Application of polysaccharide from pitaya branches

Abstract

The present invention relates to the use of a polysaccharide from pitaya branches, and relates to the field of pharmaceutical technology. The present invention provides a use of a polysaccharide from pitaya branches in the treatment of osteoporosis. The polysaccharide is an extract obtained from pitaya branches by water extraction. The polysaccharide from pitaya branches promotes the proliferation and differentiation of osteoblasts, improves bone biomechanics, and is significantly effective in preventing and treating osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, in particular to application of polysaccharide from pitaya branches. Background Art

[0002] Osteoporosis is one of the common bone degenerative diseases in the elderly population over 50 years old. Its main pathological manifestations are decreased bone mass per unit volume, thinning and sparse trabeculae, reduced bone strength, and susceptibility to fractures.

[0003] Modern medicine's treatments for osteoporosis are mainly divided into bone mineralization promoters, bone formation promoters, and bone resorption inhibitors, but these drugs have varying degrees of adverse reactions. Calcium supplements are a representative of bone mineralization promoters and one of the most basic drugs for treating osteoporosis in clinical practice. However, long-term use of calcium supplements can also lead to upper abdominal discomfort, constipation, hypercalcemia, and even kidney stones. Furthermore, recent studies have shown that the extensive use of calcium supplements increases the risk of prostate cancer. Bisphosphonates are a representative of bone resorption inhibitors. In addition to obvious gastrointestinal reactions in clinical use, adverse reactions such as fever, generalized bone pain, mandibular osteonecrosis, renal failure, and shock cannot be ignored. Therefore, seeking economical, convenient, safe, and effective anti-osteoporosis drugs is of great clinical significance. Summary of the Invention

[0004] In response to the above problems, the present invention provides a polysaccharide from pitaya branches for use in the treatment of osteoporosis. The polysaccharide from pitaya branches has the effect of promoting the proliferation and differentiation of osteoblasts, can improve the biomechanical condition of bones, and has a significant effect in preventing and treating osteoporosis.

[0005] In order to achieve the above object, the present invention provides a use of a polysaccharide from pitaya branches in treating osteoporosis. The polysaccharide is an extract obtained from pitaya branches by water extraction.

[0006] During the research process, the inventors found that the polysaccharide obtained from pitaya branches by water extraction has obvious pharmacological effects, can promote the proliferation and differentiation of osteoblasts, can increase the vertebral bone density of ovariectomized osteoporotic rats, improve bone biomechanical conditions, and has a significant effect in preventing and treating osteoporosis.

[0007] The present invention also provides a use of a polysaccharide from pitaya branches in preparing a medicine for treating osteoporosis. The polysaccharide is an extract obtained from the pitaya branches by water extraction.

[0008] The above-mentioned polysaccharide is derived from pitaya branches, has no toxic side effects, and can be used safely for a long time.

[0009] In one embodiment, the osteoporosis includes primary osteoporosis or secondary osteoporosis.

[0010] In one embodiment, the drug comprises a pharmaceutically acceptable excipient and the polysaccharide.

[0011] In one embodiment, the drug is an oral preparation or an injectable preparation.

[0012] In one embodiment, the application includes oral administration or injection of the drug, and the dosage of the polysaccharide is 1-1000 mg per kilogram of body weight.

[0013] The present invention also provides a medicine for treating osteoporosis. The medicine comprises pharmaceutically acceptable excipients and polysaccharides. The polysaccharides are extracts obtained from pitaya branches by water extraction.

[0014] In one embodiment, the auxiliary material includes at least one of the following raw materials: an excipient, a lubricant, an antioxidant, a preservative, a binder, a filler, or a thickener.

[0015] In one embodiment, the drug is in the form of an oral preparation or an injectable preparation.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention discloses a polysaccharide from pitaya branches. The polysaccharide is extracted from pitaya branches, has no toxic side effects, can be used safely for a long time, and has obvious pharmacological effects. It has the effects of promoting osteoblast proliferation and differentiation, improving bone biomechanics, and significantly preventing and treating osteoporosis. Furthermore, the raw materials for preparing the polysaccharide from pitaya branches are readily available and inexpensive. The polysaccharide freeze-dried powder is obtained by water extraction, has a simple preparation process, requires few steps, and has high production efficiency. The polysaccharide is safe and convenient for patients to use, can be used in a variety of dosage forms, alleviates symptoms, and relieves patients' pain, has significant therapeutic effects, and is inexpensive. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of the effect of pitaya branch polysaccharide on MC3T3-E1 cell proliferation in Example 2 (comparison of pitaya branch polysaccharide dose groups with the blank group) 1） P<0.05, 2） P < 0.01);

[0019] Figure 2 This is the alizarin red staining result of pitaya branch polysaccharide promoting bone mineralization in Example 3;

[0020] Figure 3 For the semi-quantitative detection of the effect of pitaya branch polysaccharide on the osteogenic differentiation of MC3T3-E1 cells in Example 3 (comparison of each dose group of pitaya branch polysaccharide with the blank group 1） P<0.05, 2）P<0.01). DETAILED DESCRIPTION

[0021] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0023] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; and the experimental methods, unless otherwise specified, are all conventional experimental methods in the art.

[0024] Example 1

[0025] A polysaccharide from pitaya branches.

[0026] The preparation method of the polysaccharide is specifically as follows:

[0027] 1. Slice the red pitaya branches and dry them under reduced pressure until the quality is constant;

[0028] 2. Soak and extract with petroleum ether and 95% ethanol for 4 hours successively to remove fat-soluble components such as pigments and small molecular components, and evaporate the solvent to obtain the pretreated sample;

[0029] 3. Accurately weigh the pretreated sample, add water at a material-liquid ratio of 1:20, place it in a pressure cooker extraction device, and extract at 121 degrees for 30 minutes;

[0030] 4. Concentrate the supernatant to 1 / 5 of the original volume, add 5 times the volume of anhydrous ethanol, precipitate for 24 hours, and centrifuge to obtain the precipitate;

[0031] 5. Dissolve the precipitate with an appropriate amount of deionized water to remove insoluble proteins, and dialyze with deionized water to remove small molecule impurities and freeze-dried polysaccharide components.

[0032] Example 2

[0033] Effects of polysaccharides from pitaya branches on the proliferation of mouse osteoblast precursor cells MC3T3-E1.

[0034] 1. Cell culture.

[0035] Mouse osteoblast precursor cells MC3T3-E1 were seeded in DNEM high-glucose medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Incubation conditions were set at 37°C and 5% CO2, with medium replacement every 48 hours.

[0036] 2. CCK-8 cell proliferation assay.

[0037] MC3T3-E1 cells were plated in a 96-well plate at a density of 5000 cells / well, and wells without cells were set as blank controls. Each experimental group was set up with 6 replicates, and the volume of culture medium in each well was 100 μL. After adhesion, the culture medium was replaced, and the new culture medium contained different doses of pitaya branch polysaccharides. At the time points of 24 h, 48 h, and 72 h of culture, 10 μL of CCK-8 detection solution was added to each well, and the cells were incubated in a 37°C incubator for 2 h. The absorbance value (A) was measured at 450 nm using an enzyme reader, and the average value of each replicate well was calculated and then inserted into the formula: relative cell viability (%) = A 处理组 / A 对照组 ×100%.

[0038] 3. Statistical processing.

[0039] The results were statistically analyzed using SPSS 12.0 software, and the differences between the groups were detected using one-way analysis of variance. The difference was statistically significant when P < 0.05.

[0040] 4. Results.

[0041] The results of CCK-8 showed that all doses of pitaya branch polysaccharide could significantly promote the proliferation of mouse osteoblast precursor cells MC3T3-E1, whether cultured for 24h, 48h or 72h. Figure 1 shown.

[0042] Example 3

[0043] Effects of pitaya branch polysaccharides on osteoblast differentiation of MC3T3-E1 cells.

[0044] 1. Alizarin red staining was used to detect the cell mineralization ability.

[0045] MC3T3-E1 cells were seeded in 24-well plates at a density of 5000 cells / well. When the cell confluence reached 60-70%, the DNEM high-glucose medium was discarded and OriCell MC3T3-E1 complete osteogenic differentiation medium (purchased from Saiye Biotechnology) containing different concentrations of pitaya branch polysaccharides was added for continued culture. Fresh OriCell MC3T3-E1 complete osteogenic differentiation medium was replaced every three days. After 15 days of induction, cells were stained with alizarin red. The medium was discarded, and the cells were washed twice with PBS. The cells were fixed with 4% neutral formaldehyde for 30 minutes, the formaldehyde was discarded, and the cells were rinsed twice with PBS. Alizarin red staining was performed for 5 minutes, the alizarin red was discarded, and the cells were rinsed three times with PBS. The orange-red calcified nodules were observed under an inverted microscope. To quantify the mineralized area, the stained samples were dissolved in 10 mmol / L sodium phosphate aqueous solution containing 10% cetylpyridinium chloride, and the absorbance at 562 nm was measured using a microplate reader.

[0046] 2. Statistical processing.

[0047] The results were statistically analyzed using SPSS 12.0 software, and the differences between the groups were detected using one-way analysis of variance. The difference was statistically significant when P < 0.05.

[0048] 3. Results.

[0049] Depend on Figure 2 、 3 It can be seen that compared with the blank control group, under the intervention of pitaya branch polysaccharide, MC3T3-E1 osteoblasts can form more calcium nodules, and more red mineralized deposits can be seen after alizarin red staining.

[0050] Example 4

[0051] Effects of pitaya branch polysaccharide intervention on bone mineral density and bone biomechanical indices in osteoporosis model rats.

[0052] 1. Experimental animals.

[0053] Eighty 6-month-old SPF female SD rats weighing (350 ± 30) g were purchased from the Experimental Animal Center of Guangdong Medical University with the license number SCXK (Yue) 2018-0008.

[0054] 2. Animal grouping, modeling and drug administration.

[0055] Sixty rats were randomly divided into a surgical group (n=45) and a sham-operated group (n=15). The rats were acclimated for one week with free access to water and food. All animals were anesthetized intraperitoneally with 10% chloral hydrate solution. The rats were placed in the supine position on the operating table with their limbs immobilized. The lower midsection of the abdomen was routinely prepared and disinfected. The abdominal cavity was entered layer by layer along the midline. The Y-shaped uterus was located. The bilateral "mulberry-like" ovarian tissue was exposed within the fat mass below the kidneys. The surrounding fat was separated, and the ovaries were removed. The surgical incisions were then sutured layer by layer. In the sham-operated group, only the fat surrounding the bilateral ovaries was removed; other surgical procedures remained unchanged. Postoperatively, penicillin was administered intramuscularly at 80,000 units daily for three consecutive days to prevent infection. Twelve weeks after surgery, five rats were randomly selected from each group and sacrificed under intraperitoneal anesthesia. The right femur was removed, and surrounding soft tissue was removed. Bone mineral density (BMD) was measured using a bone densitometer to verify the success of the model. After successful modeling, 40 rats remained in the surgical group and 10 in the sham-operated group. The remaining 40 model rats were randomly divided into a model group and low-, medium-, and high-dose groups of pitaya branch polysaccharide, with 10 rats in each group. The low-, medium-, and high-dose groups were given pitaya branch polysaccharide at 25, 50, and 100 mg / (kg·d), respectively. The sham-operated and model groups were gavaged with normal saline once daily for three consecutive months. At the end of the experiment, the rats were sacrificed by cardiac exsanguination and immediately sampled.

[0056] 3. Bone mineral density test.

[0057] The right femur was stripped of muscle and soft tissue, wrapped with gauze dipped in saline, wrapped in tin foil, and placed in a small bag.

[0058] The samples were frozen at -20°C. Bone mineral density (BMD) was measured in vitro using a dual-energy X-ray absorptiometry instrument. The whole femur was used for measurement, and the measurement results were analyzed using the accompanying software.

[0059] 4. Determination of biomechanical parameters.

[0060] The left femur was freed of muscle and soft tissue, wrapped with gauze soaked in saline, then wrapped in tin foil, placed in a small bag, and frozen at -20°C. Biomechanical testing was then performed on an MTS electronic universal testing machine. The femur underwent three-point bending tests with a support span of 18 mm, a midpoint as the compression point, a loading rate of 5.0 mm / min, a temperature of 23°C, and a humidity of 60% to 70%. Parameters such as maximum load, breaking load, elastic load, and elastic modulus were determined.

[0061] 5. Statistical processing.

[0062] The results were statistically analyzed using SPSS 12.0 software, and the differences between the groups were detected using one-way analysis of variance. The difference was statistically significant when P < 0.05.

[0063] 6. Results.

[0064] Compared with the sham operation group, the femoral BMD content of the rats in the model group was significantly decreased; compared with the model group, the femoral BMD content of the rats in the pitaya branch polysaccharide groups at all doses was significantly increased, see Table 1.

[0065] Compared with the sham operation group, the maximum load, breaking load, elastic load and elastic modulus of the model group were significantly reduced; compared with the model group, the four biomechanical parameters of the pitaya branch polysaccharide groups at each dose showed an upward trend, but the differences were not significant, see Table 2.

[0066] Table 1 Effects of pitaya branch polysaccharides on BMD in postmenopausal osteoporosis rats ( , n=10)

[0067]

[0068] Note: Compared with sham operation group 1） P<0.05, 2） P<0.01; compared with the model group 3） P<0.05, 4） P<0.01.

[0069] Table 2 Effects of pitaya branch polysaccharide on biomechanical parameters of left femur in postmenopausal osteoporosis rats ( , n=10)

[0070]

[0071] Note: Compared with sham operation group 1） P<0.05, 2） P<0.01; compared with the model group 3） P<0.05, 4） P<0.01.

[0072] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0073] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A use of a polysaccharide from pitaya branches in the preparation of a medicament for treating osteoporosis, characterized in that: The polysaccharide is an extract obtained from dragon fruit branches through water extraction.

2. The use according to claim 1, characterized in that The osteoporosis includes primary osteoporosis or secondary osteoporosis.

3. The use according to claim 1, characterized in that The medicine comprises pharmaceutically acceptable excipients and the polysaccharide.

4. The use according to claim 3, characterized in that The medicine is an oral preparation or an injection preparation.

5. The use according to claim 1, characterized in that The application includes oral administration or injection of the drug, and the dosage of the polysaccharide is 1-1000 mg per kilogram of body weight.

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