Application of dendrobium candidum stem / leaf polysaccharide

Abstract

The application of the Dendrobium candidum stem / leaf polysaccharide in preparing medicines or health foods for preventing and treating ulcerative colitis. The Dendrobium candidum stem / leaf polysaccharide obtained by extracting stems or leaves of specific Dendrobium candidum species by a specific extraction process has a higher content and a better prevention and treatment effect on ulcerative colitis, and can significantly alleviate the phenomenon of sudden weight loss of mice, reduce the DAI value of the colon tissue of ulcerative colitis mice, alleviate the abnormal atrophy or hypertrophy of the immune organs of mice, and restore the colon length. The Dendrobium candidum stem / leaf polysaccharide obtained by the application has the characteristics of definite curative effect, and has a good market development and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Dendrobium officinale stem / leaf polysaccharides, and more specifically to the application of Dendrobium officinale stem / leaf polysaccharides in the preparation of drugs or health foods for the prevention and treatment of ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that begins with inflammation of the rectal mucosa and extends proximally in a continuous manner. In the 21st century, UC has become a globally prevalent disease with an increasing incidence. Many factors can drive the pathogenesis of UC, such as genetic susceptibility and chronic immune-mediated intestinal inflammation. Currently, UC is incurable, and patients typically require lifelong medication. Conventional drug treatments can produce serious side effects due to systemic administration and drug redistribution within the body. Moreover, this traditional systemic treatment strategy does not target the site of inflammation, thus yielding only limited efficacy.

[0003] The main symptoms of colonic ulcer (UC) include abdominal pain, diarrhea, and weight loss. During the active phase of UC, the intestinal mucosa exhibits diffuse inflammation, with abundant neutrophils and eosinophils infiltrating the epithelium, crypts, and lamina propria. The mucosa is congested and edematous, and the colonic mucosa surface shows erosion and ulceration. UC lesions are generally limited to the mucosa and submucosa, not involving the muscularis propria or serosa. Clinical symptoms are primarily abdominal pain, diarrhea, and bloody, mucous stools, and it easily leads to intestinal perforation, anemia, and colon cancer.

[0004] Currently, the main medications for treating ulcerative colitis (UC) are anti-inflammatory drugs, immunosuppressants, biologics, and antibiotics, which can only help relieve symptoms. Long-term use of these drugs can lead to serious side effects, including steroid dependence and secondary infections. Therefore, there is an urgent need to develop new, highly effective, and low-toxicity drugs for UC treatment. Dendrobium officinale has long been used as a functional food; polysaccharides are its main active ingredient, exhibiting various pharmacological properties, including anti-inflammatory, immunomodulatory activities, and therapeutic effects on liver fibrosis. This could provide a new, highly effective, and low-toxicity strategy for UC treatment.

[0005] There are also some existing technologies that utilize Dendrobium officinale to treat ulcerative colitis. For example, Chinese Patent No. CN114796398 A discloses the application of Dendrobium officinale flower extract in the preparation of drugs to improve ulcerative colitis; another example is Chinese Patent No. CN114404525A, which discloses a Dendrobium officinale composition, its preparation method and application. This composition consists of the following drug components in parts by weight: 10-50 parts Dendrobium officinale and 5-30 parts Asparagus, which has a good preventive and therapeutic effect on ulcerative colitis.

[0006] However, there are no reports in the existing technology on the role of Dendrobium officinale stems / leaves and Dendrobium officinale stems / leaves polysaccharides in the prevention and treatment of ulcerative colitis, or their use in drugs or functional foods for treating this disease. Summary of the Invention

[0007] In view of the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for the application of Dendrobium officinale stem / leaf polysaccharides in the preparation of drugs or health foods for the prevention and treatment of ulcerative colitis.

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

[0009] This invention provides the application of Dendrobium officinale stem / leaf polysaccharides in the preparation of drugs or health foods for the prevention and treatment of ulcerative colitis.

[0010] Furthermore, the application of the Dendrobium officinale stem / leaf polysaccharide in the preparation of drugs or health foods that alleviate rapid weight loss caused by ulcerative colitis.

[0011] Furthermore, the application of the Dendrobium officinale stem / leaf polysaccharide in the preparation of drugs or health foods that reduce the DAI value of colonic tissue caused by ulcerative colitis.

[0012] Furthermore, the application of the Dendrobium officinale stem / leaf polysaccharide in the preparation of drugs or health foods for relieving abnormal atrophy or swelling of immune organs caused by ulcerative colitis and restoring colon length.

[0013] Furthermore, the Dendrobium officinale mentioned is a cultivated variety from Zhejiang Province, specifically Shenglan No. 8.

[0014] Furthermore, the Dendrobium officinale stem / leaf polysaccharide is prepared through the following steps:

[0015] The stems / leaves of Dendrobium officinale were homogenized with water at a ratio of 1:15, and then extracted by shaking in a constant temperature water bath at 45-85℃ for 40 minutes. After filtration through four layers of gauze, the supernatant was collected by centrifugation at 4000r / min for 10 minutes. Five times the volume of ethanol was added, and the mixture was allowed to stand at 4℃ for 12 hours before being filtered through a 2000-mesh filter cloth to obtain the precipitate.

[0016] The precipitate was redissolved in distilled water and dialyzed for 72 hours using an 8000-14000 Da dialysis bag, with the water changed every 4 hours during the process.

[0017] After dialysis, the samples were freeze-dried under vacuum for 72 hours to obtain Dendrobium officinale stem polysaccharide and Dendrobium officinale leaf polysaccharide.

[0018] Another aspect of the present invention provides a drug or health food for the prevention and treatment of ulcerative colitis, characterized in that its active ingredient is Dendrobium officinale stem / leaf polysaccharide.

[0019] The beneficial effects of this invention are as follows: By employing a specific extraction process on the stems or leaves of a particular variety of Dendrobium officinale, this invention yields Dendrobium officinale stem / leaf polysaccharides with a higher content and better preventive and therapeutic effects on ulcerative colitis. It can significantly alleviate rapid weight loss in mice, reduce the DAI value of colonic tissue in mice with ulcerative colitis, alleviate abnormal atrophy or swelling of immune organs in mice, and restore colon length. The Dendrobium officinale stem / leaf polysaccharides obtained by this invention have definite therapeutic effects and possess promising market development and application prospects. Attached Figure Description

[0020] Figure 1 The graph shows the daily variation of mouse body weight and disease activity index (DAI) in Example 2;

[0021] Figure 2 The weight of the mice in Example 3;

[0022] Figure 3 This refers to the daily variation of the Disease Activity Index (DAI) in Example 3;

[0023] Figure 4 The thymus index in Example 3;

[0024] Figure 5 The spleen index in Example 3;

[0025] Figure 6 The length of the colon in Example 3;

[0026] Figure 7 This refers to the expression of inflammatory factors in Example 3;

[0027] Figure 8 This refers to the expression of intestinal barrier-related factors in Example 3;

[0028] In the figure, DOPY represents the leaf polysaccharide of Dendrobium officinale, and DOPJ represents the stem polysaccharide of Dendrobium officinale. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0030] Example 1: Preparation of polysaccharides from Dendrobium officinale stems / leafs

[0031] Take Dendrobium officinale stems and leaves and water in a 1:15 ratio to homogenize, add to a 45-85℃ constant temperature water bath and shake to extract for 40 min, then filter through four layers of gauze, centrifuge at 4000 r / min for 10 min to collect the supernatant, add 5 times the volume of ethanol, let stand at 4℃ for 12 h, and then filter through a 2000 mesh filter to obtain the precipitate.

[0032] The precipitate was redissolved in distilled water and dialyzed for 72 hours using an 8000-14000 Da dialysis bag, with the water changed every 4 hours during the process.

[0033] After dialysis, the sample was freeze-dried under vacuum for 72 hours to obtain Dendrobium officinale stem polysaccharide / Dendrobium officinale leaf polysaccharide.

[0034] Example 2: Comparison of Structure-Activity Relationships among Different Varieties of Dendrobium officinale

[0035] SPF-grade male C57BL / 6 mice (License No.: SCXK(Su)2018-0008, 7 weeks old, 22±2g) were purchased from Jiangsu Jicui Biotechnology Co., Ltd. All animal protocols were approved by the Animal Care and Use Committee of the Animal Center of Zhejiang Academy of Agricultural Sciences (License No.: SYXK(Zhe)2020-0022). Eighty-four mice were randomly divided into 14 groups (n=6). The housing conditions were: temperature 25℃, humidity 40%-60%, 12-hour light / 12-hour dark cycle. After one week of acclimatization, the groups were named control group (Con), model group (DSS), 1Y100 group (DOP1Y, 100mg / kg), 1Y200 group (DOP1Y, 200mg / kg), 2Y100 group (DOP2Y, 100mg / kg), 2Y200 group (DOP2Y, 200mg / kg), 3Y100 group (DOP3Y, 100mg / kg), and 3Y200 group (Con, Model ... The mice were divided into four groups: DOP3Y (200 mg / kg), 1J100 (DOP1J, 100 mg / kg), 1J200 (DOP1J, 200 mg / kg), 2J100 (DOP2J, 100 mg / kg), 2J200 (DOP2J, 200 mg / kg), 3J100 (DOP3J, 100 mg / kg), and 3J200 (DOP3J, 200 mg / kg). The experiment lasted 7 days. Mice had free access to food and water. The Con group was provided with purified water without DSS, while the other groups were provided with purified water containing 3% DSS. Mice in each DOP group were easily administered the corresponding dose of polysaccharide via gavage daily.

[0036] like Figure 1As shown, the effects of stem and leaf polysaccharides from *D. 1* (DOP1 group), *D. 3* (DOP2 group), and *D. 8* (DOP3 group) on ulcerative colitis (UC) mice were investigated. The results showed that after DSS treatment, the mice gradually lost weight. Compared with the model group, other polysaccharides had no significant effect on weight loss. The weight loss of Y100 and J200 in the DOP3 group was even lower, indicating that Y100 and J200 could slow down the trend of weight loss in UC mice. The DAI index is an important indicator for evaluating UC models in mice. This index assesses the severity of the disease by observing the daily fecal state and weight changes of mice; a higher DAI score indicates a more severe condition. The DAI score of the DSS group was significantly higher than that of the model group, while the DAI scores of Y100 and J200 in the DOP3 group were lower than those of the model group, indicating that these polysaccharides can alleviate the symptoms of UC mice and have a therapeutic effect.

[0037] Example 3: Ulcerative Colitis Model in Mice Induced by Dendrobium officinale Stem / Leaf Polysaccharides 1. Animal Experiment Design

[0038] SPF-grade male Balb / c mice (License No.: SCXK(Su)2018-0008, 7 weeks old, 20±2g) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All animal protocols were approved by the Animal Care and Use Committee of the Animal Center of Zhejiang Academy of Agricultural Sciences (License No.: SYXK(Zhe)2020-0022). The mice were randomly divided into 5 groups (n=12), and the feeding conditions were the same as in Example 2. After one week of acclimatization, the Con group was given a standard diet and purified water, and was given physiological saline (10 mL / kg) by gavage once a day for 14 days. The DSS group was given a standard diet and purified water for the first 7 days, and then 3% (w / v) DSS was added to the drinking water for 7 days from days 8 to 14 to establish the UC model. The positive control group (SASP) was given physiological saline by gavage for the first 7 days, and SASP (400 mg / kg) by gavage for the next 7 days. Mice in the DOP group were given DOP solution (DOPY 50 mg / kg, DOPJ 150 mg / kg) by gavage daily from days 1 to 14, and 3% (w / v) DSS was added to the drinking water for days 8 to 14. On day 15, all mice were given purified water. On day 16, the mice were sacrificed, blood was collected, and after dissection, the spleen and thymus were weighed, the length of the colon between the ileocecal junction and the anal margin was measured, and the corresponding serum indicators were determined.

[0039] DOPY is a leaf polysaccharide from group DOP3, and DOPJ is a stem polysaccharide.

[0040] 2. Evaluation of the Disease Activity Index (DAI)

[0041] DAI is determined by assessing changes in weight loss, diarrhea status, and fecal bleeding using a reported methodology.

[0042] DAI = (Body Mass Index + Stool Shape + Bleeding) / 3

[0043] Table 1. Scoring criteria for the Disease Activity Index (DAI) in mice.

[0044]

[0045] 3. Results Analysis

[0046] 1) Weight

[0047] like Figure 2 As shown: Except for the control group, other mice experienced a rapid and sustained decrease in body weight after 4-5 days of drinking DSS, consistent with successful model establishment. The positive treatment group had no effect on alleviating the weight loss in mice. Both polysaccharide groups significantly alleviated the weight loss in mice, with stem polysaccharide being superior to leaf polysaccharide.

[0048] 2) Disease Activity Index (DAI)

[0049] like Figure 3 As shown, compared with the control group (Con), the DAI value of the DSS group was significantly higher, while the DAI scores of the positive control group (SASP) and the two Dendrobium polysaccharide groups were significantly lower. The polysaccharide group score was lower than that of the SASP group in the first 13 days, and then they were similar in the 15th day.

[0050] 3) Organ Index

[0051] like Figure 4 As shown, compared with the control group, the thymus index in the DSS group was significantly lower, indicating thymus atrophy. The positive control group showed no significant improvement, while the polysaccharide group significantly increased the thymus index, with the stem polysaccharide group reaching normal values. Figure 5 As shown, the spleen index in the DSS group was significantly increased, indicating splenomegaly. The positive control group had no effect on this and showed an increasing trend. The polysaccharide group significantly reduced the spleen index, and the index in the leaf polysaccharide group was even lower. This indicates that the positive control group had no effect on alleviating splenomegaly and thymic atrophy in mice, while Dendrobium polysaccharides had a significant effect.

[0052] 4) Colon length

[0053] like Figure 6 As shown, the colon length in the model group was significantly shorter than that in the control group, while the colon length in the positive drug group was significantly longer than that in the model group. The colon length in both polysaccharide groups was longer than that in the model group.

[0054] 5) Inflammatory factors

[0055] Inflammatory cytokines, especially TNF-α, IL-1β, and IL-6, play a crucial role in the development of colitis (UC). TNF-α is an important driver of intestinal inflammation; IL-1β is produced by monocytes and intestinal epithelial cells; blocking IL-1β signaling can improve colitis and related colorectal cancer; and IL-6 can exert a pro-inflammatory effect by inhibiting T cell apoptosis. Figure 7 The results showed that the levels of TNF-α, IL-1β, and IL-6 in the model group were higher than those in the control group. The expression levels of TNF-α, IL-1β, and IL-6 were reduced in the positive drug and polysaccharide treatment groups. Among them, leaf polysaccharide was superior to stem polysaccharide, which was superior to the positive drug, indicating that DOP has anti-inflammatory activity.

[0056] 6) Intestinal barrier analysis

[0057] One of the pathogenesis mechanisms of ulcerative colitis (UC) is the disruption of the intestinal barrier, allowing harmful bacteria to enter and disrupting the balance between luminal antigens and mucosal immunity. Tight junction protein ZO-1 (occludin) plays a crucial role in maintaining intestinal barrier function, protecting the intestine from allergens, toxins, and pathogens. The main component of intestinal mucus is a high-molecular-weight glycoprotein called mucin (Muc2), which acts as a protective physical barrier to prevent harmful substances from reaching the surface of epithelial cells. Figure 8 It is evident that the expression of ZO-1, occludin, and Muc2 was significantly reduced in the DSS group, indicating the damage of DSS to the integrity of the colonic membrane. The expression of ZO-1 in the colonic tissue of the positive drug SASP and leaf polysaccharide groups was significantly increased. The positive drug SASP had the most significant restorative effect on occludin expression, and stem polysaccharide also had an effect. The expression level of Muc2 in both the positive drug group and the polysaccharide group was significantly higher than that in the model group. This suggests that Dendrobium officinale polysaccharides help restore the integrity of the intestinal barrier in colonic tissue, and that leaf polysaccharides and stem polysaccharides have different mechanisms of action.

[0058] In summary, the Dendrobium officinale stem polysaccharides and Dendrobium officinale leaf polysaccharides of this application can significantly alleviate the phenomenon of rapid weight loss in mice, reduce the DAI value of colon tissue in mice with ulcerative colitis, alleviate the abnormal atrophy or swelling of immune organs in mice, restore colon length, reduce the expression of inflammatory factors and repair the intestinal barrier, and can be used to prevent and treat ulcerative colitis and repair the intestinal barrier.

Claims

1. Application of Dendrobium officinale leaf polysaccharide in the preparation of drugs for the prevention and treatment of ulcerative colitis, wherein the Dendrobium officinale is a cultivated variety in Zhejiang Province, specifically Shenglan No. 8, and the Dendrobium officinale leaf polysaccharide is prepared through the following steps: Dendrobium officinale leaves and water were homogenized at a ratio of 1:15, and then extracted by shaking in a constant temperature water bath at 45-85℃ for 40 min. After filtration through four layers of gauze, the mixture was centrifuged at 4000 r / min for 10 min. The supernatant was collected, and 5 times the volume of ethanol was added. After standing at 4℃ for 12 h, the mixture was filtered through a 2000 mesh filter cloth to obtain the precipitate. The precipitate was redissolved in distilled water and dialyzed for 72 hours using an 8000-14000 Da dialysis bag, with the water changed every 4 hours during the process. After dialysis, the sample was freeze-dried under vacuum for 72 h to obtain Dendrobium officinale leaf polysaccharide; The polysaccharide from Dendrobium officinale leaves can improve the rapid weight loss and increased disease activity index caused by ulcerative colitis, restore colon length, and reduce pathological damage to colon tissue.

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