CN103622980A - Application of cistanche phenylethanoid glycoside compound to preparation of drugs for treating osteoporosis and drug composition containing cistanche phenylethanoid glycoside compound - Google Patents
Application of cistanche phenylethanoid glycoside compound to preparation of drugs for treating osteoporosis and drug composition containing cistanche phenylethanoid glycoside compound Download PDFInfo
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Abstract
The invention discloses an application of a cistanche phenylethanoid glycoside compound to preparation of drugs for treating osteoporosis and a drug composition which contains the cistanche phenylethanoid glycoside compound and is used for preventing and treating the osteoporosis.
Description
Technical field
The present invention relates to the new purposes of Herba Cistanches phenethyl alcohol glycoside compounds, particularly a kind of Herba Cistanches phenethyl alcohol glycoside compounds is as application and the pharmaceutical composition of preparation treatment osteoporosis agents.
Background technology
Osteoporosis (Osteoporosis, OP) is a kind of commonly encountered diseases frequently-occurring disease, and it is to take that bone amount reduces, the microstructure degeneration of bone is feature, a kind of general skeletal diseases that causes the fragility of bone to increase and be easy to occur fracture.Along with the average life span extends, aged tendency of population develops rapidly, and osteoporotic sickness rate is more and more higher, and at present, the whole world approximately has 200,000,000 people to suffer from osteoporosis, and osteoporosis has become the important topic of medical research as worldwide disease.Therefore, find the effectively medicine for the treatment of osteoporosis disease and become study hotspot.
Herba Cistanches is the important the kidney invigorating class Chinese medicine of China, often by prescription therapeutic osteoporosis, such as Chinese patent medicine " Herba Cistanches is good for kidney ball ", Herba Cistanches, Herba Cynomorii, Stamen Nelumbinis, Radix Rehmanniae Preparata, Poria, Semen Cuscutae, Herba Epimedii etc., consists of, and cures mainly insufficiency of kidney-YANG osteoporosis; " YUBIAO BUSHEN WAN " is comprised of Air Bladder pseudosciaenae seu Acipenser, Herba Cistanches, Radix Angelicae Sinensis, Tong Fructus Tribuli, Radix Morindae Officinalis, Semen Cuscutae etc., cures mainly osteoporosis.(the Te-Mao Li such as Taiwan's scholars Te-Mao Li, Hsin-Chih Huang, Chen-Ming Su, Tin-Yun Ho, Chi-Ming Wu, Wen-Chi Chen, Yi-Chin Fong and Chih-Hsin Tang. Cistanche deserticola extract increases bone formation in osteoblasts [J]. Journal of Pharmacy and Pharmacology, 2012, 64 (6): 897-907.) the external effect with raising osteoblast alkali phosphatase (ALP) activity of report Herba Cistanches crude extract, strengthen the expression of bone morphogenesis protein-2 (BMP-2) He Guqiao (OPN) albumen, thereby promoted bone formation, in body, there is the effect that treatment mice removal ovary causes osteoporosis.Another domestic scholars was once built (Zeng Jianchun, Fan Yueguang, Liu Jianren, Zeng Yirong, Yi Chunzhi, the Yan Liang such as spring.The experimentation of Herba Cistanches Contained Serum inducing bone mesenchymal stem cell to osteoblast differentiation.China's bone injury, 2010,23 (8): 606-609.) confirming that mesenchymal stem cells MSCs can be to osteoblast differentiation under Herba Cistanches induction, seed cell and the inducible factor as organizational project has good application prospect to treatment osteoporosis, nonunion respectively.
Yet, Herba Cistanches comprises multiple compounds, for example, the compound in Herba Cistanches has: phenethyl alcohol glycosides, iridoid and glycoside thereof, wooden lipid and glycoside thereof, which kind of the compound composition not explicitly pointing out in prior art in Herba Cistanches has the effect for the treatment of osteoporosis.
Summary of the invention
The invention provides the new application of Herba Cistanches phenethyl alcohol glycoside compounds, Herba Cistanches phenethyl alcohol glycoside compounds is as the application of preparation treatment osteoporosis agents.
Also be necessary to provide a kind of osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds.
Herba Cistanches phenethyl alcohol glycoside compounds is as an application for preparation treatment osteoporosis agents, and wherein, Herba Cistanches phenethyl alcohol glycoside compounds is category-A, and the chemical structure of general formula of category-A is:
Wherein, R1: hydroxyl, methoxyl group;
R2: hydroxyl, methoxyl group;
R3: hydroxyl, methoxyl group, acetyl group;
R4: hydroxyl, methoxyl group.
Herba Cistanches phenethyl alcohol glycoside compounds is as an application for preparation treatment osteoporosis agents, and wherein, Herba Cistanches phenethyl alcohol glycoside compounds is category-B, and the chemical structure of general formula of category-B is:
Wherein, R1: hydroxyl, methoxyl group;
R2: hydroxyl, methoxyl group;
R3: hydroxyl, methoxyl group, acetyl group;
R4: hydroxyl, methoxyl group;
R5: glucosyl group, rhamanopyranosyl, aralino.
A kind of osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds, this pharmaceutical composition comprises adjuvant, active component major ingredient, the weight ratio of active component major ingredient and adjuvant is 1:99～99:1, active component major ingredient comprises category-A Herba Cistanches phenethyl alcohol glycoside compounds, and the chemical structure of general formula of category-A is:
Wherein, R1: hydroxyl, methoxyl group;
R2: hydroxyl, methoxyl group;
R3: hydroxyl, methoxyl group, acetyl group;
R4: hydroxyl, methoxyl group.
A kind of osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds, this pharmaceutical composition comprises adjuvant, active component major ingredient, the weight ratio of active component major ingredient and adjuvant is 1:99～99:1, active component major ingredient comprises category-B Herba Cistanches phenethyl alcohol glycoside compounds, and the chemical structure of general formula of category-B is:
Wherein, R1: hydroxyl, methoxyl group;
R2: hydroxyl, methoxyl group;
R3: hydroxyl, methoxyl group, acetyl group;
R4: hydroxyl, methoxyl group;
R5: glucosyl group, rhamanopyranosyl, aralino.
Above-mentioned Herba Cistanches phenethyl alcohol glycoside compounds is as the application of preparation treatment osteoporosis agents and the osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds, that inventor is in Herba Cistanches phenylethanoid glycoside research process, the phenethyl alcohol glycoside compounds of having prepared q.s from Herba Cistanches and Cistanche Tubulosa, through external skeletonization, in osteoclast and body, retinoic acid causes osteoporosis rat animal experiment, confirmed that category-A Herba Cistanches phenethyl alcohol glycoside compounds and category-B Herba Cistanches phenethyl alcohol glycoside compounds have the osteoporotic effect of control, and prepare and contain category-A, the osteoporotic pharmaceutical composition of control of category-B Herba Cistanches phenethyl alcohol glycoside compounds.
Accompanying drawing explanation
Fig. 1 is the total bone density figure of each group rat.
The specific embodiment
Inventor causes osteoporosis rat animal experiment with a plurality of Herba Cistanches phenethyl alcohol glycoside compounds that extract the q.s being separated to through retinoic acid in external skeletonization, osteoclast and body, confirm that Herba Cistanches phenylethanoid glycoside has the effect of osteoporosis, the Herba Cistanches phenethyl alcohol glycoside compounds with the effect of osteoporosis has A, B two classes, wherein:
The chemical structure of general formula of category-A is:
Wherein, R1: hydroxyl (OH), methoxyl group (OMe); R2: hydroxyl (OH), methoxyl group (OMe); R3: hydroxyl (OH), methoxyl group (OMe), acetyl group (Ac); R4: hydroxyl (OH), methoxyl group (OMe).Further, A can be any one in different verbascoside (Isoacteoside), pipe flower glycosides B (Tubuloside B), Herba Plantaginis glycosides C (Plantainoside C), different boschnaloside C (Isocistanoside C), the new glycosides of different Radix campsis (Isocampneoside), and the structural formula of above-claimed cpd is as follows:
The chemical structure of general formula of B is:
Wherein, R1: hydroxyl (OH), methoxyl group (OMe); R2: hydroxyl (OH), methoxyl group (OMe); R3: hydroxyl (OH), methoxyl group (OMe), acetyl group (Ac); R4: hydroxyl (OH), methoxyl group (OMe); R5: glucosyl group (Glc), rhamanopyranosyl (Rha), aralino (Ara).B can be verbascoside (Acteoside), 2 '-acetyl verbascoside (2 '-acetylacteoside), cistanoside A (Cistanoside A), boschnaloside B (Cistanoside B), boschnaloside C (Cistanoside C), boschnaloside D (Cistanoside D), tubulosideA (Tubuloside A), pipe flower glycosides E (Tubuloside E), Saline Cistanche Herb glycosides D (Salsaside D), Saline Cistanche Herb glycosides E (Salsaside E), Osmanthuside B, poliumoside (Poliumoside), Cistantubuloside A (Cistanbuloside A), Cistanche Tubulosa glycosides B (Cistanbuloside B), Cistanche Tubulosa glycosides C (Cistanbuloside C), Cistansinenside A. (Cistansinenside A), 2 '-acetyl sweet-scented osmanthus glycosides (2 '-acetyllosmanthuside), 2 '-acetyl poliumoside (2 '-acetylpoliumoside), ligustrin A 3 '-α-L-pyrans rhamnoside (Syringalide A 3 '-alpha-rhamnopyanoside), different ligustrin A 3 '-α-L-pyrans rhamnoside (Isosyringalide A 3 '-alpha-rhamnopyanoside), Kankanoside H, Kankanoside J, Kankanoside K, Arenarisoide, Wiedemanninoside C, the new glycosides I of Radix campsis (Campneoside I), any one in the new glycosides II of Radix campsis (Campneoside II), the structural formula of above-claimed cpd is as follows:
Further, above-mentioned A, B two class Herba Cistanches phenethyl alcohol glycoside compounds all can be made the osteoporotic pharmaceutical composition of control, are specially:
The osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds, this pharmaceutical composition comprises adjuvant, active component major ingredient, the weight ratio of active component major ingredient and adjuvant is 1:99～99:1.Adjuvant is any acceptable excipient, for example starch, lactose, microcrystalline Cellulose, dextrin, hyprolose, polyvinylpolypyrrolidone, pregelatinized Starch, Pulvis Talci, magnesium stearate, micropowder silica gel, sodium lauryl sulphate, Tween 80, hydrogenated vegetable wet goods on medicament.Active component major ingredient comprises category-A Herba Cistanches phenethyl alcohol glycoside compounds.Further, active component major ingredient also comprises category-B Herba Cistanches phenethyl alcohol glycoside compounds, and the weight ratio of category-A Herba Cistanches phenethyl alcohol glycoside compounds and category-B Herba Cistanches phenethyl alcohol glycoside compounds is 20:80～80:20.
The osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds, this pharmaceutical composition comprises adjuvant, active component major ingredient, the weight ratio of active component major ingredient and adjuvant is 1:99～99:1.Adjuvant is any acceptable excipient, for example starch, lactose, microcrystalline Cellulose, dextrin, hyprolose, polyvinylpolypyrrolidone, pregelatinized Starch, Pulvis Talci, magnesium stearate, micropowder silica gel, sodium lauryl sulphate, Tween 80, hydrogenated vegetable wet goods on medicament.Active component major ingredient comprises category-B Herba Cistanches phenethyl alcohol glycoside compounds.Further, active component major ingredient also comprises category-A Herba Cistanches phenethyl alcohol glycoside compounds, and the weight ratio of category-B Herba Cistanches phenethyl alcohol glycoside compounds and category-A Herba Cistanches phenethyl alcohol glycoside compounds is 20:80～80:20.
In Herba Cistanches phenethyl alcohol glycoside compounds, different substituted radicals has directly determined the active power of compound and has had or not, as (Yang Jianhuas such as Yang Jianhuas, Hu Junping, hot Na. Ka Simu, stifled year is raw. the structure activity study of six kinds of phenethyl alcohol glycosides compound with oxidation resistance activity in Cistanche Hoffmgg. et Link plants. and Chinese crude drug, 2009,32 (7): 1067-1069.) research different chemical structures Phenylethanoid glycosides antioxidant activity there are differences; the replacement of acetyl group on phenolic hydroxyl group number on its activity and aglycon and phenylpropenoyl, phenolic hydroxyl group link position, the sterically hindered size of compound, center 2 position of glucose; and the α on styrene acyl group, beta unsaturated ketone structure fragment has relation.Therefore, the inventor carries out animal experiment in cell in vitro or body by the phenethyl alcohol glycoside compounds to preparing from Herba Cistanches and screens, and confirms the effect that they have osteoporosis, and concrete experiment sieving process is as follows:
Embodiment 1: prepare verbascoside (acteoside); Different verbascoside (isoacteoside); The sweet A of Herba Cistanches (cistanoside A); 2 '-2'-acetylosmanthuside (2 '-acetylosmanthuside); Pipe flower glycosides B (tubuleside B).
Get dry medical material 30 kg of Yongning Ningxia Herba Cistanches planting base cultivation Herba Cistanches, with 60% ethanol 100 L soaking at room temperature 48 h, reflux, extract, 3 times, each 3 h, merge extractive liquid,, drying under reduced pressure obtains concentrated solution.Concentrated solution is successively with petroleum ether, water-saturated n-butanol extraction, abandon petroleum ether part, by concentrated rear macroporous adsorbent resin, successively water, 10% ethanol, 30% ethanol, 50% ethanol, 95% ethanol elution crossed of n-butanol portion, discard water elution liquid, collect respectively different concentration ethanol eluent.
1,10% ethanol elution is carried out to silica gel column chromatography; with methylene chloride-methanol solvent system eluting; collect respectively methylene chloride-methanol 3:1 and 1:1 eluent; and through gel filtration chromatography; reconcentration and recrystallization, obtain 2 '-2'-acetylosmanthuside (2 '-acetylosmanthuside), 620 mg (purity >95%).
2,30% ethanol elution is carried out to silica gel column chromatography, with methylene chloride-methanol solvent system eluting, collect respectively methylene chloride-methanol 5:1 and 2:1 eluent, and through gel filtration chromatography, reconcentration and recrystallization, obtain respectively verbascoside (acteoside) 3420 mg (purity >95%), the sweet A of Herba Cistanches (cistanoside A) 2140 mg (purity >95%) and different verbascoside (isoacteoside) 1330 mg (purity >95%).
3,50% ethanol elution is carried out to silica gel column chromatography, with methylene chloride-methanol solvent system eluting, collect respectively methylene chloride-methanol 6:1 and 3:1 eluent, and through gel filtration chromatography, reconcentration and recrystallization, must manage flower glycosides B (tubuleside B) 530 mg (purity >95%).
Preparation and the said method of all the other compounds are basic identical.
Embodiment 2: the experiment of inside and outside anti-osteoporosis activity
One, cell in vitro activity experiment
The In vitro cell experiment method of Herba Cistanches phenethyl alcohol glycoside compounds is basic identical, and details are as follows as example to take verbascoside, different verbascoside, cistanoside A, 2 '-2'-acetylosmanthuside and pipe flower glycosides B:
1, osteoblast (Osteoblast, OB) experiment
Animal: newborn 1 day SD rat (brood) ，You Ningxia Medical University Experimental Animal Center provides.
Main agents:
α-MEM culture medium, II Collagenase Type, superfine hyclone, trypsin ，Wei U.S. Gibco company product; Dexamethasone is purchased from U.S. Sigma company; NaCl, Na
2hPO4, NaH
2pO
4, NaHCO
3deng reagent, be domestic analytical pure.
Medicine: verbascoside (acteoside), different verbascoside (isoacteoside), the sweet A of Herba Cistanches (cistanoside A), 2 '-2'-acetylosmanthuside (2 '-acetylosmanthuside) and pipe flower glycosides B (tubuleside B) are prepared by embodiment 1.Obtain solution is as follows:
Get above-mentioned 5 kinds of tested medicines appropriate, accurately weighed, with DMSO dissolving, being mixed with concentration is respectively 10
-2the mother solution of mol/L, is diluted to 10 with the α-MEM containing 10% hyclone respectively before use again
-7, 10
-8, 10
-9the tested medicinal liquid of mol/L.
1) be prepared into osteocyte:
Get 5 of the new life SD rats of 1 day, get its calvarium lid bone, shred, 37 ℃ of digestion 30 min of 0.25% trypsin, then with 37 ℃ of digestion 1 h of II Collagenase Type of 0.05% trypsin and 3 mg/mL, collection supernatant, through nylon net filter, collect filtrate, centrifugal 10 min of 1000 rpm, abandon supernatant, collecting cell, is fresh osteoblast, puts 37 ℃ of cultivations routinely by the α-MEM culture medium containing 10% hyclone, after 24 h, change liquid, within later every 3 days, change a subculture.
2) osteoblastic proliferation test:
Getting routinely 96 well culture plates, is 2 * 10 by concentration
4the osteoblast suspension of individual/mL is inoculated in 96 well culture plates with 100 μ L/ holes, puts 37 ℃ and cultivates 24 h, absorbs the culture medium in each hole, and blank group adds the α-MEM culture medium 100 μ L/ holes containing 10% hyclone; Totally 15 groups of tested medicine groups, adding respectively concentration is 10
-9, 10
-8, 10
-75 kinds of tested pharmaceutical culture medium 100 μ L/ holes of mol/L, every group of each hole is provided with 5 multiple holes, continues to put 37 ℃ of incubators and cultivates, and observes the impact of drug effect 24 h on cell proliferation.Before detection, during 4 h, every hole adds 20 μ L MTT solution, puts into incubator and continues to hatch 4 h, take out culture plate, abandoning supernatant, every hole adds 150 μ L dimethyl sulfoxines (DMSO), concussion 5-10 minute, detects OD value by microplate reader in 570 nm places.Result shows, compares with blank group, and the high, medium and low dosage group of each tested medicine all can significantly promote osteoblastic proliferation (P < 0.05～0.001), and concrete data are in Table 1.
3) osteoblast alkali phosphatase (ALP) activity test:
Get osteoblast and take cell suspension 100 μ L/ holes that concentration is 2 * 104/mL and be inoculated in 96 well culture plates and put 37 ℃ and cultivate 24 h, absorb the culture medium in each hole, blank group adds the α-MEM culture medium 100 μ L/ holes containing 10% hyclone; Totally 15 groups of tested medicine groups, adding respectively concentration is 10
-9, 10
-8, 10
-75 kinds of tested pharmaceutical culture medium 100 μ L/ holes of mol/L, every group of each hole is provided with 5 multiple holes, continuing to put 37 ℃ of incubators cultivates, within the 4th day, change containing the culture medium of relative medicine once again, be cultured to the 6th day, it is the diethanolamine 100 μ L of 50 mmol/L that each hole is gone to add concentration after culture medium, the paranitrophenol disodium hydrogen phosphate 50 μ L of 2.5 mmol/L, 37 ℃ were reacted after 30 minutes, then used the NaOH100 μ L/ hole cessation reaction of 0.3 mol/L, by microplate reader, in wavelength 405 nm places, recorded OD value.P-nitrophenyl phenol solution with variable concentrations is made standard curve in the OD value at 405 nm places, is drawn the nmol number of the paranitrophenol that every hole discharges by standard curve, and the nmol number of the paranitrophenol that every hole discharges represents the activity of ALP.Table 1 data show, compare the effect (P < 0.05～0.001) of the raising osteoblast ALP activity of verbascoside, different verbascoside, cistanoside A, pipe flower glycosides B and the equal showed different of the high, medium and low dosage group of 2 '-acetyl sweet-scented osmanthus glycosides with blank group.
The effect of table 1 Herba Cistanches phenethyl alcohol glycoside compounds to osteoblastic proliferation and ALP enzymatic activity thereof
Compare * P < 0.05, * * P < 0.01, * * * P < 0.001. with blank
2, osteoclast (Osteoclast, OC) experiment
Animal: newborn 3 days SD rat (brood) ，You Ningxia Medical University Experimental Animal Center provide.
Medicine: the same.
Main agents:
α-MEM culture medium, II Collagenase Type, superfine hyclone, trypsin are U.S. Gibco company product; The two hydroxy-vitamine Ds of 1,25-
3(1,25-(OH)
2vitaminD
3), dexamethasone is purchased from U.S. Sigma company; It is domestic analytical pure that sodium potassium tartrate tetrahydrates etc. are domestic analytical reagent.
1) prepare osteoclast:
Choose 5 of the SD rats of newborn 3 d, separated tibia, with osteoclast inducing culture (containing 10
-8mol/L 1,25-(OH)
2-VD
3, 10
-7α-MEM culture medium of mol/L dexamethasone and 10% hyclone, lower same) rinse medullary cavity, the cell in bone marrow is gone out, collect flushing liquor, the centrifugal supernatant of abandoning, washes cell 2 times with PBS buffer, obtains fresh medullary cell, and it is myelomonocyte.By fresh medullary cell and the 3rd generation osteoblast be jointly suspended in osteoclast inducing culture and become cell suspension, wherein osteoblastic concentration is 1 * 10
5individual/mL, the concentration of myelomonocyte is 1 * 10
6individual/mL, by cell suspension inoculation in 96 well culture plates, every hole 100 μ L, in 37 ℃, 5% CO
2in incubator, cultivate, within every 3 days, change a not good liquor, after 8 days, osteoclast is ripe by Differentiation of Bone Marrow Cells.
2) osteoclast Tartrate resistant acid phosphatase (TRAP) activity test:
Get the above-mentioned osteoclast that has been inoculated in differentiation and maturation in 96 orifice plates, absorb the culture medium in each hole, blank group adds osteoclast inducing culture 100 μ L/ holes, totally 15 groups of tested medicine groups, adding respectively concentration is 10
-9, 10
-8, 10
-75 kinds of tested pharmaceutical culture medium 100 μ L/ holes of mol/L, establish 5 multiple holes for every group, continuing to put 37 ℃ of incubators cultivates, cultivate after 48 h, culture medium is abandoned in each hole, add 20 μ L0.1%Triton X-100 room temperature smudge cells 15 min, add again 100 μ L reactant liquors (0.4 g p-nitrophenyl disodium hydrogen phosphate, after deionized water dissolving, add 2.0 g sodium potassium tartrate tetrahydrates, be dissolved in water to 150 mL, HCl regulates pH to 3.5, add again water and be settled to 200 mL), in 37 ℃ of reaction 30 min, the NaOH cessation reaction that adds rapidly 100 μ L 1 mol/L, by microplate reader, in wavelength 405 nm places, measure its OD value.P-nitrophenyl phenol solution with variable concentrations is made standard curve in the OD at 405nm place value, is drawn the nmol number of the paranitrophenol that every hole discharges by standard curve, and the nmol number of the active paranitrophenol generating by every hole osteoclast of TRAP represents.The results are shown in Table 2.From table 2, compare with blank group, verbascoside, different verbascoside, cistanoside A, pipe flower glycosides B and the high, medium and low dosage group of 2 '-acetyl sweet-scented osmanthus glycosides have the effect (P < 0.05～0.001) that suppresses osteoclast TRAP enzymatic activity.
The effect of table 2 Herba Cistanches phenethyl alcohol glycoside compounds to osteoclast TRAP enzymatic activity
Compare * P < 0.05, * * P < 0.01, * * * P < 0.001. with blank
Two, zoopery
The interior animal experiment method of Herba Cistanches phenethyl alcohol glycoside compounds is basic identical, take verbascoside, details are as follows as example for cistanoside A and 2 '-2'-acetylosmanthuside:
Animal: 72 of SD rats, clean level, female, hero half and half, body weight 250 ± 20 g, purchased from Ningxia Medical University's zoopery center.
Medicine and reagent: positive control drug: Alendronate sodium sheet (Fosamax), Merck Sharp & Dohme Italia SPA produces, lot number: (110873).Tested medicine: verbascoside, the sweet A of Herba Cistanches and 2 '-2'-acetylosmanthuside embodiment, 1 preparation.Retinoic acid crude drug is purchased from Sigma company.
The preparation of medicine: above positive control drug and each tested medicine are made into respectively the concentration needing as solvent by 0.5% Sodium Tvlose.
Experimental technique: get 72 of SD rats, be divided at random 9 groups, 8 every group, that is: negative control group (giving equal-volume 0.5% CMC-Na); Model control group (giving equal-volume 0.5% CMC-Na); Positive drug group (giving Alendronate sodium sheet, 1 mg/kg, 1 time weekly); Verbascoside high and low dose group (dosage is respectively 40,10 mg/kg/d); Cistanoside A high and low dose group (dosage is respectively 40,10 mg/kg/d); 2 '-2'-acetylosmanthuside high and low dose group (dosage is respectively 40,10 mg/kg/d).After medicine being dissolved in to 0.5%CMC-Na by above-mentioned dosage, fill with hello administration (10 mL/kg), negative and model control group gavage equal-volume 0.5%CMC-Na.
Except negative control group, all the other are respectively organized rat every morning gavage and give 70 mg/kg/d retinoic acid, afternoon positive control drug group and be respectively subject to reagent group rat by grouping dosage gastric infusion respectively, once a day, after successive administration 14 days, the retinoic acid of stopping using, positive control drug group and continued by the dosage gastric infusion to 30 day that divides into groups by reagent group rat.After last 1 administration, every rat is placed in respectively to metabolic cage, water is can't help in fasting, within the 2nd, 3 days, carries out urine collecting, and the time is 3 days 8:00 a.m. of the 2nd day 20:00 a.m. to the.Collect after urine, every rats by intraperitoneal injection 10% chloral hydrate is anaesthetized rear right common femoral artery and is got blood, the centrifugal serum that obtains after standing 2 h.Peel off rapidly hind leg right side femur and tibia, wherein tibia meat is rejected, and femur and tibia are wrapped with masking foil.It is standby that above urine, serum and each tissue samples are all placed in 20 ° of C refrigerators in time.
1, bone index determining
Rat peels off rapidly right side femur after putting to death, and utilizes dual-energy x-ray borne densitometers to measure total bone density (t-BMD) of right side femur, detects data and all uses
± SD represents, statistical analysis carries out one factor analysis of variance with spss l6.0 software.Significant difference is expressed as * p<0.05, * * p<0.01 and * * * p<0.001.Each treated animal bone density BMD testing result is shown in Fig. 1; As seen from Figure 1, each administration group is compared with model control group, all can significantly improve bone density (p<0.05, p<0.01), i.e. it is effective that verbascoside, cistanoside A and 2 '-acetyl sweet-scented osmanthus glycosides control retinoic acid causes osteoporosis rat.
2, urine Biochemical Indexes
After the last administration of rat, water is can't help in fasting, collects 12 h urines, measures Ca and P content, the results are shown in following table 3.
Table 3 respectively organize calcium in rat urine (Ca) and phosphorus (P) content (n=8,
± SD)
Compare * P < 0.05, * * P < 0.01, * * * P < 0.001. with model control group
As shown in table 3, after ovariectomized rats, in urine, Ca and P level significantly raise, and bone calcium phosphorus is lost serious.With model control group comparison, verbascoside, cistanoside A and 2 '-acetyl sweet-scented osmanthus glycosides high and low dose group urine Ca and P level significantly reduce (p <0.01, p <0.001), in prevention retinoic acid rat bone tissue, the loss of calcium and phosphorus, prevents osteoporotic generation.
Claims (10)
1. Herba Cistanches phenethyl alcohol glycoside compounds is as an application for preparation treatment osteoporosis agents, and wherein, Herba Cistanches phenethyl alcohol glycoside compounds is category-A, and the chemical structure of general formula of category-A is:
Wherein, R1: hydroxyl, methoxyl group;
R2: hydroxyl, methoxyl group;
R3: hydroxyl, methoxyl group, acetyl group;
R4: hydroxyl, methoxyl group.
2. Herba Cistanches phenethyl alcohol glycoside compounds as claimed in claim 1, as the application of preparation treatment osteoporosis agents, is characterized in that: category-A is any one in different verbascoside, pipe flower glycosides B, Herba Plantaginis glycosides, different boschnaloside C, the new glycosides of different Radix campsis.
3. Herba Cistanches phenethyl alcohol glycoside compounds is as an application for preparation treatment osteoporosis agents, and wherein, Herba Cistanches phenethyl alcohol glycoside compounds is category-B, and the chemical structure of general formula of category-B is:
Wherein, R1: hydroxyl, methoxyl group;
R2: hydroxyl, methoxyl group;
R3: hydroxyl, methoxyl group, acetyl group;
R4: hydroxyl, methoxyl group;
R5: glucosyl group, rhamanopyranosyl, aralino.
4. Herba Cistanches phenethyl alcohol glycoside compounds as claimed in claim 3, as the application of preparation treatment osteoporosis agents, is characterized in that: B be verbascoside, 2 '-acetyl verbascoside, cistanoside A, boschnaloside B, boschnaloside C, boschnaloside D, tubulosideA, pipe flower glycosides E, Saline Cistanche Herb glycosides D, Saline Cistanche Herb glycosides E,
osmanthuside B , any one in poliumoside, Cistantubuloside A, Cistanche Tubulosa glycosides B, Cistanche Tubulosa glycosides C, Cistansinenside A., 2 '-acetyl sweet-scented osmanthus glycosides, 2 '-acetyl poliumoside, ligustrin A 3 '-α-L-pyrans rhamnoside, different ligustrin A 3 '-α-L-pyrans rhamnoside, Kankanoside H, Kankanoside J, Kankanoside K, Arenarisoide, Wiedemanninoside C, the new glycosides I of Radix campsis, the new glycosides II of Radix campsis.
5. the osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds, it is characterized in that: this pharmaceutical composition comprises adjuvant, active component major ingredient, the weight ratio of active component major ingredient and adjuvant is 1:99～99:1, active component major ingredient comprises category-A Herba Cistanches phenethyl alcohol glycoside compounds, and the chemical structure of general formula of category-A is:
Wherein, R1: hydroxyl, methoxyl group;
R2: hydroxyl, methoxyl group;
R3: hydroxyl, methoxyl group, acetyl group;
R4: hydroxyl, methoxyl group.
6. the osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds as claimed in claim 5, is characterized in that: category-A is any one in different verbascoside, pipe flower glycosides B, Herba Plantaginis glycosides, different boschnaloside C, the new glycosides of different Radix campsis.
7. the osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds as claimed in claim 5, it is characterized in that: active component major ingredient also comprises category-B Herba Cistanches phenethyl alcohol glycoside compounds, the weight ratio of category-A Herba Cistanches phenethyl alcohol glycoside compounds and category-B Herba Cistanches phenethyl alcohol glycoside compounds is 20:80～80:20, and the chemical structure of general formula of category-B Herba Cistanches phenethyl alcohol glycoside compounds is:
Wherein, R1: hydroxyl, methoxyl group;
R2: hydroxyl, methoxyl group;
R3: hydroxyl, methoxyl group, acetyl group;
R4: hydroxyl, methoxyl group;
R5: glucosyl group, rhamanopyranosyl, aralino.
8. the osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds, this pharmaceutical composition comprises adjuvant, active component major ingredient, the weight ratio of active component major ingredient and adjuvant is 1:99～99:1, active component major ingredient comprises category-B Herba Cistanches phenethyl alcohol glycoside compounds, and the chemical structure of general formula of category-B is:
Wherein, R1: hydroxyl, methoxyl group;
R2: hydroxyl, methoxyl group;
R3: hydroxyl, methoxyl group, acetyl group;
R4: hydroxyl, methoxyl group;
R5: glucosyl group, rhamanopyranosyl, aralino.
9. the osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds as claimed in claim 8, is characterized in that: B be verbascoside, 2 '-acetyl verbascoside, cistanoside A, boschnaloside B, boschnaloside C, boschnaloside D, tubulosideA, pipe flower glycosides E, Saline Cistanche Herb glycosides D, Saline Cistanche Herb glycosides E,
osmanthuside B , any one in poliumoside, Cistantubuloside A, Cistanche Tubulosa glycosides B, Cistanche Tubulosa glycosides C, Cistansinenside A., 2 '-acetyl sweet-scented osmanthus glycosides, 2 '-acetyl poliumoside, ligustrin A 3 '-α-L-pyrans rhamnoside, different ligustrin A 3 '-α-L-pyrans rhamnoside, Kankanoside H, Kankanoside J, Kankanoside K, Arenarisoide, Wiedemanninoside C, the new glycosides I of Radix campsis, the new glycosides II of Radix campsis.
10. the osteoporotic pharmaceutical composition of control that contains Herba Cistanches phenethyl alcohol glycoside compounds as claimed in claim 8, it is characterized in that: active component major ingredient also comprises category-A Herba Cistanches phenethyl alcohol glycoside compounds, the weight ratio of category-B Herba Cistanches phenethyl alcohol glycoside compounds and category-A Herba Cistanches phenethyl alcohol glycoside compounds is 20:80～80:20, and the chemical structure of general formula of category-A Herba Cistanches phenethyl alcohol glycoside compounds is:
Wherein, R1: hydroxyl, methoxyl group;
R2: hydroxyl, methoxyl group;
R3: hydroxyl, methoxyl group, acetyl group;
R4: hydroxyl, methoxyl group.
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