Traditional Chinese medicine composition for treating ulcerative colitis

Abstract

This invention provides a traditional Chinese medicine composition for treating ulcerative colitis, belonging to the field of traditional Chinese medicine pharmaceutical technology. The composition is obtained through rational formulation based on the principles of syndrome differentiation and treatment in traditional Chinese medicine, specifically composed of Forsythia suspensa, Lilium brownii, Lysimachia christinae, Sesamum indicum, Prickly ash, Pueraria lobata, Centella asiatica, Viola yedoensis, Houttuynia cordata, Portulaca oleracea, Litsea cubeba, Cirsium japonicum, Schizonepeta tenuifolia, Morus alba root, Lophatherum gracile, Lygodium japonicum, and Capsella bursa-pastoris. This invention's composition exhibits significant anti-inflammatory and de-inflammatory effects, and can repair and improve the integrity of colonic tissue structure, enhance intestinal barrier function, and achieve both symptomatic and root-cause treatment of ulcerative colitis. Simultaneously, this invention's composition has a high efficacy and cure rate in treating ulcerative colitis, and significantly alleviates symptoms such as diarrhea, bloody stools, abdominal pain, abdominal distension, anal burning, and tenesmus, greatly reducing patient suffering.

Description

Technical Field

[0001] This invention relates to traditional Chinese medicine manufacturing technology, specifically to a traditional Chinese medicine composition for treating ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is one of the two main forms of inflammatory bowel disease. UC is a chronic, nonspecific inflammatory disease caused by the interaction of multiple factors, including genetic background, immunity, psychological factors, and environment. It primarily affects the mucosal layer of the colon, rarely involving the entire colon. It typically occurs in the rectum and sigmoid colon, progressing proximally. The severity of the disease varies, it is prone to relapse, and it is difficult to cure completely. The main clinical symptoms of UC include persistent or recurrent diarrhea, abdominal pain, bloody and mucous stools, and tenesmus. In severe cases, varying degrees of extraintestinal manifestations and systemic symptoms may appear, severely impacting the patient's physical and mental health.

[0003] The pathogenesis of ulcerative colitis (UC) is not fully understood, but genetic susceptibility, immune imbalance, gut microbiota, and environmental factors are considered to be closely related to its occurrence. Although modern medicine has improved treatment options for ulcerative colitis in recent years, there is still no cure. Clinically, ulcerative colitis is mainly treated with antibiotics, immunotherapy, and hormone therapy. Sulfonamide drugs, such as mesalazine, are widely used due to their rapid symptom relief and strong anti-inflammatory effects, but they also have drawbacks including high relapse rates, drug resistance, difficulty in achieving a cure, high cost, and significant adverse reactions.

[0004] Based on the clinical characteristics of ulcerative colitis (UC), including persistent and recurrent abdominal pain, diarrhea, bloody and mucous stools, and tenesmus, it can be categorized under the traditional Chinese medicine (TCM) terms of "dysentery" and "chronic diarrhea." In recent years, TCM, a brilliant treasure of traditional Chinese medicine, has made some progress in the treatment of UC, with numerous TCM formulas for UC treatment published. For example, publication numbers CN105012827A and CN102258720A both disclose a TCM formula for treating ulcerative colitis; however, these formulas suffer from unclear pharmacological effects and insignificant therapeutic efficacy. Therefore, this invention aims to find a TCM composition with a definite therapeutic effect on UC. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] In view of the shortcomings of the prior art, the present invention provides a traditional Chinese medicine composition for treating ulcerative colitis.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a traditional Chinese medicine composition for treating ulcerative colitis, the composition comprising the following raw materials in parts by weight: Forsythia suspensa 10-30 parts, Lilium brownii 10-30 parts, Lysimachia christinae 5-20 parts, Sesamum indicum 5-20 parts, Opuntia ficus-indica 1-10 parts, Pueraria lobata 5-20 parts, Centella asiatica 5-20 parts, Viola yedoensis 5-20 parts, Houttuynia cordata 5-20 parts, Portulaca oleracea 1-10 parts, Litsea cubeba 5-20 parts, Cirsium japonicum 5-20 parts, Schizonepeta tenuifolia 5-20 parts, Morus alba root 1-10 parts, Lophatherum gracile 5-20 parts, Lygodium japonicum 1-10 parts, and Capsella bursa-pastoris 1-10 parts.

[0010] Furthermore, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 15-30 parts of Forsythia suspensa, 15-25 parts of Lilium brownii, 8-16 parts of Lysimachia christinae, 8-16 parts of Sesamum indicum, 4-8 parts of Opuntia ficus-indica, 8-16 parts of Pueraria lobata, 8-16 parts of Centella asiatica, 8-16 parts of Viola yedoensis, 8-16 parts of Houttuynia cordata, 4-8 parts of Portulaca oleracea, 8-16 parts of Litsea cubeba, 8-16 parts of Cirsium japonicum, 8-16 parts of Schizonepeta tenuifolia, 3-7 parts of Morus alba root, 8-16 parts of Lophatherum gracile, 3-7 parts of Lygodium japonicum, and 4-8 parts of Capsella bursa-pastoris.

[0011] Furthermore, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 26 parts forsythia, 24 parts lily, 12 parts creeping privet, 10 parts wild sesame, 5 parts cactus, 12 parts kudzu root, 10 parts centella asiatica, 14 parts wild violet, 14 parts houttuynia cordata, 6 parts purslane, 12 parts mountain licorice, 10 parts large thistle, 10 parts schizonepeta, 6 parts mulberry root, 14 parts light bamboo leaf, 4 parts lygodium japonicum, and 6 parts shepherd's purse.

[0012] The traditional Chinese medicine composition provided by this invention is used to prepare a drug for treating ulcerative colitis. The traditional Chinese medicine composition can be prepared into a dosage form that is acceptable to humans in medicine, including but not limited to any one of oral liquid, decoction, granules, pills, enema liquid, capsules, suppositories, and lyophilized powder.

[0013] (III) Beneficial Effects

[0014] This invention utilizes the principles of TCM syndrome differentiation and treatment, and is rationally formulated. The formula includes: Forsythia suspensa, bitter and slightly cold, entering the lung, heart, and small intestine meridians, possessing the effects of clearing heat and detoxifying, reducing swelling and dissipating nodules, and is effective in treating carbuncles and boils. The *Rihuazi Materia Medica* records that it "clears the small intestine, drains pus, treats boils, and relieves pain"; Lilium brownii, sweet and cold, entering the heart and lung meridians, possessing the effects of moistening the lungs and relieving cough, clearing the heart and calming the mind. The *Shennong Bencao Jing* states that it "treats abdominal distension due to pathogenic factors, heart pain, and promotes urination and defecation." Because the lungs and large intestine are internally and externally related, treating the lungs is equivalent to treating the large intestine, and clearing the lungs is equivalent to clearing the intestines; Lysimachia christinae, bland and cool in nature, entering the large intestine meridian... The five herbs enter the intestines and liver meridians, possessing the effects of clearing heat and dampness, cooling blood and detoxifying. They are effective in treating dysentery, diarrhea and other diseases caused by internal heat and toxins. Mountain sesame is bitter, slightly sweet and cold, entering the bladder and large intestine meridians. It has the effects of relieving exterior symptoms, clearing heat, reducing swelling and detoxifying. According to *Fujian Folk Herbal Medicine*, it excels at removing blood stasis and promoting new tissue growth, eliminating carbuncles and detoxifying. Cactus is bitter and astringent, cold in nature, entering the heart, stomach and intestines meridians. It has the effects of promoting qi and blood circulation, clearing heat and detoxifying. *Guangxi Traditional Chinese Medicine* records that it "stops diarrhea and treats enteritis and diarrhea." These five herbs work synergistically to clear heat and detoxify, drain pus and stop diarrhea, and treat enteritis; they are the principal herbs. Kudzu root is sweet, pungent, and cool in nature, and enters the spleen and stomach meridians. It has the effects of raising yang and relieving muscle tension, promoting rash eruption and stopping diarrhea. The *Rihuazi Materia Medica* states that it can treat chest and diaphragm heat, stop bloody dysentery, clear the small intestine, drain pus and break up blood stasis. Clinically, it is commonly used to treat various intestinal diseases. Centella asiatica is bitter, pungent, and cold in nature, and enters the liver, spleen, and kidney meridians. It has the effects of clearing heat and dampness, detoxifying and reducing swelling. Clinically, it is often used for damp-heat jaundice, heatstroke diarrhea, carbuncles, and boils. The *Mindong Materia Medica* records that it "treats heatstroke, abdominal pain and distension." Wild violet is bitter, pungent, and cold in nature, and enters the heart and liver meridians. It has the effects of clearing heat and detoxifying, cooling blood and reducing swelling. Clinically, it is often used for boils, carbuncles, and carbuncles. Houttuynia cordata is pungent and slightly cold in nature, and enters the lung and large intestine meridians. It has the effects of clearing heat and detoxifying, eliminating carbuncles and draining pus, and promoting diuresis and relieving strangury. Clinically, it is often used for lung abscess with purulent sputum, cough with phlegm and heat, dysentery, strangury, carbuncles and boils. The "Diannan Materia Medica" says: "It treats lung abscess with cough with purulent blood, foul-smelling sputum, heat toxin in the large intestine, and hemorrhoids." Purslane is sour and cold in taste, and enters the liver and spleen meridians. It has the effects of clearing heat and detoxifying, dispersing blood and reducing swelling. Clinically, it is often used for dysentery with purulent blood, carbuncles and malignant sores. The "Essentials of Herbal Properties" says: "It treats dysentery, clears heat and toxins, and washes hemorrhoids and boils." Litsea cubeba is bitter and warm in taste. It has the effects of dispelling wind and cold, regulating qi and relieving pain. Clinically, it can be used to treat amoebic dysentery. All the above herbs are assistant herbs, which enhance the efficacy of the principal herbs in clearing heat, detoxifying, eliminating carbuncles and draining pus.Thistle (Cirsium japonicum) is sweet and cool in nature, entering the liver, spleen, and kidney meridians. It has the effects of cooling the blood and stopping bleeding, removing blood stasis and reducing swelling. Clinically, it is used for hematuria, hematochezia, traumatic bleeding, carbuncles and boils. The *Yilin Zuanyao* records that it "treats intestinal wind and intestinal abscess." Schizonepeta tenuifolia (Schizonepeta tenuifolia) is pungent and slightly warm, entering the lung and liver meridians. It has the effects of relieving exterior syndromes and dispelling wind, and promoting rash eruption. Clinically, it is often used for colds and early-stage sores. After being charred, it is used to treat hematochezia. Mulberry root is slightly bitter and cold in nature, entering the liver meridian. It has the effects of clearing heat and calming the mind, dispelling wind and unblocking the meridians. Clinically, it can be used to relieve abdominal pain. Lophatherum gracile (Lophatherum gracile) is sweet, bland, and cold, entering the heart, stomach, and small intestine meridians. It has the effects of clearing heat and relieving irritability, and promoting diuresis. It is used for feverish thirst, painful and scanty urination, and sores on the mouth and tongue. Symptoms: Lygodium japonicum (Hydroxylon ammodendron) is sweet and salty in taste, cold in nature, and enters the bladder and small intestine meridians. It clears heat and dampness, promotes urination and relieves pain, and is used for urinary tract infections, urinary stones, hematuria, chyluria, and painful urination. The *Handbook of Commonly Used Chinese Herbal Medicines* states that it treats urinary tract infections, enteritis, and dysentery. Shepherd's purse (Capsella bursa-pastoris) is sweet and warm in nature, non-toxic, and enters the hand, spleen, and foot meridians. It has the effects of harmonizing the spleen and promoting diuresis, stopping bleeding and improving eyesight. Clinically, it is often used to treat dysentery, edema, gonorrhea, etc. The above are all adjuvant herbs, enhancing the heat-clearing and detoxifying effects of the principal and assistant herbs. This invention's herbal composition combines heat-clearing, detoxifying, carbuncle-reducing, pus-draining, pain-relieving, and diarrhea-stopping effects into one formula, with specific and potent efficacy, significantly improving the clinical symptoms of ulcerative colitis.

[0015] The herbal composition of this invention can effectively reduce the levels of inflammatory factors in the serum and colonic tissue of mice with ulcerative colitis and inhibit the expression of pro-inflammatory cytokines, exhibiting significant anti-inflammatory and de-inflammatory effects. Furthermore, it can repair and improve the structural integrity of colonic tissue and enhance intestinal barrier function, achieving a comprehensive treatment of ulcerative colitis. Simultaneously, the herbal composition of this invention has a high efficacy and cure rate in treating ulcerative colitis, and significantly alleviates symptoms such as diarrhea, bloody stools, abdominal pain, abdominal distension, anal burning, and tenesmus, greatly reducing patient suffering. Attached Figure Description

[0016] Figure 1 Changes in serum TNF-α, IL-1β, IL-6, and IL-8 levels. (* indicates comparison with the control group, ** indicates P<0.01;) # To compare with the model group, # For P<0.05, ## (P < 0.01)

[0017] Figure 2 mRNA expression results of IL-1β, IL-6, IL-8 and IFN-γ in colon tissue. (* indicates comparison with control group, ** indicates P<0.01; # indicates comparison with model group, # indicates P<0.05, ## indicates P<0.01, ### indicates P<0.001)

[0018] Figure 3Changes in disease-related acute liver injury (DAI) in mice of different groups. *Compared with the control group, *P<0.05, **P<0.01; #Compared with the model group, #P<0.05, ##P<0.01

[0019] Figure 4 HE of colon tissue in each group; Bar: 200×.

[0020] Figure 5 Expression of TNF-α, IL-1β, IL-6 and IL-8 in serum. (* indicates comparison with control group, * indicates P<0.05, * indicates P<0.01; # indicates comparison with model group, # indicates P<0.05) Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0022] Example 1

[0023] A traditional Chinese medicine composition for treating ulcerative colitis, the composition comprising the following raw materials in parts by weight: 10 parts forsythia, 1 part lily bulb, 5 parts creeping privet, 5 parts wild sesame, 2 parts cactus, 5 parts kudzu root, 5 parts centella asiatica, 5 parts wild violet, 5 parts houttuynia cordata, 2 parts purslane, 5 parts mountain licorice, 5 parts large thistle, 5 parts schizonepeta, 1 part mulberry root, 5 parts light bamboo leaf, 1 part lygodium japonicum, and 1 part shepherd's purse.

[0024] The traditional Chinese medicine composition can be prepared into a dosage form acceptable to the human body, including but not limited to any one of oral liquid, decoction, granules, pills, enema liquid, capsules, suppositories, and lyophilized powder.

[0025] Example 2

[0026] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 30 parts forsythia, 30 parts lily, 20 parts creeping privet, 20 parts wild sesame, 10 parts cactus, 20 parts kudzu root, 20 parts centella asiatica, 20 parts wild violet, 20 parts houttuynia cordata, 10 parts purslane, 20 parts mountain licorice, 20 parts large thistle, 20 parts schizonepeta, 10 parts mulberry root, 20 parts light bamboo leaf, 10 parts lygodium japonicum, and 10 parts shepherd's purse.

[0027] Example 3

[0028] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 18 parts forsythia, 14 parts lily, 8 parts creeping privet, 10 parts mountain sesame, 4 parts cactus, 12 parts kudzu root, 8 parts centella asiatica, 7 parts wild violet, 15 parts houttuynia cordata, 3 parts purslane, 6 parts mountain licorice, 8 parts large thistle, 12 parts schizonepeta, 4 parts mulberry root, 10 parts light bamboo leaf, 4 parts lygodium japonicum, and 2 parts shepherd's purse.

[0029] Example 4

[0030] The difference between this embodiment and Embodiment 1 is that,

[0031] In summary, the embodiments of the present invention have the following beneficial effects: 15 parts of Forsythia suspensa, 15 parts of Lilium brownii, 8 parts of Lysimachia christinae, 8 parts of Sesamum indicum, 4 parts of Opuntia ficus-indica, 8 parts of Pueraria lobata, 8 parts of Centella asiatica, 8 parts of Viola yedoensis, 8 parts of Houttuynia cordata, 4 parts of Portulaca oleracea, 8 parts of Litsea cubeba, 8 parts of Cirsium japonicum, 8 parts of Nepeta cataria, 3 parts of Morus alba root, 8 parts of Lophatherum gracile, 3 parts of Lygodium japonicum, and 4 parts of Capsella bursa-pastoris.

[0032] Example 5

[0033] The difference between this embodiment and Embodiment 1 is that,

[0034] In summary, the embodiments of the present invention have the following beneficial effects: 30 parts of Forsythia suspensa, 25 parts of Lilium brownii, 16 parts of Lysimachia christinae, 16 parts of Sesamum indicum, 8 parts of Opuntia ficus-indica, 16 parts of Pueraria lobata, 16 parts of Centella asiatica, 16 parts of Viola yedoensis, 16 parts of Houttuynia cordata, 8 parts of Portulaca oleracea, 16 parts of Litsea cubeba, 16 parts of Cirsium japonicum, 16 parts of Nepeta cataria, 7 parts of Morus alba root, 16 parts of Lophatherum gracile, 7 parts of Lygodium japonicum, and 8 parts of Capsella bursa-pastoris.

[0035] Example 6

[0036] The difference between this embodiment and Embodiment 1 is that,

[0037] In summary, the embodiments of the present invention have the following beneficial effects: 22 parts of Forsythia suspensa, 22 parts of Lilium brownii, 10 parts of Lysimachia christinae, 10 parts of Sesamum indicum, 6 parts of Prickly pear cactus, 10 parts of Pueraria lobata, 10 parts of Centella asiatica, 10 parts of Viola yedoensis, 10 parts of Houttuynia cordata, 6 parts of Portulaca oleracea, 10 parts of Litsea cubeba, 10 parts of Cirsium japonicum, 10 parts of Nepeta cataria, 5 parts of Morus alba root, 10 parts of Lophatherum gracile, 5 parts of Lygodium japonicum, and 6 parts of Capsella bursa-pastoris.

[0038] Example 7

[0039] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 26 parts forsythia, 24 parts lily, 12 parts creeping privet, 10 parts wild sesame, 5 parts cactus, 12 parts kudzu root, 10 parts centella asiatica, 14 parts wild violet, 14 parts houttuynia cordata, 6 parts purslane, 12 parts mountain licorice, 10 parts large thistle, 10 parts schizonepeta, 6 parts mulberry root, 14 parts light bamboo leaf, 4 parts lygodium japonicum, and 6 parts shepherd's purse.

[0040] Experimental Example 1

[0041] Due to the large amount of experimental data in this invention, the prescriptions were adjusted and tested multiple times. Here, only the therapeutic effects of some of the prescriptions on a mouse model of ulcerative colitis are listed and compared with the prescriptions of this invention.

[0042] Formula 1: Forsythia 26 parts, Lilium 24 parts, Lysimachia christinae 12 parts, Sesamum indicum 10 parts, Cactus 5 parts, Kudzu Root 12 parts, Centella asiatica 10 parts, Viola yedoensis 14 parts, Houttuynia cordata 14 parts, Portulaca oleracea 6 parts, Litsea cubeba 12 parts, Cirsium japonicum 10 parts, Nepeta cataria 10 parts, Mulberry Root 6 parts, Lophatherum gracile 14 parts, Lygodium japonicum 4 parts, Capsella bursa-pastoris 6 parts.

[0043] Formula 2: 12 parts creeping privet, 10 parts wild sesame, 5 parts cactus, 12 parts kudzu root, 10 parts centella asiatica, 14 parts wild violet, 14 parts houttuynia cordata, 6 parts purslane, 12 parts mountain licorice, 10 parts large thistle, 10 parts catnip, 6 parts mulberry root, 14 parts light bamboo leaves, and 6 parts shepherd's purse.

[0044] Formula 3: Forsythia 26 parts, Lilium 24 parts, Kudzu Root 12 parts, Centella Asiatica 10 parts, Wild Violet 14 parts, Houttuynia cordata 14 parts, Purslane 6 parts, Litsea cubeba 12 parts, Cirsium japonicum 10 parts, Nepeta cataria 10 parts, Mulberry Root 6 parts, Lophatherum gracile 14 parts, Lygodium japonicum 4 parts, Shepherd's Purse 6 parts.

[0045] Formula 4: Forsythia 26 parts, Lilium 24 parts, Creeping Japonica 12 parts, Sesamum indicum 10 parts, Cactus 5 parts, Centella asiatica 10 parts, Viola yedoensis 14 parts, Litsea cubeba 12 parts, Cirsium japonicum 10 parts, Nepeta cataria 10 parts, Mulberry root 6 parts, Lophatherum gracile 14 parts, Lygodium japonicum 4 parts, Capsella bursa-pastoris 6 parts.

[0046] Formula 5: Forsythia 26 parts, Lilium 24 parts, Lysimachia christinae 12 parts, Sesamum indicum 10 parts, Cactus 5 parts, Kudzu Root 12 parts, Houttuynia cordata 14 parts, Portulaca oleracea 6 parts, Litsea cubeba 12 parts, Schizonepeta tenuifolia 10 parts, Mulberry Root 6 parts, Lophatherum gracile 14 parts, Lygodium japonicum 4 parts.

[0047] Formula 6: Forsythia 26 parts, Lilium 24 parts, Creeping Japonica 12 parts, Sesamum indicum 10 parts, Cactus 5 parts, Kudzu Root 12 parts, Centella Asiatica 10 parts, Wild Violet 14 parts, Houttuynia cordata 14 parts, Purslane 6 parts, Thistle 10 parts, Nepeta cataria 10 parts, Lygodium japonicum 4 parts, Shepherd's Purse 6 parts.

[0048] Weigh each raw material according to the above-mentioned proportions, and boil it twice in 8 times its weight of ddH2O (w / v), 1 hour each time. Centrifuge the extract, and prepare lyophilized powder from the supernatant using a freeze dryer. Redissolve the lyophilized powder with ddH2O and vortex at room temperature to obtain the traditional Chinese medicine preparations of each formulation, and store them at 4°C for later use.

[0049] 1. Materials and Methods

[0050] 1.1 Animal modeling, grouping, and drug administration

[0051] Male SPF-grade C57BL / 6J mice, weighing (21±2) g, were selected and acclimatized for 7 days before the experiment began. Mice were randomly assigned to three groups according to body weight: a control group, a model group (3% DSS), and experimental groups 1-6, with 8 mice in each group. Treatments were as follows: the control group received deionized water, while the model and experimental groups 1-6 received free access to a 3% DSS solution for 7 days, with the DSS solution replaced with fresh solution every 2 days. Drug administration began on the day of model establishment. The control and model groups received an equal volume of physiological saline, while experimental groups 1-6 received the corresponding herbal decoction (10 ml / kg) prepared according to formulas 1-6 via gavage. Treatment was simultaneous with model establishment and lasted for 7 days.

[0052] 1.2 Obtaining mouse serum and intestinal tissue

[0053] Mice were fasted for 12 hours but allowed free access to water. Before blood collection, mice were anesthetized with pentobarbital, and 0.5-1 ml of blood was collected from the heart using the cardiac sampling method. After blood collection, the mice were allowed to stand at room temperature for 2 hours, centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected and aliquoted into 200 μl microcentrifuge tubes and stored at -80°C for later analysis. Mice were euthanized by cervical dislocation after blood collection. The mice were placed ventrally, and their skin was moistened with 75% alcohol. The abdominal skin was gently lifted with forceps, and the abdomen was cut open. The colon tissue was then harvested, rinsed with physiological saline to remove feces, and then cut into small pieces. These pieces were placed in fixative and cryovials, respectively. The cryovials were immediately placed in liquid nitrogen and stored at -80°C for later analysis.

[0054] 1.3 ELISA detection of serum IL-1β, IL-6, IL-8 and TNF-α levels

[0055] The methods for determining the levels of serum pro-inflammatory factors IL-1β, IL-6, IL-8, and TNF-α are as follows: 30 minutes before the experiment, bring the reagent kit to room temperature. Wash the plate three times before adding the sample. Add 100 μl of sample to each well, seal the plate, and incubate at 37°C for 90 minutes. After incubation, remove all liquid from the plate, blot dry on thickly stacked absorbent paper, add 250 μl of washing buffer per well using a multi-pipette, let stand for 30 minutes, remove all liquid from the plate, blot dry on thickly stacked absorbent paper, and wash the plate four times. Add 100 μl of biochemical antibody working solution to each well, seal the plate, and incubate at 37°C for 60 minutes. Blot and wash the plate four times. Add 100 μl of enzyme conjugate working solution to each well, seal the plate, and incubate at 37°C for 30 minutes. Blot and wash the plate five times. Add 100 μl of chromogenic substrate to the reaction wells, seal the plate, and incubate at 37°C in the dark for 15 min. Then add 50 μl of stop solution and immediately measure the OD value at 450 nm using a microplate reader.

[0056] 1.4 RT-qPCR detection of inflammatory factors expression in colon tissue

[0057] RNA was extracted from colon tissue and reverse transcribed into cDNA using a reverse transcription kit. The expression levels of inflammatory factors IL-1β, IL-6, IL-8, and IFN-γ mRNA in colon tissue were detected by RT-qPCR. The real-time quantitative PCR amplification program was as follows: 95℃ pre-denaturation for 12 min; 95℃ denaturation for 15 sec, 64℃ extension for 1 min, 5 cycles; 95℃ denaturation for 15 sec, 58℃ extension for 1 min, 40 cycles. Experimental results were analyzed according to 2... −∆∆Ct The calculations were performed, and the results were standardized to GAPDH expression levels.

[0058] 1.5 Data Statistics and Analysis

[0059] Statistical analysis was performed using SPSS 21.0 software. Results are expressed as mean ± standard deviation (Mean ± SD). Graphpad Prism 8.0 software was used for statistical analysis and graphing. One-way ANOVA was used to compare differences between groups, and Tukey's test was used for multiple comparisons. P < 0.05 was considered statistically significant.

[0060] 2 Results

[0061] 2.1 Effects of the herbal composition of the present invention on inflammatory factors in mouse serum

[0062] The results are as follows Figure 1As shown, compared with the blank control group, the serum levels of TNF-α, IL-1β, IL-6, and IL-8 in the model group mice were significantly increased, and this was statistically significant. After gavage administration of different formulations of traditional Chinese medicine, the serum levels of TNF-α, IL-1β, and IL-6 in mice were significantly reduced, with formulation 1 (the traditional Chinese medicine composition of this invention) showing the most significant reduction. The serum IL-8 level in mice also decreased to some extent after administration compared with the model group, with formulation 1 showing the most significant reduction. Therefore, formulation 1 can reduce the expression of pro-inflammatory factors in mice with ulcerative colitis.

[0063] 2.1 Effects of the herbal composition of the present invention on inflammatory factors in mouse colon tissue

[0064] The results are as follows Figure 2 As shown, compared with the blank control group, the mRNA expression of IL-1β, IL-6, IL-8, and IFN-γ in the colon of model group mice was significantly increased. After gavage administration of different formulations of traditional Chinese medicine, the mRNA expression of IL-1β, IL-6, IL-8, and IFN-γ in the colon of mice was significantly decreased, with the most significant decrease observed in formulation 1 (the traditional Chinese medicine composition of this invention). Therefore, formulation 1 can reduce the expression of pro-inflammatory factors in mice with ulcerative colitis.

[0065] 3. Conclusion

[0066] The above experimental results demonstrate that the herbal composition of this invention (i.e., formulation 1) can effectively reduce the levels of inflammatory factors in the serum and colonic tissue of mice with ulcerative colitis, exhibiting a significant therapeutic effect on the mouse model of ulcerative colitis. Comparison with formulations 2-6 shows that the components in the herbal composition of this invention interact and synergistically exert their effects; the herbal composition lacking some of its ingredients cannot achieve the desired anti-inflammatory and de-inflammatory effects on the mouse model of ulcerative colitis. The herbal composition of this invention is obtained through rational combination based on the principles of syndrome differentiation and treatment in traditional Chinese medicine; each ingredient is indispensable.

[0067] Experimental Example 2

[0068] Animal experiments

[0069] 1. Materials and Methods

[0070] 1.1 Establishment, grouping, and administration of experimental animal models

[0071] SPF-grade male C57BL / 6J mice, weighing (21±2) g, were selected and acclimatized to a 12h / 12h light / dark cycle environment at 25±2℃ and 55±3% relative humidity for 7 days. The mice were randomly divided into four groups: a blank control group, a model group (3% DSS), a high-dose group of the herbal composition of this invention (hereinafter referred to as the high-dose group), and a low-dose group of the herbal composition of this invention (hereinafter referred to as the low-dose group), with 8 mice in each group. The blank control group received deionized water, while the model group and all treated groups had free access to a 3% DSS solution for 7 days, with the DSS solution being replaced with fresh solution every 2 days. Drug administration began on the day of model establishment. The blank control group and model group were administered 0.2 mL of physiological saline by gavage daily, while the experimental groups were administered the herbal composition of this invention by gavage daily. The high-dose group was administered at a dose of 54 g / kg, and the low-dose group at a dose of 27 g / kg by gavage. After 7 days of continuous gavage, treatment was synchronized with model establishment. After 7 days, the administration of sterilized DSS water was discontinued, and the mice were fasted for 24 hours but allowed free access to water before sampling.

[0072] The preparation method of the traditional Chinese medicine composition of this invention is as follows: Weigh the following raw materials according to the following weight proportions: Forsythia suspensa 26 parts, Lilium brownii 24 parts, Lysimachia christinae 12 parts, Sesamum indicum 10 parts, Opuntia ficus-indica 5 parts, Pueraria lobata 12 parts, Centella asiatica 10 parts, Viola yedoensis 14 parts, Houttuynia cordata 14 parts, Portulaca oleracea 6 parts, Litsea cubeba 12 parts, Cirsium japonicum 10 parts, Schizonepeta tenuifolia 10 parts, Morus alba root 6 parts, Lophatherum gracile 14 parts, Lygodium japonicum 4 parts, and Capsella bursa-pastoris 6 parts. Boil twice in 8 times the amount of ddH2O (w / v), 1 hour each time. Centrifuge the extract, and prepare lyophilized powder from the supernatant using a freeze-drying apparatus. Redissolve the lyophilized powder in ddH2O and vortex at room temperature to obtain the traditional Chinese medicine composition of this invention, and store at 4°C for later use.

[0073] 1.2 Obtaining mouse serum

[0074] Mice were fasted for 12 hours but allowed free access to water. Before blood collection, mice were anesthetized with pentobarbital, and 0.5-1 ml of blood was collected from the heart using the cardiac blood collection method. After blood collection, the mice were allowed to stand at room temperature for 2 hours, centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected, aliquoted into 200 μl microcentrifuge tubes, and stored at -80℃ for later testing.

[0075] 1.3 Changes in the Disease Activity Index in Mice

[0076] The animals' health status was recorded daily, including weight changes, stool shape, and presence of bloody stool. (Disease Activity Index, DAI) DAI = (Weight Loss Score + Stool Characteristics Score + Bloody Stool Score) / 3. The scoring criteria are shown in Table 1.

[0077] surface Disease Activity Index Scoring Table

[0078]

[0079] 1.4 Histopathological observation of colon tissue

[0080] Approximately 1 cm of distal colon tissue was harvested from mice and fixed in 4% paraformaldehyde solution. After paraffin embedding, sections were prepared and stained with hematoxylin and eosin for histopathological observation.

[0081] 1.5 ELISA Detection of Serum IL-1β, IL-6, IL-8 and TNF-α Levels

[0082] The methods for determining the levels of serum pro-inflammatory factors IL-1β, IL-6, IL-8, and TNF-α are as follows: 30 minutes before the experiment, bring the reagent kit to room temperature. Wash the plate three times before adding the sample. Add 100 μl of sample to each well, seal the plate, and incubate at 37°C for 90 minutes. After incubation, remove all liquid from the plate, blot dry on thickly stacked absorbent paper, add 250 μl of washing buffer per well using a multi-pipette, let stand for 30 minutes, remove all liquid from the plate, blot dry on thickly stacked absorbent paper, and wash the plate four times. Add 100 μl of biochemical antibody working solution to each well, seal the plate, and incubate at 37°C for 60 minutes. Blot and wash the plate four times. Add 100 μl of enzyme conjugate working solution to each well, seal the plate, and incubate at 37°C for 30 minutes. Blot and wash the plate five times. Add 100 μl of chromogenic substrate to the reaction wells, seal the plate, and incubate at 37°C in the dark for 15 min. Then add 50 μl of stop solution and immediately measure the OD value at 450 nm using a microplate reader.

[0083] 1.6 Data Processing and Statistics

[0084] Statistical analysis was performed using SPSS 21.0 software. Results are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used to compare differences between groups, and Tukey's test was used for multiple comparisons. P < 0.05 was considered statistically significant.

[0085] 2 Results

[0086] 2.1 Effect of the herbal composition of the present invention on the disease activity index of DSS-induced mice

[0087] The results are as follows Figure 3 In the control group, the score did not change significantly during the experiment, while in other groups, the disease activity index (DAI) gradually increased with the extension of the modeling time. The scores of each treatment group were significantly lower than those of the model group, especially the high-dose group (P<0.01). These results indicate that the herbal composition of this invention can reduce the disease DAI in UC mice.

[0088] 2.2 Improvement of the effect of the traditional Chinese medicine composition of the present invention on the pathological changes of DSS-induced colon tissue in mice

[0089] The results are as follows Figure 4 In the control group, the colonic mucosal epithelial tissue of mice was intact, with a clear structure, neatly arranged epithelial cells, and no lesions. The DSS mouse model group exhibited significant characteristics of colitis, including severe mucosal ulceration, loss of crypts, inflammatory cell infiltration, and goblet cell depletion. After treatment with the herbal composition of this invention, these pathological changes in the colonic tissue were improved, with the high-dose group showing the most significant improvement. In the high-dose group, the colonic mucosal epithelium of mice was essentially normalized, the degree of inflammatory cell infiltration was significantly reduced, there was no congestion or edema, and the degree of intestinal villus breakage was also significantly reduced. In the low-dose group, the intestinal villus breakage phenomenon was slightly improved, and the degree of inflammation was mildly reduced. These results indicate that the herbal composition of this invention has a significant improving effect on the damaged mouse colon and a prominent protective effect on the integrity of the colonic tissue structure.

[0090] 2.3 Effects of the herbal composition of this invention on the expression of pro-inflammatory factors in colonic tissue

[0091] like Figure 5 As shown, compared with the control group, the expression of TNF-α, IL-1β, IL-6, and IL-8 in the serum of model group mice was significantly increased. After treatment with the traditional Chinese medicine composition of the present invention, the levels of TNF-α, IL-1β, IL-6, and IL-8 in mouse serum gradually decreased with increasing dosage, and statistical differences were observed in each treatment group. This result indicates that the traditional Chinese medicine composition of the present invention can reduce the expression of pro-inflammatory factors in mice with ulcerative colitis.

[0092] 3. Conclusion

[0093] In summary, the herbal composition of this invention has a significant therapeutic effect on mice with ulcerative colitis, and can repair and improve the integrity of colon tissue structure, enhance intestinal barrier function, and inhibit the expression of pro-inflammatory cytokines.

[0094] Experimental Example 3

[0095] clinical trials

[0096] 1. General Information

[0097] This study included 148 patients with chronic ulcerative colitis from our outpatient and inpatient departments. Patients were randomly divided into two groups using a random number table. The control group consisted of 70 patients (40 males and 30 females), aged 20–60 years (mean age 35.77±4.31 years), with a disease duration of 0.5–10 years (mean duration 4.92±1.09 years). The observation group consisted of 78 patients (40 males and 38 females), aged 22–60 years (mean age 38.12±3.44 years), with a disease duration of 1–12 years (mean duration 5.05±2.01 years). There were no statistically significant differences in baseline characteristics between the two groups (P > 0.05), indicating comparability.

[0098] 2 Diagnostic criteria

[0099] Western medicine diagnosis: Meets the relevant diagnostic criteria of the "Consensus Opinion on Diagnosis and Treatment of Inflammatory Bowel Disease (2018, Beijing)".

[0100] The TCM diagnosis was based on the diagnostic criteria of the "Expert Consensus Opinion on the Diagnosis and Treatment of Ulcerative Colitis in Traditional Chinese Medicine (2010)". The syndrome was classified as spleen deficiency and damp-heat type, with symptoms including: abdominal pain and diarrhea, tenesmus, mucus and bloody stools; accompanied by poor appetite, fatigue, red tongue with teeth marks and yellow and greasy coating, and deep and thready pulse.

[0101] 3 Selection Criteria

[0102] Inclusion criteria: ① All patients met the above diagnostic criteria; ② Both groups of patients were confirmed by histopathology; ③ Informed consent was obtained.

[0103] Exclusion criteria: ① Patients with toxic megacolon or those who have undergone surgery; ② Pregnant or lactating women; ③ Patients with other digestive diseases.

[0104] 4 methods

[0105] The observation group took the traditional Chinese medicine composition described in this invention, specifically: 26 parts forsythia, 24 parts lily, 12 parts creeping privet, 10 parts wild sesame, 5 parts cactus, 12 parts kudzu root, 10 parts centella asiatica, 14 parts wild violet, 14 parts houttuynia cordata, 6 parts purslane, 12 parts mountain licorice, 10 parts large thistle, 10 parts schizonepeta, 6 parts mulberry root, 14 parts light bamboo leaf, 4 parts lygodium japonicum, and 6 parts shepherd's purse. Each ingredient was weighed according to the above weight proportions, washed, and then decocted in 3-4 times the amount of pure water for 25 minutes to obtain approximately 200 mL of decoction. It was taken once a day, 100 mL each after breakfast and dinner.

[0106] The control group received oral mesalazine enteric-coated tablets 0.5g, three times a day.

[0107] Both the observation group and the control group underwent a treatment course of 2 weeks, and the treatment was repeated for 4 consecutive courses.

[0108] 5 Observation Indicators

[0109] Traditional Chinese Medicine (TCM) syndrome score: Based on the efficacy criteria of the "Expert Consensus Opinion on TCM Diagnosis and Treatment of Ulcerative Colitis (2010)," diarrhea, bloody stools, abdominal pain, abdominal distension, anal burning, and tenesmus were scored. The scores were 0, 2, 4, and 6 points respectively, from mild to severe. The higher the score, the more severe the clinical symptoms.

[0110] 6. Efficacy Standards

[0111] (1) Clinical efficacy of Western medicine

[0112] Western medicine references the "Consensus Opinion on Diagnosis and Treatment of Inflammatory Bowel Disease (2018)" for efficacy.

[0113] Complete remission: No symptoms of colitis, colonoscopy showed that the colonic mucosa had basically recovered.

[0114] Significant effect: Colitis symptoms were significantly improved, and microscopic examination showed significant improvement in inflammation.

[0115] Effective: Symptoms of colitis improved, and microscopic examination showed mild inflammation.

[0116] Ineffective: Colitis symptoms did not improve, and endoscopic examination showed no changes.

[0117] (2) Clinical efficacy of traditional Chinese medicine

[0118] The efficacy of TCM treatment is based on the "Expert Consensus Opinions on the Diagnosis and Treatment of Ulcerative Colitis with Traditional Chinese Medicine (2010)".

[0119] Record the degree of relief of diarrhea, bloody stools, abdominal pain, abdominal distension, anal burning, and tenesmus. Each symptom is graded as follows: 0 points for none, 2 points for mild, 4 points for moderate, and 6 points for severe. The efficacy is evaluated based on the efficacy index, which is calculated as: Efficacy Index = (Total score before treatment - Total score after treatment) / Total score before treatment × 100%.

[0120] Complete remission: efficacy index score greater than or equal to 95%.

[0121] Significant efficacy: The efficacy index score is 60%~94%.

[0122] Effective: The efficacy index score is 20%~59%.

[0123] Ineffective: The efficacy index score is less than or equal to 20%.

[0124] 7. Data Statistics and Analysis

[0125] Data were processed using SPSS 20.0 software. Quantitative data were expressed as mean ± standard deviation (Mean ± SD) and analyzed using t-tests. Categorical data were expressed as percentages (%) and analyzed using chi-square tests. 2 The test was performed, and P < 0.05 was considered statistically significant.

[0126] 8 Treatment Results

[0127] As shown in Table 2, there was no significant difference in TCM symptom scores between the observation group and the control group before treatment (P>0.05). After treatment, the TCM symptom scores of both the observation group and the control group were significantly lower than before treatment (P<0.05). Specifically, the TCM symptom scores of the observation group after treatment were lower than those of the control group after treatment, with the scores for symptoms such as bloody stools, abdominal pain, anal heat, and tenesmus being significantly lower in the observation group than in the control group (P<0.05).

[0128] Table 2 Comparison of TCM syndrome scores before and after treatment in the two groups (scores, Mean±SD)

[0129]

[0130] Note: For each symptom index in the observation group and the control group, *compared to before treatment,* P<0.05; for each symptom index, # Compared with the control group, #P<0.05.

[0131] As shown in Tables 2 and 3, the effective rate of treatment in the observation group reached 100%, and the cure rate of treatment in the observation group reached 71-77%, which was significantly higher than the cure rate of treatment in the control group (40-43%).

[0132] Table 3 Comparison of clinical efficacy between the two groups of Western medicine (cases, percentages)

[0133]

[0134] Table 4 Comparison of clinical efficacy between the two groups of traditional Chinese medicine (%)

[0135]

[0136] The above results indicate that the traditional Chinese medicine composition of this invention has a significantly better therapeutic effect on ulcerative colitis than the Western medicine mesalazine enteric-coated tablets, with a higher therapeutic efficacy and cure rate. Furthermore, the traditional Chinese medicine composition of this invention has a significant relieving effect on symptoms such as diarrhea, bloody stools, abdominal pain, abdominal distension, anal burning, and tenesmus, greatly reducing the suffering of patients.

[0137] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A traditional Chinese medicine composition for treating ulcerative colitis, characterized in that, The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: forsythia 10-30 parts, lily 10-30 parts, ground cover 5-20 parts, mountain sesame 5-20 parts, cactus 1-10 parts, radix puerariae 5-20 parts, centella asiatica 5-20 parts, wild shepherd's purse 5-20 parts, houttuynia cordata 5-20 parts, spilanthes acmella 1-10 parts, beilschmiedia 5-20 parts, cirsium japonicum 5-20 parts, schizonepeta 5-20 parts, mulberry root 1-10 parts, ginkgo biloba 5-20 parts, kelp 1-10 parts, shepherd's purse 1-10 parts. ​ 2. The traditional Chinese medicine composition for treating ulcerative colitis according to claim 1, characterized in that, The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: forsythia 15-30 parts, lily 15-25 parts, ground cover 8-16 parts, mountain sesame 8-16 parts, cactus 4-8 parts, radix puerariae 8-16 parts, centella asiatica 8-16 parts, wild shepherd's purse 8-16 parts, houttuynia cordata 8-16 parts, spilanthes acmella 4-8 parts, beilschmiedia 8-16 parts, cirsium japonicum 8-16 parts, schizonepeta 8-16 parts, mulberry root 3-7 parts, ginkgo biloba 8-16 parts, kelp 3-7 parts, shepherd's purse 4-8 parts. 3.The traditional Chinese medicine composition for treating ulcerative colitis according to claim 1, characterized in that, The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: forsythia 26 parts, lily 24 parts, ground cover 12 parts, mountain sesame 10 parts, cactus 5 parts, radix puerariae 12 parts, centella asiatica 10 parts, wild shepherd's purse 14 parts, houttuynia cordata 14 parts, spilanthes acmella 6 parts, beilschmiedia 12 parts, cirsium japonicum 10 parts, schizonepeta 10 parts, mulberry root 6 parts, ginkgo biloba 14 parts, kelp 4 parts, shepherd's purse 6 parts.

4. The traditional Chinese medicine composition for treating ulcerative colitis according to any one of claims 1 to 3, wherein, The traditional Chinese medicine composition is used for preparing a medicine for treating ulcerative colitis, and is prepared into any one of oral liquid, water decoction, granules, pills, enema liquid, capsules and suppositories.

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