and transporters in the jejunum.

[0160] In summary, Fufang Ma Ren Granules may improve constipation through multiple metabolic pathways, including amino acid metabolic pathways, short-chain fatty acid metabolic pathways, lipid metabolic pathways, and energy metabolic pathways.

[0161] 3. Clinical effect evaluation

[0162] 3.1 Case Source

[0163] Jiangsu Provincial Hospital of Traditional Chinese Medicine

[0164] 3.2 Western medicine diagnostic criteria

[0165] ① Must include 2 or more of the following

[0166] a. More than 1 / 4 of bowel movements feel strained

[0167] b. More than one-quarter of bowel movements are dry or hard stools (Bristol Stool Consistency Scale Type 1-2)

[0168] c. Feeling of incomplete bowel movements for more than 1 / 4 of the time

[0169] d. More than 1 / 4 of bowel movements experience anorectal obstruction / blockage

[0170] e. More than 1 / 4 of bowel movements require manual assistance

[0171] f. Spontaneous bowel movements less than 3 times per week

[0172] ②Rarely loose stools occur when laxatives are not used

[0173] Symptoms must have occurred for at least 6 months before diagnosis and must have met the diagnostic criteria for more than 3 months.

[0174] 3.3 Inclusion criteria

[0175] 1. Meet the Western medical diagnosis of functional constipation

[0176] 2.18 years old ≤ age < 75 years old

[0177] 3. Sign the informed consent form and voluntarily participate in this trial

[0178] 3.4 Exclusion criteria

[0179] 1. History of allergy to traditional Chinese medicine or its main ingredients, or drug-induced liver injury

[0180] 2. Unwilling to accept Chinese medicine treatment

[0181] 3. Patients diagnosed with intestinal stenosis caused by organic lesions of the rectum or colon (such as tumors, Crohn's disease, colon polyps, intestinal tuberculosis)

[0182] 4. Patients with serious primary diseases of the heart, liver, kidney and hematopoietic system, or mental illness

[0183] 5. Patients who are participating in clinical trials of other drugs

[0184] 3.5 Research Methods

[0185] Basic treatment and other treatment instructions: routine treatment of constipation

[0186] Treatment group: Fufang Maren Granules, 2-3 times a day, 4 packets each time, taken with hot water

[0187] Control group: conventional treatment for constipation

[0188] Treatment course: 14 days

[0189] The applicant refers to Example 1 and conducts the following debugging scheme for the dosage of the formula:

[0190] Table 14

[0191] 3.6 General Information

[0192] Subject's name, hospital number, trial start date, demographic characteristics: gender, age, height, weight

[0193] Vital signs: temperature, resting heart rate, respiration, blood pressure (systolic and diastolic) after 10 minutes of rest

[0194] 3.7 Observation indicators

[0195] Main efficacy indicators: defecation status, constipation symptom assessment form

[0196] Secondary efficacy indicators: presence of shortness of breath, fatigue, nausea and vomiting, dizziness, headache, diarrhea and abdominal pain, etc.

[0197] 3.8 Data Processing

[0198] SPSS statistical software was used for analysis, and P < 0.05 was used to determine whether the difference was statistically significant.

[0199] Table 15

[0200] Application Example 1

[0201] Admission conditions (brief medical history, positive physical signs, relevant laboratory and instrumental examination results):

[0202] On March 14, 2019, the patient underwent laparoscopic left hemicolectomy, laparoscopic right hemicolectomy, rectal anastomosis, and loop ileostomy at our hospital under general anesthesia for intestinal obstruction. Postoperative pathology: 1. Intestinal mucosal tissue showed chronic inactive enteritis, with dilated and congested submucosal blood vessels, local lymphoid tissue hyperplasia with lymphoid follicle formation, and "intestinal tubes" indicating chronic inactive enteritis. 2. No abnormalities were found in the upper and lower resection margins or omental tissue of both intestinal segments. 3. Chronic appendicitis with local lumen atresia. 4. 16 perienteric lymph nodes showed reactive hyperplasia. On September 24, 2019, the patient underwent laparoscopy, enterostomy closure, partial small bowel resection, end-to-side small bowel anastomosis, and intestinal adhesion lysis under general anesthesia. One year after surgery, the patient developed difficulty defecating, with soft stools every 2-3 days. Constipation recurred, with significant abdominal distension. The patient was admitted to our department for further treatment for constipation. On admission, the patient's spirits remained normal, but his physical strength declined, his appetite was poor, his abdominal distension was intermittent, his stools were difficult to pass, and he had one stool every 2-3 days. He slept normally, had significant weight loss, and had difficulty urinating. Physical examination revealed a flat abdomen with normal abdominal breathing. No varicose veins were observed. A 5-cm surgical scar was present in the right lower abdomen. The abdomen was soft, tender beneath the xiphoid process, without rebound tenderness, no fluid wave thrill, no gurgling sound, and no liver or spleen palpable below the ribs. No masses were palpable throughout the abdomen. Murphy's sign was negative. McBurney's point was not tender. The kidneys were not palpable. Percussion revealed a hepatic dullness boundary, negative shifting dullness, and no tenderness. Bowel sounds were normal, with no vascular murmurs.

[0203] Hospitalization process:

[0204] After the patient was admitted to the hospital, relevant examinations were actively completed. Blood routine showed: high-sensitivity C-reactive protein <0.50 mg / l, white blood cell count 5.05×10 9 / L, red blood cell count 4.00×10 12 / L, hemoglobin 129g / L, platelets 175×10 9 / L. Whole abdominal CT scan: 1. Postoperative changes after subtotal colectomy, with structural disorder of the lower abdominal bowel; follow-up with enhanced abdominal CT scan is recommended; 2. Chronic cholecystitis; multiple liver cysts; bilateral renal cysts; similar to the previous (2020-09-21); 3. Prostate enlargement with calcification; atherosclerosis of the abdominal aorta and bilateral iliac arteries; and thoracolumbar vertebral degeneration. After confirming the diagnosis of constipation (Qi and blood deficiency syndrome) according to Traditional Chinese Medicine (TCM), constipation according to Western Medicine, and intestinal obstruction, and excluding obvious surgical contraindications, the patient underwent a laparoscopic rectal resection (level 4), laparoscopic enterostomy (level 3), and ileo-rectal anastomosis (level 2) on September 1, 2022. The patient was safely returned to the ward and received symptomatic treatment, including anti-infective, anti-inflammatory, analgesic, fluid, and nutritional support. The constipation symptoms were significantly relieved after taking Compound Ma Ren Granules. The patient is recovering well. Postoperative pathology revealed the following (rectal resection specimen): 1. Chronic inflammation of the intestinal mucosa with localized polypoid hyperplasia, submucosal interstitial edema, and vascular proliferation, dilation, and congestion. 2. No abnormalities were observed at the upper and lower resection margins. 3. Fifteen perienteric lymph nodes were found, demonstrating lymphoid hyperplasia. Auxiliary examinations were unremarkable, and the patient was discharged.

[0205] Discharge status (cured, improved, not cured, not treated, discharged without doctor's advice, other): cured

[0206] The patient is currently in good condition, with stable vital signs, no noticeable incision pain, and can get out of bed freely. The abdominal drainage tube has been removed. He has no chills or fever, no nausea or vomiting, no abdominal pain or bloating, no diarrhea or constipation, no palpitations or chest tightness, no heart palpitations or shortness of breath, and is taking semi-liquid food and sleeping well. His stoma is passing gas and bowel movements, and his urination is normal. He has met the discharge criteria and, after consulting with his superior physician, has been discharged.

[0207] Application Example 2

[0208] Admission conditions (brief medical history, positive physical signs, relevant laboratory and instrumental examination results):

[0209] The patient developed defecation difficulties without obvious cause more than 8 months ago, and the symptoms worsened more than 1 month ago. Each time he had a bowel movement, he had to use a suppository to defecate. The stool was unformed and sticky, and he had to have it once a day, accompanied by abdominal distension in the left lower abdomen, intermittent lower abdominal pain, occasional nausea, no vomiting, no chills or fever, and no black stools or bloody stools. On June 23, 2021, he underwent an upper abdominal CT scan in our hospital. The results showed that the horizontal segment of the duodenum was narrowed, the proximal duodenum was dilated, and the superior mesenteric artery compression syndrome was to be excluded. On June 25, 2021, an upper gastrointestinal tract barium meal angiography was performed in our hospital. The results showed that the intestinal cavity in the horizontal part of the duodenum was slightly dilated, and external pressure was seen below the intestinal cavity. On June 29, 2021, an upper abdominal magnetic resonance imaging scan + MRCP + enhancement was performed in our hospital. The results showed that the proximal lumen of the horizontal segment of the duodenum was dilated, and duodenal congestion and superior mesenteric artery compression syndrome were considered possible. On 2021-07-14 and 2022-02-13, a whole abdominal CT scan with superior mesenteric artery reconstruction and a whole abdominal CT plain scan were performed at Nanjing Drum Tower Hospital. The results suggested that the superior mesenteric artery compression syndrome might be considered. On 2021-09-17 and 2021-09-24, the abdominal artery CTA and upper gastrointestinal tract angiography were performed at the Second Affiliated Hospital of Nanjing Medical University, respectively. The results suggested that duodenal stasis and superior mesenteric artery compression syndrome might be considered. Self-treatment with Chinese medicine, Cistanche oral liquid, hemp seed and other drugs was not effective. For further diagnosis and treatment, the patient came to our outpatient clinic and was later admitted to the hospital for "constipation". At the time of admission, the patient was in good spirits and normal physical strength, with abdominal distension and discomfort in the left lower abdomen, poor appetite, and poor sleep. He had lost nearly 20 kilograms in weight in the past year and had normal urination. Physical examination: The abdomen was flat and slightly concave, with normal abdominal breathing. No abdominal varicose veins were observed. The abdomen was soft and nontender, with no rebound tenderness, fluid wave tremors, or splashing sounds. The liver and spleen were not palpable below the ribs. No masses were palpable throughout the abdomen. Murphy's sign was negative. McBurney's point was nontender. The kidneys were not palpable. Percussion revealed a hepatic dullness border, negative shifting dullness, and no tenderness. Bowel sounds were normal, with no vascular bruits. Rectal examination revealed no significant abnormalities.

[0210] Hospitalization process:

[0211] After admission, relevant tests were completed, and the auxiliary examination results showed: comprehensive urine analysis (urine): white blood cell count 43 / μL↑; blood routine + high-sensitivity C-reactive protein (blood): white blood cell count 3.25×10 9 / L↓, red blood cell count 3.38×10 12 / L↓, monocyte percentage 8.9%↑, absolute neutrophil count 1.82×10 9 / L↓, hematocrit 0.331↓, mean platelet volume 11.2 fL↑; six coagulation test panels (plasma): D-dimer (DD) 0.56 mg / L↑; six TM panels (serum): ferritin 373.50 ng / mL↑; four liver and kidney function and lipid profiles (serum): potassium 3.30 mmol / L↓, serum bicarbonate 20.4 mmol / L↓, uric acid 447 μmol / L↑, phosphorus 0.84 mmol / L↓, low-density lipoprotein cholesterol 1.98 mmol / L↓; four thrombosis test panels (TAT+TM+PIC+t-PAIC) (plasma): thrombin-antithrombin III complex 8.13 ng / mL↑; glycated hemoglobin measurement, pre-transfusion screening, and routine stool analysis were normal. A 12-channel electrocardiogram revealed: 1. sinus rhythm; 2. T wave changes. A high-resolution chest CT scan (HRCT) with reconstruction and a whole-abdomen CT with enhanced contrast revealed: dilatation of the horizontal duodenum, dilatation of the common bile duct and pancreatic duct, and thickening of the proximal jejunum. Further examination is recommended. Multiple cysts are present in the liver and kidneys. Mild dilatation of the bronchi of both lungs, emphysema in both lungs, and multiple small nodules in both lungs. Annual follow-up is recommended. Multiple arteriosclerosis and degenerative changes in the thoracic and lumbar spine are also present. [Barium enema colonography] Examination revealed: significant gas accumulation and dilatation in the ileum, bradyperistalsis, and a large number of fecal stones remaining in the intestinal lumen. Please refer to clinical data. The transverse colon is lengthy. Treatment is comprehensive, including intravenous fluid support, parenteral nutrition, acupuncture, oral administration of Compound Ma Ren Granules, and enema of Red and Yellow Mixture.

[0212] Discharge status (cured, improved, not cured, not treated, discharged without doctor's advice, other): improved

[0213] The patient's general condition is currently good, all vital signs are stable, abdominal distension and constipation have improved compared to before, and there is no chills or fever, nausea or vomiting, no palpitations or chest tightness, no heart palpitations or shortness of breath, etc. At the request of the patient and his family, and after consulting the superior physician, he was discharged from the hospital.

[0214] Application Example 3

[0215] Admission information (brief medical history, positive physical signs, relevant laboratory and instrumental test results): Six months ago, the patient developed difficulty defecating without obvious cause, accompanied by decreased frequency of anal flatus and defecation, nausea, vomiting, acid reflux and belching. During this period, she was diagnosed with constipation (outlet obstruction) at the Affiliated Hospital of Henan University of Traditional Chinese Medicine. On March 31, 2022, a defecography showed: rectocele; prolapse of the anterior rectal wall mucosa; rectal dilatation; anal canal stenosis; and rectal tortuosity. She underwent hemorrhoidectomy, but no significant improvement was seen after the operation. On June 27, 2022, a full abdominal CT scan was performed at the Second Hospital of Jilin University, which showed: thickening of the gallbladder wall, enlargement of the left adrenal gland, suspected redundant sigmoid colon, pelvic effusion, and an intrauterine device. Colonoscopy showed: chronic colitis; changes in the rectal mucosa. No treatment was given during this period. Today, for further diagnosis and treatment, she was admitted to the department with a presumed diagnosis of "constipation" from the outpatient department. On admission, the patient had stopped passing gas or defecation for four days. His spirits were good, his physical strength was normal, his appetite was poor, his sleep was normal, his weight had not changed significantly, and his urination was normal. Physical examination: The abdomen was flat, his abdominal breathing was normal, there were no varicose veins in the abdominal wall, his abdomen was soft, there was no tenderness in the entire abdomen, no rebound tenderness, no fluid wave tremor, no splashing sound, and the liver and spleen were not palpable below the ribs. No masses were palpable in the entire abdomen. Murphy's sign was negative. McBurney's point was not tender. The kidneys were not palpable. Percussion revealed a dullness border in the liver, negative shifting dullness, and no tenderness. Bowel sounds were normal, and no vascular murmurs were heard. Digital rectal examination (left lateral decubitus position): The skin around the anus was intact, without redness, tenderness, or lumps. There were no anal fissures or external hemorrhoids. The digital rectal examination revealed no significant abnormalities.

[0216] Hospitalization process: After admission, the patient underwent active examinations. A CT scan of the whole abdomen (2022-07-12 14:31, this hospital) revealed: The stomach was well-filled, with no significant abnormal thickening of the wall. The liver was normal in morphology and size, with uniform liver parenchyma density, and no dilatation of the intrahepatic bile duct. The gallbladder, pancreas, spleen, and kidneys were normal in morphology, size, and density. There were no significant enlarged lymph nodes around the abdominal aorta. The bladder was well-filled, with no significant abnormal thickening of the wall; an IUD shadow was seen in the uterus, with a small amount of fluid in the pelvic cavity, and no abnormally enlarged lymph nodes. The bone margins of some thoracic and lumbar vertebrae were pointed. An electrocardiogram (12-channel electrocardiogram) was performed in the electrocardiogram room (2022-07-12 16:30, this hospital) and revealed: 1. Sinus rhythm 2. Normal-range electrocardiogram. (2022-07-05 09:00, this hospital) Complete blood test + high-sensitivity C-reactive protein (blood): high-sensitivity C-reactive protein <0.50 mg / L, white blood cell count 3.43×10 9 / L, red blood cell count 3.90×10 12 / L, hemoglobin 120g / L, platelets 230×10 9 / L, neutrophil percentage 40.8%; pre-transfusion screening revealed no significant abnormalities in six coagulation tests, six TM tests, and four thrombosis tests. After a definitive diagnosis and exclusion of significant surgical contraindications, the patient underwent laparoscopic subtotal colectomy on July 14, 2022. The patient returned safely to the ward and received symptomatic treatment, including anti-infectives, anti-inflammatory analgesics, Compound Ma Ren Granules, fluid replacement, and nutritional support. The patient is now recovering well. Postoperative pathology (subtotal colectomy specimen): 1. Examination of the intestinal wall tissue revealed no significant abnormalities in the mucosal structure. The muscular wall of the dilated segment was thinner; the muscularis propria was thicker in the slightly narrowed segment. No significant loss of submucosal or intramural ganglia was observed. 2. No significant abnormalities were observed in the intestinal wall at the superior and inferior resection margins. 5. Chronic appendicitis. 6. Twenty-seven perineural lymph nodes showed lymphoid hyperplasia. Immunohistochemistry (wax blocks 5, 6, and 8): S-100 (positive for nerves), calretinin (scattered positive for intramural and submucosal ganglion cells). Note: Please consider clinical considerations. Auxiliary examination results are unremarkable, and the patient and family have requested discharge.

[0217] Discharge status (cured, improved, not cured, not treated, discharged without doctor's advice, other): cured

[0218] The patient is currently in good condition, with stable vital signs, intermittent hiccups and belching, no noticeable incision pain, and can move freely out of bed. The abdominal drainage tube has been removed. He has no chills or fever, no nausea or vomiting, no abdominal pain or bloating, no diarrhea or constipation, no palpitations or chest tightness, no heart palpitations or shortness of breath, and is taking a semi-liquid diet and sleeping well. He has flatulence and bowel movements through his stoma, and his urination is normal. He has met discharge criteria and, after consulting with his superior physician, has been discharged.

[0219] 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 in the scope of protection of the present invention.

Claims

1. A laxative pharmaceutical composition, characterized in that: The invention comprises 10-300 parts of stir-fried hemp seeds, 10-200 parts of stewed prunes seeds, 10-200 parts of stir-fried black sesame seeds and 10-180 parts of stir-fried radish seeds; the hemp seeds are the dried mature seeds of the mulberry plant Cannabis sativa L.; the prunes seeds are the dried mature seeds of the rosaceae plants Prunus humilis Bge., Prunus japonica Thunb. or Prunus peduncμLata Maxim.; the black sesame seeds are the dried mature seeds of the Sesamum indicum L. of the Sesamaceae plant; and the radish seeds (Raphanus sativus L.) are the dried mature seeds of the radish of the cruciferous plant.

2. The laxative pharmaceutical composition according to claim 1, characterized in that: Also includes 10-200 servings of peach powder.

3. The laxative pharmaceutical composition according to claim 2, characterized in that: Also included are 10-300 portions of compound honey powder.

4. The laxative pharmaceutical composition according to claim 3, characterized in that: Including 14.1kg of fried hemp seeds, 9.4kg of stewed Prunus mume seeds, 9.4kg of fried black sesame seeds, 7.05kg of fried radish seeds, 7.5g of peach powder and 15g of compound honey powder.

5. The method for preparing the laxative pharmaceutical composition according to claim 4, characterized in that: According to the proportion, stir-fried hemp seeds, stewed prunes, stir-fried black sesame seeds and stir-fried radish seeds are put into a frying bag, and water is added for frying; after the extraction is completed, the mixture is concentrated, and then maltodextrin (MD15) is added and stirred until dissolved; the concentrate is spray-dried to obtain dry powder, and then compound honey powder and peach powder are added for granulation.

6. The method for preparing the laxative pharmaceutical composition according to claim 5, characterized in that: According to the proportion, 14.1kg of fried hemp seeds, 9.4kg of stewed lily seeds, 9.4kg of fried black sesame seeds and 7.05kg of fried radish seeds were weighed, and the medicinal materials were put into a frying bag for frying; the amount of water added in the first extraction was 12 times the volume of the medicinal materials, and the extraction time was 2 hours; the amount of water added in the second extraction was 12 times the volume of the medicinal materials, and the extraction time was 1 hour; after the extraction was completed, the concentration process was entered, the concentration time was 140min, the mesh number of the concentrated liquid was 200 mesh, and the concentrated liquid was 49 The measured specific gravity is 1.0332, and the weight of the concentrate is 46.94 kg. Then, 2.8 kg of maltodextrin (MD15) is added and stirred until dissolved. The concentrate is spray dried, and the inlet temperature is set to 175 ° C, the exhaust temperature is set to 95 ° C, the air delivery temperature is set to 45.4 ° C, and the peristaltic pump speed is set to 50 rpm. A total of 12.76 kg of dry powder is obtained. 2.5 g of dry powder, 15 g of compound honey powder and 7.5 g of peach powder are taken in proportion and mixed and granulated to obtain compound hemp seed granules.

7. The method for preparing the laxative pharmaceutical composition according to claim 5 or 6, characterized in that: The preparation method of stewed Prunus mume kernel is as follows: clean Prunus mume kernel is taken, placed in 10 times the amount of boiling water, heated and boiled for 5 minutes, and when the seed coat is slightly swollen, taken out, placed in cold water for a while, taken out, the seed coat and seed kernel are rubbed apart, dried, and the seed coat is sieved out.

8. Use of the composition prepared according to claim 7 in the preparation of laxative drugs.

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