CN103989732B - Beggarweed extract and the general flavone for therefrom separate and mountain naphthalene glycosides and its medical usage - Google Patents

Abstract

The invention relates to the technical field of medicines, and relates to the application of the extract of the leeches and the total flavonoids and kaempferrin monomer compounds of the leeches isolated therefrom in the preparation of medicines or foods for the prevention and treatment of osteoporosis. The present invention extracts and separates the dried whole grass of M. chinensis as raw material, obtains the extract of M. spp., prepares the total flavonoids of M. spp. from the extract, and separates kaempferin monomer compound. Through in vivo and in vitro experiments, it has been shown that the extracts of M. chinensis, total flavonoids or monomeric compounds of kaempferol have good anti-osteoporosis biological activity, so they can be used to prepare medicines for preventing and treating osteoporosis. or food. The preparation method of the invention is simple, and provides a new drug source for anti-osteoporosis.

Description

Extract from leeches and total flavonoids and kaempferols isolated therefrom and their medicinal uses

technical field

The invention relates to the technical field of medicines, and relates to the application of the extract of the leeches and the total flavonoids and kaempferrin monomer compounds of the leeches isolated therefrom in the preparation of medicines or foods for the prevention and treatment of osteoporosis. This application is a divisional application with the patent application number 201210587958.6.

Background technique

Osteoporosis (Osteoporosis, OP) is a common disease frequently-occurring disease. At present, there are about 200 million people in the world suffering from osteoporosis, and its incidence rate has jumped to the seventh among various common diseases in the world. At present, chemical drugs for treating osteoporosis mainly use bone formation stimulators such as estrogen bone resorption inhibitors or androgen, and long-term use has relatively large side effects. Finding effective extracts, effective parts and monomer compounds from natural plants to treat osteoporosis with less side effects has become a research hotspot.

Podocarpium podocarpum (DC.) Yang et Huang or Desmodiumpodocarpum DC. is a plant of the genus Podocarpium podocarpum (DC.) of the leguminous family Papilionaceae (Papilionaceae, Leguminosae) and is born at an altitude of 700 to 1000 meters In dense forests or streams in valleys, it is distributed in eastern and southwestern my country. The root, leaf and whole herb of Yuanling Yeshan leech are used as medicine, which has the function of expelling cold and stopping bleeding, and is mainly used for acute rheumatic arthralgia, bruises, knife wounds, etc. Materia Medica [M]. Shanghai Science and Technology Press, 3341-3343.). Modern pharmacological studies have shown that M. chinensis has analgesic, antipyretic and anti-inflammatory effects (Zhu, Z.Z., Ma, K.J., Ran, X., Zhang, H., Zheng, C.J., Han, T., Zhang , Q.Y., Qin, L.P., 2010. Analgesic, anti-inflammatory and antipyretic activities of the petroleum ether fraction from the ethanol extract of Desmodium podocarpum. Journal of ethnopharmacology 133, 1126-1131.). At present, there are only literature reports on the isolation of 5 compounds from the variant Podocarpium podocarpum (DC.) Yang et Huang var.oxyphyllum (DC.) Yang et Huang: β-sitosterol, Triacontanol, Suberone, Oleanolic Acid, and Pheophytin (Ma, X.Q., Zheng, C.J., Hu, C.L., Rahman, K., Qin, L.P., 2011. The genus Desmodium (Fabaceae) -traditional uses in Chinese medicine, phytochemistry and pharmacology. J. Ethnopharmacol. 138, 314-332.). So far, there has been no report on the prevention and treatment of osteoporosis by the extracts of M. chinensis, total flavonoids or monomeric compounds of kaempferenin.

Contents of the invention

The present invention provides a kind of extract of M. rotundum and the total flavonoids and kaempferin monomer compounds of M. rotifer isolated therefrom, as well as the extract of M. rotundum, the total flavonoids or kaempferol Application of the naphthalene glycoside monomer compound in the preparation of medicines or food for the prevention and treatment of osteoporosis.

The present invention extracts and separates the dried whole grass of M. chinensis as raw material, obtains the extract of M. spp., prepares the total flavonoids of M. spp. from the extract, and separates kaempferin monomer compound. The preparation method is as follows:

1. Preparing the extract of Cyclocus chinensis

Decoct or reflux the dried whole herb of the dry Rhizoma chinensis with water or an aqueous ethanol solution with a volume percentage not exceeding 95%, preferably 60 to 80% ethanol, preferably 80% ethanol, and concentrate the extract under reduced pressure To the extract that has no alcohol taste, the extract of M. chinensis is obtained.

2. Preparation of total flavonoids from Lemonia chinensis

Purify the above-prepared Miccus chinensis extract by macroporous adsorption resin or polyamide column chromatography, preferably macroporous adsorption resin, first elute with water and 20% ethanol aqueous solution, discard the eluent, and then use 30~ Elute with 80% ethanol, collect the eluate, preferably collect the eluate with 30% to 60% ethanol, concentrate and dry under reduced pressure, and obtain the total flavonoids of M. chinensis. Taking kaempferin as a control, the total flavonoid content was determined to be 52-65% by ultraviolet spectrophotometry (341nm).

3. Preparation of kaempferolin monomer compound

After dissolving the above-prepared extract of Lemonia chinensis with water into a suspension, it is extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence, and the n-butanol extract is concentrated under reduced pressure into a liquid extract , mix the sample with silica gel (100-200 mesh), put it on the column by dry method, elute with dichloromethane:methanol in different ratios of 10:1-1:1, and collect dichloromethane:methanol in the ratio of 10:1-5 The eluent of : 1 was concentrated under reduced pressure, dissolved with a small amount of methanol to separate out yellow crystals, and washed with methanol to obtain a yellow powder, that is, the kaempferrin monomer compound, and the content determined by HPLC was 95-99%. The chemical structural formula of kaempferrin monomer compound is as follows:

Where rha is rhamnose.

Through in vivo and in vitro pharmacological tests, it was shown that the extracts of M. chinensis, total flavonoids or monomeric compounds of kaempferrin all have the effect of anti-osteoporosis. Therefore, it can be used to prepare drugs or food for preventing and treating osteoporosis.

The present invention has simple preparation method of the extract of leech leech, total flavonoids of leech leech or kaempferrin monomer compound, and obvious anti-osteoporosis effect, so it can be used to prepare medicine or food for preventing and treating osteoporosis. The invention provides a new drug source for anti-osteoporosis.

Description of drawings

Figure 1 is the BMD of animals in each group, where vs model control group, *P<0.05, **P<0.05

Figure 2 is the ALP activity in the serum of animals in each group, where vs model control group, *P<0.05

Figure 3 is the TRAP activity in the serum of each group of animals, where vs model control group, *P<0.05,

**P<0.01, ***P<0.001.

detailed description

The present invention will now be described in detail in conjunction with the accompanying drawings and embodiments, and the scope of protection of the present invention is not limited to the embodiments.

Embodiment 1 prepares the extract and kaempferenin monomer compound of Rhizoma chinensis

20 kg of the whole herb of M. chinensis, crushed. It is decocted in conventional water, the ratio of solid to liquid is 1:15 (v/v), the extraction time is 2 hours, and the number of extractions is 3 times. The extract was filtered hot, and the filtrates were combined and evaporated to dryness under reduced pressure to obtain 3 kg of extract extract, with an extract yield of 15%. Suspend the extract extract with 1 times the amount of water (3L), and successively add petroleum ether (3L×3), dichloromethane (3L×3), ethyl acetate (3L×3) and n-butanol (3L×3 ) equal-volume extraction, take the n-butanol extract and concentrate it under reduced pressure to form a liquid extract, add 1.5 times of column chromatography silica gel and mix well, load the sample by dry method, wash with dichloromethane:methanol at a ratio of 10:1 to 5:1 Eluate and elute, collect the eluate, evaporate to dryness, dissolve with a small amount of methanol, place at room temperature overnight for crystallization, filter out the crystals, and then recrystallize with methanol to obtain 14 g of kaempferrin. The purity of kaempferrin was determined to be 98.5% by HPLC.

Embodiment 2 prepares the total flavonoids of Cyclops chinensis

15kg of dried whole grass of Cyclops chinensis, crushed, added 8 times the volume of 95% ethanol, reflux extraction, the extraction time was 3 hours, and the number of extractions was 3 times. The extract is filtered hot, and the filtrates are combined and then concentrated under reduced pressure to a liquid extract, which is the extract of M. chinensis. Add the extract of Lemonia chinensis to the pretreated AB-8 macroporous adsorption resin, wherein the weight ratio of macroporous resin to crude drug of the original medicinal material is 1:1. First eluted with 5 times of column water until colorless, then eluted with 20% ethanol, 30% ethanol, 60% ethanol, 80% ethanol in turn, collected 30-60% of the eluate, and washed After dehydrating and evaporating to dryness, 18.1 g of total flavonoids of M. chinensis were obtained. Taking the kaempferol prepared in Example 1 as a contrast, the total flavonoid content in the total flavonoids of the leech locust was determined by ultraviolet spectrophotometry (341nm) to be 58.4%.

Example 3: Preparation of the extract of the leech and the total flavonoids of the leech

15kg of dried whole grass of Leaf chinensis, crushed, added 8 times the volume of 80% ethanol, reflux extraction, the extraction time is 3 hours, and the number of extractions is 3 times. After hot filtration, the filtrates are combined and then concentrated under reduced pressure to a liquid extract, which is the extract of M. chinensis. Add the extract of Leedia chinensis to the pretreated polyamide resin of 60-100 mesh, wherein the weight ratio of the resin to the crude drug of the original medicinal material is 1:1. First eluted with water until it was colorless, then eluted with 10% ethanol, 30% ethanol, 60% ethanol, and 80% ethanol in sequence, and collected the eluents eluted with 30% ethanol, 60% ethanol, and 80% ethanol respectively. Taking kaempferol as a control, the total flavonoid content was measured by ultraviolet spectrophotometry (341nm), and the results showed that the total flavonoid content in 30%, 60%, and 80% ethanol eluted sites were 56.6%, 42.3%, and 40.5%, respectively. Concentrate the eluate from the elution site of 60-80% ethanol and then enrich it with polyamide resin again, first elute with 5 times column volume of 10% ethanol to remove impurities, then elute with 30% ethanol, collect and collect The content of total flavonoids measured by spectrophotometry (341nm) was 62.4%.

Anti-osteoporosis activity experiment in vivo and in vitro

1. In vitro cell experiments

1. Osteoblast (OB) experiment

Animals: newborn 1-day-old SD rats (littermates), provided by the Experimental Animal Center of Second Military Medical University.

Drugs: the extracts of M. chinensis, total flavonoids and monomeric compounds of kaempferenin are prepared from Examples 1, 2 and 3. Prepare the solution as follows:

Dissolve the extract of M. chinensis in DMSO to prepare a solution with a concentration of 1 mg/mL, and dilute it with α-MEM containing 10% fetal bovine serum to 400, 100, and 25 μg/mL before use; (referred to as extract culture base);

Dissolve the total flavonoids of M. chinensis in DMSO to prepare a solution with a concentration of 1 mg/mL, and dilute it with α-MEM containing 10% fetal bovine serum to 10, 1, 0.1 μg/mL before use; (referred to as containing total flavone medium);

Dissolve the kaempferrin monomer compound in DMSO to prepare a solution with a concentration of 10 ^-2 mol/L, and dilute it with α-MEM containing 10% fetal bovine serum to 10 ^-8 , 10 ^-9 , 10 ^{- 10} mol/L. (referred to as kaempferol-containing medium).

Main reagents:

α-MEM medium, type II collagenase, special grade fetal bovine serum, and trypsin are products of Gibco in the United States;

Dexamethasone was purchased from Sigma, USA;

Reagents such as NaCl, Na ₂ HPO ₄ , NaH ₂ PO ₄ , and NaHCO ₃ were all domestic analytical grade.

experimental method:

1) Preparation of osteoblasts:

The cranial parietal bones of 5 newborn SD rats were taken, shredded, digested with 0.25% trypsin at 37°C for 30min, and then digested with 0.05% trypsin and 3mg/mL type II collagenase at 37°C for 1h. Collect the supernatant, filter it through a nylon mesh, collect the filtrate, centrifuge at 1000rpm for 10min, discard the supernatant, collect the cells, which are fresh osteoblasts, and place them in α-MEM medium containing 10% fetal bovine serum at 37°C as usual After culturing, the medium was changed after 24 hours, and the medium was changed every 3 days thereafter.

2) Osteoblast proliferation test:

The experiment was divided into 11 groups, namely negative control group, positive control group, high, medium and low dose groups of extract, high, medium and low dose groups of total flavonoids and high, medium and low dose groups of kaempferolin.

Take a 96-well culture plate as usual, inoculate 100 μL/well of the osteoblast suspension with a concentration of ² ×10 cells/mL in the 96-well culture plate, culture at 37°C for 24 h, and suck out the culture medium in each well. 100 μL/well of α-MEM medium containing 10% fetal bovine serum was added to the negative control group; 100 μL/well of α- ^MEM medium containing 10% fetal bovine serum was added to the positive control group at wells; extract high, medium and low dose groups were added with 100 μL/well of extract medium with concentrations of 400, 100 and 25 μg/mL; total flavonoids were added with concentrations of 10, 1 and 0.1 μg/mL 100 μL/well of total flavonoid culture medium; 100 μL/well of kaempferol medium with concentrations of 10 ^-10 , 10 ^-9 , and 10 ^-8 mol/L were added to the high, medium and low dose groups of kaempferol, respectively. There are 5 duplicate wells in the wells, and continue to culture in a 37°C incubator to observe the effect of drug action on cell proliferation for 24 hours. 4 hours before detection, add 20 μL of MTT solution to each well, put it in the incubator and continue to incubate for 4 hours, take out the culture plate, discard the supernatant, add 150 μL of dimethyl sulfoxide (DMSO) to each well, shake for 5-10 minutes, The OD value was detected at 570nm with a microplate reader. The results are shown in Table 1. It can be seen from Table 1 that, compared with the negative control group, the extract, total flavonoids, and high, medium, and low dose groups of kaempferol could significantly promote osteoblast proliferation (P<0.05).

3) Osteoblast alkaline phosphatase (ALP) activity test:

The experiment was divided into 11 groups, namely negative control group, positive control group, high, medium and low dose groups of extract, high, medium and low dose groups of total flavonoids and high, medium and low dose groups of kaempferolin.

Take osteoblasts and inoculate 100 μL/well of a cell suspension with a concentration of 2×10 ⁴ cells/mL in a 96-well culture plate, culture at 37°C for 24 hours, suck out the medium in each well, and add 10% fetal bovine Serum α-MEM medium 100 μL/well; positive control group was added 100 μL/well α-MEM medium containing 10% fetal bovine serum with a concentration of 10 ^-8 mol/L genistein; extracts of high, medium and low Add 100 μL/well of extract medium with concentrations of 400, 100, and 25 μg/mL to the dose group; add 100 μL/well of total flavonoid medium with concentrations of 10, 1, and 0.1 μg/mL to the high, medium, and low dose groups of total flavonoids; Add 100 μL/well of kaempferrin culture medium with a concentration of 10 ^-10 , 10 ^-9 , and 10 ^-8 mol/L to the kaempferolin high, medium, and low dose groups respectively, and each well in each group has 5 duplicate wells, and continue Place in a 37°C incubator for culture, and change the medium containing the corresponding drug once on the 4th day. After culturing until the 6th day, add 100 μL of diethanolamine with a concentration of 50 mmol/L and 2.5 mmol/L p-Nitrate to each well after removing the medium. 50 μL of disodium phenol phosphate, reacted at 37°C for 30 minutes, then terminated the reaction with 0.3 mol/L NaOH 100 μL/well, and measured the OD value at a wavelength of 405 nm with a microplate reader. Use the OD value of p-nitrophenol solutions of different concentrations at 405nm as a standard curve, and obtain the nmol number of p-nitrophenol released from each hole from the standard curve, and the nmol number of p-nitrophenol released from each hole represents the concentration of ALP. Activity (see Table 1). It can be seen from Table 1 that, compared with the negative control group, the high-dose group of the extract of M. chinensis, the high-dose group of total flavonoids of M. edulis and the high-dose group of kaempferin all have the effect of increasing the ALP activity of osteoblasts. (P<0.05).

2. Osteoclast (OC) experiment

Animals: Newborn 3-day-old SD rats (littermates), provided by the Experimental Animal Center of Second Military Medical University.

Drugs: Same as above.

Main reagents:

α-MEM medium, collagenase type II, special grade fetal bovine serum, and trypsin are products of Gibco in the United States;

1,25-Dihydroxyvitamin D ₃ (1,25-(OH) ₂ VitaminD ₃ ) and dexamethasone were purchased from Sigma, USA;

Potassium sodium tartrate, etc. are all domestic analytical pure reagents are domestic analytical pure.

Osteoclast induction medium: this is α-MEM medium containing 10 ^-8 mol/L 1,25-(OH) ₂ -VD ₃ , 10 ^-7 mol/L dexamethasone and 10% fetal bovine serum.

Dissolve the extract of Lemonia chinensis in DMSO to prepare a solution with a concentration of 1 mg/mL, and dilute it with osteoclast induction medium to 400, 100, and 25 μg/mL before use; (abbreviated as extract osteoclast induction medium );

Dissolve the total flavonoids of M. chinensis with DMSO to prepare a solution with a concentration of 1 mg/mL, and dilute it with osteoclast induction medium to 10, 1, 0.1 μg/mL before use; (abbreviated as total flavonoids osteoclast induction medium);

Dissolve kaempferrin monomer compound in DMSO to prepare a solution with a concentration of 10 ^-2 mol/L, and dilute it with osteoclast induction medium to 10 ^-8 , 10 ^-9 , 10 ^-10 mol/L before use . (referred to as kaempferolin osteoclast induction medium).

experimental method:

1) Preparation of osteoclasts:

Select 5 newborn 3-day-old SD rats, separate the tibia, wash the bone marrow cavity with osteoclast induction medium, wash out the cells in the bone marrow, collect the washing liquid, discard the supernatant by centrifugation, and wash the cells with PBS buffer for 2 Once, fresh bone marrow cells are obtained, which are bone marrow mononuclear cells. Suspend fresh bone marrow cells and third-generation osteoblasts in osteoclast induction medium to form a cell suspension, in which the concentration of osteoblasts is ¹ ×105 cells/mL, and the concentration of bone marrow mononuclear cells is 1 ×10 ⁶ cells/mL, inoculate the cell suspension in a 96-well culture plate, 100 μL per well, culture in a 5% CO ₂ incubator at 37°C, change the liquid every 3 days, and after 8 days, the bone marrow cells differentiate into mature of osteoclasts.

2) Osteoclast-resistant tartrate acid phosphatase (TRAP) activity test:

The experiment was divided into 11 groups, namely negative control group, positive control group, high, medium and low dose groups of extract, high, medium and low dose groups of total flavonoids and high, medium and low dose groups of kaempferolin.

Aspirate the culture medium of each well of mature osteoclasts in the above-mentioned 96-well plate, add osteoclast induction medium 100 ^μL /well to the negative control group; Cell induction medium 100 μL/well; extract high, medium and low dose groups were added with 400, 100, 25 μg/mL extract osteoclast induction medium 100 μL/well; total flavonoids high, medium and low dose groups were added Total flavone osteoclast induction medium with concentrations of 10, 1, and 0.1 μg/mL was 100 μL/well; kaempferin high-, medium-, and low-dose groups were added with concentrations of 10 ^-10 , 10 ^-9 , 10 ^-8 mol/well, respectively. L kaempferrin osteoclast induction medium 100 μL/well, set 5 duplicate wells for each group, and continue to culture in a 37°C incubator. After 48 hours of culture, the medium was discarded in each well, and 20 μL of 0.1% Triton X- Break the cells at 100°C for 15 minutes, then add 100 μL of reaction solution (0.4g disodium p-nitrophenyl phosphate, dissolve in deionized water, add 2.0g potassium sodium tartrate, add water to dissolve to 150mL, adjust the pH to 3.5 with HCl, add water to volume to 200 mL), react at 37°C for 30 min, quickly add 100 μL of 1 mol/L NaOH to terminate the reaction, and measure the OD value at a wavelength of 405 nm with a microplate reader. Use the OD value of different concentrations of p-nitrophenol solutions at 405nm as a standard curve, and obtain the nmol number of p-nitrophenol released from each well from the standard curve, and use the p-nitrophenol produced by osteoclasts in each well for TRAP activity. expressed in nmol. The results are shown in Table 1. It can be seen from Table 1 that, compared with the negative control group, ^25-400 μg/mL of the extract of M. chinensis, ^0.1-10 μg/mL of total flavonoids of M. All kaempferrins have the effect of inhibiting the activity of osteoclast TRAP (P<0.05～0.001).

Table 1. Effects of the extracts, total flavonoids and kaempferin on OB and OC cells (n=5)

***P<0.001, **P<0.01, *P<0.05, compared with the negative control group.

2. Animal experiments

animal:

3-month-old female SD rats, weighing 300±20g. purchased from Shanghai Slack Experimental Animal Co., Ltd. License number: SCXK (Shanghai) 2012-0002.

Drugs and Reagents:

The extract, kaempferin, and total flavonoids of M. chinensis are prepared from Examples 1, 2, and 3.

Positive control drug: Estradiol Valerate Tablets (Procara), France DELPHARM Lille S.A.S. Guangzhou Branch of Bayer Healthcare Co., Ltd., batch number: (224A2).

experimental method:

72 SD rats were taken and randomly divided into 9 groups, 8 in each group, namely:

Sham group (SHam);

Model control group (OVX);

Estradiol valerate positive control group (E2, 1mg/kg/d);

High-dose group of leech extract (PEH, 300mg/kg/d);

Low-dose group of leech extract (PEL, 100mg/kg/d);

High-dose group of total flavonoids of leech leech (PFH, 100mg/kg/d);

Low-dose group of total flavonoids of M. chinensis (PFL, 30mg/kg);

Kaempferoside high-dose group (PH, 30mg/kg/d);

Kaempferoside low dose group (PL, 8mg/kg/d).

Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate 0.3mL/kg, and the abdominal incision was made. Except for the sham operation group, the bilateral ovaries of the other 8 groups were removed under aseptic conditions as osteoporosis model rats. In the sham operation group, only The abdomen is cut open without removing the ovaries. According to the above dosage, the drug was dissolved in 0.5% CMC-Na and administered by feeding (10 mL/kg) for 12 consecutive weeks. The sham operation group and the model control group were fed with equal volume of 0.5% CMC-Na.

1. Determination of bone markers

After the rats were sacrificed, the right femur was quickly stripped, and the total bone mineral density (t-BMD) of the right femur was measured by dual-energy X-ray absorptiometry. said, statistical analysis using spss l6.0 software for one-way analysis of variance. Significant differences are indicated as *p<0.05, **p<0.01 and ***p<0.001. The BMD detection results of animals in each group are shown in Figure 1.

It can be seen from Fig. 1 that, except the low-dose group of the extract of the leech leech, the other administration groups can significantly increase the bone mineral density compared with the model control group (p<0.05, p<0.01).

2. Determination of serum biochemical indicators

After the last intragastric administration, the rats were fasted for 12 hours (water could not be restrained), and then the rats were anesthetized, and blood was taken from the femoral artery to measure serum TRAP and ALP levels. The results are shown in Fig. 2 and Fig. 3, Fig. 2 represents each ALP activity in the serum of animals in each group, Figure 3 shows the activity of TRAP in the serum of animals in each group.

It can be seen from Figure 2 that after 12 weeks of administration, the ALP content in the model control group was increased compared with the sham operation group, which is a characteristic of high-transition osteoporosis. Compared with the sham operation group, the ALP content of the total flavone high-dose group (PFH) and the kaempferolin high-dose group (PH) significantly increased (p<0.05), indicating that bone formation was promoted; as shown in Figure 3, compared with the model Compared with the control group, each administration group can significantly inhibit the activity of TRAP enzyme in ovariectomized rats (p<0.05～p<0.001), indicating that all of them can inhibit bone resorption.

3. Determination of urine biochemical indicators After the last administration, the rats were fasted without food and water, and the urine was collected for 12 hours to measure the contents of Ca, P and Cr. The results are shown in Table 2 below.

Table 2 Effects of each administration group on urine biochemical indicators in ovariectomized rats

As shown in Table 2, after ovariectomized rats, the Ca/Cr level in urine was significantly increased, and bone calcium loss was severe. Compared with the model control group, the urinary Ca/Cr levels in the sham operation group and each administration group were significantly reduced (p<0.001), preventing the loss of calcium in the bone tissue of ovariectomized rats; Both high and low doses of kaempferol can significantly reduce the P/Cr level in the urine of ovariectomized rats, and prevent the loss of phosphorus in the bone tissue of ovariectomized rats.

The above-mentioned experiments show that the extracts, total flavonoids or kaempferrin monomer compounds of the present invention have anti-osteoporosis activity, so they can be used to prepare medicines or foods for preventing and treating osteoporosis.

Claims (1)

1. a kind of application of mountain naphthalene glycosides monomeric compound in preventing and treating medicine for treating osteoporosis is prepared, it is characterised in that the kaempferia galamga glycosides The preparation method of monomeric compound comprises the following steps：

1) the ethanol water routinely decocting of circle common podocarpium root or leaf herb water or percent by volume no more than 95% will be dried Boil or refluxing extraction, extract solution is concentrated under reduced pressure into the medicinal extract without alcohol taste, obtain round common podocarpium root or leaf extract；

2) said extracted thing water dissolves are extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol successively into after suspension Take, butanol extraction liquid is concentrated under reduced pressure into flowing soaking paste, with the silica gel mixed sample of 100~200 mesh, dry method upper prop, with dichloromethane Alkane：Methyl alcohol is that 10: 1~1: 1 different proportion is eluted, and collects dichloromethane：Methyl alcohol is 10: 1~5: 1 eluent, is subtracted Concentration is pressed, yellow crystals are separated out after being dissolved with a small amount of methyl alcohol, yellow powder, i.e. mountain naphthalene glycosides monomer chemical combination are obtained after being cleaned with methyl alcohol Thing, its chemical structural formula is as follows：

Wherein rha is rhamnose.