CN101366721B - Bulk medicament for treating orthopedic disorders and preparation method thereof - Google Patents
Bulk medicament for treating orthopedic disorders and preparation method thereof Download PDFInfo
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- CN101366721B CN101366721B CN2008100303600A CN200810030360A CN101366721B CN 101366721 B CN101366721 B CN 101366721B CN 2008100303600 A CN2008100303600 A CN 2008100303600A CN 200810030360 A CN200810030360 A CN 200810030360A CN 101366721 B CN101366721 B CN 101366721B
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Abstract
The invention discloses a bulk drug for treating orthopaedic diseases. The bulk drug is Chinese teasel root saponin VI and is prepared by extracting traditional Chinese medicinal materials which contain the Chinese teasel root saponin VI; the weight percentage content of the Chinese teasel root saponin VI occupies 95.0 to 99.9 percent of the total amount of an extract of the Chinese teasel root saponin VI; and the chemical name of the bulk drug is 3-O-alpha-L-arabopyranose ivy sapogenin-28-O-beta-D-glucopyranose(1-6)-beta-D-glucopyranoside. The invention also discloses a method for preparing the bulk drug - the Chinese teasel root saponin VI. The method comprises the following steps of extraction, concentration, separation of the total saponin, decolorization and purification and refinement. The preparation method has the advantages that the preparation method has high extraction rate and low cost, is safe and environment-friendly and is suitable for industrialized mass production. The invention also provides application of the bulk drug in preparing medicines for preventing and treating the orthopaedic diseases including osteoporosis, fracture and osteoarthritis.
Description
Technical field
The present invention relates to a kind of crude drug for the treatment of orthopaedic disease, the invention still further relates to a kind of preparation method for the treatment of the crude drug of orthopaedic disease simultaneously.
Background technology
Radix Dipsaci is the dry root of Dipsacaceae plant Radix Dipsaci Dipsacus asperoides.Y.Cheng et T.M.Ai.Beginning is stated from Shennong's Herbal, classify as top grade, put down in writing its " bitter in the mouth, tepor, main typhoid fever, tonifying for the deficiency, incised wound, carbuncle and ulcer, folding falls, reuniting the fractured tendons and bones, married woman breast is difficult.Obey physical strength profiting for a long time.A Long Dou, one belongs to folding." the Li Shizhen (1518-1593 A.D.) meaning " Radix Dipsaci, genus folding, synthetism, all also with the merit name." name visible Radix Dipsaci just to be the orthopedics department key medicine from it from ancient times.Modern pharmacopeia record: " bitter in the mouth, suffering, tepor.Return liver, kidney channel.Invigorating the liver and kidney, bone and muscle strengthening, continuous folding is hindered, and ends metrorrhagia.Be used for soreness of the waist and knees, rheumatic arthralgia, metrorrhagia, vaginal bleeding during pregnancy, injury from falling down.Prepared RADIX DIPSACI with yellow rice wine is used for rheumatic arthralgia more, injury from falling down ".It serves to show that Radix Dipsaci is from ancient times for effective medicine of orthopaedic disease.
Recent studies shows in the Radix Dipsaci there be main chemical compositions: saponins, iridoids, alkaloid, volatilization wet goods composition.The inventor can reach 10.0%～16.0% to the mensuration content of many batches of Radix Dipsaci input total saponins of Hubei or Sichuan product, and wherein based on asperosaponin VI content, asperosaponin VI content is no less than 2.0% in the version Chinese Pharmacopoeia regulation Radix Dipsaci medical materials in 2005.Asperosaponin VI claims akebin D again, does not exist only in the Radix Dipsaci medical material, also exists in Caulis Akebiae, Fructus Akebiae, the Flos Lonicerae, and wherein content is also higher in the Caulis Akebiae.Though from the Radix Dipsaci medical material, get more than 20 kind of saponin constituent so far, do not see the preparation method report of the high-purity asperosaponin VI that is applicable to suitability for industrialized production, there is not the people that high-purity asperosaponin VI is reported in the orthopaedic disease application facet yet.The Radix Dipsaci medical material is usually used in soreness of waist and knee joint, rheumatic arthralgia, traumatic injury and antiabortive in traditional medicine, modern pharmacology studies show that Radix Dipsaci has effects such as promoting bone injury healing, osteoporosis, defying age, antibiotic, anti-inflammatory and antalgic, immunomodulating.Asperosaponin VI as the Radix Dipsaci medicine just in one of the representative composition of effective ingredient, the highly purified asperosaponin VI of prepared by suitable reaches to the requirement of an international kind new medicine, will be expected to become a domestic and international original new drug.
Summary of the invention
The purpose of this invention is to provide a kind of crude drug for the treatment of orthopaedic disease---asperosaponin VI.
Another object of the present invention provides a kind of Environmental Safety, extraction ratio height, can be used for the preparation method of the asperosaponin VI of suitability for industrialized production.
The crude drug asperosaponin VI of treatment orthopaedic disease is contained the medicinal material extract preparation of asperosaponin VI and is got by Radix Dipsaci or other, and its percetage by weight accounts for 95.0%～99.9% of extract total amount.
The invention provides a kind of crude drug for the treatment of orthopaedic disease, this crude drug is asperosaponin VI, extract preparation and get by the Chinese crude drug that contains asperosaponin VI, the percetage by weight content of asperosaponin VI accounts for 95.0～99.9% of asperosaponin VI extract total amount, its chemical name is 3-O-α-L-arabopyranose helexin-28-O-β-D-Glucopyranose. (1-6)-β-D-pyranglucoside, and chemical structural formula is:
Orthopaedic disease of the present invention is meant osteoporosis, fracture, osteoarthritis or hyperosteogeny.
The Chinese crude drug that contains asperosaponin VI of the present invention is meant Radix Dipsaci, Caulis Akebiae or Fructus Akebiae.
Crude drug of the present invention can be made solid preparation or liquid preparation with mixing acceptable accessories, wherein said solid preparation is tablet, capsule, granule, drop pill, patch or injection lyophilized powder, and described liquid preparation is oral liquid, soft capsule, unguentum or injection.
The present invention also provides a kind of effective ways that extract asperosaponin VI from Chinese crude drug, and asperosaponin VI weight percentage is up to 95.0～99.9% in the extraction gained sample, and this purity belongs to very high scope in the present technique field.This extracting method comprises the following steps:
1) extracts, concentrates: get the Chinese crude drug that contains asperosaponin VI,, filter, get extracting solution A with lower alcohol or aqueous lower alcohol extraction; With gained extracting solution A reclaim under reduced pressure alcohol, being concentrated into relative density is 1.20～1.40, gets concentrated solution B;
2) concentrated solution is handled: concentrated solution B thin up is become every milliliter of solution that contains 0.3～1.5g raw medicinal herbs, add alkali and regulate pH value to 7.0～10.0, left standstill 4～36 hours, filtration or centrifugal divides and gets supernatant, gets solution C;
3) separate total saponins: solution C is splined on the chromatographic column that macroporous resin is housed, circulation absorption 2～3 times, wash with water to nearly neutrality behind 0.5～5.0% the alkali liquor eluting with 2～3 times of amount column volumes, 10～40% ethanol elutions of 2～4 times of amounts of reuse column volume discard eluent; With the 50-95% ethanol elution of 4～8 times of amount column volumes, collect eluent at last, get total saponins extracting solution D;
4) decolouring: with total soap extracting solution D peroxidating aluminum or decolorizing resin post or adopt activated carbon to decolour; With the total saponins extracting solution reclaim under reduced pressure alcohol after the decolouring, concentrate, drying gets asperosaponin VI crude product;
5) purification, refining asperosaponin VI: asperosaponin VI crude product water or aqueous lower alcohol are made solution, further adopt column chromatographic isolation and purification or adopt opposed polarity solvent fractional extraction purification refine promptly to get asperosaponin VI.
In the above-mentioned preparation method, the carbon number of lower alcohol is C1～C4 in described step 1) and the step 5), and concentration is X, 0%＜X≤100%, preferred alcohol of the present invention and n-butyl alcohol; Extracting method in the described step 1) is decocting method, heating reflux method, solvent extraction method or percolation; Described step 2) and the alkali liquor in the step 3) be NaOH, KOH, Ca (OH)
2, K
2CO
3, Na
2CO
3, NaHCO
3, one or more the mixture in ammonia and the triethylamine; The used macroporous resin of separation total saponins is one or both among SA-1, SA-2, ADS-5, ADS-7, ADS-8, AB-8 and the D201 in the described step 3), the preferred SA-2 of the present invention, SA-2 and ADS-5; Process for purification refine in the described step 5) can adopt one of following method or mix and use:
1) asperosaponin VI crude product is dissolved with eluent, be splined on the polydextran gel column chromatography for separation behind the centrifugal or microfiltration, eluent is 5～80% alcohol, the employing fraction collector is collected, the master is contained asperosaponin VI flow point collect, merge reclaim under reduced pressure alcohol, concentrate drying promptly gets and makes with extra care asperosaponin VI;
2) asperosaponin VI crude product is dissolved with eluent, be splined on reversed phase column chromatography behind the centrifugal or microfiltration and separate, eluent is 20～90% alcohol, the employing fraction collector is collected, and the master is contained asperosaponin VI flow point collect, and merges, reclaim under reduced pressure alcohol, concentrate drying promptly gets and makes with extra care asperosaponin VI;
3) asperosaponin VI crude product water is made every milliliter of solution that contains 50～300mg crude product, with adjusting PH with base value to 8.0～11.0, for several times earlier with the ethyl acetate that contains alcohol, dichloromethane or chloroform extraction; Reuse n-butyl alcohol or water are full to close n-butanol extraction for several times, merges butanol extraction liquid; Reuse water backwash butanol extraction liquid 2～4 times; Placed 12～48 hours reclaim under reduced pressure n-butyl alcohol to 1/5～1/3 back of original volume, remove precipitation, continue to concentrate, dry, promptly get asperosaponin VI, be lower than 95% as purity, adopting aqueous carbon number is that lower alcohol or the mixed organic solvents of C1～C4 carries out recrystallization, promptly gets and makes with extra care asperosaponin VI.
The present invention studies the step poly-1 in the preparation process that draws total saponins) employing n-butyl alcohol or the full n-butanol extraction that closes of water, adopt the full positive alcohol extract of Heshui backwash of water or n-butyl alcohol, concentrating n-butanol extracting liquid does near, be dissolved in water and make every milliliter of medical material solution that is equivalent to 0.3～1.5g, remove insoluble matter, be splined on SA-1 or ADS-2 macroporous resin again, carry out total saponins and separate, asperosaponin VI content can reach 85.0%～95% in the gained total saponins.Because it is still very big to extract the present technical difficulty of single active ingredient from Chinese medicine or natural drug,, " medicine registration management way " regulation can declare Chinese medicine one kind new medicine greater than 90% so extracting single component purity from Chinese medicine or natural drug.Preparation method of the present invention just can reach the requirement of a class medicine in the 4th step, but for improving drug quality control, keep clinical application safety and effectiveness, be necessary very much further to improve the purity of crude drug, so preparation method of the present invention increases the step poly-5) purification refine asperosaponin VI, purpose is that acquisition purity reaches the asperosaponin VI more than 98%.
The present invention adopts mass-spectrometric technique and nuclear magnetic resonance technique, and VI carries out structural identification to gained crude drug asperosaponin, and the result shows: this product is a white powder, ESIMS m/z927.5[M-H]
-.
1H-NMR (C
5D
5N) δ: 6.25 (1H, d, J=8.2Hz, Glc1-H), 5.42 (1H, brs, H-12), 5.03 (1H, d, J=7.7Hz, Glc2-H), 4.97 (1H, d, J=7.2Hz, Ara-H), 3.18 (1H, dd, J=4.0,13.8Hz, H-3), 1.17 (3H, s, CH
3), 1.12 (3H, s, CH
3), 0.98 (3H, s, CH
3), 0.93 (3H, s, CH
3), 0.87 (3H, s, CH
3), 0.86 (3H, s, CH
3).
13C-NMR (C
5D
5N) I that sees the following form.Above data consistent with Asperosaponin VI (asperosaponin VI) spectrum data of bibliographical information (Ma Shuancheng, Chen Dechang, Zhao Haijie. Chinese yam is known sub-chemical constitution study (III), research and development of natural products, 1998,10 (3): 49-51).
Table I Asperosaponin VI and bibliographical information
13C-NMR data (C
5D
5N, δ)
| C | Asperosaponin?VI | Data in literature | C | Asperosaponin?VI | Data in |
| 1 | 38.5 | 38.76 | 26 | 17.8 | 17.54 |
| 2 | 25.7 | 26.00 | 27 | 25.7 | 26.00 |
| 3 | 81.6 | 81.87 | 28 | 176.2 | 176.48 |
| 4 | 43.1 | 43.18 | 29 | 32.7 | 33.01 |
| 5 | 47.3 | 48.13 | 30 | 23.3 | 23.61 |
| 6 | 47.8 | 48.15 | Ara | ? | ? |
| 7 | 32.2 | 32.48 | 1 | 106.3 | 106.60 |
| 8 | 39.6 | 39.87 | 2 | 72.8 | 73.06 |
| 9 | 46.7 | 47.55 | 3 | 74.4 | 74.66 |
| 10 | 36.6 | 36.89 | 4 | 69.3 | 69.59 |
| 11 | 23.5 | 23.81 | 5 | 66.6 | 66.93 |
| 12 | 122.6 | 123.00 | Glc-1 | ? | ? |
| 13 | 143.8 | 144.11 | 1 | 95.3 | 95.62 |
| 14 | 41.8 | 42.06 | 2 | 73.5 | 73.82 |
| 15 | 28.0 | 28.25 | 3 | 78.4 | 78.66 |
| 16 | 23.0 | 23.29 | 4 | 70.6 | 70.87 |
| 17 | 17.2 | 16.96 | 5 | 77.6 | 77.90 |
| 18 | 41.3 | 41.62 | 6 | 69.0 | 69.32 |
| 19 | 45.8 | 46.15 | Glc-2 | ? | ? |
| 20 | 30.4 | 30.67 | 1 | 104.9 | 105.23 |
| 21 | 33.6 | 33.89 | 2 | 74.8 | 75.10 |
| 22 | 32.4 | 32.75 | 3 | 78.0 | 78.34 |
| 23 | 64.2 | 64.42 | 4 | 71.2 | 71.45 |
| 24 | 13.2 | 13.54 | 5 | 78.1 | 78.40 |
| 25 | 15.9 | 16.17 | 6 | 62.3 | 62.58 |
Preparation method of the present invention has solved a suitability for industrialized production high-purity asperosaponin VI difficult problem, the contrast prior art has had tangible innovation, at first the asperosaponin VI purity of purification that this programme extracts improves, can reach more than 95%, can reach more than 98.5% through purified asperosaponin VI purity, more help the quality control of clinical application; Preparation method of the present invention can select to adopt reagent almost non-toxic or that toxicity is less to extract purification, the preparation efficiency height, and the medical material utilization rate is good, and production is given birth to originally lower, more environmental protection.
The present invention proposes asperosaponin VI drug application preparation aspect preparation treatment osteoporosis, fracture, osteoarthritis or hyperosteogeny, be to be main component with asperosaponin VI raw material, add the pharmaceutics acceptable auxiliary, make pharmaceutics and can accept dosage form.The indication adjuvant comprises microcrystalline Cellulose, ethyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, sodium polyacrylate, lactose, mannitol, glucose, starch, dextrin, carboxymethyl starch sodium, calcium sulfate, Pulvis Talci, magnesium stearate, sodium chloride etc.The indication dosage form can be tablet, capsule, granule, drop pill, injection lyophilized powder, oral liquid, patch, unguentum or injection.
The present invention has also further carried out relevant pharmaceutical research to extracting gained asperosaponin VI raw material (ASP-VI) medicine, and conclusive evidence asperosaponin VI is one of effective ingredient of traditional orthopaedics medicine Radix Dipsaci, and result of the test is as follows:
(1) ASP-VI can improve the mice threshold of pain in various degree, and the pain that 150mg/kg, 300mg/kg group causes the mice thermostimulation has analgesic activity, and is dosage correlation.Formalin causes the pain result of the test and shows that ASP-VI75mg/kg, 150mg/kg, three dosage of 300mg/kg have certain effect to reducing II phase reaction pain integration, compare with matched group wherein that all there were significant differences, action effect with and positive control drug suitable, positive drug and ASP-VI all do not have obvious effect to the I phase reaction.
(2) the chronic inflammatory disease test shows that ASP-VI can suppress the rat granulation tissue hyperplasia that cotton balls stimulates, compare with negative control group, ASP-VI90mg/kg, 180mg/kg all have significant difference (P＜0.01), prompting ASP-VI obviously has inhibitory action to the rat chronic proliferative inflammation, and effect is better than positive control drug.
(3) adopt rat to remove ovary and duplicate estrogen deficiency type osteoporosis and osteoporotic joint model, ASP-VI270mg/kg and XDS300mg/kg group can obviously be improved whole body bone density and the femur hardness of ovariectomy rat after the treatment in 8 weeks, shows that ASP-VI and XDS have obvious therapeutic action to estrogen deficiency type osteoporosis.The do not fracture femoral bmd of osteoporosis model group rat of the bone density of osteoporosis rat right side femur fracture model group rat is all obviously low, treatment group osteoporotic fracture rat femur bone density all has increase after treating for 8 weeks, and wherein ASP-VI270mg/kg, 90mg/kg dosage group and XDS300mg/kg group fractured bones femur density are apparently higher than model group.Measuring of osteoporotic fracture rat, the result shows that fracture rat callus calcium, phosphorus content all are starkly lower than sham operated rats, after the treatment of 8 weeks, ASP-VI270mg/kg, 90mg/kg dosage group, XDS order 300mg/kg, 100mg/kg dosage group callus calcium obviously increase; ASP-VI and XDS high dose group callus phosphorus content all significantly raise, but the ASP-VI effect is better than XDS.
Above-mentioned experimental result shows that fully medicine of the present invention all has obvious therapeutic action to osteoporosis, fracture, and pain, inflammation clinical symptoms that fracture, osteoarthritis, hyperosteogeny etc. in the orthopaedic disease are caused have obvious therapeutic action.Experimental result prompting ASP-VI not only can improve the whole body bone density of sclerotin pine rat, bone density and the effect of being improved of callus calcium, phosphorus content to fracture, and the ASP-VI effect obviously is better than XDS, and prompting APS-VI is the main effective ingredient of Radix Dipsaci saponin, and purity increases curative effect and also increases.As seen crude drug of the present invention can be applicable to prepare osteoporosis, osteoarthritis, hyperosteogeny and fracture treatment of diseases medicine.
Description of drawings
Fig. 1 is Asperosaponin VI ESI (Neg) mass spectrum
Fig. 2 is Asperosaponin VI1H-NMR spectrum
Fig. 3 is Asperosaponin VI 1H-NMR spectrum (amplifying spectrum)
Fig. 4 is Asperosaponin VI 1H-NMR spectrum (amplifying spectrum)
Fig. 5 is an Asperosaponin VI 13C-NMR spectrum
Fig. 6 is Asperosaponin VI 13C-NMR spectrum (amplifying spectrum)
Fig. 7 is Asperosaponin VI 13C-NMR spectrum (amplifying spectrum)
Fig. 8 is an Asperosaponin VI DEPT spectrum
Fig. 9 is AsperosaponinVI DEPT spectrum (amplifying spectrum)
Figure 10 is that sample purity detects the liquid phase collection of illustrative plates behind the purification
The specific embodiment
The present invention will be described below to enumerate specific embodiment and pharmacodynamics test.Embodiment only is used for that the invention will be further described, does not represent protection scope of the present invention, and nonessential modification and adjustment that other people make according to the present invention still belong to protection scope of the present invention.
Get 1 kilogram of Radix Dipsaci, use 8L70% ethanol extraction 2 times, refluxed merge extractive liquid, at every turn 2 hours, concentrating under reduced pressure reclaims ethanol to there not being the alcohol flavor, and thin up becomes every milliliter to contain 1.0g medical material liquid, transfers pH to 8～9 with 5% calcium hydroxide suspension, left standstill 12 hours, and got supernatant, filter, get filtrate.Filtrate is added on the chromatographic column of 1.0kg SA-2 macroporous resin, after circulation was adsorbed 2 times, with the 1.0%KOH eluting of 3L, the flushing of reuse water was used the 3L30% ethanol elution instead to nearly neutrality, discard preceding twice eluent; Use the 7L60% alcohol flushing at last, collect eluent, use 0.5% decolorizing with activated carbon, filter, decompression recycling ethanol, concentrated, dry, get light yellow asperosaponin VI crude product 80.9 grams.
Asperosaponin VI crude product is added the solution that water is mixed with 800ml,, contain 5% alcoholic acid ethyl acetate extraction 3 times with equal-volume earlier with 5%KOH adjust pH to 9.0～10.0; Reuse equal-volume water is full to close n-butanol extraction 4 times, merges butanol extraction liquid.Behind the full Heshui backwash butanol extraction liquid of reuse n-butyl alcohol 3 times, add 60 ℃ of insulations of 0.5% activated carbon decolouring in 4 hours, filter, filtrate decompression reclaims n-butyl alcohol to 2400ml, and standing over night is got supernatant and continued to concentrate, drying promptly gets asperosaponin VI28.3g, purity 96.8%.
Get 1 kilogram of Radix Dipsaci, pulverize, begin percolation after closing the n-butyl alcohol soaked overnight with 6L water is full, collect n-butyl alcohol, with the full water extraction of equal-volume n-butyl alcohol 3 times, the reclaim under reduced pressure n-butyl alcohol is done near, and thin up becomes every milliliter to contain 0.8g medical material liquid, left standstill 12 hours, filter, get filtrate, filtrate is added on the chromatographic column of 1.5kg ADS-5 macroporous resin, after the circulation absorption 3 times, with the 0.5%NaOH eluting of 3L, the flushing of reuse water is used the 3L30% ethanol elution instead to nearly neutrality, discard preceding twice eluent, use the 5L70% alcohol flushing at last, collect 70% ethanol elution, after the D208 decolorizing resin decolours excessively, decompression recycling ethanol, concentrate, drying gets faint yellow asperosaponin VI crude product 52.6 grams.
With 200g reversed-phase column ODS column packing (Grace, 50 μ m) with after the methanol soaked overnight, the dress post (5.0cm (i.d.) * 80cm), treat that the post bed is stable after, change the solvent system in the post into 50% methanol, solvent is put done again to packing layer, standby.Taking by weighing asperosaponin VI crude product 5g is dissolved in 50% methanol of about 20ml, slowly be added on packing layer on along post jamb sample liquid with suction pipe, open plunger, after treating that sample liquid is infiltrated the post bed, use 2000ml50% methanol successively, the 2000ml70% methanol-eluted fractions, every 100ml is a fraction, with Rotary Evaporators be recycled to do after, the reuse dissolve with methanol shifts out.With TLC test sample purity, and merge, obtaining HPLC altogether, to detect purity be 99.2% sample asperosaponin VI2.4725g.
Embodiment 3
Get 1 kilogram of Radix Dipsaci, pulverize,, begin percolation after 15 hours with the 6L80% soak with ethanol with the 80% ethanol moistening of 1 times of amount, 5ml/min, collect percolate, concentrating under reduced pressure reclaims ethanol to nothing alcohol flavor, add water to 3000ml, left standstill 4 hours, and filtered, get filtrate, filtrate is added on the chromatographic column of 1.2kg ADS-8 macroporous resin, after the circulation absorption 3 times, with the water elution of 4L, reuse 4L30% ethanol elution, discard eluent, discard preceding twice eluent, reuse 4L90% alcohol flushing is collected 90% ethanol elution, the decolouring of 90% ethanol elution peracidity alumina column (6.0cm * 8.0cm), decompression recycling ethanol concentrates, vacuum drying gets little yellow asperosaponin VI crude product 51.3 grams.
Get above-mentioned gained asperosaponin VI crude product 8g 60ml65% dissolve with ethanol, cross the microporous filter membrane of 0.45 μ m, be splined on sephadex lh-20 post (diameter 8cm, long 120cm) chromatography.Eluent is 65% ethanol, and flow velocity 0.5cm/h adopts fraction collector to collect, and will contain asperosaponin VI flow point and collect.Every 100ml is a fraction, with Rotary Evaporators be recycled to do after, the reuse dissolve with ethanol shifts out, with TLC test sample purity, and merge, decompression recycling ethanol concentrates, cold drying promptly gets 5.2177g white asperosaponin VI powder, purity 98.7%.
Embodiment 4
Get 1 kilogram of Caulis Akebiae, add the 10L soak with ethanol and begin percolation after 12 hours, collect percolate, concentrating under reduced pressure reclaims ethanol to there not being the alcohol flavor, thin up becomes every milliliter to contain 1.0g medical material liquid, left standstill 24 hours, and filtered, filtrate is added on the chromatographic column of 1.2kg AB-8 macroporous resin, after the circulation absorption 3 times, with the 5.0%Na of 4L
2CO
3Eluting, the flushing of reuse purified water discards eluent, reuse 4L90% alcohol flushing to neutral reuse 3L20% alcohol flushing, collect 90% ethanol elution, add 60 ℃ of insulations of 0.3% activated carbon 30min decolouring, filter, decompression recycling ethanol, concentrated, dry, get asperosaponin VI crude product 69.4 grams.
Get above-mentioned gained asperosaponin VI crude product 10g and become every milliliter to contain saponin 200mg, be splined on sephadex lh-20 post (diameter 8cm, long 120cm) chromatography behind the centrifugal or microfiltration with the 50ml50% dissolve with ethanol.Eluent is 50% ethanol, and flow velocity 0.6cm/h adopts fraction collector to collect, and the master is contained asperosaponin VI flow point collect.Every 100ml is a fraction, with Rotary Evaporators be recycled to do after, the reuse dissolve with ethanol shifts out, with TLC test sample purity, and merge, decompression recycling ethanol concentrates, cold drying promptly gets 6.7716g akebin D (asperosaponin VI), purity 98.7%.
Embodiment 5
Get 1 kilogram of Fructus Akebiae, pulverize, 2 times of water gaging moistenings of water began percolation with full the closing of 6L water after n-butyl alcohol soaked liquid, concentrating under reduced pressure, and reclaiming n-butyl alcohol to relative density is 1.20 (50 ℃), thin up becomes every milliliter to contain 1.0g medical material liquid, with 5%Ca (OH)
2Left standstill 6 hours adjust pH to 8.0～9.0, filters, get filtrate, filtrate is added on the chromatographic column of 1.0kgASD-5 macroporous resin, after circulation is adsorbed 3 times, use the 2L0.8%KaOH eluting, the reuse water elution is used the 4L30% ethanol elution instead near neutral, discard eluent, use the 6L80% ethanol elution at last, collect 80% ethanol elution, after the decolouring of peracidity alumina column, decompression recycling ethanol, concentrated, dry, get asperosaponin VI crude product 56.3 grams.
With 200g reversed-phase column ODS column packing (Grace, 50 μ m) with after the methanol soaked overnight, the dress post (5.0cm (i.d.) * 80cm), treat that the post bed is stable after, change the solvent system in the post into 60% methanol, solvent is put done again to packing layer, standby.Taking by weighing asperosaponin VI crude product 5g is dissolved in 60% methanol of about 20ml, slowly be added on packing layer on along post jamb sample liquid with suction pipe, open plunger, after treating that sample liquid is infiltrated the post bed, use 2000ml60% methanol successively, the 2000ml75% methanol-eluted fractions, every 100ml is a fraction, with Rotary Evaporators be recycled to do after, the reuse dissolve with methanol shifts out.With TLC test sample purity, and merge, get asperosaponin VI1.6987g, purity is 97.4%.
Embodiment 6
Feed intake by 1000 preparation units, get Radix Dipsaci saponin VI100g, glucose 120g, mannitol 220g, sodium sulfite 10g, make 1000 bottles of injection lyophilized powders altogether, specification: every bottle contains Radix Dipsaci saponin VI100mg.
Take by weighing on-off total saponin as well as, glucose, mannitol, the sodium sulfite of recipe quantity, add injection water 5000ml, 50 ℃ of stirring and dissolving; Add 0.2% injection activated carbon, about 30 minutes of 50 ℃ of insulated and stirred; Sand core funnel filters; Add the injection water to 6000ml; With 0.22 μ m membrane filtration; Measure intermediate content, fill; Lyophilization; Cover plug, roll lid, get 926 bottles of injection on-off total saponin as well as lyophilized powders, every bottle contains saponin VI98.2mg.
Embodiment 7
Feed intake by 1000 preparation units, get Radix Dipsaci saponin VI250g, hydroxypropyl methylcellulose 100g, microcrystalline Cellulose 30g, lactose 60g, micropowder silica gel 5g, make 1000 altogether, specification: every contains Radix Dipsaci saponin VI250mg.
Above-mentioned supplementary material is prior mistake 100 mesh sieves respectively, take by weighing on-off total saponin as well as, hydroxypropyl methylcellulose, microcrystalline Cellulose, lactose by recipe quantity, mix homogeneously, with 80% medicinal alcohol system soft material, to cross 24 mesh sieves and granulate, drying is 4 hours under 60 ℃ of conditions, 20 mesh sieve granulate, add micropowder silica gel, tabletting, promptly.
Feed intake by 1000 preparation units, get Radix Dipsaci saponin VI300g, starch 80g, carboxymethyl starch sodium 8g, magnesium stearate 5g, make 1000 capsules altogether, specification: every contains Radix Dipsaci saponin VI300mg.
Above-mentioned supplementary material is prior mistake 80 mesh sieves respectively, take by weighing Radix Dipsaci saponin VI, starch by recipe quantity, carboxymethyl starch sodium, mix homogeneously is with 60% medicinal alcohol system soft material, crossing 24 mesh sieves granulates, drying is 4 hours under 60 ℃ of conditions, and 20 mesh sieve granulate add magnesium stearate, filled capsules gets 952 of Radix Dipsaci saponin VI capsules.
Feed intake by 1000 preparation units, get Radix Dipsaci saponin VI50g, Macrogol 4000 100g, polyethylene glycol 6000 30g, make 1000 drop pill altogether, specification: every contains Radix Dipsaci saponin VI50mg.
Take by weighing recipe quantity Radix Dipsaci saponin VI and add 60ml80% ethanol, 60 ℃ of stirred in water bath to melt and dissolved state, Macrogol 4000 and polyethylene glycol 6000 that other gets recipe quantity dissolve in water-bath, gradation adds Radix Dipsaci saponin VI ethanol liquid, mixing after dissolving, moves to fast and is preheated in the constant temperature drilling pill device reservoir, drip system, get 910 of Radix Dipsaci saponin VI drop pill.
Asperosaponin VI chemical structural formula conclusive evidence
White powder, ESIMS m/z927.5[M-H]
-.
1H-NMR (C
5D
5N) δ: 6.25 (1H, d, J=8.2Hz, Glc1-H), 5.42 (1H, brs, H-12), 5.03 (1H, d, J=7.7Hz, Glc2-H), 4.97 (1H, d, J=7.2Hz, Ara-H), 3.18 (1H, dd, J=4.0,13.8Hz, H-3), 1.17 (3H, s, CH
3), 1.12 (3H, s, CH
3), 0.98 (3H, s, CH
3), 0.93 (3H, s, CH
3), 0.87 (3H, s, CH
3), 0.86 (3H, s, CH
3).
13C-NMR (C
5D
5N) Table II as follows.Above data consistent with the Asperosaponin VI spectrum data of bibliographical information (Ma Shuancheng, Chen Dechang, Zhao Haijie. Chinese yam is known sub-chemical constitution study (III), research and development of natural products, 1998,10 (3): 49-51).Each collection of illustrative plates is seen accompanying drawing 1～10.Its chemical structural formula is as follows:
Table II Asperosaponin VI and bibliographical information
13C-NMR data (C
5D
5N, δ)
| C | Asperosaponin?VI | Data in literature | C | Asperosaponin?VI | Data in |
| 1 | 38.5 | 38.76 | 26 | 17.8 | 17.54 |
| 2? | 25.7? | 26.00? | 27? | 25.7? | 26.00? |
| 3 | 81.6 | 81.87 | 28 | 176.2 | 176.48 |
| 4 | 43.1 | 43.18 | 29 | 32.7 | 33.01 |
| 5 | 47.3 | 48.13 | 30 | 23.3 | 23.61 |
| 6 | 47.8 | 48.15 | Ara | ? | ? |
| 7 | 32.2 | 32.48 | 1 | 106.3 | 106.60 |
| 8 | 39.6 | 39.87 | 2 | 72.8 | 73.06 |
| 9 | 46.7 | 47.55 | 3 | 74.4 | 74.66 |
| 10 | 36.6 | 36.89 | 4 | 69.3 | 69.59 |
| 11 | 23.5 | 23.81 | 5 | 66.6 | 66.93 |
| 12 | 122.6 | 123.00 | Glc-1 | ? | ? |
| 13 | 143.8 | 144.11 | 1 | 95.3 | 95.62 |
| 14 | 41.8 | 42.06 | 2 | 73.5 | 73.82 |
| 15 | 28.0 | 28.25 | 3 | 78.4 | 78.66 |
| 16 | 23.0 | 23.29 | 4 | 70.6 | 70.87 |
| 17 | 17.2 | 16.96 | 5 | 77.6 | 77.90 |
| 18 | 41.3 | 41.62 | 6 | 69.0 | 69.32 |
| 19 | 45.8 | 46.15 | Glc-2 | ? | ? |
| 20 | 30.4 | 30.67 | 1 | 104.9 | 105.23 |
| 21 | 33.6 | 33.89 | 2 | 74.8 | 75.10 |
| 22 | 32.4 | 32.75 | 3 | 78.0 | 78.34 |
| 23 | 64.2 | 64.42 | 4 | 71.2 | 71.45 |
| 24 | 13.2 | 13.54 | 5 | 78.1 | 78.40 |
| 25 | 15.9 | 16.17 | 6 | 62.3 | 62.58 |
Pharmacodynamics test
One, test material
1, is subjected to reagent and contrast medicine
Asperosaponin VI (Asperosaponin VI, ASP-VI), by self-control, lot number: 071012, purity 99.2%, on-off total saponin as well as (XDS), lot number 070821, total saponin content 84.5% (wherein containing ASP-VI67.6%).Acetaminophen, Huanqiu Pharmaceutical Co., Ltd., Guangdong produces, lot number 080102; Celestial clever bone is protected capsule, and Guizhou Tongjitang Pharmaceutical Co., Ltd produces, lot number 070306.With preceding solution or the suspension that is made into desired concn with purified water.
3, reagent and instrument
Formalin, Guangzhou Chemical Reagent Factory production, lot number 20060704-11; Acetone, Luoyang City's chemical reagent factory, lot number: 060413; Hydrochloric acid, new east station of Guangzhou reddening factory, lot number 070703913; Sodium hydroxide, Tianjin Da Mao chemical reagent factory, lot number 20070822.The RB100 hot-plate instrument, Chengdu Tai Meng instrument Science and Technology Ltd. produces; The BS-124S electronic balance, Sai Duolisi Instr Ltd. in Beijing produces; DPX-L type dual intensity X line bone density calibrating instrument, Lunar Co.U.S.A; INSTRON-1122 type universal testing machine, Britain; Slide gauge (Chinese Jinhua); 80-1 type centrifugal precipitation mechanism (Chinese Shanghai); UV1201 ultraviolet-uisible spectrophotometer (Beijing Rayleigh Analytical Instrument Co.,Ltd).
3, animal
Kunming mice, the quality certification (Guangdong check and affirm word) 2006A063 number; The SD rat, the quality certification (Guangdong check and affirm word) provides by Zhongshan University's Experimental Animal Center for 2006A064 number.
Two, statistical disposition
Adopt the SPSS statistical software to carry out the t check of self comparing before and after between group variable analysis or the administration.
Three, test method and result
1, ASP-VI anti-inflammatory and analgesic effect research
1.1ASP-VI influence to the thermic pain
Employing hot plate method test is licked metapedes with mice and is showed as pain, measures the threshold of pain in advance, gets the threshold of pain and is the mice of 10～30s and participate in the experiment.Get 50 of qualified female kunming mices, be divided into 5 groups at random, 10 every group.Be respectively negative control group (giving the isometric(al) purified water), 300mg/kg acetaminophen group, ASP-VI75mg/kg, 150mg/kg, three dosage groups of 300mg/kg.Each organizes all gastric infusion 1 time, 0.2ml/10g.Measured the mice threshold of pain respectively in 1 hour before the administration and after the administration, greater than 60s, calculate with 60s as the threshold of pain.The results are shown in Table 1.
Table 1ASP-VI to the influence of the mice threshold of pain (X ± s, n=10)
| Group | Dosage (mg/kg) | Pain threshold (s) before the administration | Pain threshold after the administration (s) |
| Negative control group | ? | 18.9±5.14 | 19.8±6.63 |
| The |
300 | 19.2±6.21 | 38.3±10.27** |
| The ASP-VI |
75 | 18.5±5.58 | 21.5±9.79 |
| Dosage group among the ASP- |
150 | 19.6±6.42 | 28.4±10.24* |
| The ASP-VI |
300 | 18.3±5.87 | 32.2±11.20** |
[0093]Annotate: compare with cloudy matched group,
*P＜0.05,
*P＜0.01
By table 1 as seen, ASP-VI can improve the mice threshold of pain in various degree, and middle and high dosage group has significant difference (P＜0.01 or P＜0.05), and prompting ASP-VI has analgesic activity to the pain that the mice thermostimulation causes.Its analgesic activity is with positive control drug acetaminophen no significant difference, and is dosage correlation.
1.2 to the influence of formalin to pain and inflammation
60 male mices are divided into five groups at random, i.e. negative control group (giving the isometric(al) purified water), acetaminophen group, XDS group, the basic, normal, high dosage group of ASP-VI.Normal raise after one day every group of gastric infusion 0.2ml/10g behind the fasting feedwater 12h.Behind the administration 1h, in the mice left hind foot sole of the foot subcutaneous injection 1% formalin solution 20ul of portion, and timing, place the glass container of diameter 20cm immediately, respectively the cumulative time of 0～5min (I phase) and the interior mice pain reaction of 20～30min (II mutually) after the record injection formalin, mark.Methods of marking: lick, sting or trembled sufficient 3 minutes, carry foot 2 minutes, walked lamely 1 fen, walked about 0 minute freely.Draw respectively and respectively organize rat I phase and II accumulative total score value mutually.Accumulation score value=(0t1+1t2+2t3+3t4)/(t1+t2+t3+t4) [t1, t2, t3, t4 are respectively 0,1,2,3 minute persistent period (s)].The results are shown in Table 2.
The table 2ASP-VI to mice formalin cause the pain influence (X ± s, n=10)
Annotate: compare with cloudy matched group,
*P＜0.05,
*P＜0.01
By table 2 visual data as can be seen, the high, medium and low dosage of ASP-VI has certain effect to reducing II phase reaction pain integration, compares with matched group wherein that all there were significant differences, and action effect is better than positive control drug.Positive drug and ASP-VI all do not have obvious effect to the I phase reaction.
1.3 influence to the rat chronic inflammation
50 of rats, male and female half and half are divided into 5 groups at random, 10 every group.Be respectively negative control group (giving the isometric(al) purified water), acetaminophen 180mg/kg group, ASP-VI45mg/kg, 90mg/kg, three dosage groups of 180mg/kg.Under the shallow fiber crops of ether, the cropping sterilization, in hypogastric region medisection skin, it is subcutaneous that the sterilization cotton balls of heavy 50mg is implanted the both sides groin, and skin suture is sterilized then, and whole process is the sterile working.From the preceding 3 days beginning gastric infusions of performing the operation, 1.0ml/100g, every day 1 time, continuous 10 days.Put to death rat on the 11st day, and carefully peeled off the granulation tissue of taking out cotton balls and parcel, 60 ℃ of dryings were weighed after 12 hours, deducted the raw cotton ball weight and were granulation tissue weight, and the result represents with the contained granulation tissue weight of every 100g body weight (bilateral).The results are shown in Table 3.
Table 3ASP-VI to the influence of rat chronic proliferative inflammation (X ± s, n=10)
| Group | Dosage (mg/kg) | Granulation tissue weight (mg/100g body weight) |
| Negative control group | - | 51.8±14.9 |
| The |
180 | 32.6±8.9** |
| The ASP-VI |
45 | 40.3±13.5 |
| Dosage group among the ASP- |
90 | 35.5±9.7** |
| The ASP-VI |
180 | 29.4±9.2** |
Annotate: compare with cloudy matched group,
*P＜0.05,
*P＜0.01
By table 3 as seen, ASP-VI suppresses the rat granulation tissue hyperplasia that cotton balls stimulates, compare with negative control group, ASP-VI height, middle dosage group and positive controls all have significant difference (P＜0.01), prompting ASP-VI obviously has inhibitory action to the rat chronic proliferative inflammation, and effect is better than positive control drug.
2ASP-VI is to the influence of osteoporosis rat union of fracture
2.1 experimental technique
The model copy laboratory animal is selected 60 of 3 monthly age SD female rats for use, osteoporosis model duplicate the reference literature method (Zhao Yongfang, Shen Peizhi, Xu Yu etc. XIANLING GUBAO JIAONANG causes the influence of osteoporotic bone amount to removal ovary, traditional Chinese medical science bonesetting, 2000; 12 (5): 11～12.The Qi Chen chief editor. herbal medicine efficacy research thinking and method. Beijing: the People's Health Publisher, 2005:957), 3% pentobarbital intraperitoneal anesthesia is got lumbar vertebra both sides otch and is entered the abdominal cavity dorsal part, complete excision bilateral ovaries, hemostatic suture under the aseptic condition.Other gets 10 rats and is made as sham operated rats, except that not extracing the ovary, operates with method.After the stitching, the normal raising.After 3 months, osteoporosis rat is anaesthetized with chlore-ammonia ketone, and biliographic data duplicates right side femur fracture model (Li Xiaolin, Luo Xinle, Yu Nansheng etc. furrow fish calcitonin is to the influence of osteoporosis rat union of fracture. Chinese osteoporosis magazine, 2003,59 (2): 111-113; The minister in ancient times is luxuriant, freezes calculon, Zou Zhipeng. and Radix Dipsaci is to the biomechanics experiment research of osteoporosis rat union of fracture influence, Chinese medicine physical magazine, 2002,19 (3): 159-161).
Administration is divided into 6 groups with raising at random with the modeling rat, be respectively osteoporosis model group (OPS), osteoporotic fracture model group (OPF), osteoporotic fracture+celestial clever bone is protected 600mg/kg group (OPFG), osteoporotic fracture+ASP-VI270mg/kg group (OPFA-H), osteoporotic fracture+ASP-VI90mg/kg organizes (OPFA-L), and osteoporotic fracture+XDS450mg/kg group (OPFX-H), osteoporotic fracture+XDS150mg/kg organizes (OPFX-L).Each all administration on the same day after modeling of group, sham operated rats (SHAM), OP group and OPF group rat oral gavage method are given clothes purified water, 1.0ml/100g body weight, every day 1 time, medicine group, dosage preparation respective concentration in accordance with regulations, press the 1.0ml/100g body weight, irritate stomach every day 1 time, 8 weeks of continuous use.Each organizes the rat sub-cage rearing, and free picked-up standard rat feed is freely drunk distilled water.
Each treated animal of observation index and assay method is all put to death in 8 weeks, carries out following index observing.
(1) mensuration of the curved intensity (FBS) of bone density (MBD) and fracture
Before the execution, rats by intraperitoneal injection 5% chlore-ammonia ketone injection 10mg/100g, when rat presents the lasting 10min of stable lethargic state when above, place under the probe of DPX-L type dual intensity X line borne densitometers (Lunar Co.U.S.A), use bone densitometry software and carry out whole body and fracture scanning, measure the bone density of whole body bone density and fracture femur.
Rat is put to death after surveying bone density, take off the and arranged on left and right sides femur, measure the width in femur stage casing and calculate sectional area with slide gauge earlier respectively, after femur is placed on the support of universal testing machine (span=25mm), speed with 5mm/min presses down the femur stage casing, the pressure loading that record is maximum calculates bending strength (pressure loading of maximum on the unit area of section) at last.
(2) mensuration of callus calcium, phosphorus content
With measure FBS fracture the right side then femur with acetone concussion dehydration 12 hours, vacuum drying (105 ℃, weigh after 24h), specimen is ground into fine particulate, place 105 ℃ of hydrolysis 12h of 6N hydrochloric acid again, specimen cooling back transfers to pH value to 6.0 with NaOH.With methyl thymol blue colorimetric method for determining callus TC (Ca); With the direct determination of color callus of peacock green phosphorus content;
Computing formula: Ca (concentration of specimens mmol/L)=Abs * 2.5 * 100/0.3087
P (concentration of specimens mmol/L)=Abs * 1.29 * 200/0.1888
2.2 result
2.2.1 medicine is to the influence of rat bone density (MBD) and the curved intensity (FBS) of fracture
By following table 4 as seen after treating for 8 weeks, osteoporosis rat whole body bone density all has raising in various degree, the GMBD of ASP-VI high dose group, positive drug group and XDS high dose group is apparently higher than the osteoporosis model group, but also far away than the sham operated rats level, may be short relevant with administration time.Sham-operation rat that the bone density of right side femur fracture model group rat is not more fractured or the femoral bmd of osteoporosis module rat are all obviously low, curative osteoporotic fracture rat femur bone density all has increase after treating for 8 weeks, and wherein ASP-VI high and low dose group, XDS high dose group and positive drug group right side bone femur density are apparently higher than the osteoporotic fracture model group.Prompting ASP-VI not only can improve the whole body bone density of sclerotin pine rat, the bone density of fracture is also had the raising effect, and effect is better than XDS and positive drug.
Table 4 is respectively organized rat body bone density and right side femur bone densitometry result (X ± s, n=8～10)
| Grouping | Dosage (mg/kg) | Whole body bone density (g/cm 2)? | Right side femoral bmd (g/cm 2)? |
| SHAM | - | 0.402±0.0261 | 0.257±0.0083 |
| OPS | - | 0.337±0.0385 ΔΔ | 0.232±0.0113 ΔΔ |
| OPF | - | - | 0.203±0.0099 ##ΔΔ |
| OPFG | 600 | 0.373±0.0343 * | 0.238±0.0107 ▲▲ |
| OPFA- |
90 | 0.360±0.0318 | 0.236±0.0111 ▲ |
| OPFA-H | 270 | 0.386±0.0262 ** | 0.249±0.0082 ▲▲ |
| OPFX- |
150 | 0.351±0.0395 | 0.218±0.0113 |
| OPFX-H | 450 | 0.371±0.0319 * | 0.237±0.0107 ▲▲ |
Annotate: compare with osteoporosis sham operated rats (SHAM),
The Δ ΔP＜0.01; Compare with osteoporotic fracture sham operated rats (OPS);
##P＜0.01;
Compare with the OPS group,
*P＜0.05,
*P＜0.01; Compare with the OPF group,
▲P＜0.05,
▲ ▲P＜0.01;
All be lower than sham operated rats in various degree by following table 5 visible osteoporosis rat femur bending strengths, the right femur that fracture takes place is more remarkable.After treating for 8 weeks, ASP-VI high and low dose group, the fl bending strength that XDS high dose group and positive drug group are fractured is apparently higher than the osteoporosis model group.Sham-operation rat that the femur bending strength of the model group rat of right side Fracture of femur is not more fractured or osteoporosis module rat are all obviously low, curative osteoporotic fracture rat femur bending strength all has increase after treating for 8 weeks, and wherein ASP-VI high dose group, XDS high dose group and positive drug group are apparently higher than the osteoporotic fracture model group.
Table 5 is respectively organized rat fl and right femur anti-reflecting bending strength determining result (X ± s, n=8～10)
| Grouping | Dosage (mg/kg) | Fl bending strength (N/mm 2) | Right femur bending strength (N/mm 2) |
| SHAM | - | 22.26±2.58 | 22.26±2.58 |
| OPS | - | 16.12±3.08 ΔΔ | 16.49±2.27 ΔΔ |
| OPF | - | - | 12.78±1.89 ##ΔΔ |
| OPFG | 600 | 19.16±2.85 ** | 14.92±1.87 ▲ |
| OPFA- |
90 | 18.93±2.67 * | 14.13±1.93 |
| OPFA-H | 270 | 20.11±2.64 ** | 15.35±2.02 ▲▲ |
| OPFX- |
150 | 17.44±3.21 | 13.44±1.56 |
| OPFX-H | 450 | 19.38±2.79 ** | 14.75±2.13 ▲ |
Annotate: compare with osteoporosis sham operated rats (SHAM),
The Δ ΔP＜0.01; Compare with osteoporotic fracture sham operated rats (OPS);
##P＜0.01;
Compare with the OPS group,
*P＜0.05,
*P＜0.01; Compare with the OPF group,
▲P＜0.05,
▲ ▲P＜0.01;
2.2.2 medicine is to the influence of rat callus calcium, phosphorus content
All be starkly lower than the sham operated rats of not fracturing by following table 6 visible osteoporotic fracture rat callus calcium, phosphorus content.After the treatment of 8 weeks, ASP-VI high and low dose group, XDS high and low dose group and positive drug group callus calcium obviously increase; ASP-VI high dose group, XDS high dose group and XIANLING GUBAO JIAONANG group callus phosphorus content all significantly raise.Prompting ASP-VI and XDS are to the obvious therapeutic action of osteoporotic fracture rat, and the APS-VI effect obviously is better than XDS.
The variation of table 6 callus calcium and phosphorus content (X ± s, n=8～10)
| Grouping | Dosage (mg/kg) | Calcium (mg) | Phosphorus (mg) |
| OPS | - | 56.33±1.16 | 32.11±1.76 |
| OPF | - | 43.67±1.56 ## | 22.16±2.86 ## |
| OPFG | 600 | 51.29±1.93 ** | 27.10±2.82 ** |
| OPFA- |
90 | 49.52±1.28 ** | 25.15±2.33 * |
| OPFA-H | 270 | 57.18±2.45 ** | 30.63±2.07 ** |
| OPFX- |
150 | 46.61±1.73 ** | 23.89±2.31 |
| OPFX-H | 450 | 50.73±2.20 ** | 28.24±1.90 ** |
Annotate: compare with osteoporotic fracture sham operated rats (OPS);
##P＜0.01; Compare with the OPF group,
*P＜0.05,
*P＜0.01
Four, conclusion
Test adopts the present invention to extract the pharmacological research that gained asperosaponin VI (APS-VI) carries out the orthopaedic disease aspect, observes the pharmacologically active of APS-VI, for clinical practice provides foundation.Result of the test shows:
(1) ASP-VI can improve the mice threshold of pain in various degree, and the pain that 150mg/kg, 300mg/kg group causes the mice thermostimulation has obvious analgesic activity, and its analgesic activity is close with the positive control drug acetaminophen, and is dosage correlation.Formalin causes bitterly, and result of the test shows that three dosage of 75mg/kg, 150mg/kg, 300mg/kg of ASP-VI all have reduction to reducing II phase reaction pain integration, compare with matched group that all there were significant differences, action effect with and positive control drug suitable, positive drug and ASP-VI all do not have obvious effect to the I phase reaction.
(2) the chronic inflammatory disease result of the test shows that ASP-VI can suppress the rat granulation tissue hyperplasia that cotton balls stimulates, compare with negative control group, ASP-VI90mg/kg, 180mg/kg and positive controls all have significant difference (P＜0.01), prompting ASP-VI obviously has inhibitory action to the rat chronic proliferative inflammation, and effect is better than positive control drug.
(3) adopt rat to remove ovary and duplicate estrogen deficiency type osteoporosis and osteoporotic fracture model, ASP-VI270mg/kg and XDS300mg/kg group can obviously be improved whole body bone density and the femur hardness of ovariectomy rat after the treatment in 8 weeks, shows that ASP-VI and XDS have obvious therapeutic action to estrogen deficiency type osteoporosis.
The do not fracture femoral bmd of osteoporosis model group rat of the bone density of osteoporosis rat right side femur fracture model group rat is all obviously low, curative osteoporotic fracture rat femur bone density all has increase after treating for 8 weeks, and wherein ASP-VI270mg/kg, 90mg/kg dosage group and XDS450mg/kg group fractured bones femur density are apparently higher than model group.Osteoporotic fracture rat callus calcium, phosphorus are measured, the result shows that fracture rat callus calcium, phosphorus content all are starkly lower than sham operated rats, after the treatment of 8 weeks, ASP-VI270mg/kg, 90mg/kg dosage group, XDS450mg/kg, 150mg/kg dosage group callus calcium obviously increase; ASP-VI, XDS high dose group and XIANLING GUBAO JIAONANG group callus phosphorus content all significantly raise.
This result of the test prompting ASP-VI not only can improve the whole body bone density of sclerotin pine rat, bone density and the effect of being improved of callus calcium, phosphorus content to fracture, but the ASP-VI effect obviously is better than XDS, and prompting APS-VI is the main effective ingredient of Radix Dipsaci saponin, and purity increases curative effect and also increases.
Above-mentioned experimental result shows that fully medicine ASP-VI of the present invention has an effect of obviously controlling to osteoporosis, fracture, and pain such as osteoarthritis, hyperosteogeny, fracture in the orthopaedic disease or inflammation clinical symptoms are had obvious therapeutic action.
Claims (7)
1. preparation method for the treatment of the crude drug of orthopaedic disease, this crude drug is asperosaponin VI, it is characterized in that, comprises the following steps:
1) extracts, concentrates: get the Chinese crude drug that Radix Dipsaci, Caulis Akebiae or Fructus Akebiae contain asperosaponin VI,, filter, get extracting solution A with lower alcohol or aqueous lower alcohol extraction; With gained extracting solution A reclaim under reduced pressure alcohol, being concentrated into relative density is 1.20～1.40, gets concentrated solution B;
2) concentrated solution is handled: concentrated solution B thin up is become every milliliter of solution that contains 0.3～1.5g raw medicinal herbs, add alkali and regulate pH value to 7.0～10.0, left standstill 4～36 hours, filtration or centrifugal divides and gets supernatant, gets solution C;
3) separate total saponins: solution C is splined on the chromatographic column that macroporous resin is housed, used macroporous resin is one or both among SA-1, SA-2, ADS-5, ADS-7, ADS-8, AB-8 and the D201, circulation absorption 2～3 times, wash with water to nearly neutrality behind 0.5～5.0% the alkali liquor eluting with 2～3 times of amount column volumes, 10～40% ethanol elutions of 2～4 times of amounts of reuse column volume discard eluent; With the 50-95% ethanol elution of 4～8 times of amount column volumes, collect eluent at last, get total saponins extracting solution D;
4) decolouring: with total soap extracting solution D peroxidating aluminum or decolorizing resin post or adopt activated carbon to decolour; With the total saponins extracting solution reclaim under reduced pressure alcohol after the decolouring, concentrate, drying gets asperosaponin VI crude product;
5) purification, refining asperosaponin VI: asperosaponin VI crude product water or aqueous lower alcohol are made solution, further adopt column chromatographic isolation and purification or adopt opposed polarity solvent fractional extraction purification refine promptly to get asperosaponin VI, used opposed polarity solvent fractional extraction is meant earlier with the ethyl acetate that contains alcohol, dichloromethane or chloroform extraction for several times, reuse n-butyl alcohol or water are full to close n-butanol extraction for several times, merges butanol extraction liquid.
2. the preparation method of the crude drug of treatment orthopaedic disease according to claim 1 is characterized in that, the carbon number of lower alcohol is C1～C4 in described step 1) and the step 5), and concentration is X, 0%＜X≤100%.
3. the preparation method of the crude drug of treatment orthopaedic disease according to claim 1 is characterized in that, the extracting method in the described step 1) is decocting method, heating reflux method, solvent extraction method or percolation.
4. the preparation method of the crude drug of treatment orthopaedic disease according to claim 1 is characterized in that, described step 2) and step 3) in alkali liquor be NaOH, KOH, Ca (OH)
2, K
2CO
3, Na
2CO
3, NaHCO
3, one or more the mixture in ammonia and the triethylamine.
5. the preparation method of the crude drug of treatment orthopaedic disease according to claim 1 is characterized in that, the process for purification refine in the described step 5) adopts one of following method or mixes and use:
1) asperosaponin VI crude product is dissolved with eluent, be splined on the polydextran gel column chromatography for separation behind the centrifugal or microfiltration, eluent is 5～80% alcohol, the employing fraction collector is collected, the master is contained asperosaponin VI flow point collect, merge reclaim under reduced pressure alcohol, concentrate drying promptly gets and makes with extra care asperosaponin VI;
2) asperosaponin VI crude product is dissolved with eluent, be splined on reversed phase column chromatography behind the centrifugal or microfiltration and separate, eluent is 20～90% alcohol, the employing fraction collector is collected, and the master is contained asperosaponin VI flow point collect, and merges, reclaim under reduced pressure alcohol, concentrate drying promptly gets and makes with extra care asperosaponin VI;
3) asperosaponin VI crude product water is made every milliliter of solution that contains 50～300mg crude product, transfer p H-number to 8.0～11.0, for several times earlier with the ethyl acetate that contains alcohol, dichloromethane or chloroform extraction with alkali; Reuse n-butyl alcohol or water are full to close n-butanol extraction for several times, merges butanol extraction liquid; Reuse water backwash butanol extraction liquid 2～4 times; Placed 12～48 hours reclaim under reduced pressure n-butyl alcohol to 1/5～1/3 back of original volume, remove precipitation, continue to concentrate, dry, promptly get asperosaponin VI, be lower than 95% as purity, adopting aqueous carbon number is that lower alcohol or the mixed organic solvents of C1～C4 carries out recrystallization, promptly gets and makes with extra care asperosaponin VI.
6. according to the preparation method of the crude drug of each described treatment orthopaedic disease of claim 2-5, it is characterized in that, the percetage by weight content of the asperosaponin VI of gained accounts for 95.0～99.9% of asperosaponin VI extract total amount, its chemical name is 3-O-α-L-arabopyranose helexin-28-O-β-D-Glucopyranose. (1-6)-β-D-pyranglucoside, and chemical structural formula is:
7. the preparation method of the crude drug of treatment orthopaedic disease according to claim 6 is characterized in that, described orthopaedic disease is meant osteoporosis, fracture, osteoarthritis or hyperosteogeny.
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| CN101444552B (en) * | 2008-12-31 | 2011-08-31 | 重庆市中药研究院 | Chinese medicinal preparation for treating osteoporosis |
| CN101564405B (en) * | 2009-05-26 | 2012-05-30 | 赵全成 | Application of total aglycone of himalayan teasel roots and single-component hederagenin in medicaments preparing Alpha-glucosidase inhibitor |
| CN102526134A (en) * | 2010-12-24 | 2012-07-04 | 苏州宝泽堂医药科技有限公司 | Preparation method of dipsacus total saponins and teasel saponins VI |
| CN102180937B (en) * | 2011-03-28 | 2012-10-03 | 江苏神华药业有限公司 | Method for preparing enriched and refined akebin D with macroporous absorption resin |
| CN102199184B (en) * | 2011-04-07 | 2013-03-06 | 江西本草天工科技有限责任公司 | Hederasaponin derivative, preparation method of salts thereof and application thereof to resisting tumor |
| WO2017088080A1 (en) | 2015-11-25 | 2017-06-01 | Natural Response S.A. | Method for obtaining saponins from plants |
| CN108159063A (en) * | 2018-01-24 | 2018-06-15 | 刘丽宏 | Application of the akebiasaponin D in the drug for promoting cartilage of osteoarthritis reparation is prepared |
| CN109575101A (en) * | 2018-12-28 | 2019-04-05 | 湖南津湘制药有限公司 | A kind of highly effective extraction method of teasel root saponin(e VI |
| CN111330085B (en) * | 2020-02-27 | 2024-02-13 | 华东理工大学 | Traditional Chinese medicine regulation type composite active bone scaffold and preparation method and application thereof |
| CN112156101B (en) * | 2020-09-18 | 2021-11-05 | 成都体育学院 | Application of dipsacus asperoides VI in preparation of medicine for treating tendon injury |
| CN115501241A (en) * | 2022-06-07 | 2022-12-23 | 西北工业大学 | Application of dipsacus asperoides VI in medicine for regulating intestinal flora and inhibiting osteoporosis |
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