Application of cantharis in promoting osteoblast differentiation

Abstract

This invention relates to the application of *Avicennia marina* fruit extract in promoting osteoblast differentiation, and to the application of natural active ingredients in the prevention and treatment of bone metabolic diseases. The *Avicennia marina* fruit extract of this invention can effectively promote osteoblast differentiation and mineralization, and can be used to prepare drugs that improve osteoporosis, fracture healing, and other diseases. The *Avicennia marina* fruit extract has significant osteoblast differentiation-promoting activity, and since it is derived from green, natural, safe, and reliable plant raw materials, it shows good potential for industrial development.

Description

Technical Field

[0001] This invention relates to the field of application technology of natural active ingredients in the prevention and treatment of bone metabolic diseases, and to the application of extract of the fruit of the 'Sea-petaled' 'Sea-petaled' in promoting osteoblast differentiation. Background Technology

[0002] Osteoporosis is a systemic bone disease characterized by decreased bone mass and deterioration of bone microstructure, which easily leads to increased bone fragility and a higher incidence of fractures. The pathogenesis of this disease is complex, and insufficient bone formation is one of the causes. In normal bone metabolism, bone formation and bone resorption maintain a dynamic balance, sustaining bone metabolism. When osteoblast function is impaired, osteogenic differentiation weakens, mineralization capacity decreases, leading to a decline in bone formation capacity, resulting in a gradual reduction in bone mass and eventually developing into osteoporosis. The incidence of bone loss is increasing, but its symptoms are often insidious, diagnosis and treatment are delayed, existing drugs have limited efficacy, and many potential adverse reactions. Therefore, finding green, safe, and natural active ingredients that can promote osteoblast differentiation and enhance mineralization function is of great significance for the prevention and treatment of osteoporosis caused by insufficient bone formation.

[0003] The MC3T3-E1 cell line is an osteogenic progenitor cell derived from mouse skull and possesses excellent osteogenic differentiation potential. Under suitable induction conditions, MC3T3-E1 cells can differentiate into mature osteoblasts. In the early stages of differentiation, MC3T3-E1 cells can synthesize and secrete osteogenic markers such as alkaline phosphatase (ALP); in the terminal stage of differentiation, MC3T3-E1 cells can form alizarin red-positive mineralized nodules. Due to its well-defined origin, stable growth, and controllable differentiation characteristics, MC3T3-E1 cells have been widely used to evaluate the effects of drugs, natural active ingredients, or gene regulation on osteogenic differentiation and bone formation function, making it an important in vitro model for studying bone formation mechanisms and developing anti-osteoporosis treatment strategies.

[0004] Mangroves are woody plants that grow in the intertidal zone of tropical and subtropical coasts. Among them, the evergreen mangrove *Sonneratia caseolaris*, belonging to the genus *Sonneratia* of the family Lythraceae, is a highly salt-tolerant species. It comprises six species and three varieties: *Sonneratia apetta* Buch.-Ham., *Sonneratia caesolaris*, *Sonneratia ×hainanensis*, *Sonneratia alba*, *Sonneratia ×gulngai*, *Sonneratia ovata*, *Sonneratia urama*, *Sonneratia agriffithii*, and *Sonneratia lanceolata*. The first six species are mainly distributed in Hainan, Guangdong, and Guangxi provinces of China. Affected by various factors such as soil hypoxia, salinization, and strong ultraviolet radiation, sea sang has a unique metabolic system and environmental adaptability, enabling it to survive in harsh environments and produce unique secondary metabolites.

[0005] In tropical / subtropical coastal regions, *Avicennia marina* (sea mulberry) is often used as both food and medicine due to its rich nutritional value and wide range of pharmacological activities. In parts of Africa, Myanmar, and India, the juice of *Avicennia marina* can be made into beverages, with the amount added up to 30% of the total volume. Currently, there are no reports on the effects of *Avicennia marina* fruit extract on bone metabolism. It is well known that osteoblasts play a crucial role in the formation, repair, and reconstruction of bone tissue during bone formation. With age, especially in postmenopausal women or the elderly, osteoblast function gradually declines, making them highly susceptible to osteoporosis. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an application of a petalless Avicennia marina fruit extract in the preparation of an osteoblast differentiation promoter. This petalless Avicennia marina fruit extract effectively promotes osteoblast differentiation and mineralization, and can be used to prepare drugs that improve osteoporosis, fracture healing, and other diseases. The petalless Avicennia marina fruit extract exhibits significant osteoblast differentiation-promoting activity, and since it is derived from green, natural, safe, and reliable plant raw materials, it demonstrates excellent potential for industrial development.

[0007] This invention provides the application of an extract of the fruit of the arborescentis in the preparation of an osteoblast differentiation promoter. The preparation method of the extract includes: using a water-alcohol mixture as an extractant to extract the extract from the fruit of the arborescentis.

[0008] In one embodiment, the preparation method of the arborescentis fruit extract includes the following steps: crushing the arborescentis fruit, extracting it using a water-alcohol mixture as an extractant, collecting the filtrate, concentrating it, and obtaining the arborescentis fruit extract. The extractant comprises ethanol and water in a volume ratio of (7-10):1.

[0009] In one embodiment, the extraction temperature is 25-30°C, and the extraction time is 24-96 hours.

[0010] The present invention also provides an osteoblast differentiation promoter comprising a natural active ingredient and pharmaceutically acceptable excipients, wherein the natural active ingredient is an extract of the fruit of the 'Avicennia marina' in the application described herein.

[0011] In one embodiment, the excipients include at least one of the following: excipients, lubricants, antioxidants, preservatives, binders, fillers, or thickeners.

[0012] The present invention also provides the application of the extract of the fruit of the arborescentis in the preparation of a drug for treating osteoporosis, wherein the extract of the arborescentis is the extract of the fruit of the arborescentis used in the above application.

[0013] The present invention also provides a medicament for treating osteoporosis, comprising a natural active ingredient and pharmaceutically acceptable excipients, wherein the natural active ingredient is an extract of the fruit of the 'Avicennia marina' in the application described herein.

[0014] The present invention also provides the use of a petalless Sorrelius fruit extract in the preparation of a medicament for promoting fracture healing, wherein the petalless Sorrelius fruit extract is the same as the petalless Sorrelius fruit extract used in the above application.

[0015] The present invention also provides a medicament for promoting fracture healing, comprising a natural active ingredient and pharmaceutically acceptable excipients, wherein the natural active ingredient is an extract of the fruit of the 'Avicennia marina' in the application described herein.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention relates to the application of an extract of the fruit of *Avicennia marina* (a type of flowering mulberry) in the preparation of an osteoblast differentiation promoter. This extract effectively promotes osteoblast differentiation and mineralization, and is suitable for preparing drugs to improve osteoporosis, fracture healing, and other diseases. The extract of *Avicennia marina* fruit exhibits significant osteoblast differentiation-promoting activity, and because it is derived from green, natural, safe, and reliable plant materials, it demonstrates good potential for industrial development. Attached Figure Description

[0017] Figure 1A schematic diagram showing the cell viability of MC3T3-E1 cells after treatment with different concentrations of SAE for 24h, 48h, and 72h. Figure 2 A schematic diagram showing the effect of different concentrations of SAE on the osteogenic differentiation activity of MC3T3-E1 cells; Figure 3 This is a schematic diagram showing the effect of different concentrations of SAE on the mineralization ability of MC3T3-E1 cells. Detailed Implementation

[0018] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0020] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0021] Example I. Preparation and verification experiments of extract from the fruit of the petalless Sinensis hainanensis.

[0022] 1. Preparation of extract from the petalless fruit of the sea mulberry Accurately weigh 500g of fresh, unsegmented sea mulberry fruit, wash and crush. After crushing, extract with 5L of a water-ethanol mixture (ethanol:water = 9:1) at room temperature for 24h (repeat 3 times). Collect the extract filtrate, pass it through a 200-mesh sieve, combine the filtrates, place them in a rotary evaporator, concentrate, and freeze-dry to obtain 15g of brown amorphous powder. Store at -20℃ for later use.

[0023] 2. Cell Culture Osteoblastic progenitor cells MC3T3-E1 were seeded in α-MEM medium (containing 10% fetal bovine serum and 1% P / S) and cultured at 37°C and 5% CO2 until 80% cell confluence was achieved.

[0024] 3. Cell viability assay The effect of Sinapis alba fruit extract (SAE) on the growth of MC3T3-E1 cells was detected using a CCK-8 assay kit. MC3T3-E1 cells (5 × 10⁻⁶) were then divided into two groups. 3 Cells / wells were seeded in 96-well plates and treated with six different concentrations of SAE (0.01, 0.1, 1, 10, 100, 500, and 1000 μg / mL) for 24, 48, and 72 h, respectively, with six replicates per group. Then, 10 μL CCK-8 reagent was added to each well, and the plates were incubated at 37°C in a 5% CO2 humidified incubator for 2 h, with absorbance measured at 450 nm.

[0025] 4. Alkaline phosphatase (ALP) assay For in vitro differentiation of MC3T3-E1, MC3T3-E1 cells in good growth condition (5.0 × 10⁻⁶) were selected. 4 Cells / wells were seeded in 24-well plates. MC3T3-E1 cells were maintained in α-MEM medium. When the confluence reached 80%, the medium was removed with PBS and replaced with osteogenic differentiation medium (α-MEM, 10% fetal bovine serum, 1% P / S, 50 μg / mL ascorbic acid and 10 mM β-carotene) containing different concentrations of SAE (0.1, 1, 10 μg / mL). Glyceryl phosphate disodium hydrate, dexamethasone 1×10 -8 Osteogenic differentiation was induced for 7 days with an osteogenic differentiation medium (mol / L), which was changed every 2 days. At the end of day 7, the medium was removed, and the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and washed three times with PBS. The cells were stained with ALP staining solution in the dark at room temperature, washed with ultrapure water, and observed under a microscope.

[0026] 5. Alizarin Red Staining Determination To investigate the effect of Sinapis alba fruit extract (SAE) on the mineralization of MC3T3-E1 cells, Alizarin Red staining was performed 21 days after the late osteogenic differentiation stage. MC3T3-E1 cells in good growth condition (5.0 × 10⁻⁶) were collected. 4 Cells / wells were seeded in 24-well plates and maintained in α-MEM medium. When the confluence reached 80%, the complete medium was removed with PBS and replaced with osteogenic differentiation medium (α-MEM) containing different concentrations of SAE (0.1, 1, 10 μg / mL). MEM, 10% fetal bovine serum, 1% P / S, 50 μg / mL ascorbic acid and 10 mM β Glyceryl phosphate disodium hydrate, dexamethasone 10 -8Cells were induced to differentiate using a medium (mol / L) for 21 days, with the medium changed every 2 days. At the end of day 21, the medium was removed, and the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and washed three times with PBS. Cells were then stained with 0.2% alizarin red at room temperature for 30 min. After washing with ultrapure water, the cells were observed under a microscope.

[0027] II. Verification of Experimental Results: 1. Security Analysis: The results are as follows Figure 1 As shown.

[0028] Figure 1 The effects of different concentrations of SAE (0, 0.01, 0.1, 1, 10, 100, 500, 1000 μg / mL) on cell viability at different treatment times (24 h, 48 h, 72 h) were demonstrated. The results showed that SAE at lower concentrations (≤100 μg / mL) and shorter treatment times (24–48 h) had no significant effect on cells, but high concentrations (≥500 μg / mL) at longer treatment times (72 h) caused a decrease in cell viability. Generally, the effective concentration of the test substance in cell experiments is below 100 μg / mL. Therefore, the extract of *Sinapis alba* (a type of fruit) will not cause adverse effects on the body in practical applications.

[0029] 2. Alkaline phosphatase (ALP) assay results: Figure 2 The results showed that SAE significantly enhanced alkaline phosphatase activity under osteogenic induction conditions, exhibiting a concentration-dependent enhancement trend, with 10 μg / mL being the optimal concentration, suggesting that it has a good promoting effect in the early osteogenic differentiation process.

[0030] 3. Alizarin Red staining results: Figure 3 The results showed that SAE effectively enhanced the mineralization capacity of MC3T3-E1 cells in a concentration-dependent manner. As the SAE concentration increased from 0.1 μg / mL to 10 μg / mL, the formation of mineralized nodules gradually increased, suggesting that SAE plays a positive role in promoting calcification deposition in the later stages of osteogenic differentiation.

[0031] 4. Experimental conclusions: SAE significantly promoted osteogenic differentiation of MC3T3-E1 pre-osteoblasts, with effects observed in two key stages: early osteogenic stage (enhanced ALP activity) and late osteogenic stage (increased mineralization nodules), exhibiting a good dose-dependent response. These results provide experimental evidence for the potential of *Sonneratia natans* extract as a candidate drug for promoting bone regeneration or improving osteoporosis.

[0032] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0033] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of a Sonchus oleraceus fruit extract in the preparation of a bone-forming cell differentiation promoter, the Sonchus oleraceus fruit extract being prepared by a method comprising: It was extracted from the fruit of the arborescentis using a water-alcohol mixture as the extractant.

2. The application according to claim 1, characterized in that, The preparation method of the extract of the petalless Sorrelia agallocha fruit includes the following steps: crushing the petalless Sorrelia agallocha fruit, extracting it using a water-alcohol mixture as an extractant, collecting the filtrate, concentrating it, and obtaining the extract of the petalless Sorrelia agallocha fruit. The extractant comprises ethanol and water in a volume ratio of (7-10):

1.

3. The application according to claim 2, characterized in that, The extraction temperature is 25-30℃, and the extraction time is 24-96h.

4. An osteoblast differentiation promoter, characterized in that, It includes natural active ingredients and pharmaceutically acceptable excipients, wherein the natural active ingredient is the extract of the fruit of the arborescentis in any of the applications described in claims 1-3.

5. The osteoblast differentiation promoter according to claim 4, characterized in that, The excipients include at least one of the following: excipients, lubricants, antioxidants, preservatives, binders, fillers, or thickeners.

6. The application of an extract of the fruit of the araliacea purpurea in the preparation of a drug for treating osteoporosis, characterized in that, The extract of the petalless Sorrel fruit is the same as the extract of the petalless Sorrel fruit used in any of the applications described in claims 1-3.

7. A drug for treating osteoporosis, characterized in that, It includes natural active ingredients and pharmaceutically acceptable excipients, wherein the natural active ingredients are the extract of the fruit of the arborescentis in any of the applications described in claims 1-3.

8. The use of an extract of the fruit of the araliacea purpurea in the preparation of a medicament for promoting fracture healing, characterized in that, The extract of the petalless Sorrel fruit is the same as the extract of the petalless Sorrel fruit used in any of the applications described in claims 1-3.

9. A drug for promoting fracture healing, characterized in that, It includes natural active ingredients and pharmaceutically acceptable excipients, wherein the natural active ingredients are the extract of the fruit of the arborescentis in any of the applications described in claims 1-3.

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