Total alkaloids of Coptis chinensis Nepalensis and preparation method and application thereof

Abstract

The present invention discloses total alkaloids of Coptis chinensis, a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine. The Coptis chinensis medicinal material is crushed and extracted, and the extract is concentrated to obtain an extract, and the obtained extract is dissolved with acid, filtered, enriched and purified to obtain a purified product of total alkaloids of Coptis chinensis. The total alkaloid content of Coptis chinensis prepared by the preparation method of the present invention can reach 49%, and has a good therapeutic effect on ulcerative colitis in mice induced by dextran sodium sulfate (DSS). It can significantly alleviate the weight loss of mice with ulcerative colitis induced by DSS, reduce the DAI score, prolong the colon length, and reduce the degree of inflammatory infiltration of colon tissue; regulate biochemical indicators such as inflammation and oxidative stress in colon tissue; and also have a certain regulatory effect on intestinal flora.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to total alkaloids of Coptis chinensis from Nepal, and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is a common inflammatory bowel disease with clinical manifestations such as diarrhea, abdominal pain, bloody stools, weight loss, and vomiting. In recent years, the incidence of UC has been increasing year by year in emerging industrialized countries. Due to its complex etiology, recurrent symptoms, and long duration, it is difficult to cure. Currently, the most common treatments for UC are mainly drug therapy, including glucocorticoids, aminosalicylic acids, immunosuppressants, and biologics. However, many drugs are limited in clinical application and have unstable clinical efficacy. They may even lead to a series of problems such as hepatotoxicity, drug dependence, and recurrent disease after drug withdrawal. In addition, some biologics are expensive and unaffordable for ordinary patients. Therefore, seeking new drugs for the treatment of UC with precise efficacy, few side effects, and reasonable prices has become an urgent clinical need.

[0003] Nepalese Corydalis hendersonii (C. hendersonii) is a perennial herbaceous plant of the genus Corydalis in the family Papaveraceae. Its dried whole herb is used medicinally as Nepalese Corydalis. It is primarily distributed in western Xinjiang, western Qinghai, and central to western Tibet, China, and is a traditional Tibetan medicine with a long history of use. It has an aromatic aroma, cool properties, and a bitter taste. It has the effects of clearing heat and inflammation, cooling blood, and stopping diarrhea. It also has antipyretic, analgesic, anti-inflammatory, and antioxidant properties, and is used to treat high-altitude polycythemia vera. Modern research indicates that Nepalese Corydalis contains various alkaloids, including berberine, papaverine, styraxine, and protopine. Currently, there are numerous methods for preparing and applying Nepalese Corydalis alkaloids. However, in the field of traditional Chinese medicine chemistry, different preparation methods and steps result in different compositions and pharmacological effects of the resulting preparations. Summary of the Invention

[0004] The purpose of the present invention is to provide total alkaloids of Coptis chinensis, a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a preparation method of total alkaloids of Coptis nepalensis. The method comprises the following steps: crushing and extracting a Coptis nepalensis medicinal material, concentrating the extract to obtain an extract, dissolving the obtained extract with acid solution, filtering, enriching and purifying the extract, and thus obtaining a purified product of the total alkaloids of Coptis nepalensis.

[0007] Preferably, the method comprises the following steps:

[0008] (1) Grinding and sieving the medicinal material of Nepalese Coptis chinensis to obtain a powder, ultrasonically extracting it 2-4 times, filtering, combining the filtrate, and concentrating under reduced pressure to obtain a Nepalese Coptis chinensis crude extract;

[0009] (2) dissolving the crude extract of Coptis chinensis with hydrochloric acid and filtering to obtain an acidic solution;

[0010] (3) dynamically adsorbing the acidic solution using macroporous resin XDA-8;

[0011] (4) After adsorption, the macroporous resin is subjected to impurity removal and elution, and the solvent is recovered to obtain the final total alkaloids of Coptis chinensis.

[0012] Preferably, the solvent for ultrasonic extraction is acidic ethanol with a pH of 1-2.

[0013] Preferably, the volume concentration of the ethanol is 70-90%.

[0014] Preferably, the mass fraction of the hydrochloric acid is 0.5%-2%.

[0015] Preferably, the impurity removal is performed by washing with 10-40 BV of water; and the elution is performed by eluting 10-40 BV of ethanol with a volume concentration of 70-90%.

[0016] The present invention also provides the total alkaloids of Coptis chinensis obtained by the preparation method.

[0017] The present invention also provides application of the total alkaloids of Coptis chinensis in preparing medicine for treating ulcerative colitis.

[0018] Preferably, the ulcerative colitis includes dextran sulfate sodium-induced ulcerative colitis.

[0019] The present invention also provides the use of the total alkaloids of Coptis chinensis in preparing a medicine for inhibiting the expression of IL-6, IL-1β and TNF-α.

[0020] The present invention also provides the use of the total alkaloids of Coptis chinensis in preparing a medicine for promoting IL-10 expression.

[0021] The present invention also provides application of the total alkaloids of Coptis chinensis in preparing a medicine for promoting the release of SOD.

[0022] The present invention also provides application of the total alkaloids of Coptis chinensis in preparing a medicine for inhibiting the release of MDA and MPO.

[0023] The present invention discloses the following technical effects:

[0024] The present invention provides total alkaloids of Coptis nepalensis having the effect of treating ulcerative colitis. The total alkaloid content of Coptis nepalensis prepared by the preparation method of the present invention can reach 49%, and the Coptis nepalensis has a good therapeutic effect on ulcerative colitis in mice induced by dextran sodium sulfate (DSS). The Coptis nepalensis can significantly alleviate weight loss, reduce the DAI score, prolong the colon length, and reduce the degree of inflammatory infiltration in colon tissue of mice with DSS-induced ulcerative colitis; regulate biochemical indicators such as inflammation and oxidative stress in colon tissue; and also have a certain regulatory effect on intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 The effect of total alkaloids from Coptis chinensis on the body weight of UC model mice;

[0027] Figure 2 The effect of total alkaloids from Coptis chinensis on the disease activity index (DAI) of UC model mice;

[0028] Figure 3 The effect of total alkaloids from Coptis chinensis on colon length in UC model mice, where A is the result of colon length photographed and B is the statistical result of length.

[0029] Figure 4 The effect of total alkaloids from Coptis chinensis on the inflammatory infiltration of colon tissue in UC model mice;

[0030] Figure 5 Effects of total alkaloids from Coptis chinensis on biochemical indices of inflammatory stress in UC model mice, A and B respectively represent the effects of total alkaloids from Coptis chinensis on biochemical indices of inflammatory stress, including IL-6, IL-1β, TNF-α, and IL-10 in colon tissues of UC model mice;

[0031] Figure 6 are the effects of total alkaloids from Coptis chinensis on the biochemical indices of oxidative stress in UC model mice, where AC are the effects of total alkaloids from Coptis chinensis on the biochemical indices of oxidative stress in UC model mice, namely SOD, MDA and MPO;

[0032] Figure 7 This is the effect of total alkaloids from Coptis chinensis on the intestinal flora of UC model mice. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0039] Example 1

[0040] 100g of dried Coptis chinensis (Nepalese Corydalis) is ground and sieved to obtain a powder. The powder is extracted with acidic ethanol (pH 1-2) at a volume concentration of 70-90%. Ultrasonic extraction is performed 2-4 times, and the filtrates are filtered. The filtrates are combined and concentrated under reduced pressure to obtain a crude extract of total Coptis chinensis alkaloids. The obtained total Coptis chinensis alkaloids are dissolved in 0.5%-2% hydrochloric acid and filtered. The resulting acid solution is passed through XDA-8 macroporous resin for dynamic adsorption. After adsorption, the macroporous resin is rinsed with 10-40 BV of water, then eluted with 10-40 BV of 70-90% ethanol by volume. The solvent is recovered to obtain a purified Coptis chinensis alkaloid extract. Using acid dye colorimetry combined with UV-visible spectrophotometry, with berberine as the reference, the test results indicate that the total alkaloid content of Coptis chinensis alkaloids can reach 49%.

[0041] Example 2

[0042] 1. Ulcerative colitis mouse modeling: Using the internationally accepted ulcerative colitis modeling method, C57BL / 6 mice were adaptively reared for one week and then allowed to freely drink a 3% DSS solution prepared in sterile water for 7 days to induce an ulcerative colitis mouse model. The successful establishment of the ulcerative colitis mouse model was then verified using indicators such as changes in mouse weight, stool morphology and degree of blood in stool, colon length, and colon tissue pathological changes.

[0043] 2. Experimental methods: Fifty-six SPF healthy male C57BL / 6 mice weighing 18-22 g were adaptively fed for 7 days and randomly divided into 7 groups: blank group (Blank), model group (Model), Guchang Zhixie pill group (Pill), 5-aminosalicylic acid group (5-ASA), high-dose nepali total alkaloids group (High), medium-dose nepali total alkaloids group (Medium), and low-dose nepali total alkaloids group (Low); the blank group was given free access to sterilized water, and the other groups were given free access to 3% nepali total alkaloids per day. The mice were treated with sterile DSS water, and all other conditions were the same. The day of modeling was the day of administration. The blank and model groups were given the vehicle solvent by gavage at 0.1 mL / 10 g body weight. The Guchang Zhixie Wan group was given 200 mg / kg / d of Guchang Zhixie Wan by gavage at 0.1 mL / 10 g body weight. The 5-aminosalicylic acid group was given 200 mg / kg / d of 5-ASA by gavage at 0.1 mL / 10 g body weight. The high-dose, medium-dose, and low-dose groups were given 200 mg / kg / d, 100 mg / kg / d, and 50 mg / kg / d of total alkaloids from Coptis chinensis (Coleoptera: Corydalis) by gavage at 0.1 mL / 10 g body weight. The mice were administered once daily for 7 consecutive days. After the last dose, they were fasted for 12 hours, but water was not allowed. The body weight, stool morphology, and degree of blood in the stool were recorded daily during the 7 days of the experiment.

[0044] Seven days after administration, the mice were killed, their blood and colon tissues were collected and processed, and the colon length was recorded by photographing. Hematoxylin-eosin staining (H&E staining) was used to observe the pathological changes of colon tissue. Enzyme-linked immunosorbent assay (ELISA) was used to determine biochemical indicators such as colon tissue inflammation and oxidative stress. The contents of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor (TNF-α), interleukin-10 (IL-10), superoxide dismutase (SOD), malondialdehyde (MDA), and myeloperoxidase (MPO) were determined according to the instructions of each kit (IL-6, IL-1β, TNF-α are pro-inflammatory factor indicators, and IL-10 is an anti-inflammatory factor indicator).

[0045] Experimental results:

[0046] Effects of total alkaloids from Coptis chinensis on body weight in UC model mice Figure 1 From the 5th day, except for the blank group, all other groups showed significant weight loss, with the model group showing the most significant weight loss. The weight loss in each group treated with the total alkaloids of Coptis chinensis was alleviated. Therefore, the total alkaloids of Coptis chinensis preparation for treating ulcerative colitis of the present invention can significantly alleviate the weight loss caused by ulcerative colitis.

[0047] Effects of total alkaloids from Coptis chinensis on disease activity index (DAI) in UC model mice Figure 2 The DAI score table for evaluating UC model mice is shown in Table 1. The DAI score of mice is composed of the percentage of body weight loss, stool morphology score, and blood in stool score.

[0048] Calculation formula: DAI = (percentage of body weight loss + stool shape score + blood in stool score) / 3.

[0049] Table 1 Disease Activity Index (DAI) score for ulcerative colitis

[0050]

[0051] Note: (+) indicates mild severity, and (++) indicates severe severity.

[0052] Depend on Figure 2 It can be seen that as the number of days of administration increases, compared with the model group, each administration group of Coptis chinensis total alkaloids can significantly alleviate the increase in DAI score, indicating that the Coptis chinensis total alkaloid preparation for treating ulcerative colitis of the present invention significantly reduces the DAI score of UC mice.

[0053] Depend on Figure 3It can be seen that compared with the blank group, the colon length of the mice in the model group was significantly shortened, while the groups administered with total alkaloids of Coptis chinensis obtained certain relief, indicating that the total alkaloid preparation of Coptis chinensis for treating ulcerative colitis of the present invention significantly prolonged the colon length of UC mice.

[0054] Depend on Figure 4 It can be seen that the colon tissue of the blank group mice had intact mucosa and normal villi, and there was no inflammatory cell infiltration or mucosal erosion. Compared with the blank group, the colon tissue morphology of the model group mice changed significantly, with a large number of inflammatory cells infiltrating, a significant decrease in the number of goblet cells and crypts, edema in the submucosal area of ​​the colon, incomplete intercellular spaces, and severe damage to the colon mucosal barrier. After treatment with the high-dose group of Coptis chinensis total alkaloids, the colon tissue morphology of the UC mice was significantly improved, inflammatory cell infiltration was significantly reduced, and the intestinal mucosal barrier was relatively intact. Therefore, it can be concluded that the Coptis chinensis total alkaloids preparation of the present invention for treating ulcerative colitis significantly reduces the degree of inflammatory infiltration of colon tissue caused by ulcerative colitis.

[0055] Depend on Figure 5 It can be seen that compared with the model group, each administration group of Nepalese Corydalis total alkaloids can significantly inhibit the release of proinflammatory factors IL-6, IL-1β, and TNF-α, and show a dose-dependent manner, among which the Nepalese Corydalis total alkaloids high-dose group has the best inhibitory effect; compared with the model group, each administration group of Nepalese Corydalis total alkaloids can significantly promote the release of anti-inflammatory factor IL-10, and show a dose-dependent manner, among which the Nepalese Corydalis total alkaloids high-dose group has the best promoting effect. It can be seen that the Nepalese Corydalis total alkaloids prepared by the preparation method of the present invention exert their medicinal effects by inhibiting proinflammatory factors and promoting the release of anti-inflammatory factors. The Nepalese Corydalis total alkaloids preparation for treating ulcerative colitis of the present invention can significantly alleviate the imbalance of inflammatory stress biochemical indicators caused by ulcerative colitis.

[0056] Depend on Figure 6 As can be seen from the comparison with the blank group and the model group, each group of the total alkaloids of Nepalese Corydalis can promote the release of SOD and inhibit the release of MDA and MPO, thereby exerting its effect. This shows that the total alkaloids of Nepalese Corydalis preparation for treating ulcerative colitis of the present invention can significantly alleviate the imbalance of oxidative stress biochemical indicators caused by ulcerative colitis.

[0057] Depend on Figure 7 It can be seen that the total alkaloid preparation of Coptis chinensis exerts a therapeutic effect by regulating the ratio of harmful and beneficial bacteria in the mouse intestine, indicating that the total alkaloid preparation of Coptis chinensis for treating ulcerative colitis of the present invention can significantly regulate the imbalance of intestinal flora caused by ulcerative colitis.

[0058] In summary, it can be seen that the total alkaloids of Coptis chinensis prepared by the preparation method of the present invention can significantly alleviate the weight loss of mice with DSS-induced ulcerative colitis, reduce the DAI score, prolong the colon length, and reduce the degree of inflammatory infiltration of colon tissue; regulate biochemical indicators such as inflammation and oxidative stress in colon tissue; and regulate the intestinal flora.

[0059] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A use of total alkaloids of Coptis chinensis in the preparation of a medicament for treating ulcerative colitis, characterized in that: The preparation method of the total alkaloids of Nepalese Coptis chinensis comprises the following steps: crushing and extracting the medicinal material of Nepalese Coptis chinensis, concentrating the extract to obtain an extract, dissolving the obtained extract with acid solution, filtering, enriching and purifying, thereby obtaining a purified product of the total alkaloids of Nepalese Coptis chinensis; The following steps are involved: (1) Crush and sieve the Nepalese Corydalis medicinal material to obtain powder, perform ultrasonic extraction 2-4 times, filter, combine the filtrate, and concentrate under reduced pressure to obtain the Nepalese Corydalis crude extract; (2) dissolving the crude extract of Nepalese Coptis chinensis with hydrochloric acid and filtering to obtain an acidic solution; the mass fraction of the hydrochloric acid is 0.5%-2%; (3) dynamically adsorbing the acidic solution using macroporous resin XDA-8; (4) After adsorption, the macroporous resin is subjected to impurity removal and elution, and the solvent is recovered to obtain the final total alkaloids of Coptis chinensis; The impurity removal is performed by washing with 10-40 BV of water; and the elution is performed by eluting 10-40 BV of ethanol with a volume concentration of 70-90%.

2. The use according to claim 1, characterized in that The total alkaloids of Coptis chinensis can treat ulcerative colitis by inhibiting the expression of IL-6, IL-1β and TNF-α, promoting the expression of IL-10, promoting the release of SOD, and inhibiting the release of MDA and MPO.

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