Epimedium extract short peptide and its application in promoting bone growth

Abstract

The application discloses a short peptide extracted from Epimedium and application of the short peptide in promoting bone growth. The short peptide is extracted from traditional Chinese medicinal material Epimedium, and a mechanism of the short peptide for improving bone-related diseases is analyzed. It is found that a short peptide with a sequence of VPIVMHDYLTGGFTANTSL can promote proliferation of osteoblasts by activating a phosphorylation pathway of MAPK, thereby laying a certain foundation for research on a new drug for improving bone-related diseases.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to the improvement of bone diseases by active short peptides extracted from traditional Chinese medicine. Background Technology

[0002] Osteoporosis is defined as a metabolic bone disease characterized by decreased bone strength, which increases the risk of fractures. Bone strength varies depending on bone mass and bone quality. After bone growth, repeated bone resorption by osteoclasts and bone remodeling by osteoblasts occur, with 20%–30% of bone being replaced by new bone each year. If this remodeling balance is disrupted, with bone resorption exceeding bone formation, bone mass decreases. Furthermore, bone quality decreases due to changes in bone microstructure, the accumulation of bone-forming components such as collagen from micro-trauma and microfractures.

[0003] Bone mass and bone quality decline with age. This decline can be caused by a combination of factors, including hormonal imbalances due to decreased estrogen levels in women, deficiencies in calcium, vitamin D, vitamin K, and reduced nutrient absorption; lack of exercise; smoking; alcohol consumption; endocrine disorders; and conditions such as diabetes and rheumatoid arthritis, which are known to be caused by medications such as steroids.

[0004] In patients with osteoporosis, even minor impacts such as falls increase the risk of fractures, especially in the elderly, as fractures often require more care. Therefore, improving and preventing osteoporosis is crucial.

[0005] Treatment methods for osteoporosis include dietary therapy (due to calcium and protein being the main components of bones, vitamin D and vitamin K being essential for bone rebuilding, and attention to a balanced diet); exercise therapy (walking, aerobic exercise, and other moderate-intensity exercises, as well as back exercises); and drug therapy (using bisphosphate preparations such as alendronate sodium hydrate, riseralate sodium hydrate, ibanronate sodium hydrate, and zoledronic acid hydrate, selective estrogen receptor modulators such as raloxifene hydrochloride, SERM preparations such as denorsmab, and anti-RANKL receptor preparations).

[0006] In addition, consuming foods that prevent or improve abnormal bone metabolism can effectively increase bone density, such as ascorbic acid protein hydrolysate, ascorbic acid derivatives like magnesium ascorbate and ascorbic acid-2-glucoside, and shark cartilage protein. Currently, reports indicate that ethanol-water extracts of Cassia tora seeds (formerly used in traditional medicine) and solvent extracts selected from these mixtures have an inhibitory effect on bone resorption.

[0007] However, it is known that bisphosphonate preparations, for example, used as treatments for osteoporosis, not only absorb bone but also inhibit bone formation. Hormone preparations, such as parathyroid hormone preparations, may exhibit hormone-like effects in addition to promoting bone formation and increasing bone mass. Therefore, there is still a need for research and development of effective drugs or health products for bone formation in the current technology.

[0008] Epimedium, also known as Fairy Spirit, is a perennial herb belonging to the genus Epimedium in the family Berberidaceae. There are approximately 59 species worldwide, with about 48 species found in China, making it one of the major distribution areas for Epimedium species globally. The 2020 edition of the Chinese Pharmacopoeia lists five species as authentic Epimedium: Epimedium brevicornum Maxim, Epimedium sagittatum (Sieb. et Zucc.) Maxim, Epimedium pubescens Maxim, Epimedium wushanense TSYing, and Epimedium koreanum Nakai. The areas of distribution of Epimedium resources in Chinese medicine are, in descending order: Epimedium sagittatum (Sieb. et Zucc.) Maxim, Epimedium brevicornum Maxim, Epimedium acuminatum Maxim, Epimedium koreanum Nakai, Epimedium pubescens Maxim, Epimedium leptorrhizum Maxim, and Epimedium wushanense. TSYing divides the country into four production areas, ranked by importance: Northeast Region, which contains only one species, Epimedium brevicornu from Korea, and is of the highest quality; Northwest and North China Region, mainly Epimedium, with relatively stable component content; East and South China Region, mainly Epimedium sagittatum, but with significant differences in quality within the species; Southwest Region, in Guizhou Province, the mainstream variety is Epimedium coarse hair, followed by Epimedium qianlingense, and then Epimedium wushanense. Epimedium qianlingense is not used medicinally. In Sichuan Province, the mainstream varieties are Epimedium coarse hair and Epimedium pubescens. This region has a large output, but the varieties are complex, and attention should be paid to morphological identification.

[0009] Currently, there are literature reports that traditional Chinese medicine compositions containing Epimedium can be used to treat bone hyperplasia, such as WO2022247640A1, but there are no reports on the relevant mechanisms. There are also reports on the extraction of effective components from Epimedium in the prior art, such as CN115671215A, but no literature reports on the effects of peptides formed by the cleavage of plant proteins contained in Epimedium on osteoblasts and bone hyperplasia. This application is expected to provide research in this area and provide a certain basis for the further development and use of Epimedium as a traditional Chinese medicine. Summary of the Invention

[0010] To overcome the shortcomings of existing technologies, this invention selects traditional Chinese medicinal herbs recorded in traditional Chinese medicine as having improving and repairing effects on bone hyperplasia as the analytical objects. It screens short peptide molecules with biochemical functions among them, analyzes whether they have an effect on improving bone-related diseases, and attempts to explain their mechanism of action in improving bone-related diseases. Specifically, this invention provides:

[0011] In a first aspect, a short peptide selected from Epimedium is provided, the sequence of which is a polypeptide molecule containing VPIVMHDYLTGGFTANTSL and less than 100 amino acid residues in length; preferably, the short peptide contains VPIVMHDYLTGGFTANTSL and less than 50 amino acid residues in length.

[0012] Secondly, a short peptide selected from Epimedium is provided, characterized in that it is a short peptide having at least one and at most three conserved amino acid substitutions compared to the polypeptide described in VPIVMHDYLTGGFTANTSL. Preferably, the sequence of the short peptide is as follows:

[0013] VPIVMHDYLTGGFTANTSL.

[0014] A third aspect provides a pharmaceutical composition comprising the short peptides described in the first and second aspects and a pharmaceutically acceptable carrier.

[0015] Fourthly, the use of the short peptides described in the first and second aspects in the preparation of formulations that promote osteoblast proliferation is provided.

[0016] Fifthly, the use of the short peptides described in the first and second aspects in the preparation of medicaments for improving bone-related diseases is provided; preferably, the bone-related diseases are selected from osteoporosis, degenerative osteoarthritis, and femoral head necrosis. Most preferably, the bone-related disease is osteoporosis. Attached Figure Description

[0017] Figure 1 Epimedium polypeptide promotes osteoblast proliferation in a concentration-dependent manner.

[0018] Figure 2 Epimedium polypeptide promotes osteoblast proliferation by stimulating phosphorylation of the MAPK pathway. Detailed Implementation

[0019] The objects and functions of the present invention, as well as the methods for achieving these objects and functions, will be explained below with reference to exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in various forms. This specification is merely intended to help those skilled in the art to comprehensively understand the specific details of the invention.

[0020] Example 1: Effect of Epimedium short peptide on osteoblast proliferation

[0021] 1. Isolation and identification of short peptides from Epimedium

[0022] Multiple short peptides extracted from Epimedium were isolated and their effects on cell proliferation were evaluated using commercially available mouse cells. The peptide with the best effect was identified by mass spectrometry, and the sequence of the peptide was obtained as: VPIVMHDYLTGGFTANTSL.

[0023] The method is summarized as follows: MC3T3-E1 is subjected to a 2×10⁻⁶ ppm using the MTT method. 4 / cm 2 The cells were seeded at a density of 1, 10, and 100 μg / mL in 96-well plates, and the proliferation of osteoblasts was measured after co-incubation for 24, 48, and 72 h with the short peptides of Epimedium. This study aimed to analyze whether Epimedium peptides have a proliferative effect on osteoblasts.

[0024] 2. Establishment of mouse primary osteoblast cell lines

[0025] Stable passaged mouse osteoblasts were constructed according to the method described in CN1662664A. The method was summarized as follows: Skull bones were excised from the skulls of 15-25 day old fetal mice, and the frontal and parietal bones (excluding sutures and periosteum) were collected. After washing, the skull bones were treated twice at 37°C with 3 mL of 1 mg / mL collagenase for 7 minutes. After discarding the supernatants from these two digestions, the skull bones were further treated twice with 3 mL of 2 mg / mL collagenase (30 minutes, 37°C). The supernatants from the two digestions were collected, centrifuged, and the cells were washed. When the cells grew into a confluent monolayer, they were passaged into 24-well plates. Growth was terminated by culturing in MEM (minimum essential medium) / 0.1% bovine serum albumin for 24 hours before harvesting.

[0026] 3. Identification of the effect of short peptides on the proliferation of mouse primary osteoblasts

[0027] Cells obtained in step 2 were subjected to the MTT assay at a concentration of 1 × 10⁻⁶. 6 / cm 2 The ephemeral peptides were seeded at a density of 10, 50, and 100 μg / mL in 96-well plates, and the proliferation of osteoblasts was measured after co-incubation for 24, 48, and 72 h at these concentrations. This analysis aimed to determine whether the ephemeral peptides had a proliferative effect on osteoblasts. The negative control was DMSO, and the positive control was the peptide composition before extraction in step 1) (results are shown in Figure 1). Figure 1 As shown in the figure, the purified polypeptide from Epimedium promotes osteoblast proliferation more effectively than the unpurified polypeptide composition, and exhibits certain time- and concentration-dependent characteristics.

[0028] Example 2: Effect of peptides on activation of the MAPK signaling pathway in osteoblasts

[0029] The effects of peptides from Epimedium on cell signaling pathways during osteoblast proliferation were detected using the method described in CN114480264A. The method is summarized as follows:

[0030] Using characteristic inhibitors of three kinases—ERK, JNK, and p38—U0126, SP600125, and SB203580, this study investigated the roles of three MAPK pathways (ERK, JNK, and p38) in the action of Epimedium polypeptide from both activation and inhibition perspectives. Simultaneously, Western blotting was used to verify the activation of key kinases in the MAPK signaling pathway and the different effects of each pathway on osteoblast function at the translational level. Based on the experimental results, further research was conducted on the pathways involving ERK, JNK, and p38 that mediate extracellular Epimedium polypeptide stimulation signals, analyzing the upstream signal transduction process of key kinases and the downstream activation of transcription factors.

[0031] 1) The activation and expression of MAPK in Epimedium peptides were detected using ERK, JNK, and p38 threonine and tyrosine dual-site phosphorylation antibodies. After stimulating osteoblasts with 100 μg / mL Epimedium peptides, total protein samples were extracted at 0, 3, 6, 12, and 24 h for Western blot analysis.

[0032] The results are as follows Figure 2 As shown, exposure to 100 μg / mL Epimedium polypeptide for 0-24 hours increased the phosphorylation levels of the ERK, JNK, and p38 signaling pathways in osteoblasts prepared in Example 1, with peak levels at 3 hours of exposure followed by a decrease. Overall, the phosphorylation levels of all three signaling pathways were promoted within 24 hours, while the inhibitor reversed the phosphorylation levels. This indicates that the polypeptide screened in this invention promotes osteoblast proliferation by activating the MAPK phosphorylation pathway.

[0033] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A short peptide selected from Epimedium, with the amino acid sequence VPIVMHDYLTGGFTANTSL.

2. A pharmaceutical composition comprising the short peptide of claim 1 and a pharmaceutically acceptable carrier.

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