CN115068558A - Application of dendrobium huoshanense extract in preparation of medicine for treating and/or preventing lower limb arteriosclerotic occlusion - Google Patents

Abstract

The application is applicable to the technical field of medicines, and provides an application of dendrobium huoshanense extract in preparing medicines for treating and/or preventing lower limb arteriosclerotic occlusive disease. The dendrobium huoshanense extract provided by the application can recover the lower limb arterial blood flow velocity of a rat with lower limb arteriosclerosis obliterans, relieve the lower limb arterial stenosis symptom of the rat with lower limb arteriosclerosis obliterans, reduce the contents of TNF-alpha, IL-1 beta and IL-6 in the serum of the rat with lower limb arteriosclerosis obliterans and reduce the contents of TC, TG and LDL-C in the serum of the rat with lower limb arteriosclerosis obliterans.

Description

Application of dendrobium huoshanense extract in preparation of medicine for treating and/or preventing lower limb arteriosclerotic occlusion

Technical Field

The application belongs to the technical field of medicines, and particularly relates to application of a dendrobium huoshanense extract in preparation of medicines for treating and/or preventing lower limb arteriosclerotic occlusion.

Background

Lower limb arteriosclerosis obliterans is a chronic arterial occlusive disease which develops on the basis of atherosclerosis, is mostly seen in lower limb arteries, and can cause intermittent claudication, rest pain, ulcer and gangrene. The lower limb arteriosclerosis obliterans mostly occur in people over 50 years old, the incidence rate is about 0.74 percent, and the health life of people is seriously threatened. Therefore, there is an urgent need to develop safe and effective drugs for treating and/or preventing arteriosclerosis obliterans of lower limbs.

The etiology of lower limb arteriosclerosis obliterans is not clear, and at present, the activation of lipid metabolism disorder and vascular inflammatory reaction is generally considered to be the most basic pathological link in the occurrence and development process of lower limb arteriosclerosis obliterans, and can promote arteriosclerosis and aggravate lumen stenosis. The targeted anti-inflammatory and hypolipidemic treatment is the main target point of the current lower limb arteriosclerosis obliterans intervention.

Dendrobium huoshanense (Dendrobium huoshanense C.Z.Tang et S.J.Cheng) is a Chinese herbal medicine which is unique in China and has various biological activities, such as oxidation resistance, anti-inflammation and the like. However, the application of dendrobium huoshanense in lower limb arteriosclerosis obliterans is not found.

Disclosure of Invention

The embodiment of the application aims to provide a new application of the dendrobium huoshanense extract, namely an application of the dendrobium huoshanense extract in preparing a medicine for treating and/or preventing lower limb arteriosclerotic occlusion.

The embodiment of the application is realized by applying the dendrobium huoshanense extract to the preparation of the medicine for treating and/or preventing the lower limb arteriosclerotic occlusive disease.

Another object of the embodiments of the present application is to provide an application of dendrobium huoshanense extract in preparing a medicament for treating and/or preventing arterial stenosis of lower limbs.

Another object of the embodiments of the present application is to provide an application of dendrobium huoshanense extract in preparing a medicament for treating and/or preventing inflammation of lower limb arteriosclerotic occlusive disease.

Another objective of the embodiments of the present application is to provide an application of dendrobium huoshanense extract in preparing a medicament for treating and/or preventing inflammatory lipid metabolic disorder of lower limb arteriosclerotic occlusion.

Another object of the embodiments of the present application is to provide an application of dendrobium huoshanense extract in preparing a medicament for treating and/or preventing femoral artery blood flow velocity of lower limb arteriosclerotic occlusion.

Another object of the embodiments of the present application is to provide an application of dendrobium huoshanense extract in preparing a medicament for treating and/or preventing atherosclerosis.

Another object of an embodiment of the present application is a pharmaceutical composition comprising an extract of dendrobium huoshanense and a pharmaceutically acceptable carrier or excipient.

Another object of an embodiment of the present application is to provide a use of the pharmaceutical composition according to the present application for the preparation of a medicament for the treatment and/or prevention of arteriosclerotic occlusive disease of the lower limbs.

Another object of an embodiment of the present application is the use of a pharmaceutical composition according to the invention for the preparation of a medicament for the treatment and/or prevention of symptoms of arterial stenosis of the lower extremities.

A further object of an embodiment of the present application is the use of a pharmaceutical composition according to the description for the preparation of a medicament for the treatment and/or prevention of atherosclerosis.

The dendrobium huoshanense extract provided by the embodiment of the application can recover the lower limb arterial blood flow velocity of a rat with lower limb arteriosclerosis obliterans, relieve the lower limb arterial stenosis symptom of the rat with lower limb arteriosclerosis obliterans, reduce the contents of TNF-alpha, IL-1 beta and IL-6 in the serum of the rat with lower limb arteriosclerosis obliterans and reduce the contents of TC, TG and LDL-C in the serum of the rat with lower limb arteriosclerosis obliterans.

Drawings

Fig. 1 is a graph showing the results of the dendrobium huoshanense extract for restoring the arterial blood flow velocity of the lower limbs of rats with lower limb arteriosclerosis obliterans provided in the embodiments of the present application;

fig. 2 is a schematic view of a dendrobium huoshanense extract for relieving lower limb arterial stenosis symptoms of rats with lower limb arteriosclerosis obliterans provided in the embodiments of the present application;

FIG. 3 is a graph showing the results of the Dendrobium huoshanense extract provided in the examples of the present application for reducing the levels of TNF- α, IL-1 β and IL-6 in the serum of rats with lower limb arteriosclerosis obliterans;

FIG. 4 is a graph showing the results of the Dendrobium huoshanense extract provided in the examples of the present application for reducing the content of TC, TG and LDL-C in serum of rats with lower limb arteriosclerosis obliterans.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

The application provides an application of a dendrobium huoshanense extract in preparing a medicine for treating and/or preventing lower limb arteriosclerosis obliterans, and is a new application of the dendrobium huoshanense extract. At present, no report indicates that the dendrobium huoshanense extract can be used for treating and/or preventing lower limb arteriosclerosis obliterans.

The application of the dendrobium huoshanense extract in preparing the medicine for treating and/or preventing lower limb arteriosclerosis obliterans is characterized in that the dendrobium huoshanense extract is obtained by extracting dendrobium huoshanense medicinal materials, and the dendrobium huoshanense extract is a dendrobium huoshanense water extract.

Further, the dendrobium huoshanense aqueous extract is a dry dendrobium huoshanense aqueous extract.

Further, the preparation method of the dry dendrobium huoshanense water extract comprises the following steps: taking 20g of dendrobium huoshanense powder, adding 500mL of distilled water, performing reflux extraction for 1h each time, and repeating for 2 times. After extraction, the water extracts were combined and filtered through 200 mesh filter cloth. The aqueous extract was concentrated to 100 ml by rotary evaporator. Freeze-drying the concentrate to powder, and weighing. 5.37g of powder was finally obtained, with an extraction rate of 26.85%.

Furthermore, the dendrobium huoshanense extract can be prepared into any pharmaceutically acceptable oral and non-oral preparation forms, such as one of granules, pills, capsules, tablets, powder, paste and oral liquid.

Plaque is formed in arterial blood vessels of lower limbs, and the resulting vascular stenosis is one of the main symptoms of arteriosclerosis obliterans of lower limbs. The application detects plaque formation in lower limb femoral artery blood vessels, and researches the influence of dendrobium huoshanense extract on plaque formation in lower limb femoral artery blood vessels of lower limb arteriosclerosis obliterans as a medicine.

The blood flow is not smooth due to the artery blockage of the lower limb, which is one of the main symptoms of arteriosclerosis obliterans of the lower limb. This application detects through the blood flow velocity to lower limbs femoral artery, studies the influence of dendrobium huoshanense extract as the medicine to lower limbs femoral artery blood flow velocity of lower limbs arteriosclerosis obliterans.

Inflammatory responses are considered to be one of the important causes of lower limb arteriosclerotic occlusion. According to the application, the content of some inflammatory factors (mainly TNF-alpha, IL-1 beta and IL-6) in serum is detected, so that the influence of the dendrobium huoshanense extract as a medicament on the inflammatory reaction of lower limb arteriosclerosis obliterans is researched.

Disorders of lipid metabolism are considered to be one of the important causes of lower limb arteriosclerotic occlusion. The application researches the influence of the dendrobium huoshanense extract as a medicament on lipid metabolism disorder of lower limb arteriosclerosis obliterans by detecting the content of some lipids (mainly TG, TC and LDL-C) in serum.

EXAMPLE 1 Material preparation

Dendrobium huoshanense is purchased from Biotech limited, Anhui Huohu Shengji. Triglyceride (TG) content detection kit, Total Cholesterol (TC) content detection kit, low-density lipoprotein (LDL) kit, high-density lipoprotein (HDL) kit, tumor necrosis factor alpha (TNF-alpha) enzyme-linked immunoassay kit, interleukin 1 beta (IL-1 beta) enzyme-linked immunoassay kit and interleukin 6(IL-6) enzyme-linked immunoassay kit are all purchased from Solebao biotechnology, Inc.

The main apparatus is as follows:

HH-4 digital display constant temperature water bath (national electric appliance Co., Ltd.); LEGEND Micro 21R type centrifuge (Thermo scientific, USA); BS210S electronic balance (Sartorius); cell incubator (HERA Cell 150); fluorescence inverted microscope (Olympus); clean bench (Sujing group Suzhou Antai air technology Co., Ltd.).

Experimental animals:

SPF grade adult male SD rats (200 +/-20) g are purchased from Hangzhou medical college, and the production license number of experimental animals is SCXK (Zhe) 2019-.

Example 2 preparation of aqueous extract of Dendrobium huoshanense

And (4) crushing the stems of the dry dendrobium huoshanense by adopting a crusher. Taking 20g of dendrobium huoshanense powder, adding 500mL of distilled water, performing reflux extraction for 1h each time, and repeating for 2 times. After extraction, the water extracts were combined and filtered through 200 mesh filter cloth. The aqueous extract was concentrated to 100 ml by rotary evaporator. Freeze-drying the concentrate to powder, and weighing. 5.37g of powder was finally obtained, with an extraction rate of 26.85%. Storing the obtained dendrobium huoshanense water extract powder at low temperature for later use.

Example 3 animal model establishment and grouping

A rat lower limb arteriosclerosis obliterans model is manufactured by adopting a method of high fat diet and femoral artery intimal injury. After 1 week of adaptive feeding, 1% sodium pentobarbital (1mL/200g) was anesthetized by intraperitoneal injection, and after anesthesia was successful, the rats were fixed on an operating table. The left hind limb was sterilized for each rat. The skin is incised from the midpoint of the groin to the infraknee longitudinal line, the femoral artery is separated and exposed to the knee popliteal artery bifurcation, the distal end and the proximal end of the femoral artery are blocked by arteriolar clamps for 1.5-2.0 cm, 1 syringe with 0.1mL is taken, a special glass puncture needle is connected to be punctured from the proximal end along the long axis of the femoral artery blood vessel, 0.2-0.3 mL of sterile distilled water for injection is slowly injected into the blood vessel at the blocked part until the blood vessel is full. And 5min later, taking down the needle and the artery clamp, stopping bleeding and suturing. Vitamin D3 was administered simultaneously at 30 uU/kg body mass and intramuscular injection into the right hind limb for 1 time/month for a total of 12 weeks.

24 adult male SD rats were randomly divided into 4 groups: sham, model, low dose, and high dose groups. Sham group: after anesthetization of the rats, the lower limb skin was incised, and the saphenous artery was separated and exposed, without saphenous endarterial injury surgery, and the surgical wound was sutured. The normal basal feed is fed for 12 weeks after the stomach is drenched with distilled water for 1 time every day. Model group: after the rats were anesthetized, the skin of the lower limbs was incised, and the femoral artery was separated and exposed, a femoral endarterium injury surgery was performed, the surgical wound was sutured, and after feeding high-fat diet for 4 weeks, gastric perfusion with distilled water was started 1 time per day, and feeding of high-fat diet was continued for 8 weeks. Low dose group: after the rats were anesthetized, the skin of the lower limbs was incised, and the femoral artery was separated and exposed, a femoral artery intimal injury operation was performed, the operation wound was closed, and after feeding high-fat diet for 4 weeks, gavage of the aqueous extract of dendrobium huoshanense was started 1 time per day (administration amount: 1mg/kg), and feeding of high-fat diet was continued for 8 weeks. High dose group: after the rats were anesthetized, the skin of the lower limbs was incised, and the femoral artery was separated and exposed, a femoral artery intimal injury operation was performed, the operation wound was closed, and after feeding high-fat diet for 4 weeks, gavage of the aqueous extract of dendrobium huoshanense was started 1 time per day (administration amount: 10mg/kg), and feeding of high-fat diet was continued for 8 weeks.

And (3) data statistics:

statistical processing SPSS 10.0 software is used for statistical processing, origin 8.0 is used for processing pictures, and data are adopted

And (3) representing that the comparison among groups adopts t test, the comparison among multiple groups of means adopts variance analysis, and when P is less than or equal to 0.05, the significant difference is represented.

Example 4 measurement of mean blood flow velocity of femoral artery of lower extremity

Rats were anesthetized with isoflurane and after anesthesia, the left hind limb of the rat was subjected to a depilatory treatment. After the hair removal was completed, a laser speckle blood flow imaging system (moorFLPI-2) was used ^TM ) And detecting the arterial blood flow condition of the lower limbs of rats in each group.

As can be seen from the experimental results shown in fig. 1, the blood flow rate in the model group was significantly reduced compared to that in the sham operation group. Compared with a model group, the dendrobium huoshanense extract can obviously increase the lower limb arterial blood flow velocity of a rat with lower limb arteriosclerotic occlusion. Therefore, the dendrobium huoshanense extract has the effect of obviously recovering the blood flow velocity of the femoral artery of the lower limb arteriosclerotic occlusion as a medicament.

Example 5 detection of plaque formation in lower extremity femoral artery vessels

The rats are anesthetized by pentobarbital sodium, the rats are sacrificed after being anesthetized, the left hind leg femoral artery is taken out, the left hind leg femoral artery is fixed in paraformaldehyde for 24 hours, the femoral artery is taken out of the fixing liquid, the tissue of the target part is flattened by a scalpel in a fume hood, and the trimmed vascular tissue and the corresponding label are placed in a dehydration box. The dewatering box is put into a hanging basket to be dewatered by gradient alcohol in the dewatering machine in sequence: 75% alcohol 4 h-85% alcohol 2 h-90% alcohol 2 h-95% alcohol 1 h-absolute ethanol I30 min-absolute ethanol II 30 min-alcohol benzene 5-10 min-xylene I5-10 min-xylene II 5-10 min-wax I1 h-wax II 1 h-wax III 1 h. Embedding the wax-soaked tissue in an embedding machine. Firstly, molten wax is put into an embedding frame, tissues are taken out from a dehydration box and put into the embedding frame according to the requirements of an embedding surface before the wax is solidified, and corresponding labels are attached. And (4) freezing and cooling at-20 ℃, taking out the wax block from the embedding frame after the wax is solidified, and trimming the wax block. The trimmed wax block was sliced on a paraffin slicer to a thickness of 4 μm. The slices float on a spreading machine at 40 ℃ warm water to flatten the tissues, the tissues are taken out by a glass slide, and the slices are baked in a 60 ℃ oven. And taking out after the water is baked, the wax is baked, and observing the lower limb arterial stenosis of each group of rats by HE staining and EVG staining.

HE staining experiment procedure:

1) paraffin section dewaxing to water: placing the slices in xylene I20 min-xylene II 20 min-absolute ethyl alcohol I10 min-absolute ethyl alcohol II 10 min-95% alcohol 5 min-90% alcohol 5 min-80% alcohol 5 min-70% alcohol 5 min-distilled water washing.

2) Hematoxylin staining of cell nucleus: slicing into Harris hematoxylin, staining for 3-8min, washing with tap water, differentiating with 1% hydrochloric acid alcohol for several seconds, washing with tap water, returning blue with 0.6% ammonia water, and washing with running water.

3) Eosin staining of cytoplasm: the sections were stained in eosin stain for 1-3 min.

4) Dewatering and sealing: placing the slices in 95% alcohol I5 min-95% alcohol II 5 min-absolute ethanol I5 min-absolute ethanol II 5 min-xylene I5 min-xylene II 5min to dehydrate and transparent in sequence, taking out the slices from xylene, slightly drying, and sealing with neutral gum.

5) Microscopic examination and image acquisition and analysis.

6) And (3) dyeing results: blue in nucleus and red in cytoplasm.

EVG staining Experimental procedure:

1) paraffin section dewaxing to water: placing the slices in xylene I20 min-xylene II 20 min-absolute ethyl alcohol I10 min-absolute ethyl alcohol II 10 min-95% alcohol 5 min-90% alcohol 5 min-80% alcohol 5 min-70% alcohol 5 min-distilled water washing.

2) Dyeing: the slices are placed into Verhoeff's dye liquor for greenhouse dyeing for 15-30min until the color is dark black.

3) Differentiation: washing with tap water, differentiating with 2% ferric trichloride solution for 10-20s, and observing with microscope that elastic fiber is black and background is gray.

4) Removing redundant iodine: washing with tap water, and treating with 5% sodium thiosulfate for 1min to remove iodine.

5) Counterdyeing: washing with tap water and slightly washing with distilled water; counter staining with Van Gieseon's solution for 3-5min, and washing with anhydrous ethanol.

6) Dewatering and sealing: placing the slices in anhydrous ethanol I5 min-anhydrous ethanol II 5 min-xylene I5 min-xylene II 5min in sequence, dehydrating and transparentizing, taking out the slices from xylene, slightly drying, and sealing with neutral gum.

7) Microscopic examination and image acquisition and analysis.

8) And (3) dyeing results: the elastic fiber and the core are black, the collagen fiber is red, and other tissues are yellow.

The experimental results shown in fig. 2 show that, compared with the sham-operated group, plaque is formed in the artery of the lower limb of the rats in the model group, which causes severe stenosis. Compared with a model group, the dendrobium huoshanense can obviously reduce plaque formed in lower limb arterial blood vessels and lower limb arterial stenosis of rats with lower limb arteriosclerotic occlusion. Therefore, the dendrobium huoshanense extract serving as the medicine has the effect of obviously relieving the lower limb arteriosclerotic occlusive disease

Example 6 detection of inflammatory factor levels

Rats were anesthetized with sodium pentobarbital, after which they were bled from the eye sockets, centrifuged, and serum collected and stored at-20 ℃ until use. The contents of TNF-alpha, IL-1 beta and IL-6 in serum are detected by using a tumor necrosis factor alpha (TNF-alpha) enzyme-linked immunoassay kit, an interleukin 1 beta (IL-1 beta) enzyme-linked immunoassay kit and an interleukin 6(IL-6) enzyme-linked immunoassay kit respectively.

As can be seen from the results of the experiment shown in FIG. 3, the levels of TNF- α, IL-1 β and IL-6 were significantly increased in the model rats as compared with those in the sham-operated rats. Compared with the model group, the dendrobium huoshanense obviously reduces the contents of TNF-alpha, IL-1 beta and IL-6 in rats with lower limb arteriosclerotic obliteration. The dendrobium huoshanense extract as a medicine has the function of obviously relieving the inflammation of the lower limb arteriosclerotic occlusive disease.

Example 7 detection of lipid levels

Rats were anesthetized with sodium pentobarbital, after which they were bled from the eye sockets, centrifuged, and serum collected and stored at-20 ℃ until use. The contents of TG, Total Cholesterol (TC) and low-density lipoprotein (LDL-C) in serum were measured using a Triglyceride (TG) content measuring kit, a Total Cholesterol (TC) content measuring kit and a LDL-C kit, respectively.

As can be seen from the results of the experiment shown in FIG. 4, the model rats had significantly increased TC, TG and LDL-C as compared with those in the sham-operated group. Compared with a model group, the dendrobium huoshanense extract can obviously reduce the content of TC, TG and LDL-C in serum of rats with lower limb arteriosclerotic occlusive disease. Therefore, the dendrobium huoshanense extract serving as the medicine has the effect of obviously relieving the inflammatory lipid metabolic disorder of the lower limb arteriosclerotic occlusive disease.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims (10)

1. Application of dendrobium huoshanense extract in preparing medicine for treating and/or preventing lower limb arteriosclerotic obliteration is provided.

2. Application of Dendrobium huoshanense extract in preparing medicine for treating and/or preventing lower limb arterial stenosis symptom is provided.

3. Application of dendrobium huoshanense extract in preparing medicine for treating and/or preventing lower limb arteriosclerotic obliteration inflammation.

4. Application of dendrobium huoshanense extract in preparing medicine for treating and/or preventing lower limb arteriosclerotic obliterans inflammation lipid metabolism disorder.

5. Application of dendrobium huoshanense extract in preparing medicine for treating and/or preventing femoral artery blood flow velocity of lower limb arteriosclerotic occlusion.

6. Application of Dendrobium huoshanense extract in preparing medicine for treating and/or preventing atherosclerosis is provided.

7. A pharmaceutical composition is characterized by comprising dendrobium huoshanense extract and a pharmaceutically acceptable carrier or excipient.

8. Use of the pharmaceutical composition according to claim 7 for the preparation of a medicament for the treatment and/or prevention of arteriosclerotic occlusive disease of the lower extremities.

9. Use of a pharmaceutical composition according to claim 7 for the preparation of a medicament for the treatment and/or prevention of symptoms of arterial stenosis of the lower extremities.

10. Use of a pharmaceutical composition according to claim 7 for the preparation of a medicament for the treatment and/or prevention of atherosclerosis.

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