Preparation method and application of dendrobium crepidatum extract
By preparing dichloromethane extract from Dendrobium Rose stem, the adverse reactions and high cost problems of existing UC therapeutic drugs are solved, and safe and efficient anti-inflammatory treatment plans are provided, which improves the intestinal barrier function and cell apoptosis of ulcerative colitis, showing significant therapeutic effects.
Patent Information
- Application Number
- CN202510507592.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-22
- Publication Date
- 2025-07-11
AI Technical Summary
The existing UC treatment drugs have many adverse reactions and are costly, and lack safe and efficient plant-derived drugs. The anti-inflammatory activity of Dendrobium rose is insufficient, which affects its application in the treatment of ulcerative colitis.
The preparation method of dichloromethane extract of dendrobium stems, including drying, crushing, ethanol ultrasonic extraction, heat reflux, pH adjustment and dichloromethane extraction, etc., is used to prepare an extract with anti-inflammatory activity.
A targeted treatment plan for ulcerative colitis is provided, showing significant anti-inflammatory activity, improving intestinal barrier function, inhibiting cell apoptosis, reducing spleen enlargement and colon shortening, and reducing inflammatory response.
Smart Images
Figure CN120285103A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method of Dendrobium crepidatum extract and its application, especially in the application of preparing drugs for treating ulcerative colitis disease. Background Art
[0002] Ulcerative colitis (UC) is a typical non-specific chronic inflammatory bowel disease, a phenotype of inflammatory bowel diseases (IBD), starting from the rectum and spreading proximally to the colon in sequence, characterized by persistent mucosal inflammation and periodic recurrence in the rectum and colon. Its incidence shows an upward trend worldwide. There are many clinical manifestations of UC, including diarrhea, abdominal pain, weight loss, bloody stools, dehydration, and it is prone to develop into colorectal cancer, thus increasing the risk of patient death, affecting millions of people globally, and bringing a huge social and economic burden. Therefore, it is necessary to find safe and effective drugs for preventing and treating UC. Currently, the main treatment method for UC is to use some drugs for treatment, and clinicians will select appropriate drugs according to the severity of the lesions of UC patients to control the development of the disease. Traditional treatment methods for UC include 5-aminosalicylic acid drugs, immunomodulators, corticosteroids, and anti-cytokine drugs. These drugs have various adverse reactions and high treatment costs. In recent years, due to the therapeutic potential shown by natural products derived from plants in the treatment of UC, especially their ability to regulate the intestinal microbiota and restore intestinal barrier function with fewer side effects, they have received attention.
[0003] Dendrobium crepidatum Lindl. ex Paxt. is a traditional Chinese medicinal material in China, with the effects of nourishing yin and benefiting the stomach, promoting the production of body fluid and relieving vexation, used for symptoms such as dry mouth and thirst, and yin injury and fluid deficiency, and has high health care and medicinal value. Although the clinical application prospects of Dendrobium crepidatum and its compound preparations are broad, there are few reports on its clinical research for treating inflammation currently, and the relevant mechanism of action research is insufficient, which has great research value. In-depth research on its anti-inflammatory activity will add new research results to the field of phytopharmacology. If the anti-inflammatory activity of Dendrobium crepidatum is fully verified, its dichloromethane extract (DCE-DCM) or its active ingredients are expected to become high-quality candidate resources for developing new drugs for treating UC.
[0004] Therefore, the present invention studies the protective effect of the dichloromethane extract of Dendrobium crepidatum on ulcerative colitis in mice, in order to provide experimental and theoretical basis for the in-depth research and clinical promotion of the anti-inflammatory effect of Dendrobium crepidatum. Summary of the Invention
[0005] The object of the present invention is to provide a preparation method and application of dendrobium crepidatum extract in view of the problems existing in the prior art.
[0006] The object of the present invention can be achieved through the following solutions:
[0007] The present invention provides a preparation method of dichloromethane extract of dendrobium crepidatum, comprising the following steps:
[0008] S1. Dry and crush the stems of dendrobium crepidatum to obtain dried and powdered stems of dendrobium crepidatum;
[0009] S2. Perform ethanol ultrasonic extraction on the dried and powdered stems of dendrobium crepidatum, then perform heat reflux treatment, and repeat the ethanol ultrasonic extraction and heat reflux treatment on the filter cake after suction filtration. The obtained filtrates are combined to obtain an ethanol extract of dendrobium crepidatum;
[0010] S3. Rotavaporize the ethanol extract of dendrobium crepidatum until there is no alcohol smell, adjust the pH to 2 - 3, adjust the pH of the filtrate after suction filtration to 9 - 9.5 with ammonia water, suspend it in ultrapure water, add dichloromethane for extraction, and rotary evaporate and freeze-dry the obtained organic layer to obtain dichloromethane extract of dendrobium crepidatum.
[0011] As an embodiment of the present invention, in step S1, drying is carried out at 50 - 60 °C until constant weight, preferably 55 °C. After crushing, it is sieved through a 60-mesh sieve.
[0012] As an embodiment of the present invention, in step S2, the mass ratio of the dried and powdered stems of dendrobium crepidatum to absolute ethanol is 1:8 - 12, preferably 1:10.
[0013] As an embodiment of the present invention, in step S2, the time for ultrasonic extraction is 2 - 3 h. The time for heat reflux is 3 - 4 h.
[0014] As an embodiment of the present invention, in step S2, the mass fraction of the ethanol solution used for ethanol ultrasonic extraction is 90% - 95%. The number of times of repeating the ethanol ultrasonic extraction and heat reflux treatment is 3 - 5 times. The mass fraction of the ethanol solution used for the last extraction is 70% - 80%.
[0015] As an embodiment of the present invention, in step S3, adjusting the pH to 2 - 3 is carried out by hydrochloric acid solution (mass fraction 10 - 15%).
[0016] As an embodiment of the present invention, in step S3, adjusting the pH to 9 - 9.5 is carried out by ammonia water (mass fraction 5 - 8%).
[0017] As an embodiment of the present invention, in step S3, the volume of dichloromethane used is the same as that of the ethanol extract of dendrobium crepidatum.
[0018] As an embodiment of the present invention, in step S3, the temperature of freeze-drying is -50 °C and the time is 72 h.
[0019] The present invention also provides an application of the dendrobium crepidatum dichloromethane extract obtained by the above preparation method in the preparation of drugs for treating ulcerative colitis.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] (1) The present invention specifically provides a more targeted treatment plan for the specific disease of ulcerative colitis.
[0022] (2) It has good anti-inflammatory activity and is expected to be used in the preparation of drugs for treating ulcerative colitis.
[0023] (3) The experimental materials are derived from plants, and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] By reading the following detailed description of the non-limiting embodiments with reference to the accompanying drawings, other features, objects and advantages of the present invention will become more apparent:
[0025] Figure 1 Effect of the dendrobium crepidatum extract prepared in Example 1 on the colon length of UC mice;
[0026] Figure 2 Effect of the dendrobium crepidatum extract prepared in Example 1 on the spleen index of UC mice;
[0027] Figure 3 Effect of the dendrobium crepidatum extract prepared in Example 1 on the colon histopathology of UC mice;
[0028] Figure 4 Effect of the dendrobium crepidatum dichloromethane extract prepared in Example 1 on the expression of mechanical barrier proteins in the colon epithelium of UC mice; wherein, a is the ZO-1 protein band and expression level of each group, and b is the Claudin-1 protein band and expression level of each group;
[0029] Figure 5 Effect of the dendrobium crepidatum dichloromethane extract prepared in Example 1 on Cleaved-Caspase-3 in UC mice; wherein, a is the Cleaved-Caspase-3 protein band diagram of each group, and b is the Cleaved-Caspase-3 protein expression level of each group. DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, providing detailed implementation manners and specific operation processes, which will help those skilled in the art to further understand the present invention. It should be noted that the protection scope of the present invention is not limited to the following embodiments. Several adjustments and improvements made on the premise of the concept of the present invention all fall within the protection scope of the present invention.
[0031] Example 1
[0032] I. Preparation of dichloromethane extract from Dendrobium crepidatum
[0033] The stems of Dendrobium crepidatum were dried to a constant weight at 55 °C, crushed, and passed through a 60-mesh sieve. 100 g of the dried powder of Dendrobium crepidatum stems was accurately weighed, ultrasonically extracted with 10 times the amount of ethanol solution (mass fraction 90%) for 2 h and then heat refluxed for 3 h. Subsequently, it was filtered by suction and the filtrate was collected. The filter cake was repeated the above extraction steps, and the extraction was carried out 2 times in total. The filter cake was collected, 10 times the amount of ethanol solution (mass fraction 75%) was added, ultrasonically extracted for 2 h and then heat refluxed for 3 h, and then filtered by suction and the filtrate was collected. The three filtrates were combined, and an equal amount of ultrapure water was added to suspend and rotary evaporate until there was no alcohol smell. 10% hydrochloric acid solution (mass fraction 10%) was added to adjust the pH to 2 - 3, and then filtered by suction to collect the filtrate. Ammonia water (mass fraction 5%) was added to the filtrate to adjust the pH to 9 - 9.5, suspended in ultrapure water, extracted with dichloromethane with the same volume as the filtrate, the organic solvent layer was recovered, rotary evaporated to 10 mL, frozen (-50 °C) and dried for 72 h to obtain DCE-DCM, and the yield of the paste was 2.1%.
[0034] II. Protective effect of dichloromethane extract from Dendrobium crepidatum on DSS-induced ulcerative colitis
[0035] 1. Method
[0036] (1) Establishment of UC model
[0037] Thirty-six SPF-grade C57BL / 6 male mice were randomly divided into 6 groups (n = 6), and the specific grouping was as follows: blank control group (CON), UC model group (DSS group), positive control group (5-ASA, 20 mg / kg), low-dose group of dichloromethane extract from Dendrobium crepidatum (DCE-DCML, 90 mg / kg), medium-dose group (DCE-DCMM, 120 mg / kg), and high-dose group (DCE-DCMH, 150 mg / kg)
[0038] The mice were adaptively fed for 7 days, during which they had free access to food and drinking water. The modeling and drug administration lasted for 7 days. In the 5-ASA group and each dose group of DCE-DCM, the corresponding concentration of drugs was administered by gavage daily, while in the CON group and DSS group, an equal amount of normal saline was administered by gavage daily. In the CON group, distilled water was given for free drinking during the experiment, and in the remaining groups, a 2.5% DSS solution was given for free drinking for one week to establish a murine ulcerative colitis model (UC).
[0039] (2) Sample collection and processing
[0040] After drug administration on the 7th day, the mice were fasted but not deprived of water. On the 8th day, the mice were dissected. Before sacrificing the mice, their body weights were recorded. After anesthesia and blood collection from the eyeballs, the mice were sacrificed by cervical dislocation. After standing at room temperature for 4 h, they were centrifuged at 3000 r / min for 10 min in a 4°C low-temperature centrifuge, and the serum was aspirated into cryotubes and stored in an -80°C refrigerator. The spleens of the mice were removed, the surface moisture was wiped dry, and after accurately weighing, the ratio of the spleen weight to the body weight was calculated, which was the spleen index. The calculation formula was as follows: The colon of the mice was completely dissected and its length was measured. The colon contents were rinsed clean with normal saline, the colon tissue was dried, and the colon 0.5 cm above the anus was selected and fixed by soaking in 4% paraformaldehyde. The remaining colon tissue was stored in a cryotube and placed in an -80°C refrigerator for later detection.
[0041] Spleen index (%) = spleen weight (g) / body weight (g) on the day of sacrificing the mice x (100%)
[0042] (3) HE staining of colon tissue
[0043] The colon tissue fixed in 4% paraformaldehyde solution for 24 h was taken out, rinsed with ethanol of different concentrations (30%, 50%, 70%, 80%, 100%) as required, and then subjected to ethanol and xylene transparency treatment. It was quickly infiltrated with pure wax of appropriate melting point and embedded to obtain tissue wax blocks. The wax blocks were sectioned after overnight storage in a 4°C refrigerator, placed in water, fished out after the sections were flat and without wrinkles, and placed in a 60°C oven overnight and stored at room temperature. The sections were dewaxed with xylene and gradient ethanol, then nuclear staining was performed using hematoxylin staining solution and differentiation solution, cytoplasmic staining was performed using eosin staining solution, and finally washed with absolute ethanol. After the sections were treated with xylene transparency, they were sealed with neutral gum, and the morphology of the colon mucosa, glandular structure, number of crypts, and changes in goblet cells were observed.
[0044] (4) Immunoblotting to detect the expression of related proteins such as epithelial mechanical barrier and apoptosis in colon tissue
[0045] Detection of protein expression by Western blot: Mouse colon tissues were lysed with RIPA lysis buffer, and protein quantification was performed using a BCA protein assay kit. Equal amounts of protein were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with tris-buffered saline with Tween (TBST) containing 5% skim milk at room temperature for 1 h, and incubated overnight at 4 °C with ZO-1 (1:1000), Cleaved-Caspase-3 (1:5000), Claudin-1 (1:1000), and β-actin primary antibodies (1:1000). The membrane was washed 3 times with TBST for 10 min each time, then incubated with the corresponding secondary antibody at 37 °C for 2 h. After incubation, ECL exposure solution was added and exposed in the dark, and the protein gray scale was analyzed using Image J software and quantified.
[0046] (5) Data statistics and analysis
[0047] SPSS 20.0 software was used for data analysis, and Graphpad Prism 8.0 software was used for drawing graphs. Measurement data were expressed as mean ± standard deviation, and one-way analysis of variance was used for comparison of samples between multiple groups. A P < 0.05 was considered statistically significant.
[0048] 2. Results
[0049] (1) Effect of dichloromethane extract of Dendrobium crepidatum on colon length in UC mice
[0050] Colon length is an important indicator reflecting the severity of ulcerative colitis. When an inflammatory response occurs in mice, it will damage the intestinal structure and intestinal villi, and the mouse colon will shorten. Moreover, the more severe the inflammatory response, the more obvious the colon shortening. Colon shortening may also lead to increased defecation frequency, aggravated abdominal pain, and malabsorption of nutrients. As Figure 1 shown, compared with the CON group, the average colon length of DSS-induced colitis mice was significantly shortened (P < 0.001). Compared with the DSS group, the phenomenon of colon shortening was inhibited after intervention with the 5-ASA group and the dichloromethane extract of Dendrobium crepidatum. Among them, the recovery of colon length in the DCE-DCMM group and the DCE-DCMH group was the most obvious (P < 0.001), indicating that the dichloromethane extract of Dendrobium crepidatum can improve ulcerative colitis in mice.
[0051] (2) Effect of dichloromethane extract of Dendrobium crepidatum on spleen index in UC mice
[0052] As the largest immune organ in the human body, the spleen plays a crucial role in the body's immunity. When the body has an inflammatory response, it will trigger an immune response, leading to splenomegaly, and the increase in the spleen is related to the infiltration of splenic giant cells. From Figure 2 It can be seen that after induction with DSS, mice will develop ulcerative colitis, resulting in a significant increase in the spleen index of mice (p<0.01). After intervention with the 5-ASA group and the dichloromethane extract of Dendrobium crepidatum, the phenomenon of splenomegaly was alleviated. Among them, the DCE-DCMM group and the DCE-DCMH group had the most obvious inhibitory effect on splenomegaly (P<0.05), indicating that the dichloromethane extract of Dendrobium crepidatum has a certain effect on the immune recovery of mice, and thus has a relieving effect on ulcerative colitis.
[0053] (3) Effect of dichloromethane extract of Dendrobium crepidatum on the pathological morphology of colon tissue in UC mice
[0054] When ulcerative colitis occurs in mice, neutrophils are recruited to the inflammatory site and are found in the intestinal crypts and at the bottom of the ulcers, forming crypt abscesses. The pathological results of the colon tissue are as Figure 3 shown. In the CON group, the goblet cells and crypts of the colon tissue of mice were intact, the glands were clear and arranged neatly, and there was no infiltration of inflammatory cells. Compared with the CON group, the colon crypt structure of DSS group mice was disordered, the goblet cells decreased or even disappeared, and the inflammatory cells infiltrated the entire layer of epithelial cells. Combined with the shortening of the mouse colon, it jointly reflected the successful establishment of the mouse ulcerative colitis model. After intervention with 5-ASA and various concentrations of DCE-DCM, the epithelial damage of the mouse colon tissue was alleviated to a certain extent, manifested as the recovery of the disordered crypt structure, the increase in goblet cells, and the reduction of inflammatory infiltration.
[0055] (4) Dichloromethane extract of Dendrobium crepidatum improves the damage of the mechanical barrier of the colon epithelium in UC mice
[0056] In the UC mouse model, the colon tissue is invaded by inflammation, resulting in damage to the intestinal barrier function. At this time, the expression levels of tight junction proteins such as ZO-1 and Claudin-1 decrease. As Figure 4 shown, compared with the CON group, the expression levels of ZO-1 and Claudin-1 proteins in the DSS group were significantly decreased (P<0.001); compared with the DSS group, the expression levels of ZO-1 and Claudin-1 proteins in the 5-ASA group and each dose group of the dichloromethane extract of Dendrobium crepidatum were significantly increased (P<0.001), indicating that the drug restores and enhances the tight junction function of the intestinal tissue through mechanisms such as protecting the intestinal barrier, anti-inflammatory and antioxidant, and promoting cell repair.
[0057] (5) Dichloromethane extract of Dendrobium crepidatum may inhibit the Caspase apoptosis pathway through the endoplasmic reticulum pathway
[0058] In a mouse model of ulcerative colitis (UC), the change in the expression level of Cleaved-Caspase-3 protein in colon tissue can reflect the dynamics of apoptosis. Intestinal inflammation induced by DSS in mice can lead to damage and apoptosis of intestinal epithelial cells. Caspase-3 is a key execution protein of apoptosis, and the increased expression of its cleaved active form, Cleaved-Caspase-3, indicates that apoptosis is activated. The increase in apoptosis will lead to a large number of deaths of intestinal epithelial cells, thereby destroying the integrity of the intestinal mucosal barrier.
[0059] As Figure 5 shown, compared with the CON group, the relative expression level of Cleaved-Caspase-3 in the DSS group was significantly increased (P<0.01); compared with the DSS group, the protein expression levels of Cleaved-Caspase-3 in the 5-ASA group and each dose group of dichloromethane extract of Dendrobium crepidatum were significantly decreased (P<0.01), which indicates that the drug may play a protective role in ulcerative colitis by inhibiting the apoptosis pathway mediated by endoplasmic reticulum stress.
[0060] 3. Conclusion
[0061] In recent years, Dendrobium crepidatum, as a natural plant product, has attracted much attention in anti-inflammatory research. DSS-induced mouse colitis is considered one of the classic experimental colitis models because it is similar to human UC in terms of etiology, pathogenesis, and treatment response, and the clinical manifestations are intestinal inflammatory infiltration accompanied by diarrhea, rectal bleeding, weight loss, and edematous colon shortening. DSS can directly damage the epithelial cells of the mouse colon, leading to the destruction of the integrity of the colonic mucosal barrier, triggering an inflammatory response, and thus inducing ulcerative colitis.
[0062] In the UC model, the colon tissue is invaded by inflammation, resulting in impaired intestinal barrier function and decreased expression levels of tight junction proteins such as Claudin and ZO-1. DCE-DCM intervention increased the expression of these proteins, indicating its improvement effect on intestinal barrier function. In addition, during the pathogenesis of UC, the damage and death of intestinal epithelial cells, especially through the apoptosis mechanism, have an important impact on the disease condition. Caspase-3, as a key execution protein of apoptosis, its activation and increased expression are important indicators of apoptosis. The study showed that after DCE-DCM intervention, the protein expression level of cleaved-Caspase-3 was significantly decreased, confirming that DCE-DCM has the effect of inhibiting apoptosis.
[0063] In summary, DCE-DCM shows a significant protective effect on the DSS-induced mouse UC model, providing a new potential treatment strategy for the treatment of UC by improving intestinal barrier function and inhibiting apoptosis.
[0064] Comparative Example 1
[0065] The preparation of the dichloromethane extract of Dendrobium crepidatum in this comparative example was basically the same as that in Example 1, except that: in the extraction step, dichloromethane was replaced with an equal amount of ethanol, but the final extract yield was lower, and the paste yield was lower than that in Example 1.
[0066] Comparative Example 2
[0067] The preparation of the dichloromethane extract of Dendrobium crepidatum in this comparative example was basically the same as that in Example 1, except that: in the extraction step, the ethanol solution was replaced with an equal amount of water, but the final extract yield was lower, and the paste yield was lower than that in Example 1.
[0068] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.
Claims
1. A preparation method of dichloromethane extract of Dendrobium crepidatum Lindl. ex Paxt., characterized in that, It includes the following steps: S1. Dry and crush the stems of Dendrobium crepidatum to obtain the dried powder of Dendrobium crepidatum stems; S2. Perform ultrasonic extraction of the dried powder of Dendrobium crepidatum stems with absolute ethanol, then conduct heat reflux treatment. The filter cake after suction filtration is repeatedly subjected to ethanol ultrasonic extraction and heat reflux treatment, and the obtained filtrates are combined to obtain the ethanol extract of Dendrobium crepidatum; S3. Rotavaporize the ethanol extract of Dendrobium crepidatum until there is no alcohol smell, adjust the pH to 2 - 3, adjust the pH of the filtrate after suction filtration to 9 - 9.5, suspend it in ultrapure water, add dichloromethane for extraction, and rotavaporize and freeze-dry the obtained organic layer to obtain the dichloromethane extract of Dendrobium crepidatum.
2. The preparation method of the dichloromethane extract of Dendrobium crepidatum Lindl. ex Paxt. according to claim 1, wherein In step S1, the drying is carried out at 50 - 60 °C until constant weight.
3. The preparation method of the dichloromethane extract of Dendrobium crepidatum Lindl. ex Paxt. according to claim 1, characterized in that, In step S2, the mass ratio of the dried powder of Dendrobium crepidatum stems to absolute ethanol is 1:8 - 12.
4. The preparation method of the dichloromethane extract of Dendrobium crepidatum Lindl. ex Paxt. according to claim 1, wherein In step S2, the time for ultrasonic extraction is 2 - 3 h.
5. The preparation method of the dichloromethane extract of Dendrobium crepidatum Lindl. ex Paxt. according to claim 1, wherein, In step S2, the time for heat reflux is 3 - 4 h.
6. The preparation method of the dichloromethane extract of Dendrobium crepidatum Lindl. ex Paxt. according to claim 1, wherein, In step S2, the mass fraction of the ethanol solution used for ethanol ultrasonic extraction is 90% - 95%.
7. The preparation method of the dichloromethane extract of Dendrobium crepidatum Lindl. ex Paxt. according to claim 1, characterized in that, The number of times of repeatedly performing ethanol ultrasonic extraction and heat reflux treatment is 3 - 5 times; the mass fraction of the ethanol solution used for the last extraction is 70% - 80%.
8. The preparation method of the dichloromethane extract of Dendrobium crepidatum Lindl. ex Paxt. according to claim 1, wherein, In step S3, the adjustment of the pH to 2 - 3 is carried out by hydrochloric acid solution.
9. The preparation method of the dichloromethane extract of Dendrobium crepidatum Lindl. ex Paxt. according to claim 1, wherein, In step S3, the adjustment of the pH to 9 - 9.5 is carried out by ammonia water.
10. Use of the dichloromethane extract of Dendrobium crepidatum obtained by the preparation method as claimed in claim 1 in the preparation of a drug for treating ulcerative colitis.