CN116617249A - Application of dendrobium candidum stem/leaf polysaccharide - Google Patents

Abstract

The dendrobium candidum stem/leaf polysaccharide is applied to the preparation of medicines or health-care foods for preventing and treating ulcerative colitis. According to the application, the stem or leaf of the dendrobium candidum of a specific variety is extracted by adopting a specific extraction process, so that the obtained stem/leaf of the dendrobium candidum has higher polysaccharide content and better control effect on ulcerative colitis, and can remarkably relieve the phenomenon of sudden weight loss of mice, reduce the DAI value of colon tissues of the mice with ulcerative colitis, relieve abnormal atrophy or swelling of immune organs of the mice and restore colon length. The stem/leaf polysaccharide of dendrobium candidum obtained by the application has the characteristics of definite curative effect and the like, and has good market development and application prospects.

Description

Application of Stem/Leaf Polysaccharide of Dendrobium officinale

technical field

The invention belongs to the technical field of biomedicine, and specifically relates to the application of the stem/leaf polysaccharide of Dendrobium officinale, and more specifically to the application of the stem/leaf polysaccharide of Dendrobium officinale in preparing medicines or health food for preventing and treating ulcerative colitis.

Background technique

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) of the colon that begins with inflammation of the rectal mucosa and extends proximally in a serial fashion. In the 21st century, UC has become a global disease with increasing incidence. Many factors can drive the pathogenesis of UC, such as genetic predisposition and chronic immune-mediated intestinal inflammation. Currently, UC is incurable and patients often need to take medication for life. When using conventional drug therapy, serious side effects may occur due to systemic administration and drug redistribution in the body. Moreover, this traditional systemic treatment strategy does not target the site of inflammation, therefore, only limited efficacy can be obtained from it.

The main symptoms of UC include abdominal pain, diarrhea and weight loss. The intestinal mucosa exhibited diffuse inflammation during the active phase of UC, and there was a large infiltration of neutrophils and eosinophils in the epithelium, crypts, and lamina propria. The mucosa is hyperemic and edematous, and the surface of the colonic mucosa is eroded and ulcerated. The lesions of UC are generally limited to the mucosa and submucosa, without involving the muscular layer and serosa. Clinical symptoms are mainly manifested by abdominal pain, diarrhea, and stool with mucus and pus and blood, which can easily lead to intestinal perforation, anemia and colon cancer.

At present, the main drugs for the treatment of ulcerative colitis (UC) are mainly anti-inflammatory drugs, immunosuppressants, biological agents and antibiotics, etc., which can only help relieve the symptoms of the disease. Long-term use of these drugs can cause some serious side effects, including steroid dependence and secondary infections. Therefore, it is urgent to develop new drugs with high efficiency and low toxicity for the treatment of UC. Dendrobium candidum has been used as a functional food. Polysaccharides are the main active ingredients of Dendrobium candidum, which can exert various pharmacological properties, including anti-inflammatory, immunomodulatory activities, and therapeutic effects on liver fibrosis, and can provide a highly effective and low-toxicity New strategies for the treatment of UC.

In the prior art, there are also some technical solutions utilizing Dendrobium candidum to treat ulcerative colitis, such as the Chinese patent whose publication number is CN114796398 A, which discloses the application of a Dendrobium candidum flower extract in the preparation of medicines for improving ulcerative colitis; For example, the Chinese patent with the publication number CN 114404525A discloses a composition of Dendrobium officinale and its preparation method and application. 30 parts, it has a good preventive effect on ulcerative colitis.

However, there is no report in the prior art on the effect of Dendrobium officinale stem/leaf and Dendrobium officinale stem/leaf polysaccharide on the prevention and treatment of ulcerative colitis and its use in medicine or functional food for treating this disease.

Contents of the invention

Aiming at the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for the application of Dendrobium officinale stem/leaf polysaccharides in the preparation of medicines or health food for the prevention and treatment of ulcerative colitis.

In order to achieve the above object, the present invention specifically adopts the following technical solutions:

One aspect of the present invention provides the application of stem/leaf polysaccharide of Dendrobium candidum in the preparation of medicine or health food for preventing and treating ulcerative colitis.

Further, the application of the stem/leaf polysaccharide of Dendrobium officinale in the preparation of medicine or health food for relieving sudden weight loss caused by ulcerative colitis.

Further, the application of the stem/leaf polysaccharide of Dendrobium officinale in the preparation of medicine or health food for reducing the DAI value of colon tissue caused by ulcerative colitis.

Further, the application of the stem/leaf polysaccharide of Dendrobium officinale in the preparation of medicine or health food for relieving abnormal atrophy or enlargement of immune organs caused by ulcerative colitis and restoring colon length.

Further, the Dendrobium candidum is a cultivated species in Zhejiang, specifically Shenglan No. 8.

Further, the stem/leaf polysaccharide of Dendrobium officinale is prepared through the following steps:

Dendrobium candidum stems/leaves and water were homogenized at a ratio of 1:15, added to a constant temperature water bath at 45-85°C, shaken and extracted for 40 minutes, then filtered through four layers of gauze, centrifuged at 4000r/min for 10 minutes to collect the supernatant, and then added 5 times the volume of ethanol. After standing at 4°C for 12 hours, it was filtered through a 2000-mesh filter cloth to obtain a precipitate;

Redissolve the precipitate in distilled water, dialyze with 8000-14000Da dialysis bag for 72h, change the water every 4h during this period;

After the dialysis, the samples were vacuum freeze-dried for 72 hours to obtain stem polysaccharides of Dendrobium officinale and leaf polysaccharides of Dendrobium officinale.

Another aspect of the present invention provides a medicine or health food for preventing and treating ulcerative colitis, which is characterized in that its active component is the stem/leaf polysaccharide of Dendrobium officinale.

The beneficial effect of the present invention is that: the present invention extracts the stems or leaves of a specific species of Dendrobium candidum by using a specific extraction process, and the stem/leaf polysaccharide content of the obtained Dendrobium candidum is higher, and has a better effect on ulcerative colitis. The preventive effect can significantly alleviate the phenomenon of sudden weight loss in mice, reduce the DAI value of colon tissue in mice with ulcerative colitis, relieve abnormal atrophy or enlargement of immune organs in mice, and restore the length of the colon. The stem/leaf polysaccharide of dendrobium candidum obtained by the invention has the characteristics of definite curative effect and the like, and has good market development and application prospects.

Description of drawings

Fig. 1 is the diurnal variation figure of mouse body weight situation and disease activity index (DAI) in embodiment 2;

Fig. 2 is the mouse body weight situation in embodiment 3;

Fig. 3 is the diurnal variation of disease activity index (DAI) in embodiment 3;

Fig. 4 is thymus index in embodiment 3;

Fig. 5 is spleen index in embodiment 3;

Fig. 6 is colon length in embodiment 3;

Figure 7 is the expression of inflammatory factors in Example 3;

Figure 8 is the expression of factors related to intestinal barrier in Example 3;

In the figure, DOPY is the leaf polysaccharide of Dendrobium candidum, and DOPJ is the stem polysaccharide of Dendrobium candidum.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments and accompanying drawings, but the protection scope of the present invention is not limited thereto.

Embodiment 1: the preparation of Dendrobium officinale stem/leaf polysaccharide

Take the stems, leaves and water of Dendrobium officinale and homogenize at a ratio of 1:15, add to a 45-85°C constant temperature water bath, shake and extract for 40 minutes, then filter with four layers of gauze, centrifuge at 4000r/min for 10 minutes to collect the supernatant, add 5 times the volume of ethanol , after standing at 4°C for 12 hours, it was filtered through a 2000-mesh filter cloth to obtain a precipitate;

Redissolve the precipitate in distilled water, dialyze with 8000-14000Da dialysis bag for 72h, change the water every 4h during this period;

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

Embodiment 2: the structure-activity relationship comparison of different varieties of Dendrobium officinale

SPF-grade C57BL/6 male mice (permit number: SCXK (Su) 2018-0008, 7 weeks old, 22 ± 2 g) were purchased from Jiangsu Jicui Biological Co., Ltd., and all animal protocols were approved by the Animal Center of Zhejiang Academy of Agricultural Sciences. Care and Use Committee Approval (license number: SYXK (Zhejiang) 2020-0022). 84 mice were randomly divided into 14 groups (n=6), and the feeding conditions were: temperature 25° C., humidity 40%-60%, 12-hour light/12-hour dark cycle. After one week of adaptive feeding, they were named as 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), 3Y200 group (DOP3Y, 200mg/kg), 1J100 group (DOP1J, 100mg/kg), 1J200 group (DOP1J, 200mg/kg) , 2J100 group (DOP2J, 100mg/kg), 2J200 group (DOP2J, 200mg/kg), 3J100 group (DOP3J, 100mg/kg) and 3J200 group (DOP3J, 200mg/kg). The whole experiment lasted for 7 days, and the mice had free access to food and water. The Con group was provided with pure water without DSS, while the other groups were provided with pure water with 3% DSS. The mice in each DOP group were orally administered with corresponding doses of polysaccharides every day.

As shown in Figure 1, the effects of stem and leaf polysaccharides of Yanhu No. 1 (DOP1 group), Yanhu No. 3 (DOP2 group) and Shenglan No. 8 (DOP3 group) on UC mice were studied respectively. The results showed that after DSS treatment , the weight of the mice gradually decreased. Compared with the model group, other polysaccharides had no significant effect on the weight loss of the mice. The weight loss of Y100 and J200 in the DOP3 group was lower, indicating that Y100 and J200 could slow down the weight loss of UC mice . The DAI index is an important index for evaluating the UC model in mice. This index evaluates the severity of the disease by observing the daily fecal status and weight changes of the mice. The higher the DAI score, the more severe the disease of the mouse. The DAI score of the DSS group was significantly higher, the DAI scores of other varieties of polysaccharides were higher than those of the model group, and the DAI scores of Y100 and J200 of the DOP3 group were lower than those of the model group, indicating that the polysaccharides of this variety can alleviate the symptoms of UC mice and have a therapeutic effect.

Example 3: Dendrobium officinale stem/leaf polysaccharide mouse model of ulcerative colitis 1. Animal experiment design

SPF-grade male Balb/c mice (permit number: SCXK (Su) 2018-0008, seven-week-old, 20 ± 2 g) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Approved by the Animal Care and Use Committee of the Hospital Animal Center (permit number: SYXK (Zhejiang) 2020-0022). The mice were randomly divided into 5 groups (n=12), and the feeding conditions were the same as in Example 2. After 1 week of adaptive feeding, the Con group was given standard diet and purified water, and normal saline (10mL/kg) was administered once a day for 14 days; the DSS group was given standard diet and purified water for the first 7 days, and the drinking water Add 3% (w/v) DSS to drinking 7 days to construct UC model; Positive control group (SASP) before 7 days intragastric administration of normal saline, after 7 days intragastric administration of SASP (400mg/kg), DOP group mice in 1- DOP solution (DOPY50mg/kg, DOPJ150mg/kg) was administered intragastrically every day for 14 days, and 3% (w/v) DSS was added to the drinking water on the 8th-14th day. On the 15th day, all the mice resumed drinking pure water, and the mice were killed on the 16th day. After blood was collected and dissected, the spleen and thymus were weighed, the length of the colon between the ileocecal junction and the anal edge was measured, and the corresponding serum indicators were determined. .

Among them, DOPY is the leaf polysaccharide of DOP3 group, and DOPJ is the stem polysaccharide.

2. Evaluation of disease activity index (DAI)

DAI was determined by evaluating changes in weight loss, diarrheal status, and fecal bleeding according to reported methods.

DAI = (body mass index + stool shape + bleeding)/3

Table 1 Mouse Disease Activity Index (DAI) Scoring Criteria

3. Result analysis

1) weight

As shown in Figure 2: Except for the control group, the weight of other mice dropped sharply and continuously after drinking DSS for 4-5 days, which was consistent with the successful modeling. The positive treatment group had no effect on alleviating the weight loss of the mice. Both polysaccharide groups significantly alleviated weight loss in mice, with stem polysaccharides being superior to leaf polysaccharides.

2) Disease Activity Index (DAI)

As shown in Figure 3, compared with the control group (Con), the DAI value of the DSS group was significantly increased, while the DAI scores of the positive control group (SASP) and the two Dendrobium polysaccharide groups were significantly reduced, and the scores of the polysaccharide group in the first 13 days It was lower than that of the SASP group, and tended to be consistent on the 15th day.

3) Organ index

As shown in Figure 4, compared with the control group, the thymus index in the DSS group was significantly reduced, suggesting that thymus atrophy occurred, and the positive drug group had no significant improvement, while the polysaccharide group could significantly increase the thymus index, and the thymus index in the stem polysaccharide group reached normal Numerical value; as shown in Figure 5, the spleen index in the DSS group increased significantly, suggesting that splenomegaly occurred. The positive drug group had no improvement effect on this, and there was an increasing trend. The polysaccharide group could significantly reduce the spleen index, and the leaf polysaccharide group had a lower index . It shows that the positive control group has no effect on alleviating splenomegaly and thymus atrophy in mice, while Dendrobium polysaccharide has obvious effect.

4) Colon length

As shown in Figure 6, the colon length of the model group was significantly lower than that of the control group, and the colon length of the positive drug group was significantly higher than that of the model group. The colon length of both polysaccharide groups was higher than that of the model group.

5) Inflammatory factors

Inflammatory cytokines, especially TNF-α, IL-1β and IL-6, play a key role in the development of UC. TNF-α is an important driver of intestinal inflammation, IL-1β is produced by mononuclear phagocytes and intestinal epithelial cells, blocking IL-1β signaling can improve colitis and related colon cancer, IL-6 can prevent T cell Apoptosis thus play a pro-inflammatory role. As shown in Figure 7, the contents of TNF-α, IL-1β and IL-6 in the model group were all higher than those in the control group, and the expression levels of TNF-α, IL-1β and IL-6 were reduced in the positive drug and polysaccharide treatment group, among which Leaf polysaccharides were better than stem polysaccharides than positive drugs, indicating that DOP has anti-inflammatory activity.

6) Intestinal barrier analysis

One of the pathogenesis of UC is to disrupt the intestinal barrier, leading to the entry of harmful bacteria and disrupting the balance between luminal antigens and mucosal immunity. Tight junction proteins (ZO-1, occludin) play a key role in maintaining the intestinal barrier function, which can protect the intestinal tract from allergens, toxins and pathogens. A major 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. It can be seen from Figure 8 that the expressions of ZO-1, occludin, and Muc2 in the DSS group were significantly reduced, indicating that DSS damaged the integrity of the colonic membrane, and the expression of ZO-1 in the positive drug SASP and leaf polysaccharide groups was significantly increased; the positive drug SASP for The recovery effect of occludin expression was the most significant, and stem polysaccharide also had an effect; the expression of Muc2 in the positive drug group and the polysaccharide group were significantly higher than that in the model group. It shows that the polysaccharides of Dendrobium candidum can help restore the integrity of the intestinal barrier in the colon tissue, and the leaf polysaccharides and stem polysaccharides have different action modes.

In summary, the stem polysaccharides of Dendrobium candidum and leaf polysaccharides of Dendrobium candidum of the present application can significantly alleviate the phenomenon of sudden weight loss in mice, reduce the DAI value of colon tissue in mice with ulcerative colitis, and alleviate the abnormal atrophy or abnormality of immune organs in mice. Swelling, restoring the length of the colon, reducing the expression of inflammatory factors and repairing the intestinal barrier can be used to prevent and treat ulcerative colitis and repair the intestinal barrier.

Claims (7)

1. The dendrobium candidum stem/leaf polysaccharide is applied to the preparation of medicines or health-care foods for preventing and treating ulcerative colitis.

2. The use according to claim 1, characterized in that the dendrobium candidum stem/leaf polysaccharide is used for preparing a medicament or health food for relieving the sudden weight loss caused by ulcerative colitis.

3. The use according to claim 1, characterized in that the dendrobium candidum stem/leaf polysaccharide is used for preparing medicines or health-care foods for reducing DAI value of colon tissues caused by ulcerative colitis.

4. The use according to claim 1, characterized in that the dendrobium candidum stem/leaf polysaccharide is used for preparing a medicament or health food for relieving abnormal atrophy or swelling of immune organs and restoring colon length caused by ulcerative colitis.

5. The use according to claim 1, characterized in that said dendrobium candidum is a cultivar of Zhejiang, in particular holly number 8.

6. The use according to any one of claims 1 to 5, characterized in that the dendrobium candidum stem/leaf polysaccharide is prepared by the following steps:

dendrobium officinale stems/leaves and water according to the proportion of 1: homogenizing at 15:1, adding into 45-85deg.C constant temperature water bath, shake extracting for 40min, filtering with four layers of gauze, centrifuging at 4000r/min for 10min, collecting supernatant, adding 5 times volume of ethanol, standing at 4deg.C for 12h, and filtering with 2000 mesh filter cloth to obtain precipitate;

redissolving the precipitate in distilled water, dialyzing 72h with 8000-14000Da dialysis bag, changing water every 4 h;

and after the dialysis is finished, carrying out vacuum freeze drying on the sample 72 and h to obtain dendrobium candidum stem polysaccharide/dendrobium candidum leaf polysaccharide.

7. A medicine or health food for preventing and treating ulcerative colitis is characterized in that the active component is dendrobium candidum stem/leaf polysaccharide.