Application of phylaephora gregata and its water extract and active ingredient in preparation of medicine for preventing and treating osteoporosis

Abstract

The application discloses application of a plant of Melothria japonica, water extract and active ingredients of the plant in preparation of drugs for preventing and treating osteoporosis. It is found for the first time that the plant of Melothria japonica can significantly promote cell proliferation, improve intracellular ALP activity, make bone formation related protein expression up-regulated, make RAGE and TNFR1 expression down-regulated and inhibit cell oxidative stress induced by AGEs, H2O2 and TNF-alpha, which indicates that the plant of Melothria japonica has a protective effect on osteoblasts, is helpful to improve osteoblast activity and promote new bone formation, and finally achieves the purpose of preventing and treating osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Melilotus arvense and its water extracts and active ingredients in the preparation of drugs for the prevention and treatment of osteoporosis. Background Technology

[0002] Osteoporosis is a metabolic bone disease primarily caused by decreased bone formation and increased bone resorption. It is well known that bone balance in the human body is mainly maintained through the coordinated function of osteoblasts and osteoclasts. Osteoclasts are primarily responsible for the resorption of aging bone, while osteoblasts are primarily responsible for the formation of new bone. When osteoblast activity decreases and the amount of new bone formation reduces, bone balance is disrupted, leading to osteoporosis.

[0003] Traditional Chinese medicine is a treasure trove for the research and development of anti-osteoporosis drugs. It has great advantages in preventing and treating osteoporosis, relieving symptoms, improving prognosis, and improving patients' quality of life. It also has the advantages of stable efficacy, long-lasting effect, and low toxicity and adverse reactions.

[0004] For example, Chinese invention patent CN114558047B discloses a traditional Chinese medicine composition for the prevention and treatment of osteoporosis. This composition includes the following raw materials in parts by weight: 300-500 parts of Epimedium, 300-400 parts of Drynaria fortunei, and 400-600 parts of urolithin A. Urolithin A significantly inhibits osteoclast activation, reduces the number and size of osteoclasts, and has a significant inhibitory effect on bone resorption and the expression of osteoclast-related genes. It significantly inhibits osteoclast proliferation and activity, slows cartilage degeneration, and promotes bone health. Furthermore, it regulates mitochondrial expression in skeletal muscle, increases cellular respiration in skeletal muscle, thereby increasing skeletal muscle strength and indirectly promoting normal bone metabolism. Epimedium and Drynaria fortunei both promote osteogenic differentiation of bone marrow mesenchymal stem cells.

[0005] There are currently no reports of using the She ethnic medicinal herb *Rhodiola rosea* for the prevention and treatment of osteoporosis. Summary of the Invention

[0006] The purpose of this invention is to provide the application of *Melastoma candida* and its water extracts and active ingredients in the preparation of osteoporosis prevention and treatment drugs, thus providing a new and effective approach for the prevention and treatment of osteoporosis.

[0007] To achieve the above-mentioned objectives, the technical solution of this invention is as follows:

[0008] Application of Melastoma can be used in the preparation of drugs for the prevention and treatment of osteoporosis.

[0009] This invention is the first to discover that, for osteoblasts induced by AGEs, H2O2, and TNF-α, *Melastoma can significantly promote cell proliferation, increase intracellular ALP activity, upregulate the expression of bone formation-related proteins, downregulate the expression of RAGE and TNFR1, and inhibit oxidative stress in cells. This indicates that *Melastoma* has a protective effect on osteoblasts, helps to improve osteoblast activity, promotes new bone formation, and ultimately achieves the goal of preventing and treating osteoporosis.

[0010] Analysis of the medicinal substances of *Melastoma dodecandrum* revealed that the water extract of *Melastoma dodecandrum* and the active ingredients contained therein are the material basis for its role in preventing and treating osteoporosis. Therefore, this invention also provides the application of the water extract of *Melastoma dodecandrum* in the preparation of osteoporosis prevention and treatment drugs, as well as the application of the active ingredients of *Melastoma dodecandrum* in the preparation of osteoporosis prevention and treatment drugs.

[0011] The water extract of *Melastoma dodecandrum* is obtained by the following method: crushing *Melastoma dodecandrum* and extracting it with water under reflux at least twice to obtain an extract; filtering each extract and combining the filtrates; concentrating and drying the filtrates to obtain the water extract of *Melastoma dodecandrum*.

[0012] The active ingredients of the herb *Rosa multiflora* include at least one of gallic acid, 3-methoxyellagic acid, protocatechuic acid, scopolamine, epicatechin-(8,7-e)-4β-(4-hydroxyphenyl)-3,4-dihydroxy-2(3H)-pyranone, [6-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-3-yl]oxy-3,4,5-trihydroxyoxan-2-yl]methyl-3,4,5-trihydroxybenzoate, rutin, luteolin, quercetin, styracin, and vitexin, more preferably containing at least vitexin.

[0013] Studies have found that 0.1 μM of isovitexin can show excellent protective effects on osteoblasts, indicating that isovitexin is the active ingredient in *Melastoma candida* for preventing osteoporosis.

[0014] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0015] (1) This invention is the first to discover that for osteoblasts induced by AGEs, H2O2 and TNF-α, Melilotus arvense can significantly promote cell proliferation, increase intracellular ALP activity, upregulate the expression of bone formation-related proteins, downregulate the expression of RAGE and TNFR1, and inhibit oxidative stress in cells. This indicates that Melilotus arvense has a protective effect on osteoblasts, helps to improve osteoblast activity, promotes new bone formation, and ultimately achieves the purpose of preventing and treating osteoporosis.

[0016] (2) This invention is the first to discover that 0.1 μM of isovitexin can show excellent protective effect on osteoblasts, indicating that isovitexin is the active ingredient of Melilotus arvense in preventing osteoporosis. Attached Figure Description

[0017] Figure 1 The effect of Melilotus spp. on AGEs-induced osteoblast survival (n=6);

[0018] Wherein, cell viability (%) represents cell viability (percentage), the same below;

[0019] Figure 2 The effect of Melilotus spp. on AGEs-induced ALP activity in osteoblasts (n=6);

[0020] Here, ALP activity represents alkaline phosphatase activity, and the same applies below;

[0021] Figure 3 To detect the effect of Melilotus arvense on the expression of bone formation-related proteins in AGEs-induced osteoblasts (n=3) by Western blot;

[0022] Figure A shows the immunoblotting results of bone formation-related proteins, Figure B shows the relative expression level analysis results of the bone formation-related protein OCN (osteocalcin), and Figure C shows the relative expression level analysis results of the bone formation-related protein RUNX2 (key osteogenic factor), and the same applies below;

[0023] Figure 4 To detect the effect of Melilotus arvense on the expression of AGEs receptor RAGE protein in AGEs-induced osteoblasts by Western blot (n=3);

[0024] Figure A shows the immunoblotting results of RAGE protein, and Figure B shows the analysis results of the relative expression level of RAGE protein, and so on.

[0025] Figure 5 Effects of Meliloti on SOD, MDA, CAT and GPx levels in AGEs-induced osteoblasts (n=6);

[0026] Figure 6 The effect of Melilotus spp. on the level of TNF-α, an inflammatory factor in osteoblasts induced by AGEs (n=3);

[0027] Figure A shows the electrophoresis results of TNF-α, and Figure B shows the analysis results of the relative expression level of TNF-α; the same applies below.

[0028] Figure 7 The effect of Melilotus spp. on H2O2-induced osteoblast survival (n=6);

[0029] Figure 8 The effect of Melilotus spp. on H2O2-induced ALP activity in osteoblasts (n=6);

[0030] Figure 9 To detect the effect of Melilotus spp. on the expression of bone formation-related proteins in H2O2-induced osteoblasts by Western blot (n=3);

[0031] Figure 10 The effect of Melilotus spp. on SOD levels in H2O2-induced osteoblasts (n=6);

[0032] Figure 11 The effect of Melilotus spp. on TNF-α-induced osteoblast survival (n=6);

[0033] Figure 12 The effect of Melilotus spp. on TNF-α-induced ALP activity in osteoblasts (n=6);

[0034] Figure 13 To detect the effect of Melilotus arvense on the expression of bone formation-related proteins in TNF-α-induced osteoblasts by Western blot (n=3);

[0035] Figure 14 To detect the effect of Melilotus spp. on TNF-α-induced TNFR1 protein expression in osteoblasts by Western blot (n=3);

[0036] Figure 15 The TIC mass spectrum of the aqueous extract of *Melastoma candida*.

[0037] Wherein, Time represents the retention period, and the same applies below;

[0038] Figure 16 HPLC chromatogram of osteoblast cell membrane induced by H2O2 modeling;

[0039] Where Intensity (mV) represents the intensity (millivolts), the same applies below;

[0040] Figure 17 HPLC chromatogram of osteoblast cell membrane in TNF-α modeling;

[0041] Figure 18 HPLC chromatogram of osteoblast cell membrane for AGEs modeling;

[0042] Figure 19 TIC mass spectrometry of melilotinib-retained components in the cell membrane of osteoblasts modeled with H2O2;

[0043] Figure 20 TIC mass spectrometry image of melilotus 'Gnaphalium' retained components in the cell membrane of osteoblasts modeled for TNF-α;

[0044] Figure 21 TIC mass spectrometry of melilotus 'Gnaphalium's' retained components in osteoblast cell membrane chromatography for AGEs modeling;

[0045] Figure 22 This is a schematic diagram of the molecular docking between isovitexin and RAGE;

[0046] Wherein, Interactions represent the type of interaction, van der Waals represent van der Waals forces, Conventional Hydrogen Bond represents conventional hydrogen bonds, Carbon Hydrogen Bond represents carbon-hydrogen bonds, and Pi-LonePair represents Pi-lone pairs of electrons, and the same applies below;

[0047] The left figure is a three-dimensional schematic diagram of the interaction between isovitexin and RAGE, and the right figure is a two-dimensional schematic diagram of the interaction between isovitexin and RAGE.

[0048] Figure 23 This is a schematic diagram of the molecular docking between isovitexin and TNFR1.

[0049] The left figure is a three-dimensional schematic diagram of the interaction between isovitexin and TNFR1, and the right figure is a two-dimensional schematic diagram of the interaction between isovitexin and TNFR1.

[0050] Figure 24 The effect of isovitexin on AGEs-induced osteoblast survival (n=6);

[0051] Figure 25 The effect of isovitexin on AGEs-induced ALP activity in osteoblasts (n=6);

[0052] Figure 26 To detect the effect of isovitexin on the expression of bone formation-related proteins in AGEs-induced osteoblasts (n=3) by Western blot.

[0053] Figure 27 To detect the effect of isovitexin on the expression of AGEs receptor RAGE protein in AGEs-induced osteoblasts by Western blot (n=3);

[0054] Figure 28 The effect of isovitexin on the levels of SOD, MDA, CAT, and GPx in AGEs-induced osteoblasts (n=6);

[0055] Figure 29The effect of isovitexin on the level of TNF-α, an inflammatory factor in osteoblasts induced by H2O2 (n=3);

[0056] Figure 30 The effect of isovitexin on H2O2-induced osteoblast survival (n=6);

[0057] Figure 31 The effect of isovitexin on H2O2-induced ALP activity in osteoblasts (n=6);

[0058] Figure 32 To detect the effect of isovitexin on the expression of bone formation-related proteins in H2O2-induced osteoblasts by Western blot (n=3);

[0059] Figure 33 The effect of isovitexin on SOD levels in H2O2-induced osteoblasts (n=6);

[0060] Figure 34 The effect of isovitexin on TNF-α-induced osteoblast survival;

[0061] Figure 35 The effect of isovitexin on TNF-α-induced ALP activity in osteoblasts;

[0062] Figure 36 To detect the effect of isovitexin on the expression of bone formation-related proteins in TNF-α-induced osteoblasts using Western blot;

[0063] Figure 37 To detect the effect of vitexin on TNFR1 protein expression in TNF-α-induced osteoblasts using Western blot. Detailed Implementation

[0064] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0065] The cells, reagents, materials, and instruments used in this embodiment are as follows:

[0066] (1) Cells: Newborn SD rats were selected, and their skull bones were taken. Blood vessels and connective tissue were removed, and the bones were placed in PBS. The bones were cut into 1mm×1mm tissue blocks with scissors and digested with trypsin in a 37℃ incubator for 30min. After centrifugation, the trypsin was discarded, and type I collagenase was added. The bones were digested in a 37℃ incubator for 2h. The digested suspension was added with an appropriate amount of PBS, centrifuged, and the supernatant was removed to obtain primary rat osteoblasts. Complete culture medium was added, the cells were resuspended, and incubated in a 37℃ 5% CO2 incubator. When the cell confluence reached 80%, the cells were passaged.

[0067] Osteoblasts were cultured in DMEM medium (containing 10% fetal bovine serum and antibiotics) at 37°C, 5% CO2, and saturated humidity. The medium was changed every 2-3 days, and cells in the logarithmic growth phase were used for experiments.

[0068] (2) Main reagents and materials: The medicinal material of *Melastoma dodecandrum* used in the experiment was obtained from the Chinese medicine warehouse of our unit. It was inspected and accepted by the Chinese medicine decoction pieces acceptance group of our unit and met the relevant provisions of the 2015 edition of the Chinese Pharmacopoeia. The embodiment of the present invention uses the water extract of *Melastoma dodecandrum*. The preparation method is as follows: 100g of *Melastoma dodecandrum* medicinal material was weighed and extracted twice with 8 times the amount of water, each time for 2 hours. The extract was filtered, the filtrates were combined, concentrated under reduced pressure, and dried under reduced pressure to obtain the water extract of *Melastoma dodecandrum*.

[0069] Calcitriol (purity: 97%, catalog number: C120126) was purchased from Aladdin Biochemical Technology Co., Ltd.; 3% hydrogen peroxide solution (National Drug Approval Number H44023919) was purchased from Guangdong Hengjian Pharmaceutical Co., Ltd.; recombinant rat tumor necrosis factor-alpha (TNF-α) (catalog number: P01436) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; anti-Runt-related transcription factor 2 (RUNX2) antibody (catalog number: PB0171), anti-advanced glycosylation end-product specific receptor antibody (Anti-RAGE / AGER Antibody, RAGE) (catalog number: BM4901), tumor necrosis factor receptor 1 antibody (Anti-TNFR1 / TNFRSF1A Antibody, TNFR1), and anti-glyceraldehyde-3-phosphate dehydrogenase (GLD) were also mentioned. The following reagents were purchased: Dehydrogenase (GAPDH) antibody (catalog number: BM3874) from Wuhan Boster Biological Engineering Co., Ltd.; Osteocalcin (OCN) antibody (catalog number: DF12303) from Jiangsu Qinke Biotechnology Research Center Co., Ltd.; MTT cell proliferation and cytotoxicity assay kit (catalog number: KGA312) and BCA protein quantification kit (catalog number: KGPBCA) from Jiangsu Kaiji Biotechnology Co., Ltd.; BIOKER pre-stained protein marker (catalog number: BK8007) from Hangzhou Baoke Biotechnology Co., Ltd.; Alkaline phosphatase assay kit (catalog number: P0321), Total SOD activity assay kit (catalog number: S0101), Catalase assay kit (catalog number: S0051), Lipid oxidation (MDA) assay kit (catalog number: S0131), and Total glutathione peroxidase assay kit (catalog number: S0059) from Beyotime Biotechnology Co., Ltd. TaKaRa Ex (Mg2+free Buffer) (Catalog No.: RR01AM) was purchased from Baori Biotechnology Co., Ltd.; Ultrapure RNA Extraction Kit (Catalog No.: CW0581) was purchased from Century Biotechnology Co., Ltd.; FastKing cDNA First Strand Synthesis Kit (Genomic De-generated) (Catalog No.: KR116) was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0070] Gallic acid (batch number: 110831-201605), protocatechuic acid (batch number: 110809-201906), protocatechuic aldehyde (batch number: 110810-201909), succinic acid (batch number: 111777-202003), luteolin (batch number: 11720-201106), neochlorogenic acid (batch number: MUST-20031002), vitexin rhamnoside (batch number: 111668-20) 0602), apigenin-7-O-β-D-glucopyranoside, isovitexin (batch number: P15J11F118584), apigenin (batch number:), kaempferol (batch number: 110861-202013), epicatechin gallate, rutin (content 91.7%, batch number: 100080-201811), chlorogenic acid (content 96.1%, batch number: 110753-202018), vitexin (content 94%). 9% purity (batch number: 111687-201704) was purchased from the National Institutes for Food and Drug Control; quercetin (98% purity, batch number: MUST-14082610) was purchased from Beijing Century Aoke Biotechnology Co., Ltd.; isovitexin (purity ≥98%, catalog number: B21544) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; chromatographic grade acetonitrile and methanol were purchased from Thermo Fisher Scientific; DMEM high glucose medium (catalog number: CR12800-S) was purchased from Zhejiang Senrui Technology Co., Ltd.; fetal bovine serum (catalog number: 11011-8611) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; MTT cell proliferation and cytotoxicity assay kit (catalog number: KGA312) was purchased from Jiangsu Kaiji Biotechnology Co., Ltd.; 0.25% trypsin digestion solution (containing EDTA and phenol red) (catalog number: G4001) and DMSO (catalog number: GC203002) were purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0071] (3) Instruments: Multiskan GO 1510 full-wavelength microplate reader (Thermo Scientific, USA); 2-16R medical centrifuge (Hunan Hengnuo Instrument Equipment Co., Ltd.); ChemiDoc TMXRS+ chemiluminescence imaging system (BioRad, USA); SW-CJ-2D double-person clean bench (Shanghai Sujing Industrial Co., Ltd.); Waters Acquity UPLC mass spectrometer (Waters, USA); Smart 2Pure UV / UF ultrapure water system (Thermo, USA); Eclipse Ts2 inverted microscope (NIKON, Japan); Spectra Max190 full-wavelength microplate reader (Molecular Devices, USA); 3111 cell culture incubator (Thermo, USA); 1388 biosafety cabinet (Thermo, USA).

[0072] In this invention, GraphPad Prism 9.0 software was used for data processing and graphing. All data are expressed as mean ± standard deviation (x ± s), and one-way ANOVA was used for comparative analysis among multiple groups of data.

[0073] Example 1: Protective effect of *Melastoma candida* on an AGEs osteoblast model

[0074] 1. Effect of Melilotus spp. on AGEs-induced osteoblast survival

[0075] Primary rat osteoblasts were subjected to a concentration of 5 × 10⁻⁶ 4 Cells were seeded at a concentration of / mL in 96-well plates and cultured for 24 hours. The culture medium was then discarded. Cells were then cultured in drug-containing medium for the corresponding time periods. Cell proliferation activity was then assessed using an MTT assay kit. Results are shown below. Figure 1 As shown.

[0076] Depend on Figure 1 It is evident that the aqueous extracts of *Melastoma candida* at concentrations of 0.01–10 mg / mL significantly promoted AGEs-induced osteoblast proliferation (P<0.01). Considering both dosage concentration and efficacy, subsequent experiments in this embodiment selected 0.01 mg / mL and 0.1 mg / mL as low and high dosage concentrations of the aqueous extract, respectively.

[0077] 2. Effects of Melilotus spp. on AGEs-induced ALP activity in osteoblasts

[0078] After primary osteoblasts reached 80% confluence, DMEM medium was replaced with osteogenic induction medium (10 nM dexamethasone, 10 mM β-249 glycerophosphate, 50 μg / mL ascorbic acid). The appropriate concentrations of *Rhizophora stylosa* water extract and calcitriol were added to the osteogenic induction medium. The medium was changed every 2-3 days. After 7 days of culture, alkaline phosphatase (ALP) activity was measured. The results are shown below. Figure 2 .

[0079] Depend on Figure 2As can be seen, compared with the normal group, the ALP activity in osteoblasts of the model group was significantly reduced (P<0.01); while compared with the model group, the ALP activity in osteoblasts of the low-dose, high-dose, and calcitriol groups was significantly increased (P<0.01); compared with the low-dose group, the ALP activity in osteoblasts of the high-dose group was also significantly increased (P<0.01), comparable to that of the calcitriol group. These results indicate that the aqueous extract of *Melastoma can increase AGEs-induced ALP activity in osteoblasts.

[0080] 3. Western blot analysis of the effect of *Melastoma candida* on the expression of AGEs-induced osteoblast-related proteins.

[0081] Cells were cultured using the same method as described in "2. ALP Activity Detection" above. Osteoblast proteins were then extracted using a lysis buffer containing a mixture of protease and phosphatase inhibitors, and quantified using a BCA protein assay kit. Proteins were separated by electrophoresis and transferred to a PVDF membrane. After blocking for 2 hours, the membrane was incubated overnight at 4°C with primary antibodies against OCN (1:2000), RUNX2 (1:2000), RAGE (1:2000), TNFR1 (1:2000), and GAPDH (1:2000). After washing, goat anti-rabbit (1:5000) was added and incubated at room temperature for 1 hour. Color development was performed using a ready-to-use ultrasensitive ECL chemiluminescence substrate, and the band grayscale was analyzed using ImageJ software (with GAPDH as an internal control).

[0082] (1) Bone formation-related proteins

[0083] Depend on Figure 3 As can be seen, compared with the control group, the expression levels of bone formation-related proteins OCN (osteocalcin) and RUNX2 (key osteogenic factor) in the model group were significantly decreased (P<0.01); while compared with the model group, the expression levels of OCN and RUNX2 in the low-dose group, high-dose group, and calcitriol group were significantly increased (P<0.01); compared with the low-dose group, the expression levels of OCN and RUNX2 in the high-dose group were significantly increased (P<0.01), comparable to the calcitriol group. These results indicate that the aqueous extract of *Melastoma can promote the expression of bone formation-related proteins in AGEs-induced osteoblasts.

[0084] (2) AGEs receptor RAGE protein

[0085] like Figure 4As shown, compared with the control group, the RAGE expression level in the model group was significantly increased (P<0.01); while compared with the model group, the RAGE expression level in the low-dose and high-dose *Melastoma candida* groups was significantly decreased (P<0.01); compared with the low-dose group, the RAGE expression level in the high-dose group was also significantly decreased (P<0.01). These results indicate that *Melastoma candida* may exert a protective effect against AGEs-induced osteoblast damage through the RAGE pathway.

[0086] 4. Effects of *Melastoma candida* on the levels of SOD, MDA, CAT, and GPx in AGEs-induced osteoblasts.

[0087] 1×10 5 Cell suspension was seeded at / mL into 6-well cell culture plates. After 24 hours, the cell culture supernatant was discarded. Then, the appropriate drug-containing culture medium was added, and the cell levels of SOD, MDA, CAT, and GPx were measured according to the kit instructions. The results are as follows: Figure 5 As shown.

[0088] Depend on Figure 5 As can be seen, compared with the normal group, the levels of SOD, CAT, and GPx in osteoblasts of the model group were significantly decreased (P<0.01), while the level of MDA was significantly increased (P<0.01). This is because the AGEs receptor RAGE was activated, leading to oxidative stress. Compared with the model group, the levels of SOD, CAT, and GPx in osteoblasts of the low and high doses of *Melastoma candidum* were significantly increased (P<0.01), while the level of MDA was significantly decreased (P<0.01). This indicates that RAGE activation causes oxidative stress damage to osteoblasts, while the aqueous extract of *Melastoma candidum* can increase the levels of SOD, CAT, and GPx in cells, decrease the level of MDA, and inhibit oxidative stress.

[0089] 5. Effects of Melilotus spp. on the levels of AGEs-induced osteoblast inflammatory factors

[0090] The level of the inflammatory factor TNF-α in cells was detected using semi-quantitative PCR. The detection method included:

[0091] Cells were cultured using the same method as described in "2. ALP Activity Detection" above. RNA was then extracted using the TRIzol method, and the RNA was reverse transcribed into cDNA using the FastKing cDNA First-Strand Synthesis Kit (Genomic De-transfer). The cDNA concentrations were calculated and diluted to unify the concentrations across all groups. The PCR was performed using TaKaRa Ex... (Mg2+free Buffer) reagents and primers (see Table 1) were used for PCR reactions at optimized concentrations.

[0092] Table 1 Primer sequences

[0093]

[0094] The amplification products were analyzed by 2% agarose gel electrophoresis and examined using a gel imaging system. GAPDH was used as an internal control gene. The results are shown below. Figure 6 .

[0095] Depend on Figure 6 As can be seen, compared with the normal group, the expression level of TNF-α mRNA in osteoblasts of the model group was significantly increased (P<0.01); while compared with the model group, the mRNA level of TNF-α in osteoblasts of low and high doses of Melilotus arvense was significantly decreased (P<0.01). This indicates that RAGE activation of AGEs receptors leads to an increase in the level of the inflammatory factor TNF-α in osteoblasts, while the aqueous extract of Melilotus arvense can reduce the TNF-α level in osteoblasts.

[0096] Example 2: Protective effect of *Rhizoma Meliloti* on H2O2 osteoblast model

[0097] 1. Effect of Melilotus spp. on AGEs-induced osteoblast survival

[0098] The experimental method is the same as in Example 1, and the experimental results are shown in [the table below]. Figure 7 .

[0099] Depend on Figure 7 It is evident that 0.01-10 mg / mL of *Melastoma candida* promoted H2O2-induced osteoblast proliferation (P<0.01), with the proliferation-promoting effect being stronger at concentrations of 0.01 mg / mL and 0.1 mg / mL. Based on these results, 0.01 mg / mL and 0.1 mg / mL were selected as the low and high doses of *Melastoma candida* in this embodiment.

[0100] 2. Effects of Melilotus spp. on AGEs-induced ALP activity in osteoblasts

[0101] The experimental method is the same as in Example 1, and the experimental results are shown in [the table below]. Figure 8 .

[0102] Depend on Figure 8 As can be seen, compared with the normal group, the ALP activity in osteoblasts of the model group was significantly reduced (P<0.01); while compared with the model group, the ALP activity in osteoblasts of the low-dose, high-dose, and calcitriol groups was significantly increased (P<0.01); and compared with the low-dose group, the ALP activity in osteoblasts of the high-dose group was also significantly increased (P<0.01). These results indicate that *Melastoma can significantly increase H2O2-induced ALP activity in osteoblasts.

[0103] 3. Western blot analysis of the effect of *Melastoma candida* on the expression of bone formation-related proteins in AGEs-induced osteoblasts.

[0104] The experimental method is the same as in Example 1, and the experimental results are shown in [the table below]. Figure 9 .

[0105] Depend on Figure 9 As can be seen, compared with the control group, the expression levels of OCN and RUNX2 in the model group were significantly decreased (P<0.01); while compared with the model group, the expression levels of OCN and RUNX2 in the low-dose group, high-dose group, and calcitriol group were significantly increased (P<0.01); and compared with the low-dose group, the expression levels of OCN and RUNX2 in the high-dose group were significantly increased (P<0.01). These results indicate that *Melastoma can protect against H2O2-induced osteoblast injury and can increase the expression levels of bone formation-related proteins OCN and RUNX2.

[0106] 4. Effects of Melilotus spp. on SOD levels in AGEs-induced osteoblasts

[0107] The experimental method is the same as in Example 1, and the experimental results are shown in [the table below]. Figure 10 .

[0108] Depend on Figure 10 As can be seen, compared with the control group, the SOD activity in the model group cells was decreased (P<0.01); while compared with the model group, the SOD activity in the low-dose, high-dose, and calcitriol groups of *Melastoma candida* was increased (P<0.01); and compared with the low-dose group, the SOD activity in the high-dose group was increased (P<0.01). These results indicate that *Melastoma candida* can inhibit H2O2-induced oxidative stress damage in osteoblasts by increasing cellular SOD activity.

[0109] Example 3: Protective effect of *Melastoma candida* on a TNF-α osteoblast model.

[0110] 1. Effect of Melilotus spp. on TNF-α-induced osteoblast survival rate

[0111] The experimental method is the same as in Example 1, and the experimental results are shown in [the table below]. Figure 11 .

[0112] Depend on Figure 11 It can be seen that 0.01-10 mg / mL of Melilotus spp. promotes TNF-α-induced osteoblast proliferation (P<0.01). Based on the comprehensive consideration of drug concentration and efficacy, 0.01 mg / mL and 0.1 mg / mL were selected as the low and high dosages of Melilotus spp. in this embodiment.

[0113] 2. Effects of Melilotus spp. on TNF-α-induced ALP activity in osteoblasts

[0114] The experimental method is the same as in Example 1, and the experimental results are shown in [the table below]. Figure 12 .

[0115] Depend on Figure 12As can be seen, compared with the normal group, the ALP activity in osteoblasts of the model group was significantly reduced (P<0.01); while compared with the model group, the ALP activity in osteoblasts of the low-dose, high-dose, and calcitriol groups was significantly increased (P<0.01); and compared with the low-dose group, the ALP activity in osteoblasts of the high-dose group was also significantly increased (P<0.01). These results indicate that *Melastoma can significantly increase TNF-α-induced ALP activity in osteoblasts.

[0116] 3. Western blot analysis of the effect of *Melastoma candida* on the expression of related proteins in AGEs-induced osteoblasts.

[0117] (1) Bone formation-related proteins

[0118] The experimental method is the same as in Example 1, and the experimental results are shown in [the table below]. Figure 13 .

[0119] Depend on Figure 13 As can be seen, compared with the control group, the expression levels of OCN and RUNX2 in the model group were significantly decreased (P<0.01); while compared with the model group, the expression levels of OCN and RUNX2 in the low-dose group, high-dose group, and calcitriol group were significantly increased (P<0.01); and compared with the low-dose group, the expression levels of OCN and RUNX2 in the high-dose group were significantly increased (P<0.01). These results indicate that *Melastoma can protect against TNF-α-induced osteoblast injury and can increase the expression levels of bone formation-related proteins OCN and RUNX2.

[0120] (2) TNFR1 protein

[0121] The experimental method is the same as in Example 1, and the experimental results are shown in [the table below]. Figure 14 .

[0122] Depend on Figure 14 As can be seen, compared with the control group, the expression level of TNFR1 in the model group was increased (P<0.01); while compared with the model group, the expression level of TNFR1 in the low-dose group of *Melastoma candida* was decreased (P<0.05), and the expression level of TNFR1 in the high-dose group of *Melastoma candida* was also decreased (P<0.01); compared with the low-dose group of *Melastoma candida*, the expression level of TNFR1 in the high-dose group of *Melastoma candida* was also decreased (P<0.01). These results indicate that *Melastoma candida* exerts a protective effect against TNF-α-induced osteoblast injury by downregulating the expression of the TNF-α receptor TNFR1.

[0123] Example 4: Cell membrane chromatography screening of active substances in *Melastoma candida* for preventing osteoporosis.

[0124] The screening of active pharmaceutical ingredients is carried out according to the following procedure:

[0125] 1. Preparation of reference standard and test solution

[0126] Accurately weigh appropriate amounts of the following reference standards: gallic acid, protocatechuic acid, protocatechuic aldehyde, succinic acid, luteolin, neochlorogenic acid, vitexin glucoside, apigenin-7-O-β-D-pyranoside, isovitexin, apigenin, kaempferol, epicatechin gallate, rutin, chlorogenic acid, vitexin, and quercetin. Prepare a 1.0 mg / mL stock solution with methanol for later use.

[0127] After crushing the *Melastoma dodecandrum*, the extract was refluxed twice with water as the extraction solution. The extracts were filtered, the filtrates were combined, concentrated, and dried. An appropriate amount of the freeze-dried powder was dissolved in 50% methanol, filtered through a 0.22 μm filter membrane, and used for chromatographic analysis.

[0128] 2. Chromatographic analysis

[0129] The reference standard and the test sample were analyzed using the following chromatographic conditions:

[0130] use A 5600 quadrupole time-of-flight mass spectrometer (Q-TOF-MS, AB SCIEX Inc.) was used, with a ZORBAX SB-C 18 column (4.6 × 100 mm, 1.8 μm). The column temperature was set to 40 °C, and the flow rate was 0.3 mL / min. The mobile phase consisted of water (A) and acetonitrile (B), and the gradient elution program was as follows: 0–1 min 10% A, 1–3 min 10–35% A, 3–5 min 35–70% A, 5–10 min 70–95% A, 10–11 min 95%, 11–11.01 min 95–10% A, and 11–16 min 10% A.

[0131] Chromatographic analysis was performed in positive and negative ion modes. The ion spray voltage was 4500 kV, the ion source temperature was 550 °C, the extinction potential was 100 V, the collision energies for positive and negative ion modes were 10 eV and -30 eV, respectively, the nebulizer gas and auxiliary gas were 50 psi, and the scan quality was set in the range of 50-1500 m / z.

[0132] The analysis results are shown in Table 2 and Figure 15 .

[0133] Table 2. Results of chemical composition analysis of *Rhizophora stylosa*

[0134]

[0135]

[0136]

[0137] From Table 2 and Figure 15As can be seen, 46 components were detected in the aqueous extract of *Rhizoma Melastoma* in this embodiment. Sixteen active ingredients were identified through comparison with standards.

[0138] 3. Preparation of osteoblast membrane chromatographic stationary phase

[0139] Primary rat osteoblasts were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum and antibiotics) at 37°C, 5% CO2, and saturated humidity. The medium was changed every 2 days, and cells in the logarithmic growth phase were used for experiments.

[0140] Cells were treated with 400 μM H2O2, 5 ng / mL TNF-α, and 5 mg / mL AGEs for 24 h, respectively. The cultured osteoblasts were washed three times with PBS, centrifuged at 1000 rpm for 10 min at low temperature, and PBS was added to form a cell suspension. The cells were then lysed using a cell disruptor. The mixture was centrifuged at 1000 rpm for 10 min at low temperature, the precipitate was discarded, and the supernatant was centrifuged at 12000 rpm for 20 min at low temperature. The precipitate was mixed with PBS and vortexed for 5 min to form a cell membrane suspension. 0.05 g silica gel was added, and the mixture was incubated overnight at 4 °C. After washing three times with PBS, the cells were centrifuged at low temperature to obtain a cell membrane chromatography (CMC) stationary phase. A cell membrane chromatography column was prepared by wet packing.

[0141] 4. Cell membrane chromatography

[0142] The cell membrane chromatography columns of the three models were connected to a high performance liquid chromatograph. The Melilotus spp. test solution was injected into the cell membrane chromatography columns respectively, and cell membrane chromatography was performed under the following conditions: the mobile phase was ultrapure water, the flow rate was 0.1 ml / min, the injection volume was 50 μL, the detection wavelength was 254 nm, and the column temperature was 37 °C. The retained components of the cell membrane chromatography were collected for later use.

[0143] 5. Mass spectrometry analysis

[0144] The following mass spectrometry detection conditions were used to analyze the chromatographically retained components of the cell membranes in each cell model:

[0145] DiKMA Endeavorsil C18 column (2.1 × 150 mm, 1.8 μm), mobile phase acetonitrile (B)-0.1 formic acid water (A), gradient elution: 0.3 mL / min, 0–1 min, 90–90% A; 1–3 min, 90–85% A; 3–6 min, 85–80% A; 6–10 min, 80–75% A; 10–12 min, 75–50% A; 12–13 min, 50–10% A; 13–13.1 min, 10–90% A; 13.1–15 min, 90% A. Flow rate: 0.3 mL / min, column temperature: 30 °C, injection volume: 3 μL.

[0146] Mass spectrometry analysis was performed in negative ion mode, with an ion spray voltage of 4500 kV, an ion source temperature of 550 °C, an extinction potential of 100 V, a collision energy of -30 eV in negative ion mode, and 50 psi for both nebulizer gas and auxiliary gas. The scanning mass was set in the range of 50-1000 m / z.

[0147] The chromatographic screening results of H2O2, TNF-α, and AGEs model cell membranes are shown in the following figures. Figure 16 , 17 18, where R0 is the HPLC chromatogram of the non-retained fraction and R1 is the HPLC chromatogram of the retained fraction. The analytical results of the retained components in the H2O2 model cell membrane chromatogram are shown in Table 3 and 18. Figure 19 The analytical results of the components retained by chromatographic analysis of the TNF-α model cell membrane are shown in Table 4 and Figure 20 The analytical results of the components retained by chromatographic analysis of the AGEs model cell membrane are shown in Table 5 and Figure 21 .

[0148] Table 3. Analytical results of components retained by chromatographic analysis of H2O2 model cell membranes.

[0149]

[0150]

[0151] Table 4. Analytical results of components retained by chromatographic analysis of TNF-α model cell membranes.

[0152]

[0153]

[0154] Table 5. Analysis results of components retained by chromatographic analysis of AGEs model cell membranes.

[0155]

[0156]

[0157] Comprehensive analysis revealed that 15 components of *Rhizophora stylosa* were retained in the cell membrane chromatogram of osteoblasts induced by H2O2, 32 components were retained in the cell membrane chromatogram of osteoblasts induced by TNF-α, and 22 components were retained in the cell membrane chromatogram of osteoblasts induced by AGEs. Eleven components were shared by all three models: gallic acid, 3-methoxyellagic acid, protocatechuic acid, scopolamine, epicatechin-(8,7-e)-4β-(4-hydroxyphenyl)-3,4-dihydroxy-2(3H)-pyranone, [6-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-3-yl]oxy-3,4,5-trihydroxyoxan-2-yl]methyl-3,4,5-trihydroxybenzoate, rutin, luteolin, quercetin, sennain, and vitexin (isovitilloside).

[0158] Example 5: Validation of the efficacy of the screening results from the cell membrane chromatography platform

[0159] 1. Molecular docking research

[0160] Molecular docking studies were performed using Sybyl-X 2.0 software. The 3D structure of isovitexin was downloaded from the Pubchem database (https: / / www.ncbi.nlm.nih.gov / pccompound / ), and its energy was optimized using ChembioOoffice software. Simultaneously, the 3D structures of RAGE (PDB: 3O3U) and TNFR1 (PDB: 7KP9) proteins were downloaded from the PDB database (https: / / www.rcsb.org / ), and molecular docking was performed using Sybyl-X 2.0 software. The interaction between the receptor and ligand was analyzed, and visualization was performed using Discovery Studio Visualizer software. The results are shown below. Figure 22 and Figure 23 As shown.

[0161] Depend on Figure 22 and Figure 23 As can be seen, the docking scores of isovitexin with RAGE and TNFR1 were 5.5471 and 8.9338, respectively. The docking scores were greater than 5, indicating that isovitexin has good affinity with both RAGE and TNFR1.

[0162] 2. Protective effect of isovitexin against AGEs-induced osteoblast damage

[0163] (1) Effect of isovitexin on AGEs-induced osteoblast survival

[0164] The experimental method was the same as in Example 1, and the results were as follows: Figure 24 As shown.

[0165] Depend on Figure 24 It can be seen that 0.1-10 μM of isovitexin has a significant promoting effect on the proliferation of osteoblasts induced by AGEs (P<0.01). Based on the comprehensive consideration of drug concentration and efficacy, this study selected 0.1 μM and 1 μM as the low and high doses of isovitexin.

[0166] (2) Effect of isovitexin on AGEs-induced ALP activity in osteoblasts

[0167] The experimental method was the same as in Example 1, and the results were as follows: Figure 25 As shown.

[0168] Depend on Figure 25 As can be seen, compared with the normal group, the ALP activity in osteoblasts of the model group was decreased (P<0.01); while compared with the model group, the ALP activity in osteoblasts of the low-dose isovitelline group, high-dose isovitelline group, and calcitriol group was increased (P<0.01); compared with the low-dose isovitelline group, the ALP activity in osteoblasts of the high-dose isovitelline group was increased (P<0.01). These results indicate that isovitelline can increase the ALP activity in osteoblasts induced by AGEs.

[0169] (3) Western blot analysis of the effect of isovitexin on the expression of osteoblast-related proteins induced by AGEs.

[0170] ① Bone formation-related proteins

[0171] The experimental method was the same as in Example 1, and the results were as follows: Figure 26 As shown.

[0172] like Figure 26 As shown, compared with the blank group, the expression levels of OCN and RUNX2 in the model group were decreased (P<0.01); compared with the model group, the expression levels of OCN and RUNX2 in the low-dose isovitelline group, high-dose isovitelline group, and calcitriol group were increased (P<0.01); compared with the low-dose isovitelline group, the expression levels of OCN and RUNX2 in the high-dose isovitelline group were increased (P<0.01). These results indicate that isovitelline has a protective effect against AGEs-induced osteoblast damage.

[0173] ②AGE receptor RAGE protein

[0174] The experimental method was the same as in Example 1, and the results were as follows: Figure 27 As shown.

[0175] like Figure 27As shown, compared with the control group, the expression level of RAGE in the model group was increased (P<0.01); compared with the model group, the expression level of RAGE in the low-dose and high-dose isovitelline groups was decreased (P<0.01); compared with the low-dose isovitelline group, the expression level of RAGE in the high-dose isovitelline group was decreased (P<0.01). These results indicate that isovitelline has a protective effect against AGEs-induced osteoblast damage by downregulating the expression of the AGEs receptor RAGE.

[0176] (4) Effects of isovitexin on the levels of SOD, MDA, CAT, and GPx in AGEs-induced osteoblasts

[0177] The experimental method was the same as in Example 1, and the results were as follows: Figure 28 As shown.

[0178] Depend on Figure 28 As can be seen, compared with the normal group, the model group showed decreased levels of SOD, CAT, and GPx in osteocytes (P<0.01) and increased levels of MDA (P<0.01); compared with the model group, the low- and high-dose isovitelline groups showed increased levels of SOD, CAT, and GPx in osteocytes (P<0.01) and decreased levels of MDA (P<0.01). These results indicate that isovitelline can increase intracellular levels of SOD, CAT, and GPx, decrease MDA levels, and inhibit oxidative stress.

[0179] (5) Effects of isovitexin on the levels of inflammatory factors in osteoblasts induced by AGEs

[0180] The experimental method was the same as in Example 1, and the results were as follows: Figure 29 As shown.

[0181] Depend on Figure 29 As can be seen, compared with the normal group, the expression level of TNF-α mRNA in osteoblasts of the model group was increased (P<0.01); compared with the model group, the mRNA level of TNF-α in osteoblasts of the low and high doses of isovitelline was decreased (P<0.01). These results indicate that RAGE activation of AGEs receptors leads to an increase in the level of the inflammatory factor TNF-α in osteoblasts, and isovitelline can reduce the TNF-α level in osteoblasts.

[0182] 3. Protective effect of isovitexin against H2O2-induced osteoblast damage

[0183] (1) Effect of isovitexin on H2O2-induced osteoblast survival rate

[0184] The experimental method was the same as in Example 1, and the results were as follows: Figure 30 As shown.

[0185] Depend on Figure 30As can be seen, compared with the blank group, the cell survival rate of the model group was decreased (P<0.01); compared with the model group, the cell survival rate of the 0.1-10 μM isovitexin administration group was increased (P<0.01), and 0.1-10 μM isovitexin promoted H2O2-induced osteoblast proliferation. Based on a comprehensive consideration of drug concentration and efficacy, 0.1 μM and 1 μM were selected as the low and high dosages of isovitexin in this embodiment.

[0186] (2) Effect of isovitexin on H2O2-induced ALP activity in osteoblasts

[0187] The experimental method was the same as in Example 1, and the results were as follows: Figure 31 As shown.

[0188] Depend on Figure 31 As can be seen, compared with the normal group, the ALP activity in osteoblasts of the model group was decreased (P<0.01); compared with the model group, the ALP activity in osteoblasts of the low-dose isovitelline group, high-dose isovitelline group, and calcitriol group was increased (P<0.01); compared with the low-dose isovitelline group, the ALP activity in osteoblasts of the high-dose isovitelline group was increased (P<0.01). These results indicate that isovitelline can increase H2O2-induced ALP activity in osteoblasts.

[0189] (3) Western blot analysis of the effect of isovitexin on the expression of bone formation-related proteins in H2O2-induced osteoblasts.

[0190] The experimental method was the same as in Example 1, and the results were as follows: Figure 32 As shown.

[0191] like Figure 32 As shown, compared with the blank group, the expression levels of OCN and RUNX2 in the model group were decreased (P<0.01); compared with the model group, the expression levels of OCN and RUNX2 in the low-dose isovitelline group, high-dose isovitelline group, and calcitriol group were increased (P<0.01); compared with the low-dose isovitelline group, the expression levels of OCN and RUNX2 in the high-dose isovitelline group were increased (P<0.01). These results indicate that isovitelline has a protective effect against H2O2-induced osteoblast injury.

[0192] (4) Effect of isovitexin on SOD levels in H2O2-induced osteoblasts

[0193] The experimental method was the same as in Example 1, and the results were as follows: Figure 33 As shown.

[0194] Depend on Figure 33As can be seen, compared with the control group, SOD activity in the model group cells was decreased (P<0.01); compared with the model group, SOD activity in the low-dose isovitelline group, high-dose isovitelline group, and calcitriol group cells was increased (P<0.01); compared with the low-dose isovitelline group, SOD activity in the high-dose isovitelline group cells was increased (P<0.01). These results indicate that isovitelline can inhibit H2O2-induced oxidative stress damage in osteoblasts by enhancing SOD activity.

[0195] 4. Protective effect of isovitexin against TNF-α-induced osteoblast damage

[0196] (1) Effect of isovitexin on TNF-α-induced osteoblast survival

[0197] The experimental method was the same as in Example 1, and the results were as follows: Figure 34 As shown.

[0198] Depend on Figure 34 It can be seen that 0.1-10 μM of isovitexin promotes TNF-α-induced osteoblast proliferation (P<0.01). Based on the comprehensive consideration of drug concentration and efficacy, this study selected 0.1 μM and 1 μM as the low and high doses of isovitexin.

[0199] (2) Effect of isovitexin on TNF-α-induced ALP activity in osteoblasts

[0200] The experimental method was the same as in Example 1, and the results were as follows: Figure 35 As shown.

[0201] Depend on Figure 35 As can be seen, compared with the normal group, the model group showed decreased intracellular ALP activity in osteoblasts (P<0.01); compared with the model group, the low-dose isovitelline group, the high-dose isovitelline group, and the calcitriol group showed increased intracellular ALP activity in osteoblasts (P<0.01); compared with the low-dose isovitelline group, the high-dose isovitelline group showed increased intracellular ALP activity in osteoblasts (P<0.01). These results indicate that isovitelline can increase TNF-α-induced intracellular ALP activity in osteoblasts.

[0202] (3) Western blot analysis of the effect of isovitexin on the expression of related proteins in TNF-α-induced osteoblasts.

[0203] ① Bone formation-related proteins

[0204] The experimental method was the same as in Example 1, and the results were as follows: Figure 36 As shown.

[0205] like Figure 36As shown, compared with the model group, the expression levels of OCN and RUNX2 were increased in the low-dose isovitelline group, high-dose isovitelline group, and calcitriol group (P<0.01); compared with the low-dose isovitelline group, the expression levels of OCN and RUNX2 were increased in the high-dose isovitelline group (P<0.01). These results indicate that isovitelline has a protective effect against TNF-α-induced osteoblast injury.

[0206] ②TNF-α receptor TNFR1 protein

[0207] The experimental method was the same as in Example 1, and the results were as follows: Figure 37 As shown.

[0208] like Figure 37 As shown, compared with the control group, the expression level of TNFR1 in the model group was increased (P<0.01); compared with the model group, the expression level of TNFR1 in the low-dose and high-dose isovitelline groups was decreased (P<0.01); compared with the low-dose isovitelline group, the expression level of TNFR1 in the high-dose isovitelline group was decreased (P<0.01). These results indicate that isovitelline has a protective effect against TNF-α-induced osteoblast injury by downregulating the expression of the TNF-α receptor TNFR1.

Claims

1. Application of water extract of Melilotus arvense in the preparation of drugs for the prevention and treatment of osteoporosis.

2. The application as described in claim 1, characterized in that, The water extract of *Melastoma dodecandrum* is obtained by the following method: crushing *Melastoma dodecandrum* and extracting it with water under reflux at least twice to obtain an extract; filtering each extract and combining the filtrates; concentrating and drying the filtrates to obtain the water extract of *Melastoma dodecandrum*.

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