A refined component of ginseng and antlers, and its preparation method and application

Abstract

A refined component of ginseng and antler, and its preparation method and application, relate to the technical field of traditional Chinese medicine, and solve the problem that long-term use of existing drugs for postmenopausal osteoporosis is prone to adverse reactions. It contains 12 ginsenoside components and 4 antler oligopeptide components. The antler and ginseng are crushed and mixed evenly, and after passing through a 10-30 mesh sieve, ethanol is refluxed for extraction; the extracts are combined, the supernatant is collected after centrifugation, and after drying, ethanol is eluted on a macroporous adsorption resin, and the eluate is collected, dried, and then precipitated on a C 18 The reverse chromatography column is eluted with ethanol, and the eluate is collected and dried to obtain the refined components of the ginseng and antler medicine pair. The invention can be applied to the preparation of medicines for postmenopausal osteoporosis.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a ginseng and antler refined component and a preparation method and application thereof. Background Art

[0002] Postmenopausal osteoporosis is a common disease related to aging, which often occurs in postmenopausal women. It is mainly due to estrogen deficiency, which leads to bone loss and changes in bone tissue structure, increasing bone brittleness and susceptibility to fractures. The pain and bone deformation caused by fractures seriously affect the physical health and quality of life of elderly women. Clinically, the diagnostic indicator of postmenopausal osteoporosis is bone mineral density (BMD), with BMD being 2.5 standard deviations or more lower than the absolute value of the peak bone mass of healthy adults of the same gender and race. The main first symptom is pain in multiple joints throughout the body, accompanied by symptoms such as general fatigue and limb numbness. It is very easy to have serious complications such as brittle fractures as clinical manifestations.

[0003] The pathogenesis of postmenopausal osteoporosis mainly includes estrogen deficiency, oxidative stress, intestinal flora imbalance and inflammation. Studies have found that estrogen can enhance the bone formation process by inhibiting oxidative stress response, inhibiting cell apoptosis, and regulating inflammatory factors. Estrogen helps to improve the body's antioxidant capacity, maintain the timely removal of reactive oxygen species (ROS), thereby reducing ROS accumulation, preventing the occurrence of oxidative stress, and preventing oxidative stress from inhibiting osteoblast differentiation, stimulating osteoclast growth and differentiation and bone absorption. At the same time, estrogen can prevent and treat postmenopausal osteoporosis by regulating the production of inflammatory factors such as TNF-α and IL-1, inhibiting the proliferation and differentiation of osteoclasts, etc.

[0004] At present, the commonly used drugs for the treatment of osteoporosis in clinic mainly include bisphosphonates, estrogens, selective estrogen receptor modulators, RANKL inhibitors and calcitonin drugs that inhibit bone resorption; parathyroid hormone drugs that promote bone formation; and vitamin D and its analogs. Although these drugs have good clinical efficacy, long-term use will produce adverse reactions to varying degrees and even endanger the patient's life. For a chronic disease such as postmenopausal osteoporosis, it is a very unfavorable treatment factor. On the contrary, traditional Chinese medicine is often used to treat chronic diseases, so exploring how to use traditional Chinese medicine to treat postmenopausal osteoporosis is a more appropriate research direction.

[0005] At present, both at home and abroad, effective ingredients for treating postmenopausal osteoporosis are constantly being explored from traditional Chinese medicine. However, due to the complex ingredients, unclear quality standards, and unclear mechanism of action, traditional Chinese medicine is difficult to produce widely, and the clinical effects of existing Chinese medicine therapeutic drugs on the market are still insufficient. In traditional Chinese medicine formulas, ginseng and deer antler are widely studied. Ginseng, known as the "king of herbs", is a local medicinal material in Jilin Province, which has the effects of greatly replenishing vitality, benefiting the kidney and strengthening yang. Deer antler is the young antler of male deer of the Cervidae family, which is densely covered with velvet and not ossified. The former is commonly known as flower deer antler, and the latter is commonly known as red deer antler, which can replenish kidney yang and benefit essence and blood. At present, there is still no research report on the ginseng and deer antler medicine pair in the treatment of postmenopausal osteoporosis. Summary of the invention

[0006] In order to solve the problem that long-term use of existing postmenopausal osteoporosis drugs is prone to adverse reactions, the present invention provides a ginseng and antler refined component and a preparation method and application thereof.

[0007] The technical solution of the present invention is as follows:

[0008] A refined component of ginseng and antler medicine pair, comprising the following components in parts by weight:

[0009] 20-O-Glucoginsenoside Rf, 2-4 parts; Ginsenoside Rg1, 21-44 parts; GinsenosideRe, 18-37 parts; Ginsenoside Rf, 6-14 parts; Ginsenoside Ra3, 3-6 parts; Ginsenoside F5, 1 part; Ginsenoside Rb1, 42-84 parts; Ginsenoside Rc, 23-48 parts; Ginsenoside Ro, 23-47 parts; Ginsenoside Rb2, 24-48 parts; Ginsenoside Rb3, 10-20 parts; Ginsenoside Rd, 11-21 parts;

[0010] Arg-Val (arginine-valine), 188-279 copies; Cys-Val-Pro (cysteine-valine-proline), 119-176 copies; Thr-Glu-Leu (threonine-glutamic acid-leucine), 46-68 copies; Tyr-Arg-Val (tyrosine-arginine-valine), 43-63 copies.

[0011] Preferably, the following components are included in parts by weight:

[0012] 20-O-Glucoginsenoside Rf, 3 parts; Ginsenoside Rg1, 35 parts; Ginsenoside Re, 29 parts; Ginsenoside Rf, 11 parts; Ginsenoside Ra3, 5 parts; Ginsenoside F5, 1 part; GinsenosideRb1, 67 parts; Ginsenoside Rc, 38 parts; Ginsenoside Ro, 37 parts; Ginsenoside Rb2, 38 parts; Ginsenoside Rb3, 16 parts; Ginsenoside Rd, 17 parts;

[0013] Arg-Val, 225 copies; Cys-Val-Pro, 142 copies; Thr-Glu-Leu, 55 copies; Tyr-Arg-Val, 51 copies.

[0014] The present invention also provides a method for preparing a refined component of ginseng and antlers, the preparation method comprising the following steps:

[0015] Crush the antler and ginseng and mix them evenly, pass through a 10-30 mesh sieve, and then reflux extract with ethanol;

[0016] The extracts were combined, centrifuged, and the supernatant was collected. After drying, the supernatant was eluted with ethanol on a macroporous adsorption resin. The eluate was collected and dried at C 18 The reversed phase chromatography column was eluted with ethanol, and the eluate was collected and dried to obtain the refined components of the ginseng and antler medicine pair.

[0017] Preferably, the particle size of the crushed velvet antler and ginseng is 20 mesh.

[0018] Preferably, the usage ratio of ginseng to deer antler is 1-3:1.

[0019] Preferably, the ethanol concentration for reflux extraction is 40% to 90%, and the reflux extraction is performed 2 to 5 times, each time for 1 to 3 hours. More preferably, the ethanol for reflux extraction is 40% to 90% ethanol with a volume 10 times the weight of the medicinal material, and the reflux extraction is performed 2 to 5 times, each time for 1 to 3 hours.

[0020] Preferably, the centrifugal speed is 4000-8000 r / min, and the centrifugal time is 10-20 min.

[0021] Preferably, the specific method of eluting with the macroporous adsorption resin comprises the following steps:

[0022] (1) The ethanol extract and the supernatant after centrifugation were dried at 30°C to 70°C, and then reconstituted with ultrapure water to prepare a solution with a concentration of 0.5-2 g / mL. The solution was repeatedly loaded 3 times at 1-3 BV / h on a D-101 macroporous adsorption resin separation column that had been activated with ethanol and eluted without ethanol, and then adsorbed for 12-24 hours;

[0023] (2) eluting the macroporous adsorption resin with ultrapure water 5 to 8 times the volume of the macroporous adsorption resin at 1 to 3 BV / h;

[0024] (3) Elute the macroporous adsorption resin with 40% to 90% ethanol at a rate of 1 to 3 BV / h in an amount of 5 to 8 times the volume of the macroporous adsorption resin, and collect the eluate.

[0025] Preferably, the C 18 The specific method of the reversed phase chromatography column comprises the following steps:

[0026] The dry paste obtained by drying the eluate after elution with the macroporous adsorption resin was re-dissolved in ultrapure water to prepare a 1-3 mg / mL solution. 18 After loading the sample onto the column at 0.5~1 mL / min for 3 times, the column was eluted with ultrapure water 5~8 times the volume of the filler at 0.5~1 mL / min, and then eluted with 40%~90% ethanol 5~8 times the volume of the filler at 0.5~1 mL / min. 18 Fill the column and collect the eluate.

[0027] The present invention also provides an application of the ginseng and antler refined components or the ginseng and antler medicine pair refined components obtained by the preparation method, which is used as an active ingredient in the preparation of a medicine for postmenopausal osteoporosis.

[0028] Compared with the prior art, the specific beneficial effects of the present invention are:

[0029] The present invention prepares a refined component capable of treating postmenopausal osteoporosis from the ginseng and deer antler medicine pair for the first time, and has a significant effect on treating postmenopausal osteoporosis, specifically manifested in a significant improvement in bone density. Among them, GinsenosideRg1 can promote the proliferation of bone marrow mesenchymal stem cells; Ginsenoside Re can promote osteoblast differentiation; Ginsenoside Rb1 can increase the survival rate and alkaline phosphatase (ALP) activity of osteoblasts; Ginsenoside Rc can promote the osteogenic differentiation of MC3T3-E1 cells by activating the classical Wnt pathway; Ginsenoside Rd participates in activating the BMP-2 / Smad signaling pathway to induce osteoblast differentiation and mineralization, and promote bone healing. In addition, ginsenosides may also prevent osteoporosis caused by estrogen reduction by simulating or enhancing the effects of estrogen in the body. For example, Ginsenoside Re can promote the proliferation of mouse bone marrow mesenchymal stem cells in vitro through the estrogen receptor and MAPK-ERK1 / 2 pathway. The oligopeptide components in deer antler have good bioavailability and permeability. They may fight oxidative stress by mediating the PI3K / AKT signaling pathway, thereby achieving bone protection in postmenopausal osteoporosis patients. They can significantly promote the bone density of ovariectomized osteoporosis model rats, increase the activity of osteoblast markers ALP and BMP2 in serum, and inhibit the activity of osteoclast marker TRACP.

[0030] In traditional Chinese medicine, there is a theory that "kidneys govern bones". Kidneys are regarded as the "foundation of innateness". Kidneys store essence, essence produces marrow, and marrow nourishes bones. Therefore, the functional state of kidneys directly affects the health of bones. Ginseng has the effects of greatly replenishing vital energy, strengthening kidney and strengthening yang, while deer antler is believed to replenish kidney yang, improve essence and blood, and strengthen tendons and bones. The present invention uses both together to enhance the tonic effect.

[0031] The preparation method provided by the present invention is clear, simple, convenient, rapid, economical, widely applicable, inexpensive and readily available, and ensures the reliability of the efficacy of traditional Chinese medicine. It is expected to be developed into a food or health product with a wide range of therapeutic effects and commercial development value using ginseng and deer antler as raw materials, and plays a positive role in promoting the development of medicines with local characteristics in Northeast China. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 HPLC-ESI-MS of the refined components of the ginseng and antler medicine pair obtained in Example 1 2 Chromatogram;

[0033] Figure 2 HPLC-ESI-MS of the refined components of the ginseng and antler medicine pair obtained in Example 2 2 Chromatogram;

[0034] Figure 3HPLC-ESI-MS of the refined components of the ginseng and antler medicine pair obtained in Example 3 2 Chromatogram;

[0035] Figure 4 Figure 2 shows the test results of femoral bone density of rats in each group of experiments. DETAILED DESCRIPTION

[0036] In order to make the technical solution of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the specification of the present invention. It should be noted that the following embodiments are only used to better understand the technical solution of the present invention and should not be understood as a limitation to the present invention.

[0037] The raw materials and instruments used in the following experiments are all known products, which were purchased from commercial products. Ginseng and deer antler were purchased from the Chinese herbal medicine market, rats were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and liquid chromatography-mass spectrometry was purchased from Waters Corporation in the United States.

[0038] Example 1

[0039] Step S1, taking a number of dry and clean ginseng and deer antler in a mass ratio of 2:1, crushing them all, passing through a 20-mesh sieve, and placing the coarse powder in a cool and dry place for later use.

[0040] Step S2, heating and refluxing the above coarse powder with 60% ethanol in a volume 10 times the weight of the medicinal material for 3 times, each time for 2 hours. Centrifuge each extract at 4000r / min for 15 minutes, retain and combine the supernatant, and dry the supernatant to obtain a dry paste.

[0041] Step S3, the ethanol extract was combined with the supernatant after centrifugation, dried at 60°C, and then re-dissolved with ultrapure water to prepare a solution with a concentration of 1g / mL. The sample was repeatedly loaded on the macroporous adsorption resin separation column at 1.5BV / h for 3 times and adsorbed for 24h. The macroporous adsorption resin was eluted with ultrapure water 6 times the volume of the macroporous adsorption resin at 2BV / h. The macroporous adsorption resin was eluted with 50% ethanol 6 times the volume of the macroporous adsorption resin at 1.5BV / h, and the eluate was collected to obtain a 60% ethanol eluate, which was dried to obtain a dry paste.

[0042] Step S4: The dry paste after drying the ethanol eluate of the macroporous adsorption resin is re-dissolved in ultrapure water to prepare a 2 mg / mL solution, and then heated at C 18 After the sample was loaded onto the column three times at 0.75 mL / min, elution was performed with ultrapure water 6 times the volume of the filler at 0.75 mL / min, and C was eluted with 60% ethanol 6 times the volume of the filler at 0.75 mL / min. 18 The filler is added, and the eluate is collected and dried to obtain the refined components of the ginseng and antler medicine pair.

[0043] The HPLC-MS / MS detection of the refined components of the ginseng and antler medicine pair obtained in this example:

[0044] (1) Preparation of test solution: Accurately weigh 1 mg of the refined component, add 1 mL of 70% methanol to dissolve it, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0045] (2) The chromatographic conditions for HPLC-MS / MS determination were as follows: Instrument: Waters Q-TOF SYNAPT G2-Si ultra-high performance liquid chromatography-high resolution mass spectrometer (Waters, USA); Chromatographic column: ACQUITY BEH C18 (50 mm×2.1 mm, 1.7 μm); Flow rate: 0.3 mL / min; Column temperature: 30°C; Injection volume: 5 μL; Mobile phase A was 0.1% formic acid, and mobile phase B was acetonitrile. Gradient elution: 0-5 min, 90%-80% A; 5-10 min, 80%-72% A; 10-25 min, 72%-68.5% A.

[0046] (3) Mass spectrometry conditions: ESI ion source, positive and negative ion modes, full information tandem mass spectrometry (MSE) mode scanning; scanning range: 50~2000Da; ion source temperature: 120℃ (positive ion mode) / 110℃ (negative ion mode); desolvation temperature: 400℃; cone gas flow rate: 50L / h; desolvation gas flow rate: 800L / h; capillary voltage: 3.0kV (positive ion mode) / 2.5kV (negative ion mode); MSE collision energy: low collision energy: 6eV, high collision energy: 20~30eV / 30~45eV.

[0047] The total ion current of the refined components of the ginseng and antler medicine pair in positive ion mode is shown in Figure 1 As shown, based on HPLC-ESI-MS 2The components of the refined fractions of the ginseng and antler medicine pair were analyzed. The information of the compounds in the refined fractions of the ginseng and antler medicine pair was preliminarily identified and shown in Table 1. 16 compounds were identified, including 12 ginsenosides: 20-O-Glucoginsenoside Rf, 3 parts; Ginsenoside Rg1, 35 parts; Ginsenoside Re, 29 parts; Ginsenoside Rf, 11 parts; Ginsenoside Ra3, 5 parts; Ginsenoside F5, 1 part; Ginsenoside Rb1, 67 parts; Ginsenoside Rc, 38 parts; GinsenosideRo, 37 parts; Ginsenoside Rb2, 38 parts; Ginsenoside Rb3, 16 parts; Ginsenoside Rd, 17 parts; and 4 kinds of velvet antler oligopeptide components: Arg-Val, 225 parts; Cys-Val-Pro, 142 parts; Thr-Glu-leu, 55 parts; Tyr-Arg-Val, 51 parts.

[0048] Table 1

[0049]

[0050] Example 2

[0051] Step S1, taking a number of dry and clean ginseng and deer antler in a mass ratio of 1:1, crushing them all, passing through a 20-mesh sieve, and placing the coarse powder in a cool and dry place for later use.

[0052] Step S2, heating and refluxing the above coarse powder with 60% ethanol in a volume 10 times the weight of the medicinal material for 3 times, each time for 2 hours. Centrifuge each extract at 4000r / min for 15 minutes, retain and combine the supernatant, and dry the supernatant to obtain a dry paste.

[0053] Step S3, the ethanol extract was combined with the supernatant after centrifugation, dried at 60°C, and then re-dissolved with ultrapure water to prepare a solution with a concentration of 1g / mL. The sample was repeatedly loaded on the macroporous adsorption resin separation column at 1.5BV / h for 3 times and adsorbed for 24h. The macroporous adsorption resin was eluted with ultrapure water 6 times the volume of the macroporous adsorption resin at 2BV / h. The macroporous adsorption resin was eluted with 50% ethanol 6 times the volume of the macroporous adsorption resin at 1.5BV / h, and the eluate was collected to obtain a 60% ethanol eluate, which was dried to obtain a dry paste.

[0054] Step S4: The dry paste after drying the ethanol eluate of the macroporous adsorption resin is re-dissolved in ultrapure water to prepare a 2 mg / mL solution, and then heated at C 18After the sample was loaded onto the column three times at 0.75 mL / min, elution was performed with ultrapure water 6 times the volume of the filler at 0.75 mL / min, and C was eluted with 60% ethanol 6 times the volume of the filler at 0.75 mL / min. 18 Filler, and collect the eluate, and after drying, obtain the refined components of the ginseng and antler medicine pair. The refined components of the ginseng and antler medicine pair obtained in this example were detected by high performance liquid chromatography-mass spectrometry. The total ion flow diagram of the refined components of the ginseng and antler medicine pair in the positive ion mode is shown in FIG. Figure 2 Based on HPLC-ESI-MS 2 The composition of the refined components of the ginseng and antler medicine pair was analyzed, and the compound components in the components were the same as those in Example 1.

[0055] Example 3

[0056] Step S1, taking a number of dry and clean ginseng and deer antler in a mass ratio of 3:1, crushing them all, passing through a 20-mesh sieve, and placing the coarse powder in a cool and dry place for later use.

[0057] Step S2, heating and refluxing the above coarse powder with 60% ethanol in a volume 10 times the weight of the medicinal material for 3 times, each time for 2 hours. Centrifuge each extract at 4000r / min for 15 minutes, retain and combine the supernatant, and dry the supernatant to obtain a dry paste.

[0058] Step S3, the ethanol extract was combined with the supernatant after centrifugation, dried at 60°C, and then re-dissolved with ultrapure water to prepare a solution with a concentration of 1g / mL. The sample was repeatedly loaded on the macroporous adsorption resin separation column at 1.5BV / h for 3 times and adsorbed for 24h. The macroporous adsorption resin was eluted with ultrapure water 6 times the volume of the macroporous adsorption resin at 2BV / h. The macroporous adsorption resin was eluted with 50% ethanol 6 times the volume of the macroporous adsorption resin at 1.5BV / h, and the eluate was collected to obtain a 60% ethanol eluate, which was dried to obtain a dry paste.

[0059] Step S4: The dry paste after drying the ethanol eluate of the macroporous adsorption resin is re-dissolved in ultrapure water to prepare a 2 mg / mL solution, and then heated at C 18 After the sample was loaded onto the column three times at 0.75 mL / min, elution was performed with ultrapure water 6 times the volume of the filler at 0.75 mL / min, and C was eluted with 60% ethanol 6 times the volume of the filler at 0.75 mL / min. 18 Filler, and collect the eluate, and after drying, obtain the refined components of the ginseng and antler medicine pair. The refined components of the ginseng and antler medicine pair obtained in this example were detected by high performance liquid chromatography-mass spectrometry. The total ion flow diagram of the refined components of the ginseng and antler medicine pair in the positive ion mode is shown in FIG. Figure 3 Based on HPLC-ESI-MS 2 The composition of the refined components of the ginseng and antler medicine pair was analyzed, and the compound components in the components were the same as those in Example 1.

[0060] After analysis, the components in the refined components of the above three embodiments are: 20-O-Glucoginsenoside Rf, 2-4 parts; Ginsenoside Rg1, 21-44 parts; Ginsenoside Re, 18-37 parts; Ginsenoside Rf, 6-14 parts; Ginsenoside Ra3, 3-6 parts; Ginsenoside F5, 1 part; Ginsenoside Rb1, 42-84 parts; GinsenosideRc, 23-48 parts; Ginsenoside Ro, 23-47 parts; Ginsenoside Rb2, 24-48 parts; Ginsenoside Rb3, 10-20 parts; Ginsenoside Rd, 11-21 parts; Arg-Val, 188-279 parts; Cys-Val-Pro, 119-176 parts; Thr-Glu-Leu, 46-68 parts; Tyr-Arg-Val, 43-63 parts.

[0061] Effect example

[0062] Experimental study on the activity of refined components of ginseng and antler medicine in treating osteoporosis in ovariectomized rats:

[0063] (1) Experimental animals:

[0064] SPF grade 8-week-old female SD rats (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were used, with a temperature of 24-26°C and a humidity of 50%-60%. They were numbered and raised, with 6 rats per cage. They were raised in barrier environment IVC cages, with bedding changed every other day, free access to water, standard feed, and weighed once a week. Adaptive feeding was performed for 7 days.

[0065] (2) Establishment of Ovariectomized Rat Model:

[0066] Rats were anesthetized by intraperitoneal injection of 40 mg / kg body weight of 2% sodium pentobarbital. After general anesthesia, the rats were placed in a supine position, fixed, and the abdomen was shaved and disinfected with 75% alcohol and iodine. The abdomen was incised, and the abdominal muscles were separated. The light pink ovaries and surrounding fat were visible in the abdominal cavity. The fat mass was gently lifted with forceps to expose the ovaries. The fallopian tubes at the lower end of the ovaries were ligated with silk thread. After ligation, the bilateral ovaries were removed, and the incision was sutured and disinfected.

[0067] Sham operation group control experiment: the same ovariectomy process, after anesthesia, sham oophorectomy was performed, bilateral ovaries were found, but not removed, and the incisions were sutured and disinfected.

[0068] The animals were placed on a heating platform to keep warm until they regained consciousness, and then returned to their cages for another 2 months.

[0069] (3) Experimental design, grouping and dosing regimen:

[0070] The rats were randomly divided into 8 groups, namely, a sham operation group (Sham), an ovariectomized (OVX) group, a medium dose group (SRZ) of the component in Example 1, a high dose group (SRG) of the component in Example 1, a medium dose group (SRDZ) of the component in Example 2, a high dose group (SRDG) of the component in Example 2, a medium dose group (SRHZ) of the component in Example 3, and a high dose group (SRHG) of the component in Example 3.

[0071] Rats in the sham group (Sham group) underwent sham surgery to remove the ovaries without removing the ovaries, and were given normal saline by gavage after surgery. Rats in the ovariectomized group (OVX group) underwent bilateral ovariectomy and were given normal saline by gavage after surgery. Rats in the medium-dose group (SRDZ, SRZ, SRHZ) underwent bilateral ovariectomy and were given the corresponding refined ginseng and antler components 1.54g / kg / d by gavage after surgery. Rats in the high-dose (SRDG, SRG, SRHG) group underwent bilateral ovariectomy and were given 3.07g / kg / d of refined ginseng and antler components by gavage after surgery. Bone density was tested 20 days after administration to all rats.

[0072] (4) Sampling and bone specimen preparation:

[0073] After the administration, the rats were anesthetized and blood was drawn from the femoral artery to kill them. The right femur was removed, the surrounding tissues were removed, and the femur was wrapped with gauze soaked in saline and stored in a -80°C refrigerator for subsequent bone density testing.

[0074] (5) Femoral bone density test:

[0075] The bone mineral density (BMD) of rat femurs was measured using an XR-36 dual-energy bone densitometer and its accompanying "The Small Subject Scout Scan" software. BMD was automatically calculated. Bone mineral density values ​​were expressed as bone mineral content per unit area (g·cm -2 ).

[0076] (6) Experimental results:

[0077] The bone density of different groups of ovariectomized rats was tested. The specific results are shown in Figure 4 .Depend on Figure 4 It can be seen that the bone density of the rats in the ovariectomized group was significantly decreased compared with that in the sham operation group (p < 0.0001), indicating that the postmenopausal osteoporosis model rats were successfully established. The average bone density of the femur of the rats in the sham group, OVX group, SRDZ group, SRDG group, SRZ group, SRG group, SRHZ group and SRHG group was 222.94 mg / cm 2、197.67mg / cm 2 、214.66mg / cm 2 、207.99mg / cm 2 、221.16 mg / cm 2 、210.99mg / cm 2 、218.2mg / cm 2 and 216.28mg / cm 2 Compared with the ovariectomized group, the medium and high dose groups of ginseng and antler refined components significantly increased the bone density of rats ( p <0.05). Although the improvement effect of the high-dose ginseng and antler group was lower than that of the medium-dose ginseng and antler group, the results can prove that the refined components of the ginseng and antler medicine pairs obtained by the preparation method in the range of (1-3):1 in different ratios have a significant effect on improving the femoral bone density of ovariectomized rats ( p <0.05), which can be used as the refined components of ginseng and antler medicine for the treatment of postmenopausal osteoporosis, providing an experimental basis for the follow-up study of the refined components of ginseng and antler medicine for anti-postmenopausal osteoporosis.

[0078] It is to be understood that the present invention is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the scope of protection of the present invention.

Claims

1. A method for preparing a refined component of a ginseng and antler medicine pair, characterized in that: The refined components of the ginseng and antler medicine pair are used as active ingredients in the preparation of a drug for postmenopausal osteoporosis. The refined components of the ginseng and antler medicine pair contain the following components in parts by weight: 20-O-Glucoginsenoside Rf, 2-4 parts; Ginsenoside Rg1, 21-44 parts; Ginsenoside Re, 18-37 parts; Ginsenoside Rf, 6-14 parts; Ginsenoside Ra3, 3-6 parts; Ginsenoside F5, 1 part; Ginsenoside Rb1, 42-84 parts; Ginsenoside Rc, 23-48 parts; Ginsenoside Ro, 23-47 parts; Ginsenoside Rb2, 24-48 parts; Ginsenoside Rb3, 10-20 parts; Ginsenoside Rd, 11-21 parts; Arg-Val, 188-279 copies; Cys-Val-Pro, 119-176 copies; Thr-Glu-Leu, 46-68 copies; Tyr-Arg-Val, 43-63 copies; The preparation method comprises the following steps: Crush the antler and ginseng and mix them evenly, pass through a 10-30 mesh sieve, and then reflux extract with ethanol; The extracts were combined, centrifuged, and the supernatant was collected. After drying, the supernatant was eluted with ethanol on a macroporous adsorption resin. The eluate was collected and dried at C 18 The reversed phase chromatography column is eluted with ethanol, and the eluate is collected and dried to obtain the refined components of the ginseng and antler medicine pair; The dosage ratio of ginseng to pilose antler is 1-3:1; The ethanol concentration for the reflux extraction was 60%, and the reflux extraction was performed 3 times, each time for 2 hours; The drying temperature is 60°C; The centrifugal speed is 4000r / min, and the centrifugal time is 15min; The specific method of eluting the macroporous adsorption resin comprises the following steps: (1) The ethanol extract was combined with the supernatant after centrifugation, dried at 60°C, and then reconstituted with ultrapure water to prepare a solution with a concentration of 1 g / mL. The solution was repeatedly loaded 3 times at 1.5 BV / h on a D-101 macroporous adsorption resin separation column that had been activated with ethanol and eluted without ethanol, and then adsorbed for 24 hours; (2) eluting the macroporous adsorption resin with ultrapure water 6 times the volume of the macroporous adsorption resin at 1.5 BV / h; (3) eluting the macroporous adsorption resin with 50% ethanol (6 times the volume of the macroporous adsorption resin) at 1.5 BV / h, and collecting the eluate; The C 18 The specific method of the reversed phase chromatography column comprises the following steps: The dry paste obtained by drying the eluate after elution with the macroporous adsorption resin was reconstituted with ultrapure water to prepare a 2 mg / mL solution, and then the AQ-C 18 After loading the sample onto the column at 0.75 mL / min for 3 times, the column was eluted with ultrapure water 6 times the volume of the filler at 0.75 mL / min, and then eluted with 60% ethanol 6 times the volume of the filler at 0.75 mL / min. 18 Fill the tube and collect the eluate.

2. A refined component of ginseng and antler medicine pair, characterized in that: The method is used to prepare the product according to claim 1.

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