Traditional Chinese medicine composition for improving constipation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of marine biological medicines, and relates to a traditional Chinese medicine composition for improving constipation as well as a preparation method and application thereof. The traditional Chinese medicine composition comprises the following raw material components in parts by weight: 5-15 parts of radix rehmanniae recen, 1-5 parts of ginseng, 2-10 parts of Chinese yam, 2-10 parts of kelp, 5-15 parts of rhizoma phragmitis, 5-15 parts of lotus leaves, 1-5 parts of platycodon grandiflorum and 5-15 parts of liquorice. The preparation method comprises the following steps: weighing the raw material components, mixing the raw material components with water according to a material-to-liquid ratio of 1: (8-12), decocting for multiple times, gradually reducing the water addition amount each time, decocting for 25-35 minutes each time, and concentrating to obtain an extracting solution with the crude drug amount of 0.8-1.2 g / mL. The traditional Chinese medicine composition which can effectively improve constipation symptoms and supplement nutritional ingredients required by human bodies and is safe and free of side effects is developed by scientific and reasonable compatibility of various traditional Chinese medicines, combination of important characteristics of marine homology of medicine and food and adoption of an advanced preparation method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of marine organisms and medicine, and particularly relates to a traditional Chinese medicine composition for improving constipation, a preparation method and application thereof. BACKGROUND

[0002] Constipation, as a common digestive system disease, seriously affects people's quality of life. According to relevant medical research, the incidence of constipation is increasing year by year worldwide, especially in the elderly and female groups. The main symptoms are decreased defecation frequency, dry and hard stool, and less stool, often accompanied by difficulty in defecation, hematochezia, anal pain and other discomfort symptoms. If the symptoms of constipation are not effectively relieved for a long time, it may cause a series of complications such as anal fissure, hemorrhoids, and anal papillitis.

[0003] At present, there are various treatment methods for constipation. In western medicine, common treatment drugs include external catharsis drugs such as Kaiselu, which mainly penetrates water in the intestinal wall into the intestinal cavity through the high osmotic pressure of glycerol, and uses the lubricating effect of glycerol to achieve defecation. However, such drugs are not suitable for use in patients with inflammatory organic intestinal disease and undiagnosed abdominal pain, and should be used with other drugs at an interval of 2 hours. In traditional Chinese medicine, some traditional Chinese medicine preparations are also used for the treatment of constipation, but they often contain rhubarb, senna leaves, and cassia seeds, which are purgative drugs. These drugs can relieve constipation to some extent, but due to the presence of anthraquinone glycosides and tannins, which have purgative and astringent effects, long-term use can easily cause secondary constipation and cause irreversible damage to the intestinal tract. Under such circumstances, it is particularly urgent to develop a safe, effective and side-effect-free product to improve constipation.

[0004] The ocean, as one of the richest resources on earth, contains a large number of medicinal organisms. Marine food-medicine homologous traditional Chinese medicine has gradually become the focus of research in the field of improving constipation due to its unique advantages. The concept of food-medicine homology has a long history in traditional Chinese medicine. These substances, which can be used as food and have medicinal effects, play an important role in protecting human health. Marine food-medicine homologous traditional Chinese medicine not only contains rich nutrients such as vitamins, minerals, and amino acids, but also contains some substances with special physiological activity, such as fucoidan and mannitol in seaweed.

[0005] Modern scientific research has found that potassium ions in seaweed can maintain potassium ion balance in the human body, and the mucus on the surface of seaweed can lubricate food in the intestinal tract, helping to improve constipation. Marine organisms such as kelp and sea tangle have also been confirmed to have mild laxative effects. Based on these characteristics of marine food-medicine homologous traditional Chinese medicine, it is of great practical significance to develop a marine food-medicine homologous traditional Chinese medicine composition for improving constipation. SUMMARY

[0006] The present application aims to solve the above-mentioned problems existing in the prior art, and provides a traditional Chinese medicine composition for improving constipation, a preparation method and application thereof.

[0007] The technical scheme of the present application is: The present application protects a traditional Chinese medicine composition for improving constipation, which comprises the following raw material components in parts by weight: rehmannia 5-15 parts, ginseng 1-5 parts, Chinese yam 2-10 parts, kelp 2-10 parts, reed root 5-15 parts, lotus leaf 5-15 parts, platycodon 1-5 parts, and licorice 5-15 parts.

[0008] The present application is aimed at the pathogenesis of constipation of yin deficiency and dryness, qi deficiency and lack of driving force, and qi stagnation and dampness obstruction, and combines the effects of nourishing yin and moistening the intestines, tonifying qi and promoting stagnation, clearing heat and reducing dampness, and selects rehmannia, ginseng, Chinese yam, kelp, reed root, lotus leaf, platycodon, and licorice to form a traditional Chinese medicine composition, which is explained as follows: The monarch drug is rehmannia, which nourishes yin and increases fluid, moistens the intestines and promotes defecation, and catalpol promotes intestinal mucus secretion and repairs intestinal mucosal barrier.

[0009] The ministerial drugs are ginseng and Chinese yam, which tonify qi and invigorate the spleen, assist intestinal driving, and polysaccharides enhance intestinal peristalsis frequency and regulate intestinal flora.

[0010] The auxiliary drugs are reed root, lotus leaf, and kelp, which clear heat, generate fluid, reduce dampness, inhibit intestinal inflammation, and improve water metabolism.

[0011] The leading drugs are platycodon and licorice, which promote lung qi and regulate various drugs, and the saponin components can stimulate intestinal nerve plexus activity.

[0012] The whole prescription promotes yin and moistens dryness (rehmannia as the monarch), tonifies qi and promotes driving (ginseng and Chinese yam as the ministers), clears heat and reduces dampness (reed root, lotus leaf, and kelp as the auxiliaries), and regulates the lung and intestines (platycodon and licorice as the leading drugs). The prescription is suitable for constipation of qi and yin deficiency with dampness (dry and hard stool accompanied by sticky and unsmooth defecation).

[0013] Any range or any specific value within the weight range of the above-mentioned traditional Chinese medicine composition formula is applicable to the present application, for example, 5-15 includes any sub-range and any specific value within it, such as 5, 6, 7, 9, 11, 13, 15, etc. any value, 1-5 includes any sub-range and any specific value within it, such as 1, 1.5, 2.3, 3.5, 4.6, 5, etc. any value; 2-10 includes any sub-range and any specific value within it, such as 2, 3, 5, 7, 8, 10, etc. any value.

[0014] Further, the traditional Chinese medicine composition comprises the following raw material components by weight: Rehmanniae Radix 10 parts, Ginseng 2 parts, Dioscoreae Rhizoma 5 parts, Ecklonia 5 parts, Phragmites communis 10 parts, Lotus Leaf 10 parts, Platycodon 2 parts, and Glycyrrhiza 10 parts.

[0015] The application also protects a preparation method of the traditional Chinese medicine composition, comprising the following steps: The raw material components are weighed, mixed with water according to a solid-liquid ratio of 1: (8-12), decocted for several times, the water amount is gradually reduced each time, each decoction time is 25-35 min, and an extract with a crude drug amount of 0.8-1.2 g / mL is obtained after concentration.

[0016] Further, the following steps are included: The raw material components are weighed according to weight parts, mixed with water according to a solid-liquid ratio of 1:10, decocted twice, the volume ratio of water used in the two times is 6:4, each decoction time is 30 min, and a traditional Chinese medicine extract with a crude drug amount of 1 g / mL is obtained after concentration.

[0017] The application protects the application of the traditional Chinese medicine composition prepared by any one of the above or the preparation method in any one of the above in the preparation of a medicine for treating or improving constipation.

[0018] Further, the traditional Chinese medicine composition is suitable for treating or improving constipation of the type of deficiency of both qi and yin with dampness.

[0019] The application also protects the application of the traditional Chinese medicine composition prepared by any one of the above or the preparation method in any one of the above in the preparation of a product with the effects of improving constipation and / or moistening the intestines and promoting defecation.

[0020] Further, the product includes food and drink.

[0021] The application protects a traditional Chinese medicine preparation, which comprises the traditional Chinese medicine composition in any one of the above and a pharmaceutically acceptable excipient.

[0022] The application has the following beneficial effects: The application provides a traditional Chinese medicine composition with the same origin as food, which is composed of Rehmanniae Radix, Ginseng, Dioscoreae Rhizoma, Ecklonia, Phragmites communis, Lotus Leaf, Platycodon and Glycyrrhiza.

[0023] The traditional Chinese medicine composition whole recipe promotes the body by nourishing yin and moistening dryness, promotes by tonifying qi, assists by clearing heat and draining dampness, and leads by regulating the lung and intestines, and is especially suitable for treating or improving constipation of the type of deficiency of both qi and yin with dampness with symptoms such as dry and hard stool accompanied by sticky and uncomfortable. Attached Figure Description

[0024] Figure 1 Changes in rat body weight; Figure 2 It is in the form of feces; Figure 3 This refers to the water content of feces. Figure 4 For intestinal propulsion rate; Figure 5 The time when the first black stool is expelled; Figure 6 The effects of traditional Chinese medicine composition on the morphology of colon tissue in SD rats; Figure 7 The effects of traditional Chinese medicine composition on the morphology of colon tissue in SD rats; Figure 8 This is the dilution curve; Figure 9 Results of α-diversity analysis in different groups; Figure 10 Results of β-diversity analysis in different groups; Figure 11 This is a schematic diagram showing the relative abundance of species in different groups. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0026] To further understand the present invention, it will be further described in conjunction with the accompanying drawings and embodiments. Example 1

[0027] The traditional Chinese medicine composition of this embodiment is made from the following raw material components in parts by weight: 10 parts Rehmannia glutinosa, 2 parts ginseng, 5 parts yam, 5 parts kelp, 10 parts reed rhizome, 10 parts lotus leaf, 2 parts platycodon grandiflorus, and 10 parts licorice.

[0028] Its preparation method includes the following steps: Weigh each raw material component according to the above weight proportions. Mix all weighed raw material components with water according to the material-to-liquid ratio of 1:10. Decoction twice, with the volume ratio of water used in the two decoctions being 6:4. Each decoction is 30 minutes long and concentrated into a Chinese herbal extract with a raw drug content of 1 g / mL. Example 2

[0029] The traditional Chinese medicine composition of the embodiment is made of the following raw material components in parts by weight: Radix Rehmanniae 5 parts, Radix Ginseng 1 part, Rhizoma Dioscoreae 2 parts, Kelp 2 parts, Rhizoma Phragmitis 5 parts, Folium Nelumbinis 5 parts, Radix Platycodonis 1 part, and Radix Glycyrrhizae 5 parts.

[0030] The preparation method thereof comprises the following steps: The above raw material components are weighed according to the above weight proportions, and the weighed raw material components are mixed with water according to a proportion of 1:8, decocted three times, and the volume ratio of water used in the three times is 6:4:3, each time for 25 min, and concentrated into a traditional Chinese medicine extract with a crude drug amount of 1 g / mL. Example 3

[0031] The traditional Chinese medicine composition of the embodiment is made of the following raw material components in parts by weight: Radix Rehmanniae 5 parts, Radix Ginseng 1 part, Rhizoma Dioscoreae 2 parts, Kelp 2 parts, Rhizoma Phragmitis 5 parts, Folium Nelumbinis 5 parts, Radix Platycodonis 1 part, and Radix Glycyrrhizae 5 parts.

[0032] The preparation method thereof comprises the following steps: The above raw material components are weighed according to the above weight proportions, and the weighed raw material components are mixed with water according to a proportion of 1:8, decocted three times, and the volume ratio of water used in the three times is 6:4:3, each time for 25 min, and concentrated into a traditional Chinese medicine extract with a crude drug amount of 1 g / mL. Example 4

[0033] The traditional Chinese medicine composition of the embodiment is made of the following raw material components in parts by weight: Radix Rehmanniae 5 parts, Radix Ginseng 1 part, Rhizoma Dioscoreae 2 parts, Kelp 2 parts, Rhizoma Phragmitis 5 parts, Folium Nelumbinis 5 parts, Radix Platycodonis 1 part, and Radix Glycyrrhizae 5 parts.

[0034] The preparation method thereof comprises the following steps: The above raw material components are weighed according to the above weight proportions, and the weighed raw material components are mixed with water according to a proportion of 1:8, decocted three times, and the volume ratio of water used in the three times is 6:4:3, each time for 25 min, and concentrated into a traditional Chinese medicine extract with a crude drug amount of 1 g / mL.

[0035] Test Example 1 Animal experiment design 1. Animal model establishment and grouping In this study, 7-week-old, healthy male SPF Sprague-Dawley (SD) rats were selected as experimental objects. All experimental animals were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were adaptively fed for one week in a standard experimental animal feeding environment (room temperature 22-24℃, humidity 50-60%, 12h / 12h light-dark cycle), during which they were allowed to freely eat and drink.

[0036] After the end of the adaptive feeding period, the rats were randomly divided into two groups according to body weight: control group and model group. The model group of rats was given Loperamide Hydrochloride solution by gavage (ig) for modeling, the dosage was 10 mg / kg, the frequency of administration was twice a day, and the modeling was continuous for 4 days. After 4 days of modeling, the rat feces morphology and quantity were observed, and after the model was successfully confirmed, the formal drug intervention was started the next day.

[0037] The successfully modeled rats were randomly assigned to the following five treatment groups (n=6 per group) according to body weight: Positive drug group: given Mosapride Citrate (positive drug) solution, the dosage was 5 mg / kg / d.

[0038] High-dose traditional Chinese medicine composition group (composition-high): given traditional Chinese medicine composition extract, the dosage was 1.62 g / kg / d.

[0039] Medium-dose traditional Chinese medicine composition group (composition-medium): given traditional Chinese medicine composition extract, the dosage was 0.8 g / kg / d.

[0040] Low-dose traditional Chinese medicine composition group (composition-low): given traditional Chinese medicine composition extract, the dosage was 0.4 g / kg / d.

[0041] Control group: only given the same volume (consistent with the gavage volume of each drug group) of normal saline for gavage.

[0042] The gavage intervention of all groups (including the control group and each treatment group) was started the day after the successful modeling verification, once a day, for 7 consecutive days. During the entire experiment, all rats were continued to be fed under standard conditions, with free access to food and water.

[0043] 2. Detection index and results Each group of experimental rats was systematically evaluated for general signs at fixed time points (usually 8:00-10:00 am) daily, and the observation period covered the entire treatment.

[0044] General behavioral observation of rats included weight, mental state, food and water intake, activity, and fecal characteristics. After treatment, the body weight, food intake, fecal water content, defecation time, and intestinal propulsion rate of SD rats were detected.

[0045] (1) General observation of rats with chronic transit constipation The general signs of rats were observed during modeling and drug administration, and no deaths occurred in any group of rats.

[0046] Compared with the control group, the model group rats were less active, had back hair and low glossiness, and had less fecal particles, which were small and granular, and the surface was dry; compared with the model group, the low, medium and high dose groups of the traditional Chinese medicine composition, and the positive drug group, the rats had increased fecal particles, increased fecal moisture, and a wet surface, and the feces were smooth and soft; at the same time, the low dose group (composition-low) had more granular feces, and the medium dose group (composition-medium), the high dose group (composition-high) and the mosapride group (positive drug) had no significant difference in fecal quantity, and the fecal surface was wet and mostly in long strips; the control group, the model group, the composition-low group, the composition-medium group, the composition-high group and the positive drug group had no significant difference in diet, water and urine, see Table 1. The weight changes of the control group, the model group, the composition-low group, the composition-medium group, the composition-high group and the positive drug group are shown in Figure 1 .

[0047] Table 1 Change in food intake of rats Days Control group Model group Composition-low Composition-medium Composition-high Positive drug 0 21.34±0.61 20.97±0.68 21.86±0.64 21.41±0.57 21.62±0.83 20.61±0.69 6 28.25±0.79 14.86±0.40 26.31±0.77 19.81±0.7 20.84±0.77 24.16±0.95 12 31.2±2.06 23.86±1.34 25.37±1.07 29.15±1.07 28.33±0.86 29.06±1.06 (2) Effect of traditional Chinese medicine composition on rat defecation (2.1) Fecal morphology Fecal characteristics are key parameters for evaluating constipation models and drug effects. Collect feces 2 hours after administration and observe the appearance characteristics of the feces (including particle size, particle number, shape and texture).

[0048] As shown in Figure 2 , the feces of normal control mice were regular oval, smooth and moderately moist; the constipation model group mice induced by loperamide hydrochloride showed constipation symptoms, including difficulty in defecation, reduced fecal particles, and dry feces. After intervention with the traditional Chinese medicine composition (composition-medium), the above constipation-related symptoms were effectively alleviated, the number of mouse feces increased, and the feces were moist, indicating that the prepared traditional Chinese medicine composition can alleviate chronic transit constipation induced by loperamide hydrochloride.

[0049] (2.2) Fecal water content Each rat was placed in a clean metabolic cage for free feeding and drinking 12 hours before fecal sample collection, and all feces excreted within 20 hours were continuously collected; 5 fresh particles with complete morphology were randomly selected from the feces of each rat and placed in a pre-weighed sterile centrifuge tube, and the wet weight was recorded using a precision 0.001 g analytical balance in a constant temperature and humidity environment (22±2℃); then it was immediately transferred to a constant temperature electric heating air drying oven, dried at 80±5℃ for 2 hours (air speed 1.0-1.5 m / s), and then placed in a desiccator to cool to room temperature, and the dry weight was accurately weighed on the same precision balance.

[0050] Fecal water content=(wet fecal weight-dry fecal weight) / wet fecal weight x 100%.

[0051] Results as shown in Figure 3 Figure 2, compared with the control group, the fecal water content of each group of rats was significantly reduced after modeling (P <0.01). After administration, compared with the control group, the fecal water content of the model group was significantly reduced (P <0.01). Compared with the model group, the fecal water content of the composition-low, composition-medium, composition-high and positive drug groups was significantly increased (P <0.01). After administration, there was no statistically significant difference between the different dose groups of traditional Chinese medicine composition and the positive drug group.

[0052] (3) Determination of rat intestinal propulsion rate The activated carbon powder method was used to determine the intestinal propulsion rate of rats.

[0053] The activated carbon powder was accurately weighed, mixed with 10% gum arabic solution at a concentration of 5% (w / v) to prepare a homogeneous suspension (ultrasonic oscillation for 5 min, and used after standing and defoaming). Before the experiment, the rats were fasted for 12 hours (free water) to exclude the interference of food residues on intestinal movement. Each rat was given 5% carbon suspension 2 mL by gavage, and after 30 min, blood was taken under anesthesia, and the rats were sacrificed by dislocation. The abdominal cavity was quickly opened, the mesenteric attachment tissue was cut off, and the whole segment of the intestinal canal from the pylorus to the anus (including the duodenum to the rectum) was completely peeled off. The whole process was kept wet with pre-cooled normal saline (4°C) to avoid tissue drying. The free intestinal canal was peeled off, and the intestinal segment from the pylorus to the anus was cut off, and the intestinal canal was gently straightened to keep it in a tension-free state.

[0054] The propulsion distance of carbon suspension from the pylorus and the total length of the intestinal canal were accurately measured. Intestinal propulsion rate = propulsion distance of carbon suspension from the pylorus (cm) / total length of the intestinal canal (cm) x 100%.

[0055] As shown in Figure 4 Figure 4, compared with the control group, the intestinal propulsion rate of the model group was significantly reduced (P <0.01). Compared with the model group, the intestinal propulsion rate of the high, medium and low dose groups of traditional Chinese medicine composition and the positive drug group was significantly increased (P <0.01). There was no statistically significant difference between the high, medium and low dose groups of traditional Chinese medicine composition and the positive drug group.

[0056] As shown in Figure 5 Figure 5, compared with the control group, the first black stool discharge time of the model group was significantly increased (P <0.01). Compared with the model group, the first black stool discharge time of the high, medium and low dose groups of traditional Chinese medicine composition and the positive drug group was significantly shortened (P <0.01). There was no statistically significant difference between the high, medium and low dose groups of traditional Chinese medicine composition and the positive drug group.

[0057] (4) Effect of the Chinese medicine composition on the histopathological staining results of the colon tissue of SD rats The colon tissue of the rats was dyed by hematoxylin-eosin (HE) according to the literature method.

[0058] Tissue fixation: the colon tissue of the rats was taken out from 4% paraformaldehyde and transferred to 10% neutral buffered formaldehyde solution (pH 7.2-7.4) for fixation for 24 hours, and fresh fixing solution was replaced every 8 hours to ensure uniform penetration.

[0059] Decalcification treatment: after fixation, the tissue was moved into 10% ethylenediaminetetraacetic acid (EDTA) decalcification solution (pH 7.0) for decalcification at room temperature (25±2°C) for 3 weeks, and the decalcification solution was replaced every 48 hours, and the end point of decalcification was verified by needle puncture method (the needle tip penetrated the tissue without resistance).

[0060] Gradient dehydration: 70% ethanol (1 h)→80% ethanol (1 h)→95% ethanol I (1 h)→95% ethanol II (1 h)→100% ethanol I (1 h)→100% ethanol II (1 h), and the water was completely replaced.

[0061] Transparency and wax immersion: xylene-ethanol mixture (1:1, 30 min)→xylene I (30 min)→xylene II (30 min) until the tissue is transparent; immersion in 60-62°C molten paraffin (3 times×1 h), and embedding in a metal mold.

[0062] Section preparation: after the wax block was trimmed, it was continuously cut by a paraffin sectioning machine, and the thickness was strictly controlled at 4-6 μm (error≤0.5 μm), and the section was flattened in a 40°C constant temperature water bath and attached to a polylysine-coated glass slide, and then baked at 60-65°C for 2 hours for solidification.

[0063] HE staining: Decalcification and hydration: xylene I / II (10 min each)→gradient ethanol (100%→70%)→distilled water; Hematoxylin staining: hematoxylin solution was immersed and dyed for 5 min, and then washed with running water; Differentiation and blueing: 1% hydrochloric acid ethanol differentiation for 2-5 seconds→running water washing→0.1% ammonia water or saturated lithium carbonate blueing for 1 min→running water washing for 10 min; Eosin re-staining: 0.5% eosin Y aqueous solution re-staining for 1-3 min→distilled water fast washing.

[0064] Dehydration and mounting: gradient ethanol dehydration (80%→100%)→xylene transparency (I / II for 3 min each); dropwise addition of neutral gum, cover with a cover glass (avoid air bubbles), and solidification at room temperature for 24 hours in the dark.

[0065] Microscopy and analysis: under optical microscope, the nucleus was dark blue, the cytoplasm and collagen fibers were pink; the integrity of the colon mucosa layer, the number of goblet cells, inflammatory cell infiltration and the morphology of the intermuscular nerve plexus were evaluated.

[0066] As shown in Figure 6 , the control group rats had no colon edema, the mucosal epithelium was intact, the mucosal microvilli was arranged in order, the epithelial cells was arranged regularly, the crypt structure was normal, the number of goblet cells was rich and arranged in order, and the muscle layer was normal. The colon mucosa layer structure of the model group rats was irregular, the epithelial cells was arranged in disorder, part of the crypts disappeared and expanded, the gland distribution was irregular, there were inflammatory cell infiltration, the number of goblet cells was reduced, the cytoplasm was obviously expanded, and part of them appeared to be broken. The colon mucosa structure of the traditional Chinese medicine composition middle dose group was complete, the goblet cells was arranged regularly without breaking, the number was more, and there was no edema between cells.

[0067] (5) ELISA detection The content of substance P (SP) in the serum of rats was determined by strictly following the principle of double antibody sandwich enzyme-linked immunosorbent assay (ELISA), and the operation process was carried out according to the kit instruction and the standardized method in the literature.

[0068] Sample pretreatment: after the rats were anesthetized, blood was taken from the abdominal aorta, and the blood was placed at room temperature for 30 min, then centrifuged at 3000 r / min for 15 min, the serum was separated and stored in sterile EP tubes at-80℃ for testing, and repeated freezing and thawing was avoided.

[0069] Coating and balancing: the 96-well plate coated with anti-SP / VIP antibody was balanced at room temperature for 30 min; Sample addition: 50 μL of standard (concentration gradient: 1000, 500, 250, 125, 62.5, 31.25 pg / mL) or diluted serum sample (1:2 diluted in sample diluent) was added to each well, and duplicate wells and blank controls (only diluent) were set; Incubation: incubate at 37℃ for 90 min, wash the plate 5 times (fill each well with washing solution, stand for 30 s, then shake dry); Enzyme-labeled antibody binding: add 100 μL of HRP-labeled detection antibody to each well, incubate at 37℃ for 60 min, and repeat the washing; Color development: add 100 μL of TMB substrate solution per well, react at 37℃ for 15 min in the dark; Termination and reading: add 50 μL of termination solution (2 M H2SO4), immediately detect the absorbance (OD value) at 450 nm wavelength on the enzyme label instrument.

[0070] Standard curve and calculation: the standard concentration was taken as the abscissa, and the average OD value was taken as the ordinate, and a four-parameter logistic regression curve (R 2≥ 0.99).

[0071] SP content in plasma of each group before and after treatment The ELISA analysis results of the present study showed that the serum SP content of the model group was reduced compared with the control group. After administration, the serum SP content of the high, medium and low dose traditional Chinese medicine composition groups was significantly increased compared with the model group (see Table 4). Figure 7 ).

[0072] (6) Intestinal flora detection The feces of each group of rats (not less than 2 g) were collected, the genomic DNA of the samples was extracted using a DNA extraction kit, and the concentration of DNA was detected by agarose gel electrophoresis and NanoDrop2000.

[0073] Using genomic DNA as a template, according to the selection of the sequencing region, the corresponding primers were used for PCR amplification, and the amplification conditions were 94°C for 5 min; 94°C for 30 s, 56°C for 30 s, 72°C for 30 s, 26 cycles; 72°C for 5 min, then stop at 4°C; The PCR product was detected by electrophoresis, 20 μL of AMPureXPbeads was added to the U-shaped plate and mixed thoroughly, and the magnetic beads were purified, then the PCR product was added, and the pipette gun was slowly blown for 10 times, mixed, and placed at room temperature for 5 min. After purification, it was used as a template for the second round of PCR, and 2 rounds of PCR amplification were performed using universal primers in a 25 μL reaction (V3V4 front primer: 343FTACGGRAGGCAGCAG; rear primer: 798RAGGGTATCTAATCCT).

[0074] According to the concentration of the PCR product, equal amounts of samples were mixed, and sequencing was performed. 5 μL of purified second round product was detected by agarose gel electrophoresis, and whether there was a band and whether the band was single was detected. The concentration of the product of the two rounds of PCR was detected by Qubit, and 1 μL of the purified second round product was detected by Qubit.

[0075] The detection results are as follows: (6.1) Sample abundance The rarefaction curve is used to estimate the microbial abundance within the sample. By comparing the rarefaction curves of different samples, the differences in species diversity between samples are shown. The rarefaction curve is flat (see Fig. 6), indicating that the amount of sequencing data in the present experiment is reasonable. Figure 8

[0076] (6.2) Effect of traditional Chinese medicine composition on intestinal flora α-diversity of rats ​Alpha diversity is a commonly used index in microbial analysis, used to assess the richness and diversity of microorganisms within a sample. Various alpha diversity parameters, such as Chao1, ACE, observed species, Goods coverage, Shannon, Simpson, etc., are used to assess the richness and diversity of the gut flora community in SD rats.

[0077] According to Figure 9 Compared with the control group, the Chao1, Shannon, and Simpons indices of the fecal flora of the model group rats showed a downward trend. Compared with the model group, the Chao1, Shannon, and Simpons indices of the high, medium, and low dose groups of the traditional Chinese medicine composition showed an upward trend. The Chao1, Shannon, and Simpons indices of the positive drug group showed a downward trend.

[0078] (6.3) Effect of Traditional Chinese Medicine Composition on Beta Diversity of Rat Intestinal Flora Beta diversity refers to the difference in species between different environmental communities (samples), and is an analysis of the similarity and difference between samples as a whole. Beta diversity is an analysis of the microbial community structure of SD rats, and dimensionality reduction analysis mainly includes principal component analysis (PCA) and principal coordinate analysis (PCoA).

[0079] (6.31) PCA analysis PCA analysis reduces the dimensionality of data with multiple features, and can observe the differences between samples or groups. Different colors represent different groups, and points of the same color represent different samples within a group. The same group is displayed in a circular form according to a 95% confidence interval. The closer the samples in the same group are, and the farther they are from other groups, the better the grouping effect. The PCA analysis results show that Figure 10 ), the distance between the control group and the model group in the figure is far, indicating that the intestinal flora after modeling has obvious differences compared with the control group. The distance between the control group and the low dose group of the traditional Chinese medicine composition and the positive drug group is closer, indicating that after the SD rats are given the low dose of the traditional Chinese medicine composition and the positive drug, their intestinal flora is closer to the fecal flora of the control group.

[0080] (6.32) Effect of Traditional Chinese Medicine Composition on Species Relative Abundance of Intestinal Flora in Different Groups Based on the phylum level species annotation results, the top 30 species in the abundance ratio were selected to generate a relative abundance column chart to investigate the species composition and proportion of each sample at different classification levels. At the phylum level, the human gut microbiota is mainly composed of several dominant phyla, including Firmicutes, Bacteroidota, Proteobacteria, Campylobacterota, Desulfobacterota, etc. Figure 11 From the phylum level, Firmicutes, Bacteroidota and Proteobacteria are the dominant phyla in the three groups. Compared with the control group, the ratio of Firmicutes / Bacteroidota in the model group decreased, and the proportion of Proteobacteria increased significantly. The high and low dose groups of the traditional Chinese medicine composition increased the ratio of Firmicutes / Bacteroidota in constipated mice, and reduced the level of Proteobacteria. It shows that from the phylum level, the traditional Chinese medicine composition provided by the present application can reverse the intestinal flora changes caused by constipation.

[0081] The above description is only the preferred embodiments of the present application, and is not a limitation on the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions described in the foregoing embodiments, or make equivalent replacements to some technical features. Any modification, equivalent replacement, modification, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A traditional Chinese medicine composition for improving constipation, characterized in that, The traditional Chinese medicine composition comprises the following raw material components in parts by weight: 5-15 parts of Rehmannia glutinosa, 1-5 parts of ginseng, 2-10 parts of Dioscorea opposita, 2-10 parts of kelp, 5-15 parts of Phragmites communis rhizome, 5-15 parts of lotus leaf, 1-5 parts of Platycodon grandiflorus, and 5-15 parts of Glycyrrhiza uralensis.

2. The traditional Chinese medicine composition for improving constipation according to claim 1, characterized in that, The traditional Chinese medicine composition comprises the following raw material components in parts by weight: 10 parts Rehmannia glutinosa, 2 parts ginseng, 5 parts Dioscorea opposita, 5 parts Laminaria japonica, 10 parts Phragmites communis, 10 parts Nelumbo nucifera, 2 parts Platycodon grandiflorus, and 10 parts Glycyrrhiza uralensis.

3. The method for preparing the traditional Chinese medicine composition according to claim 1 or 2, characterized in that, Includes the following steps: Weigh each raw material component, mix it with water at a material-to-liquid ratio of 1:(8~12), and decoct several times, gradually reducing the amount of water each time, with each decoction lasting 25~35 minutes, and concentrate to obtain an extract with a crude drug content of 0.8~1.2 g / mL.

4. The preparation method according to claim 3, characterized in that, Includes the following steps: Weigh each raw material component according to the specified weight proportions. Mix all weighed raw material components with water according to the material-to-liquid ratio of 1:

10. Decoction is performed twice, with the volume ratio of water used in the two decoctions being 6:

4. Each decoction is performed for 30 minutes, and the mixture is concentrated into a Chinese herbal extract with a raw drug content of 1 g / mL.

5. The use of the traditional Chinese medicine composition according to any one of claims 1-2 or the traditional Chinese medicine composition prepared by the preparation method according to any one of claims 3-4 in the preparation of a medicament for treating or improving constipation.

6. The application according to claim 5, characterized in that, The traditional Chinese medicine composition is suitable for treating or improving constipation caused by deficiency of both qi and yin with dampness.

7. The use of the traditional Chinese medicine composition according to any one of claims 1-2 or the traditional Chinese medicine composition prepared by the preparation method according to any one of claims 3-4 in the preparation of products having the effects of improving constipation and / or lubricating the intestines and relieving constipation.

8. The application according to claim 7, characterized in that, The products include food and beverages.

9. A traditional Chinese medicine preparation comprising the traditional Chinese medicine composition according to any one of claims 1-2 and pharmaceutically acceptable excipients.

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