CN104971086B

CN104971086B - Use of Rabbit tail grass for promoting osteogenesis or providing neuroprotection

Info

Publication number: CN104971086B
Application number: CN201410140673.7A
Authority: CN
Inventors: 李美贤
Original assignee: Taipei Medical University TMU
Current assignee: Taipei Medical University TMU
Priority date: 2014-04-09
Filing date: 2014-04-09
Publication date: 2020-12-08
Anticipated expiration: 2034-04-09
Also published as: CN104971086A

Abstract

The present invention provides a method of promoting osteogenesis, increasing bone mass, or promoting a rate of strong bone growth comprising administering to a subject an effective amount of laggera or a fraction or extract thereof. The invention also provides a method of treating and/or preventing osteoporosis and a method of providing neuroprotection comprising administering to a subject an effective amount of laggera or a fraction or extract thereof.

Description

Use of Rabbit tail grass for promoting osteogenesis or providing neuroprotection

Technical Field

The present invention relates to a method of using a plant product to promote osteogenesis or provide neuroprotection. In particular, the present invention uses laggera (Uraria) to promote osteogenesis or provide neuroprotection.

Background

Bone is the structural material of the body skeleton and is used to maintain the necessary bone mass and structure. Bone contains calcium ions (Ca2+) and plays an important role in maintaining the amount of calcium in the blood. For this reason, bone growth is a metabolic balance between the activities of osteoblasts and osteoclasts in the bone remodeling cycle. When the balance between bone resorption and bone formation is disrupted, the amount of bone tissue replaced by osteoblasts does not match the amount of bone tissue resorbed by osteoclasts, thus leading to osteoporosis, a common disease that causes loss of bone density or bone mass. Osteoblasts are bone-forming cells, which are derived from Mesenchymal Stem Cells (MSCs). Osteoblasts play a very important role in creating and maintaining skeletal architecture. Bone formation involves a complex series of events, including proliferation and differentiation of osteoblasts, ultimately leading to mineralization of the extracellular matrix. At various stages of osteoblast development, specific genes are expressed or re-expressed sequentially, for example, histone 4(histone 4) is a proliferation marker, alkaline phosphatase (ALP) is a differentiation marker and osteocalcin (osteopalcin) is a mineralization marker. Bone formation is a major clinical concern in human health in infants and children with normal bone growth, in menopausal women to prevent loss of bone mass, and in fracture patient healing and remodeling. Nutritional and pharmaceutical formulations are required in order to prevent bone loss and to increase bone formation. Therefore, in vitro models of osteoblast primary cell (osteoplast primary cell) culture have been widely used to find new drugs that induce osteoblast differentiation, matrix mineralization and new bone formation. There are many parameters that affect the expression of osteoblast phenotypes in cell culture.

Bone diseases, such as osteoporosis, are common in middle-aged or elderly women. Examples of therapeutic agents for osteoporosis include, but are not limited to, bisphosphonate products (alendronate, etidronate), hormone products (raloxifene), vitamin D products, calcitonin products, and calcium products. However, bisphosphonate products have low absorption and problematic dosing problems and may induce esophagitis. Hormone products require lifelong administration and have potential side effects such as breast cancer, uterine cancer, gallstones and thrombosis. Vitamin D products are expensive and not very effective. Calcitonin products also have problems of high cost and difficulty of administration. Calcium products have fewer side effects, but are limited to prophylaxis rather than treatment. Thus, there remains a need for new osteoporosis therapies.

Phytotherapy has been considered as a viable alternative to various diseases. U.S. patent No. 5478549 relates to a method for orally inducing and promoting calcium absorption into bone tissue of a mammal comprising administering an effective amount of a flavonol aglycone glycoside in combination with calcium nutrient. U.S. patent No. 6340703 provides a method for treating and preventing osteoporosis comprising administering to a subject in need of such treatment an effective amount of the isoflavone formononetin, optionally with one or more pharmaceutically acceptable adjuvants, carriers and/or excipients. U.S. patent No. 7122214 uses Rhizoma Drynariae Extract (RDE) as a therapeutic agent for osteoporosis. U.S. patent No. 7350914 provides a pharmaceutical or medicinal preparation comprising a mixture of plants or a mixture of active ingredients extracted from soybean (Glycine max), Coleus forskohlii, tea tree (Camellia sinensis), garlic (Allium sativum), Withania somnifera (Withania somnifera), oldenlandia diffusa (Boerhavia diffusa), and turmeric (Curcuma longa). U.S. patent No. 8153167 provides a composition derived from 6 plant materials: (i) compositions of epimedium (Herba epimedidii), (ii) psoralea (Fructus Psoraleae), (iii) rehmannia glutinosa (Radix Rehmanniae preparata), (iv) eucommia ulmoides (Cortex Eucommiae), (v) cnidium monnieri (Fructus Cnidii), (vi) astragalus membranaceus (Radix Astragali); the composition is used for treating conditions associated with osteoblasts and/or osteoclasts, such as osteoporosis and conditions associated with bone mass or menopause, obesity, glucose intolerance and diabetes. Previous studies of other traditional chinese herbal medicines with possible effects on bone formation include pueraria lobata (Wang, x., Wu, j., Chiba, h., Yamada, k., & Ishimi, Y. (2005), Puerariae radiata bone loss in confined male mold.metabolism, 54, 1536. times. 1541.), drynaria rhizome (drynaria rhizome) (Jeong, j.c., le, j.w., Yoon, c.h., Lee, y.c., Chung, k.h., Kim, m.g., Kim, C.H. (2005), Stimflammatory effects of drynaria rhizogenes, emission powders, cement, 12. 9. times. c., epime, 11. 7. 12. times. 11. p., 11. 7. eight, 11. times. 7. 9. times. 9. times. e.21. times. g., cement, 11. 7. times. g., cement, 11. 7. times. 9. times. e.7. e.g., 11. 9. e.g., cement, 11. 9. e.

Rabbit tail grass (Uraria crinita (L.) Desv.Ex DC. (Fabaceae)) is a traditional edible botanical drug that is widely distributed throughout India, Thailand, Indonesia, southern China and Taiwan of China. It has been reported that pressure ulcers can be effectively inhibited. Laggera species exhibit nitric oxide clearance and in vitro Antioxidant effects (Luo, c., Liu, a.m., Xing, w.q., Shi, g., Cao, y., Pang, j.x., & Qiu, Y.C. (2011). Antioxidant effect of vitamins from Uraria crinita. zhongguo Shiyan Fangjixue Zazhi,17,198- & 201.). Their use in treating chills, swelling, stomachache and ulcers may be due to their anti-inflammatory activity. In taiwan, people boil in water and drink the taste, fragrance, sweetness and quench thirst like ginseng tea; it has also been called "taiwan ginseng". In addition, its root is used in traditional Chinese medicine for dietary supplementation for the treatment of infantile skeletal dysplasia, and sports-related bone torsion, sprains and strains (Machida, k., Sakamoto, s., & Kikuchi, M. (2009).

However, nothing has been reported to show that Leptospermum petasitum is associated with bone diseases.

Disclosure of Invention

The present invention provides a method of promoting osteogenesis comprising administering to a subject an effective amount of laggera or a fraction or extract thereof. The invention also provides the use of a laggera species or a fraction or extract thereof for the manufacture of a medicament for promoting osteogenesis.

The present invention also provides a method of treating and/or preventing bone disorders, the method comprising administering to a subject an effective amount of chia or a portion or extract thereof. The invention also provides the use of chia or a part or extract thereof for the manufacture of a medicament for the treatment and/or prevention of bone diseases.

The invention further provides a method of providing neuroprotection comprising administering to an individual an effective amount of a laggera species part or extract thereof. The invention further provides the use of chia or a part or extract thereof for the manufacture of a medicament for providing neuroprotection.

Drawings

FIG. 1 shows ALP activity in HOb cells of an ethanolic extract of Rabbit tail (Uraria crinite; UC) and a partition fraction (partition fraction). HOb cells were seeded in 96-well plates. After 24 hours, the old medium was discarded, and the cells (100. mu.g/ml) were treated with the test extract in osteoblast differentiation medium for 72 hours. ALP activity was assessed using a p-nitrophenyl phosphate disodium substrate (p-nitrophenyl phosphate sodium substrate). Data are shown as mean ± standard deviation (n ═ 3). P <0.05 and P <0.01 compared to control group (n-3).

FIG. 2 shows the mineralization (mineralization) activity of an ethanolic extract of Leptospermum petersonii (UC) and a partition fraction (partition fraction) in HOb cells. HOb cells were plated in 24-well plates, the old medium was discarded after three days, and the cells (osteoblast differentiation medium containing ascorbic acid (50 μ g/ml) and β -glycerophosphate (10 mmol)) were treated with the test extract (100 μ g/ml) in mineralized medium for 12 days. At the end of the experiment, cultures were fixed with 75% ethanol and mineralized nodule (mineralized nodule) formation was assessed by staining with Alizarin Red-S (Alizarin Red-S). Bound staining was washed out in 10% cetylpyridinium chloride (cetylpyridinium chloride) solution and quantified using a microtiter plate reader. Data are shown as mean ± standard deviation (n ═ 3). P <0.05 and P <0.01 compared to control group (n-3).

FIG. 3 shows a schematic of the chemical structure of the components isolated from Rabbit tail.

FIG. 4 shows the HMBC correlation (H → C) of apigenin 6-C-beta-D-furanosylyl (1 → 2) -alpha-D-xylopyranoside (apigenin 6-C- (beta-D-apiofuranosyl (1 → 2) -alpha-D-xylopyranoside) (3).

FIG. 5 shows cell viability of compounds isolated from the genus Lepidium in HOb cells. HOb cells were seeded in 96-well plates. After 24 hours, the old medium was discarded, and the cells were treated with test compounds (100 μ M) in osteoblast differentiation medium for 72 hours. Cell activity was determined using an MTT assay (MTT assay) evaluation. Data are shown as mean ± standard deviation (n ═ 3). P <0.05 and P <0.01 compared to control group (n-3).

FIG. 6 shows ALP activity in HOb cells of a compound isolated from Leptospermum petersonii. HOb cells were seeded in 96-well plates. After 24 hours, the old medium was discarded, and the cells were treated with test compounds (100 μ M) in osteoblast differentiation medium for 72 hours. ALP activity was assessed using a p-nitrophenyl phosphate disodium substrate (p-nitrophenyl phosphate sodium substrate). Data are shown as mean ± standard deviation (n ═ 3). P <0.05 and P <0.01 compared to control group (n-3).

Figure 7 shows the mineralization (mineralization) activity of compounds isolated from rabbit grass in HOb cells. HOb cells were plated in 24-well plates, the old medium was discarded after three days, and the cells (osteoblast differentiation medium containing ascorbic acid (50 μ g/ml) and β -glycerophosphate (10 mmol)) were treated with the test extract (100 μ g/ml) in mineralized medium for 12 days. At the end of the experiment, cultures were fixed with 75% ethanol and mineralized nodule (mineralized nodule) formation was assessed by staining with Alizarin Red-S (Alizarin Red-S). Bound staining was washed out in 10% cetylpyridinium chloride (cetylpyridinium chloride) solution and quantified using a microtiter plate reader. Data are shown as mean ± standard deviation (n ═ 3). P <0.05 and P <0.01 compared to control group (n-3).

Detailed Description

The present invention surprisingly found that chia or an extract thereof increases osteogenesis and stimulates bone growth and repair, and provides neuroprotective effects.

Unless defined otherwise, 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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For the purposes of the present invention, the following terms are defined as follows.

As used herein, the terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

As used herein, the term "or" in the claims is intended to mean "and/or" unless specifically indicated to be either only, or unless such is the case or mutually exclusive.

As used herein, the terms "promote," "promoting," or "improving" refer to an increase in activity, response, condition, disease, or other biological parameter.

As used herein, the term "subject" includes living organisms, such as humans, monkeys, cows, sheep, horses, pigs, cows, goats, dogs, cats, mice, rats, cultured cells and transgenic species. In a preferred embodiment, the individual is a human.

As used herein, the term "administration" (or administration, administration) includes the route of administration that allows the laggera species of the present invention to perform their intended function.

As used herein, the term "treatment" refers to a method of alleviating the effects of a disease or condition. Treatment may also refer to methods of reducing the cause of the disease or disorder itself. Treatment may also be any reduction from natural levels, and may be, but is not limited to, a disease, disorder, or complete elimination of symptoms of a disease or disorder.

As used herein, the term "preventing" refers to inhibiting or alleviating the symptoms associated with osteoporosis.

As used herein, the term "effective amount" refers to an amount of laggera effective to treat and/or prevent osteoporosis or provide a neuroprotective effect.

As used herein, the term "osteoblast" or "osteogenesis" refers to the proliferation from undifferentiated stem cells and osteoblast cell lines into osteoblasts and bone tissue (e.g., the synthesis and accumulation of new bone matrix). Osteogenesis also refers to the differentiation or transdifferentiation of progenitor cells (progenitors) or precursor cells (precursor cells) into osteocytes (i.e., osteoblasts). The progenitor or precursor cells can be pluripotent stem cells, including, for example, mesenchymal stem cells. The progenitor or precursor cells can be cells that are predetermined to form an osteoblast line (e.g., pre-osteoblast cells) or cells that are not predetermined to form an osteoblast line (e.g., preadipocytes or myoblasts).

As used herein, the term "bone disease" refers to a disease associated with loss of bone tissue, such as osteoporosis.

Method or use for promoting osteogenesis and treating and/or preventing osteoporosis

In one aspect, the invention provides a method of promoting osteogenesis, the method comprising administering to a subject an effective amount of chia or a portion or extract thereof. The invention also provides the use of a laggera species or a fraction or extract thereof for the manufacture of a medicament for promoting osteogenesis.

In another aspect, the invention provides a method of increasing bone mass, the method comprising administering to a subject an effective amount of chia or a portion or extract thereof. The invention also provides the use of chia or a part or extract thereof for the manufacture of a medicament for increasing bone mass.

In another aspect, the invention provides a method of increasing or enhancing the rate of bone growth, the method comprising administering to a subject an effective amount of laggera or a part or extract thereof. The invention also provides the use of a laggera species or a fraction or extract thereof for the manufacture of a medicament for increasing or enhancing the bone growth rate.

In yet another aspect, the present invention provides a method of treating and/or preventing bone diseases, the method comprising administering to a subject an effective amount of laggera or a part or extract thereof. The invention also provides the use of chia or a part or extract thereof for the manufacture of a medicament for the treatment and/or prevention of bone diseases.

In vitro or in vivo, induction of osteogenesis may be detected using any method known in the art; for example, by detecting the expression of osteoblast specific proteins, detecting the expression of bone specific transcription factors, and detecting changes in bone density. Osteoblast-specific proteins include, for example, alkaline phosphatase (ALP), collagen type I, osteocalcin (osteopontin), and osteogenic protein (osteoponin) (Olsen et al, annu, rev, cell. dev. biol.16:191 (2000)). In some embodiments, the expression of alkaline phosphatase is measured as an indicator of osteogenesis. Bone-specific transcription factors include, for example, Cbfa1/Runx2, GSC, D1x1, D1x5, Msx1, Cart1, Hoxa1, Hoxa2, Hoxa3, Hoxb1, rae28, Twist, AP-2, MF1, Pax1, Pax3, Pax9, TBX3, TBX4, TBX5 and Brachyury (Olsen et al,2000, supra).

The methods of the invention are useful for treating bone disorders. Bone diseases including, but not limited to, osteoporosis, arthritis, osteoarthritis, periodontal disease, alveolar bone loss, osteotomy bone loss, childhood idiopathic bone loss, curvature spine and high loss may be treated according to the present invention by increasing bone mass or bone growth. Destructive bone disorders including, but not limited to, osteoporosis, osteoarthritis and osteolytic lesions, such as those caused by neoplastic disease, radiation therapy, or chemotherapy, may be treated according to the present invention.

Bone disease can be caused by conditions characterized by low bone mass, bone defects, or cartilage defects. The term "bone mass" refers to the amount of bone per unit volume. A condition characterized by low bone mass is one in which the level of bone mass is below the normal level for a particular age, as defined in "Association of frame Risk and Its Application to Screening for Postmenopausal Osteoporosis," Report of a World Health Organization Study Group, World Health Organization Technical Series 843(1994) ". A bone defect is an imbalance in the proportion of bone formation to bone resorption, such that, if unmodified, an individual will present less than expected bone, or the individual's bone will be less complete than expected. Bone defects may also result from bone fractures, from surgical intervention, or from dental or periodontal disease. Bone healing includes, but is not limited to, repair of bone defects, as occur, for example, in closed, open, and non-healing fractures. The cartilage defect is damaged cartilage, less cartilage than expected, or less complete cartilage than expected.

Conditions including conditions characterized by low bone mass are, but not limited to, primary and secondary osteoporosis, periodontal disease, alveolar bone loss, osteotomy bone loss, childhood idiopathic bone loss. Conditions characterized by low bone mass also include, but are not limited to, long-term complications of osteoporosis such as spinal curvature, high-grade loss, and prosthetic surgery.

Osteoporosis or cancellous bone is a disease characterized by a net loss of bone mass per unit volume. The consequence of this loss of bone mass and resulting fractures is the destruction of the skeleton to provide adequate structural support for the body, low bone mass and structural deterioration of bone tissue leading to bone fragility and increased fracture of the hip, spine and wrist. There were no symptoms of bone loss. Osteoporosis includes "secondary osteoporosis" such as glucocorticoid-induced osteoporosis, osteoporosis due to hyperthyroidism, osteoporosis due to immobilization, heparin-induced osteoporosis or osteoporosis due to immunosuppression. In patients with osteoporosis, the bone can become so weak that sudden tension can cause fractures or collapse of the spine. Most current osteoporosis treatments stop the continuous loss of bone mass, but fail to enhance bone formation and thus bone quality remains poor, but does not get worse.

In one embodiment, any of the above methods of the present invention includes the additional step of administering an osteogenesis promoter. Any of the agents of the above uses of the present invention further comprises an osteogenesis promoter. Preferably, the additional active agent is selected from bisphosphonates. Preferred bisphosphonates include, but are not limited to, tiludronic acid (tiludronic acid), alendronic acid (alendronic acid), zoledronic acid (zoledronic acid), ibandronic acid (ibandronic acid), risedronic acid (risedronic acid), etidronic acid (etidronic acid), clodronic acid (clodronic acid), and pamidronic acid (pamidronic acid) and pharmaceutically acceptable salts thereof. Those skilled in the art will appreciate that these compounds are generally represented in their ionic forms, for example, tiludronate (tiludronate), alendronate (alendronate), zoledronate (zoledronate), ibandronate (ibandronate), risedronate (risedronate), etidronate (etidronate), clodronate (clodronate) and pamidronate (pamidronate). Particularly preferred bisphosphonates include alendronate and risedronate.

Methods or uses for providing neuroprotection

In another aspect, the invention provides a method for providing neuroprotection comprising administering to an individual an effective amount of a laggera species part or extract thereof. The invention also provides the use of a laggera species or a fraction or extract thereof for the manufacture of a medicament for providing neuroprotection.

Neuroprotection refers to mechanisms and strategies used to prevent brain/neuron injury or degeneration in the nervous system after acute disease (e.g., stroke or brain or nervous system injury/trauma, hypoxia, spinal cord injury or peripheral nerve injury) or to prevent the consequences of chronic neurodegenerative diseases (e.g., parkinson's disease, alzheimer's disease, multiple sclerosis). The goal of neuroprotection is to reduce neuronal dysfunction/death following central nervous system injury and to attempt to maintain the greatest possible integrity of brain cell interactions, thereby producing undisturbed neural function. By providing neuroprotection, Leptospermum or a part or extract thereof can be used for the treatment of nerve injury (such as stroke, especially ischemic stroke, brain injury, hypoxia, spinal cord injury or peripheral nerve injury), and for the treatment or prevention of neurodegenerative diseases.

Leptospermum or parts or extracts thereof for use in the methods or uses of the invention

Lepidium (Uraria) is a genus of flowering plants in the family Leguminosae, Papilionaceae. It belongs to the subfamily Papilionaceae (Faboideae). In one embodiment, the genus Leptospermum is selected from the group consisting of: rabbit-tail grass (Uraria crinita (L.) Desv.ex DC.), big-leaf rabbit-tail grass (Uraria lagopodioides (L.) Desv.ex DC.), feathered rabbit-tail grass (Uraria piscta (Jacq.) Desv.ex DC.), round-leaf rabbit-tail grass (Uraria negnectaria Prain), Uraria acaulis Schl, Uraria acuminata acrinata rz, Uraria balansae Schl., Uraria barbata Lace, Uraria campula (Benth.) Lantern. G., Uraria basidia cauliflora, Uraria corda Garvee, Uraria viridula, Uraria coriaria, Uraria viridifra, Uraria viridula, Uraria viridaria viridula, Uraria viridaria viridula, Uraria viridaria viridula. Preferably, the laggera species is laggera, laggera or turfgrass. More preferably, the laggera species is laggera. Preferably, the laggera extract is an extract of laggera, ramaria, laggera or ramaria.

Whole plants of the genus laggera or all parts of plants of the genus laggera may be used in the present invention. In one embodiment, roots of plants of the genus Leptospermum are used in the present invention.

In one embodiment, extracts of laggera may be used in the present invention. Preferably, Leptospermum petiolatum, Leptospermum macrocephalum, Leptospermum petiolatum or Leptospermum rotundus is used to obtain the extract. Preferably, the laggera species is laggera. More preferably, Leptospermum petersonii is used to obtain the extract. Preferably, the extract is an organic solvent extract of whole plants of the genus Leptospermum, and more preferably the extract is an organic solvent extract of roots of the genus Leptospermum. Preferably, the extract is a methanol extract, an ethanol extract, a propanol extract, a butanol extract or a hexane extract. More preferably, the extract is an ethanolic extract. According to an embodiment of the present invention, the organic solvent extract may be further partitioned (partition) with hexane, ethyl acetate and butanol to obtain a hexane extract, an ethyl acetate extract and a butanol extract. Accordingly, the present invention provides an extract of laggera, which is produced by the steps of: extracting Leptospermum with ethanol to obtain ethanol extract, and extracting the ethanol extract with ethyl acetate or butanol to obtain ethyl acetate extract or butanol extract. The ethyl acetate extract was further partitioned to give 9 fractions. The 3 rd of these fractions was further separated and purified by Sephadex LH-20 column chromatography (chromatography) and extracted with methanol/water to give 17 fractions. The resulting fractions were further chromatographically separated. Therefore, a new compound, apigenin 6-C-beta-D-furanosylapigenin (1 → 2) -alpha-D-xylopyranoside, can be obtained. Accordingly, the present invention provides a chia extract obtained by extracting the chia to obtain an ethanol extract, and extracting the ethanol extract with ethyl acetate or butanol to obtain an ethyl acetate extract or a butanol extract.

Any extraction technique known in the art can be used to prepare the extracts of the present invention. The resulting extract can be further fractionated by chromatography. A preferred chromatography is liquid chromatography using solvent elution. Preferably, the liquid chromatography is High Performance Liquid Chromatography (HPLC) or reverse phase HPLC.

Novel compounds isolated from laggera or parts or extracts thereof

The present invention provides a novel compound isolated from the genus Lepidium or a part or extract thereof:

apigenin 6-C-beta-D-furanosyl (1 → 2) -alpha-D-xylopyranoside (apigenin 6-C- (beta-D-apiofuranosyl (1 → 2) -alpha-D-xylopyranoside)

Accordingly, the present invention provides a compound of formula (I),

wherein R is₁Are furanylapiosyl-alpha-L-arabinopyranosyl (apiosyl-alpha-L-arabinopyranosyl), furanylapiosyl-alpha 0-D-glucopyranosyl (apiosyl-alpha 1-D-glucopyranosyl), furanylapiosyl-alpha 2-D-galactopyranosyl (apiosyl-alpha 3-D-galactopyranosyl), furanylapiosyl-alpha 4-D-xylopyranosyl (apiosyl-alpha 5-D-xylopyranosyl), furanylapiosyl-alpha 6-D-arabinopyranosyl (apiosyl-alpha 7-D-arabinopyranosyl), furanylapiosyl-alpha 8-D-ribopyranosyl (apiosyl-alpha 9-D-ribopyranosyl), furanylosyl-beta-D-arabinopyranosyl (apiosyl-beta-D-arabinopyranosyl) Furanosyl- β 1-D-ribopyranosyl (apiosyl- β 2-D-ribopyranosyl), furanosyl- β 3-D-xylopyranosyl (apiosyl- β 4-D-xylopyranosyl), furanosyl- β 5-D-allopyranosyl (apiosyl- β -D-allopyranosyl), furanosyl- β -D-mannopyranosyl (apiosyl- β -D-mannopyranosyl), furanosyl- β -D-glucopyranosyl- β -D-gulosyl (apiosyl- β -D-glucopyranosyl), furanosyl- β -D-glucopyranosylbeta-D-idopyranosyl (apofuranosyl-beta-D-idopyranosyl), apiosyl-beta-D-talosyl (apofuranosyl-beta-D-talosyl), apiosyl-beta 0-L-glucopyranosyl (apofuranosyl-beta 1-L-glucopyranosyl), apiosyl-beta 2-L-galactopyranosyl (apofuranosyl-beta 3-L-galactopyranosyl), apiosyl-beta 4-L-xylopyranosyl (apofuranosyl-beta 5-L-xylopyranosyl), apiosyl-beta 6-L-arabinopyranosyl (apofuranosyl-beta 7-L-arabinopyranosyl), apiosyl-beta 8-L-ribopyranosyl (apofuranosyl-beta 9-L-ribopyranosyl), apiosyl-beta 0-L-lyxopyranosyl (apiosyl-beta 1-L-lyxopyranosyl), apiosyl-beta 2-L-pyranosylulose (apiosyl-beta 3-L-ribopyranosyl), apiosyl-beta 4-L-xylopyranosyl (apiosyl-beta 5-L-xylopyranosyl), apiosyl-beta 6-L-allopyranosyl (apiosyl-beta 7-L-allopyranosyl), apiosyl-beta 8-L-arabinopyranosyl (apiosyl-beta 9-L-amylopyranosyl), apiosyl-beta 0-L-mannopyranosyl (apiosyl-beta 1-L-mannosyl), apiosyl-alpha-L-xylopyranosyl (apiosyl-alpha-xylopyranosyl) A furanosyl- α -L-idopyranosyl (apiofuranosyl- α -L-idopyranosyl) or a furanosyl- α -L-talosyl (apiofuranosyl- α -L-talosyl).

Preferably, R1 is a furanosyl- α -L-arabinopyranosyl, a furanosyl- β -D-glucopyranosyl, a furanosyl- β -D-galactopyranosyl, a furanosyl- β -D-xylopyranosyl or a furanosyl- β -D-arabinopyranosyl. More preferably, R1 is furanosyl-alpha-L-arabinopyranosyl.

The compounds with different sugar groups of R1 in formula (I) can be modified from apigenin 6-C- β -D-furanapigenin base (1 → 2) - α -D-xylopyranoside according to methods known in the art (org. biomol. chem.,2010,8, 4451-4462). The synthetic scheme for linking the various sugar groups of R1 to apigenin (4',5, 7-trihydroxyflavone) is shown below:

formulations and methods of administration

The laggera species, fraction thereof or extract thereof may be formulated with pharmaceutically acceptable carriers, excipients, diluents and/or salts into pharmaceutical compositions or medicaments for administration.

By pharmaceutically acceptable carrier, diluent, excipient and/or salt is meant that the carrier, diluent, excipient and/or salt must be compatible with the other ingredients of the formulation, not adversely affect the therapeutic benefits of the laggera part or extract thereof, and not compromise the recipient thereof.

Administration of laggera, portions or extracts thereof, or pharmaceutical compositions thereof, for practicing the present invention can be any method of systemic and/or local delivery of a compound (e.g., at the site of a bone fracture, osteotomy, or orthopedic surgery). These methods include oral routes, parenteral routes, intraduodenal routes, and the like.

In topical applications, the laggera, a part thereof or an extract thereof, or a pharmaceutical composition thereof, is applied to the site of a fracture, osteotomy, or graft, for example, by injection of the laggera, a part thereof or an extract thereof in a suitable solvent (e.g., an oily solvent such as peanut oil) at the site of the fracture or site of bone healing, or in the case of open surgery, by topical application of, for example, a bone wax, decalcified bone powder, polymerized bone cement, bone sealant, polylactic acid, polyglycolic acid, polylactic-polyglycolic acid, etc., a part thereof or an extract thereof in, for example, a suitable carrier (e.g., bone wax, demineralized bone powder, polymerized bone cement, bone sealant, polylactic acid, polyglycolic acid, etc.). Alternatively, topical application may be by applying a solution or dispersion of the laggera, parts thereof or extracts thereof in a solution vehicle to the surface or incorporating them into solid or semi-solid implants conventionally used in bone surgery, such as Dacron mesh (Dacron-mesh), gel foams and kirschner bones (kiel bones), or prostheses (procthses).

For topical administration, the laggera species, parts or extracts thereof, or pharmaceutical compositions thereof, may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the laggera species, fractions thereof or extracts thereof used in the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying waxes, sugars such as lactose and water. Alternatively, the pharmaceutical composition may be formulated as an emulsion or cream containing the genus laggera, parts thereof or extracts thereof suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitol monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Depending on the particular condition, disorder or disease to be treated, additional therapeutic agents may be administered with the laggera species, parts thereof or extracts thereof. These additional therapeutic agents and the composition comprising chia, a portion thereof, or an extract thereof may be administered sequentially in any order as part of a multiple dose regimen (continuous or intermittent). Alternatively, those agents may be part of a single dosage form, mixed together with the laggera species, parts thereof or extracts thereof in a single composition (administered simultaneously).

For oral administration, the pharmaceutical compositions used in the present invention may be in the form of solutions, suspensions, lozenges, pills, capsules, powders, granules, semi-solids, sustained release formulations, elixirs, aerosols, and the like. Lozenges comprise various excipients such as sodium citrate, calcium carbonate and calcium phosphate together with various disintegrants such as starch, preferably potato or tapioca starch and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tableting purposes. Similar solid compositions are also useful as fillers in soft and hard-filled gelatin capsules, preferred materials in this regard also including lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are used for oral administration, the laggera species, fractions thereof or extracts thereof of the present invention may be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying and/or suspending agents, and diluents, such as, for example, water, ethanol, propylene glycol, glycerin, and various like combinations thereof.

The choice of formulation depends on a variety of factors such as the mode of drug administration (e.g., in the case of oral administration, the formulation is preferably in the form of a tablet, pill or capsule) and the bioavailability of the drug substance.

The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intramedullary and intraarticular injection and infusion. Pharmaceutical compositions for parenteral injection may comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions prior to use. The aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this regard, the sterile aqueous medium employed is readily available to those skilled in the art by standard techniques. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

The pharmaceutical compositions used in the present invention may also contain adjuvants such as, but not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Slow infusion administration is particularly useful for intrathecal or epidural routes. Many implantable or modulated rate in vivo mountable pumps for delivering compounds are known in the art. See, for example, U.S. patent No. 4619652.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth (tragacanth), and mixtures thereof.

For transdermal (e.g., topical) administration, dilute sterile aqueous or partially aqueous solutions (typically at a concentration of about 0.1% to 5%) are prepared, similar to the non-enteric solutions described above.

The pharmaceutical compositions for use in the present invention may also be administered by nasal aerosol or inhalation. Such compositions may be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as saline solutions, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the laggera species, parts thereof or extracts thereof of the present invention with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or a suppository wax (solid at room temperature and liquid at body temperature and therefore melting in the rectum or vaginal cavity and releasing the drug).

Other pharmaceutically acceptable carriers include, but are not limited to, non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or formulation aids of any type, including, but not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acids, water, partial glyceride mixtures of salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin.

Solid pharmaceutical excipients include, but are not limited to, starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, and the like. The liquid and semi-solid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols.

Methods of preparing various pharmaceutical compositions containing certain amounts of active ingredients are known or will be apparent to those skilled in the art in light of this disclosure. Other suitable Pharmaceutical excipients and formulations thereof are described in Remington's Pharmaceutical Sciences, edited by E.W. Martin, Mack Publishing Company,19th ed. (1995).

The pharmaceutical composition for use in the present invention contains 1% to 100% (by weight) of the genus Lagotis, its part or its extract, preferably 10% to 100%, 10% to 95% or 20% to 95% (by weight). In any event, the composition or formulation to be administered will determine the amount of chia, portion thereof or extract thereof contained according to the effective amount of treatment of the present invention, the condition, disorder or disease of the subject being treated.

One of ordinary skill in the art will appreciate that a pharmaceutically effective amount of laggera, parts thereof or extracts thereof can be empirically determined and used in pure form (when such forms exist), in the form of a pharmaceutically acceptable salt, ester or prodrug. The agent may be administered to a patient as a pharmaceutical composition in combination with one or more pharmaceutically acceptable excipients. It will be understood that the total daily usage of the agents or compositions of the present invention when administered to, for example, a human patient will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose for any particular patient will depend upon a variety of factors: the type and extent of cellular response to be achieved, the activity of the particular agent or composition used, the age, body weight, general health, sex and diet of the patient, the time of administration, the route of administration and rate of excretion of the agent, the duration of the treatment, the drugs of the particular agents used in combination or concomitantly, and similar factors known in the medical arts.

Administration can also be arranged in a patient-specific manner to provide a predetermined concentration of the drug in the blood, as determined by accepted and routine techniques.

Alternatively, the laggera species, fractions or extracts thereof may be used as a health food or dietary supplement with food grade carriers, excipients, diluents and/or salts. The excipients, diluents and/or salts of the above carriers may be used in the health food or dietary supplement of the present invention. The health food or dietary supplement may be in various forms including, but not limited to, tablets, capsules, caplets, powders, beverages, including milkshakes, solid foods, including snack bars, and the like.

Other suitable modifications and adaptations will be apparent to persons skilled in the relevant arts, and the methods and applications described herein may be made without departing from the spirit and scope of the invention or any embodiment thereof. The following examples are provided to illustrate but not to limit the invention.

Examples

Materials and methods

Plant material

The root of Rabbit-tailed grass (Uraria crinite) was collected from the Ministry of rural Cooperation, Taiwan, Japan, and identified by Mr. Guo Shong, department of pharmacy and Chinese medicine resources, university of medicine, Taizhong, Taiwan, China. One specimen (M-343) was stored in Taipei institute of medicine, university student's pharmaceutical research, Taiwan, China.

Reagents and chemicals

DMSO (dimethyl sulfoxide), thiazole blue tetrazolium bromide (MTT), Triton X-100(Triton X-100), p-nitrophenyl disodium phosphate matrix, Alizarin Red-S (Alizarin Red-S), cetylpyridinium chloride monohydrate (cetylpyri)dinium chloride monohydrate), ascorbic acid, beta-glycerophosphate disodium salt hydrate were purchased from Sigma-Aldrich (Sigma, st. louis, Missouri, USA). Phosphate Buffered Saline (PBS) and trypsin were purchased from Gibco (Gibco Canada inc., Burlington, ont., Canada). The gyrogram of osteoblasts was measured by JASCO P-1020digital polarimeter (JASCO P-1020digital polarimeter). The UV spectrum was measured using a Hitachi U-2800U-2800 UV/visible spectrophotometer (Hitachi U-2800UV/vis spectrometer). 1D-and 2D-Nuclear Magnetic Resonance (NMR) Spectroscopy Using a Bruker AM-500 spectrometer dimethyl sulfoxide-D6, MOH-D4, and D₂And (4) measuring the O solution. HRESI-MS and ESI-MS analyses were determined using VG Platform Electrospray ESI/MS from Cell Applications (Cell Applications, Inc., San Diego, Calif., USA). All chemicals and reagents used in this study were high grade commercial products. Other materials include BCA (bicinchoninic acid) protein assay kit (Thermo Scientific, Rockford, IL, USA) and 4-nitrophenyl phosphate disodium salt hexahydrate (4-nitrophenylphosphate sodium salt) (Alfa Aesar, Ward Hill, Massachusetts, USA).

General Experimental methods

The optical rotatory power was measured by JASCO P-1020digital polarimeter. The UV spectrum was measured using a Hitachi U-2800U-2800 UV/Vis spectrophotometer. 1D-and 2D-Nuclear Magnetic Resonance (NMR) Spectroscopy Using a Bruker AM-500 spectrometer dimethyl sulfoxide-D6, MOH-D4, and D₂And (4) measuring the O solution. HRESI-MS and ESI-MS were analyzed as VG Platform Electron ESI/MS.

Preparation and purification of crude extract

Fresh roots of Dorkish grass (14.7 kg) were extracted 3 times with aqueous ethanol (95% v/v, 120 l total). After filtration, the filtrate was evaporated and dried under vacuum. The dried extract (601.4 g) was suspended in H₂O, and partitioned successively with n-hexane, ethyl acetate (EtOAc) and n-butanol (n-BuOH) to give 78.4, 32.0, and 133.9 g of extracts, respectively. The maximum matrix mineralization activity was observed in the EtOAc and n-butanol extracts (fig. 2). Passing the EtOAc extract through dextran gelSephadex LH-20 column was eluted with 95% ethanol to give 9 fractions (UCE-1 to UCE-9). The UCE-3 fraction was further separated by Sephadex LH-20 column chromatography and eluted with MeOH/H₂And (4) carrying out O (40-100%) extraction to obtain 17 parts (UCE-3-1 to UCE-3-17). Subjecting UCE-3-7 fraction to MCI CHP-20 column chromatography with H₂Elution with O-MeOH gradient yielded 14 sub-fractions (UCE-3-7-1 to UCE-3-7-14). Compound 2(98.1 mg) UCE-3-7-4 in portions with MeOH/H₂And recrystallizing to obtain the product. The UCE-4 fraction was further separated by Sephadex LH-20 column chromatography and eluted with MeOH/H₂O (50 to 100%) to obtain 17 fractions (UCE-4-1 to UCE-4-17). UCE-4-3 and UCE-4-10 parts were passed through MCI CHP-20 column and with MeOH/H₂Extracting with 30-100% of O to obtain 8 (UCE-4-3-1 to UCE-4-3-8) and 11 sub-fractions (UCE-4-10-1 to UCE-4-10-11). Compound 11(12.0 mg) was obtained after purification by HPLC on a semi-preparative C-18 reverse flow column from UCE-4-3-2 fraction with 10% MeOH as the eluting solvent system. Compound 4(6.9 mg) was obtained after HPLC on a semi-preparative C-18 reverse flow column from UCE-4-10-2 fraction, purified using 25% MeCN as the elution solvent system. Chromatography of UCE-5 fraction on MCI CHP-20 column with H₂Elution with O-MeOH gradient yielded 24 sub-fractions (UCE-5-1 to UCE-5-24). Passing UCE-5-15 and UCE-5-16 part through Sephadex LH-20 column, eluting with MeOH/H₂Extracting with 60% of water to obtain 6 fractions (UCE-5-15-1 to UCE-5-15-6) and 10 fractions (UCE-5-16-1 to UCE-5-16-10). Compound 3(12.2 mg) and compound 1(184.5 mg) were obtained after purification by HPLC from UCE-5-15-1 and UCE-5-16-9 fractions on a semi-preparative C-18 reverse flow column with 55% MeOH as the eluting solvent system. The n-butanol extract was passed through a Diaion HP-20 column and eluted with H₂The O-MeOH gradient was eluted to give 6 subfractions (UCB-1 to UCB-6). The UCB-3, UCB-4 and UCB-5 parts are further separated by 95% EtOH elution through respective Sephadex LH-20 columns to respectively obtain 6 (UCB-3-1 to UCB-3-6), 9 (UCB-4-1 to UCB-4-9) and 6 (UCB-5-1 to UCB-5-6) parts. Subjecting UCB-3-3 and UCB-5-2 fractions to MCI CHP-20 column chromatography, and subjecting to chromatography with H₂Performing gradient extraction and separation on O-MeOH to obtain 6 (UCB-3-3-1 to UCB-3-3-6) and 8 (UCB-5-2-1 to UCB-5-2-8) sub-parts respectively. Compound 7(38.3 mg) and compound 8(15.3 mg) prepared fromUCB-3-3-2. Compound 5(58.6 mg) and compound 6(7.6 mg) were obtained after purification from UCB-3-3-3 and UCB-5-2-1 fractions by respective HPLC on a semi-preparative C-18 reverse flow column with 20% MeOH as the eluting solvent system. UCB-4-3 and UCB-4-4 parts were passed through MCI CHP-20 column and in MeOH/H₂And (4) separating and purifying O (60-100% and 20-100% respectively) to obtain 4 (UCB-4-3-1 to UCB-4-3-4) and 9 (UCB-4-4-1 to UCB-4-4-9) sub parts respectively. Compound 9(38.5 mg) was obtained after purification by HPLC on a semi-preparative C-18 reverse flow column from UCB-4-3-3 fraction using 20% MeOH as the eluting solvent system. Compound 10(13.5 mg) was obtained after purification by HPLC on a semi-preparative C-18 reverse flow column from UCB-4-4-3 fraction using 40% MeOH as the eluting solvent system.

Acid hydrolysis of Compound 3

1 mg of Compound 3 was hydrolyzed with a heated block at 80 ℃ with 6N HCl for 6-8 hours. The mixture was cooled and evaporated to remove the acid, then resuspended in milli-Q water and passed through a Millipore-GX nylon membrane before being re-analyzed. Monosaccharides of compound 3 and polysaccharide hydrolysates were separated in a High Performance Anion Exchange Chromatography (HPAEC) system (Dionex, Sunnyvale, CA) and anion exchange column (Carbopac PA-10,4.6 × 250 mm). The analysis of monosaccharides was performed using an isocratic 18mM sodium hydroxide solution at ambient temperature.

Apigenin 6-C- (. beta. -D-furanosylogliosyl (1 → 2) -alpha-D-xylopyranoside (3): yellow solid, [ alpha ]22D +21 (C0.5, MeOH); UV (MeOH). lambda.max (log):272(4.36),329(4.35) nm; 1H-NMR (DMSO-d6,500MHz) and 13C-NMR (DMSO-d6,125MHz), see Table 1; calculated HRESI-MS M/z 535.1459[ M + H ] + (C25H27O13,535.1452).

Cell culture

Human Osteoblasts (HOB) are primary cells derived from the normal femur of a 63-year-old white female and obtained from Cell Applications (San Diego, CA, USA). HOb cells were grown in Osteoblast Growth Medium (Osteoblast Growth Medium) maintained at 37 deg.C and 5% CO₂The moist environment of (2). The medium was changed every other day. To maintain exponential growth, cells were subcultured every 7 days. Hob cells for cell survival, differentiation and mineralizationAnd (4) measuring.

Cell survival assay

Cell survival of the rabbit tail extract and fractions thereof was determined using the MTT assay. HOb cells were plated at 4X 10 per well³The density of individual cells was seeded in 96-well plates. After 24 hours of inoculation, the old medium is discarded and the cells are treated with the test extract or compound. After 72 hours of incubation, the old medium was discarded and MTT reagent was added to each well and incubated for 4 hours. The medium was then discarded and 100 μ l DMSO was added to all wells and mixed well to dissolve the dark blue crystals. The absorbance at 600nm was recorded with an ELISA reader (ELISA reader).

Alkaline phosphatase (ALP) detection

Alkaline phosphatase (ALP) activity of Rabdosia extract and its fractions was measured using p-nitrophenyl phosphate (p-nitrophenyl phosphate) as a substrate. HOb cells were plated at 4X 10 per well³The density of individual cells was seeded in 96-well plates. After 24 hours of inoculation, the old Medium was discarded, and the cells in Osteoblast Differentiation Medium (Osteoblast Differentiation Medium) were treated with the test extract or compound. After 72 hours of incubation, the medium was discarded, the cells were washed twice with cold PBS and lysed with lysis buffer (PBS containing 0.1% Triton X-100). The cell protein concentration in the supernatant was measured after 1 hour of treatment with BCA (bicinchoninic acid) protein reagent at 37 ℃, the reaction was stopped by adding 1M NaOH and the absorbance at 560nm was measured. In addition, alkaline phosphatase in the supernatant was added to 0.1M NaHCO3-Na2CO3 buffer (pH 10, containing 0.1% Triton X-100, 2mM MgSO 2) at 37 deg.C₄And 6mM p-nitrophenylphosphate) was assayed after 1 hour. The reaction was stopped by addition of 1M NaOH and the absorbance at 405nm was measured. Each value was normalized to protein concentration.

Mineralised Matrix Formation analysis (Mineralized Matrix Formation Assay)

The mineralizing activity of the Rabdosia extract and its fractions was determined by alizarin red-S staining with calcium. HOb cells were plated at 7X 10 per well⁴Density of individual cellsSeeded in 96-well plates. After 3 days of inoculation, the old Medium was discarded and cells in mineralized Medium (Osteoblast Differentiation Medium) containing ascorbic acid (50. mu.g/ml) and. beta. -glycerophosphate (10mM) were treated with the test extracts or compounds for 12 days. At the end of the experiment, the cultures were fixed in 75% ethanol and the calcium deposits were stained with alizarin red-S solution (40mM, pH 4.2) for 15 minutes at room temperature. The bound staining was washed with 10% (w/v) cetylpyridinium chloride solution in 10mM sodium phosphate (pH 7.0) and quantified with an ELISA reader at 550 nm.

Statistical analysis

Student's t-test was used to determine statistically significant differences between values for each experimental group. Data are shown as mean ± standard deviation and P-value <0.05 as statistically significant difference.

Example 1Rabbit tail grass Hob cells promoting osteoblast differentiation and matrix mineralization (matrix mineralization)

The 95% ethanol (EtOH) extract from the roots of rabbit tail grass (UC) was extracted and chromatographed using a biological grade method. The 95% crude ethanol extract is suspended in H₂O and was divided in the order of n-hexane, ethyl acetate and n-butanol to give 4 fractions. The cell proliferation of the crude extract at Hob cells was evaluated using the MTT method. The results showed that none of the crude extracts was found to result in significant cytotoxicity or proliferation of HOb cells at concentrations of 100 μ g/ml or below (data not shown). ALP activity is a major marker of osteoblast differentiation. The effect of the extract on osteoblast differentiation was examined by measuring ALP activity of HOb cells. After Hob cell treatment for 72 hours, the results showed that the 100. mu.g/ml 95% ethanol crude extract and the aqueous fraction exhibited increased ALP activity of 106.51. + -. 0.44 and 119.36. + -. 5.64%, respectively (FIG. 1). In addition, the effect of the test extract on osteoblast mineralization was determined by measuring bone nodule formation in HOb cells. Histochemical alizarin red staining was performed on mineralized nodules on day 12. The results showed that the ethyl acetate fraction and the n-butanol fraction exhibited increases of 130.45 + -1.51, 123.11 + -3.28, and 146.73 + -2.85%, respectively, in 100. mu.g/ml of the 95% ethanol crude extractMineralization activity (fig. 2).

EXAMPLE 2 Bioguided isolation and characterization of active Compounds from Rabdosia Rubescens

The active ethyl acetate and n-butanol fractions were purified by various column chromatography and high performance liquid chromatography and yielded a new compound (3), as well as 10 known compounds, 2',4',5, 7-tetrahydroxyisoflavanone (dalbergioidin) (1) (duranogo, Quinones, Torres, Rosero, Gil)&Echeveri, 2002), salicylic acid (2) (Scott, 1972), vitexin (4) (Krafczyk)&Glomb,2008), tryptophan (5) ((Lee)&Phillips,1992)), maltol-3-O- β -D-glucopyranoside (maltol-3-O- β -D-glucopyranoside) (6) (Ono, Masuoka, Tanaka, Ito)&Nohara, 2001), adenosine (adenosine) (7) (Moyroud)&Strazewski,1999)，spatholosineside A(8)(Yin,Liu,Wang,Tu,Liang&Zhao,2008)，byzantionoside B(9)(Matsunami, Otsuka&Takeda,2010), (7R,8R) -threo-guaiacylglycerol-8-O-4'-sinapyl ether 7-O-beta-D-glucopyranoside ((7R,8R) -threo-guaiacylglycerol-8-O-4' -sinapyl ether 7-O-beta-D-glucopyranoside) (10) (Machida, Sakamoto&Kikuchi,2009) and thymine (thymine) (11) (Pouchert,1993) (fig. 3). These known compounds are identified by comparing their spectral data with corresponding data in the literature or with real samples (UV,¹H-NMR,¹³C-NMR,ESI-MS)。

new compound 3 is a yellow powder. 3 is represented by its positive HRESI-MS M/Z: 535.1459[ M + H] ⁺(calculated as C)₂₅H₂₇O₁₃ ⁺535.1452) is determined as C₂₅H₂₆O₁₃This is consistent with its Nuclear Magnetic Resonance (NMR) data (table 1). The UV spectrum shows absorption bands at λ max 272 and 329nm, and the NMR spectrum shows_H6.75(1H, s, H-3) and_Csignals of 102.8(C-3), 163.5(C-2) and 181.8(C-4), which indicate the presence of a flavone group. In A₂X₂In the coupling system, in_HSignals of 6.91(2H, d, J ═ 8.8Hz, H-3 ', H-5') and 7.92(2H, d, J ═ 8.8Hz, H-2 ', H-6'), and NMR spectra_CThe presence of the 116.0(C-3 ', C-5') and 128.5(C-2 ', C-6') signals suggests a monosubstituted aromatic moiety in 3 (Ring B). In Heteronuclear Multiple Bond Correlation (HMBC) spectra, due to the interaction with C-6,

the long chain of C-9 couples with an aromatic signal,_H6.51(1H, s, H-8) is designated at H-8. These data show that 3 has a backbone of 5,7, 4-trihydroxyflavone (apigenin) (5,7, 4' -trihydroxyslavone (apigenin) skexeton) (fig. 4). NMR spectra also showed two sugar signals_H2.52 to 5.25 and_C64.5 to 109.1. 3, the aqueous layer was separated by HPLC and compared to the standard to give only one glycoside: apiose (apiose). The presence of other sugars was determined by NMR, HSQC, COSY and HMBC spectroscopy. The above evidence indicates that 3 is apigenin (apigenin) substituted with a xylose and apiose (apiose) group. 6-C-glycosylation of apigenin and xylose occurs in a field (field) of luteolin (leutolin)_C100.1) was confirmed as low as about 8ppm, and the C-1' signal was in close proximity in NMR analysis_C72.1 (Dubois, Zoll, Bouillant)&Chopin, 1982). The α -configuration of D-xyloside and the β -configuration of D-apioside (D-apioside) can be deduced by the coupling constants of the isomeric protons (isomeric proton) H-1 'and H-1'. The position (C-1 '→ C-2') at which the beta-D-furanapioside (beta-D-apiofuranoside) and alpha-D-xylopyranoside (alpha-D-xylopyranoside) are linked is located by HMBC correlation (HMBC corrlation)_C198.1 with C-1' "located at_H4.27 (1H, brs) (FIG. 4). Based on the above data, the structure of 3 was determined to be apigenin 6-C- (β -D-furanapigenin (1 → 2) - α -D-xylopyranoside.

TABLE 1 preparation of Compound 3¹H and¹³NMR data of C (DMSO-d)₆500 and 125 Hz).

Example 3 survival of HOb cells by Compounds isolated from Rabdosia

HOb cells were cultured for 72 hours with compounds isolated from rabbit grass and cell survival was measured using MTT method. The results showed that only compound (5) slightly inhibited cell survival at 100. mu.M with a value of 80.43. + -. 1.11%. The results are shown in FIG. 5.

Example 4 ALP Activity on HOb cells of Compounds isolated from Rabdosia Rubescens

To investigate the effect of compounds isolated from rabbit grass on osteoblast differentiation, alkaline phosphatase (ALP) activity of HOb cells was determined. Plant component, daidzein (daidzein), was used as a positive control. Of these compounds, 4 compounds, dalbergioidin (1), apigenin 6-C- (β -D-furanapigenin (1 → 2) - α -D-xylopyranoside (3), adenosine (7) and byzantioside B (9), were found to significantly increase ALP activity at 100 μ M in HOb cells by 114.10 ± 4.41, 109.68 ± 2.20, 108.70 ± 4.14 and 114.81 ± 0.18%, respectively (fig. 6).

Example 5 mineralization of HOb cells by Compounds isolated from Rabdosia

Alizarin red staining, widely used to detect the presence of calcium in cells, was used to study the effect of compounds isolated from Leptospermum leprae on bone mineralization in HOb cells cultured for 12 days (in the presence of test compounds, ascorbic acid and β -glycerophosphate). The results showed that the novel compound 3, dalbergioidin (1), adenosine (7) and byzantioside B (9) significantly increased the formation of mineralized bone nodules in HOb cells at 100. mu.M, with data of 150.10. + -. 0.80%, 107.09. + -. 2.80%, 125.21. + -. 3.75% and 129.21. + -. 6.13%, respectively (FIG. 7). These results show that novel compound 3 exhibits the most potent mineralizing activity of human osteoblasts.

EXAMPLE 6 neuroprotection of Rabdosia extract

NGF (nerve growth factor) -induced differentiation assay of PC12 cells was used in the nerve injury test model, and 6-hydroxydopamine (6-hydroxydopamine) was used to induce nerve injury. The extract of the roots of Leptospermum pettitatum prepared as described above was used for the assay. It was found that ethyl acetate extract and n-butanol extract have excellent neuroprotective effect. Cell viability of PC12 cells damaged by 6-hydroxydopamine increased from 0.55% to 62.88 +/-1.10% (cells treated with ethyl acetate extract) and 61.54 +/-1.48% (cells treated with n-butanol extract).

Claims

1. Use of an extract of the roots of rabbit hair grass (Uraria crinita (L.) Desv.ex DC.) or of the roots of rabbit hair grass (Uraria crinita (L.) Desv.ex DC.) for the manufacture of a medicament for promoting osteogenesis or for the treatment and/or prevention of bone diseases or for providing neuroprotection,

wherein the extract is an ethanol extract, an ethyl acetate extract or a butanol extract of the ethanol extract, and

wherein the neuroprotection is for the treatment of Parkinson's disease,

wherein the bone disease is osteoporosis, alveolar bone loss, osteotomy bone loss or childhood idiopathic bone loss.

2. The use according to claim 1, wherein the extract of the roots of said grass of Ranunculus sieboldii is prepared by the steps of: extracting the roots of Leptospermum petersonii with ethanol to obtain an ethanol extract, and extracting the ethanol extract with ethyl acetate or butanol to obtain an ethyl acetate extract or a butanol extract.

3. The use according to claim 1, wherein the extract of the roots of Leptospermum petersonii is an ethyl acetate extract or a butanol extract obtained from an ethanol extract of roots of Leptospermum petersonii.

4. The use according to claim 3, wherein the ethyl acetate extract contains apigenin 6-C- β -D-furanosylose (1 → 2) - α -D-xylopyranoside.