CN1209116C - Application of akebia saponin D in preparation of medicine for proventing and treating osteo pathic - Google Patents
Application of akebia saponin D in preparation of medicine for proventing and treating osteo pathic Download PDFInfo
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Abstract
The present invention discloses the purpose of akebia saponin D, akebia total saponin containing the akebia saponin D and Radix Dipsaci akebia total saponin containing the akebia saponin D in preparation of medicine for preventing osteoporosis and / or promoting knitting. Compared with a contrast group, the present invention can obviously relieve inflammation, eliminate turgescence and ease pain, can cause the serum alkaline phosphatase of a fracture rabbit, the radius weight factor of the fracture rabbit, the cremains content of the fracture rabbit, the bone phosphorus content of the fracture rabbit and the bone mine density of the fracture rabbit to be increase evidently, and can cause osteoporosis caused by vitamin A acid, rat bone weight factor, cremains content, bone mine density, bone trabecula breadth and bone phosphorus content to be increased obviously.
Description
Technical field
The Caulis Akebiae total saponins that the present invention relates to akebin D and contain akebin D and the new purposes, especially the akebin D that contain the on-off total saponin as well as of akebin D prevent and treat purposes in the osteopathia medicine in preparation.
Background technology
The Caulis Akebiae seed is the seed of Lardizabalaceae plant Caulis Akebiae Akebia trifol iata (Thunb.) Koidz.Var.australis and threeleaf akebia A.tri foliata (Thunb.) Koidz and Caulis Akebiae A.quiuatu (Thunb.) Decne.The place of production: originate in ground such as Sichuan, Hubei, Hunan, Guangxi.Main component: akebin (Akebin) helexin, neat rattan fruit acid potassium salt etc.Character identification: the seed majority, prolate is avette, yellowish-brown or puce.Feeble QI perfume (or spice), bitter in the mouth.Nature and flavor, return: cool in nature, bitter in the mouth through, function.Liver-smoothing, qi-regulating, promoting blood circulation and stopping pain, diuresis, parasite killing.Be used for gastral cavity rib distending pain, dysmenorrhea amenorrhea, dysuria, snake bite and insect sting.
Radix Dipsaci is the dry root of Dipsacaceae plant Radix Dipsaci Dipsacus asper Wall..The place of production: main product in Sichuan, ground such as Hubei, Yunnan, Guizhou.Character identification: be elongated cylindrical, slightly flat or microbend, long 5-15cm, diameter 0.5-2cm.Appearance taupe or yellowish-brown all have the vertical wrinkle and the rill of obvious distortion, can see the hole skin and the minority mark of fibrous root of transverse fissure.Matter is soft, puts dry back hardening for a long time, frangibility.The section unevenness, skin zone's outer rim is brown, in be blackish green or brown, the woody part yellow is radial decorative pattern.Feeble QI perfume (or spice), bitter in the mouth, little sweet then puckery.Thick with bar, matter is soft, the blackish green person of section band is good.Nature and flavor, return: bitter in the mouth, sweet, hot through, function; Slightly warm in nature, invigorating the liver and kidney, bone and muscle strengthening, continuous folding is hindered.Be used for diseases such as lumbago flaccidity of the lower limbs, seminal emission, metrorrhagia, threatened abortion, injury from falling down, incised wound, carbuncle ulcer.
Osteoporosis is that a kind of destruction with low bone amount and osseous tissue micro structure is feature, causes skeleton fragility to increase and easily take place the systemic disease of fracture.Osteoporotic sickness rate has leapt to the 7th in chronic disease.Three big symptom 1. clinical manifestations of sufferers of osteoporosis face are the most common with pain: mostly are the back ache, secondly are the shoulder back of the body, cervical region or wrist ankle, pain in the time of can or standing up because of seat, upright position, clinostatism is bad during fashion.2. textured bone: the spinal bone distortion, bend over, bow-backed, stature becomes short.3. fracture: common fracture site is vertebrae (compressibility, wedge type), wrist (head of radius) and hipbone (neck of femur).Fracture is the complication of osteoporosis, and fracture of femoral neck 10-20% is in the death in 1 year of being in hospital, and half can't take care of oneself remainder.Diagnosis: mainly rely on dual intensity X line bone density meter, middle and advanced stage can rely on the X-ray film diagnosis.Clinical prevention relies on and gets sun, moves, replenishes the calcium at present.Treatment vitamin D, diphosphate, calcitonin, estrogen etc.Kidney-nourishing tcm drugs such as report Rhizoma Drynariae, Herba Cistanches are arranged, the effect that promotes knitting is arranged.
Summary of the invention
The technical problem to be solved in the present invention is effective site and/or the active ingredient that extraction separation is prevented and treated osteopathia from Chinese Chinese medicine, but so that preparation good effect, toxic and side effects treatment osteopathia little, the taking convenience life-time service especially prevent and treat osteoporosis and/or promote the medicine of knitting.
For addressing the above problem, the invention provides following technical scheme.
Akebin D and Caulis Akebiae total saponins that contains akebin D and the on-off total saponin as well as that contains akebin D are prevented and treated purposes in the osteopathia medicine in preparation.Described Caulis Akebiae total saponins contains akebin D 〉=5%; On-off total saponin as well as contains akebin D 〉=5%.Be specially the Caulis Akebiae total saponins and contain akebin D 5-95%; On-off total saponin as well as contains akebin D 5-95%.
The Caulis Akebiae total saponins also contains periearpsaponin A, B etc. except containing akebin D.
On-off total saponin as well as is except containing akebin D, and also containing the Caulis Hederae Sinensis sapogenin is the serial saponin of parent nucleus.
Aforementioned purposes of preventing and treating in the osteopathia medicine is characterized in that: prevent and treat the healing that osteopathia is meant osteoporosis and/or promotes bone injury.Described bone injury comprises fracture, bone splits.
Described purposes of preventing and treating in the osteopathia medicine, its medicine are to contain akebin D and the pharmaceutically acceptable component and/or the carrier for the treatment of effective dose; Perhaps for containing the Caulis Akebiae total saponins that contains akebin D and the pharmaceutically acceptable component and/or the carrier for the treatment of effective dose; Perhaps for containing the on-off total saponin as well as that contains akebin D and the pharmaceutically acceptable component and/or the carrier for the treatment of effective dose.
Described akebin is prevented and treated purposes in the osteopathia medicine in preparation, pharmaceutically acceptable component for can with akebin D or the on-off total saponin as well as that contains the Caulis Akebiae total saponins of akebin D or contain akebin D with unite the chemical compound or the compositions of preventing and treating osteopathia.
Described akebin is prevented and treated purposes in the osteopathia medicine in preparation, and described medicine is oral formulations, parenteral preparation or the external preparation that contains treatment effective dose akebin and pharmaceutically acceptable component and/or carrier.
Can have with the adjuvant that akebin or akebin D are mixed with various oral formulations such as tablet, hard capsule, granule: disintegrating agent such as hydroxypropyl starch, hyprolose, carboxymethyl starch sodium, carboxymethylcellulose calcium, polyvinylpolypyrrolidone, cross-linking sodium carboxymethyl cellulose etc.; Filler such as lactose, sucrose, mannitol, microcrystalline Cellulose, dextrin, starch, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, calcium carbonate, cyclodextrin, micropowder cellulose etc.; Wetting agent and binding agent are as pregelatinized Starch, polyvidone, sodium carboxymethyl cellulose, hypromellose; Lubricant such as Pulvis Talci, stearic acid, magnesium stearate, calcium stearate, micropowder silica gel, hydrogenated vegetable oil, Macrogol 4000 and 6000; Wetting agent is as sodium lauryl sulphate, Tween 80; Framework material such as hypromellose, ethyl cellulose etc.The oil phase that can be mixed with soft capsule with akebin of the present invention or akebin D can be soybean oil, PEG400, Oleum Gossypii semen, Oleum Arachidis hypogaeae semen, Oleum Sesami, Semen Maydis oil, Fructus Canarii albi wet goods; Also can add solubilizing agent or cosolvent and antioxidant etc., also can select following adjuvant for use: solvent such as PEG400, Oleum Gossypii semen, Oleum Arachidis hypogaeae semen, Oleum Sesami, Semen Maydis oil, Fructus Canarii albi wet goods; Solubilizing agent or cosolvent are as Tween 80, polyoxyethylene ether Oleum Ricini, benzyl benzoate, ethyl lactate etc.; Antioxidant is as propyl gallate, t-butyl phenol (BHT), vitamin E etc.The ratio of gelatin, glycerol and water can suitably be regulated in the glue shell, is advisable 1: 0.3～0.4: 0.7～1.4 as gelatin/glycerin/water three's ratio, also can add other compositions in the glue shell, as antiseptic P-hydroxybenzoic acid first, second, third, butyl ester etc.; Plasticizer such as sorbitol etc.; Stabilizing agent such as arabic gum etc.; Opacifier such as titanium dioxide, barium sulfate, precipitated calcium carbonate etc.
The drawing explanation
Fig. 1, rabbit radius fracture resistence force assay device
Fig. 2, rabbit radius bending moment figure
The specific embodiment
Medical material. Fructus Akebiae (Caulis Akebiae seed) is commercially available with Radix Dipsaci.Meet every standard under the pharmacopeia continuous item respectively through check.
The plant origin of Caulis Akebiae seed and formal name used at school thereof: the Caulis Akebiae seed is the seed of Lardizabalaceae plant Caulis Akebiae Akebia trifoliata (Thunb.) Koidz.Var.australis and threeleaf akebia A.tri foliata (Thunb.) Koidz and Caulis Akebiae A.quiuatu (Thunb.) Decne.Radix Dipsaci is the dry root of Dipsacaceae plant Radix Dipsaci Dipsacus asper Wall..
Embodiment 1
Akebin D and contain the extraction separation of the Caulis Akebiae total saponins of akebin D
Get Caulis Akebiae seed 100g, be ground into coarse powder, with defat with petroleum ether, volatilize petroleum ether after, with with 85% alcohol reflux three times, each 2 hours, add 6 times of amount ethanol, extracting liquid filtering at every turn, merging filtrate, decompression recycling ethanol is closely dried, adds water and forms suspension, uses n-butanol extraction three times, merge butanol extraction liquid, the reclaim under reduced pressure n-butyl alcohol gets n-butanol extract to doing, and adds water 100ml, by the D-101 macroporous resin column, with till being washed to sugar-free reaction, the ethanol elution of reuse 70%, eluate be Caulis Akebiae total saponins (containing akebin D 5% approximately) (being called for short sample 1).Sample 1 is carried out silica gel column chromatography,, get sample 2 (containing akebin D 55% approximately) with chloroform-methanol (1: 1) eluting; Sample 2 is carried out silica gel column chromatography once more,, get sample 3 (containing akebin D 95% approximately), promptly get akebin D 1 gram with chloroform-methanol (1: 1) eluting.
Embodiment 2
Akebin D and contain the extraction separation of the on-off total saponin as well as of akebin D
Get Radix Dipsaci medical material 100 gram, be ground into coarse granule (Semen Glycines is big), with 85% alcohol reflux three times, each 2 hours, add 6 times of amount ethanol, extracting liquid filtering, merging filtrate at every turn, decompression recycling ethanol is closely dried, add water and form suspension, use n-butanol extraction three times, merge butanol extraction liquid, the reclaim under reduced pressure n-butyl alcohol is to doing, residue with water dissolution after, with petroleum ether defat repeatedly, the aqueous solution after the defat, concentrate, separate out precipitate, precipitate washs with little water, after the drying, get on-off total saponin as well as crude product, i.e. sample 4 (containing akebin D 5% approximately); Sample 4 is used recrystallizing methanol, get sample 5 (containing akebin D 55% approximately); With sample 5 usefulness methanol recrystallization repeatedly, sample 6 be akebin D 4 grams (akebin D content is about 95%).
Akebin D formal name used at school is 3-O-α-L-arabopyranose-helexin 28-O-β-D-Glucopyranose. (1 → 6)-β-D-glucopyranosyl ester glycoside.
Molecular formula: C
47H
76O
18, M=952 (M+Na
+)
Physicochemical constant: mp is 207-210 ℃ of (recrystallizing methanol) [α]
D 1a+ 14.9 ℃ (C0.25 MeOH), is soluble in methanol.
Akebin D: white powder (methanol).mp207-210.5℃。[α]D19+14.9℃(C 0.25，MeOH)。
Referring to: Zhang Yongwen, the chemical constitution study Acta Pharmaceutica Sinica of Xue Zhi Radix Dipsaci, 1991; 26 (9): 676～681; Phytochemistry.1989; 28:644; Yang Shangjun, Wu Zhihang waits the research II China Medicine University journal of triterpene saponin in the Radix Dipsaci, 1993; 24 (5): 276～280.
Embodiment 3
Akebin D and Caulis Akebiae total saponins that contains akebin D and the pharmacodynamic study that contains the on-off total saponin as well as of akebin D
In fact, along with the content increase of akebin D in the Caulis Akebiae total saponins, when it contained 95% akebin D, purer akebin D was generally just thought in the present technique field.
Referring to: Wang Yitao, Wang Jiakui, Yang Kui etc., the pharmaceutical research of Caulis Akebiae, Pharmacology and Clinics of Chinese Materia Medica, 1996 (3): 20-23; Chai Benpu, Tang Xueming, the ultrastructural studies of experimental union of fracture, Chinese journal of orthopedics, 1982,2 (2): 117; All copper water, Liu Xiaodong, Zhourong's Chinese, Rhizoma Drynariae is to the influence of rat experiment bone injury healing, Chinese herbal medicine, 1994,25 (5): 249-250; Liu Yushun, Zhou Jinshui, Lin Baokai etc., bone knitting powder is to the experimentation of rabbit fracture, Fujian Chinese medicine, 1994,25 (3): 19.
The applicant has studied the main pharmacodynamics effect of Caulis Akebiae total saponins (containing 5-95% akebin D), though only selected to contain the drug effect data of the Caulis Akebiae total saponins of 5,55 and 95% akebin D in the following description, this should not produce restriction to technical solution of the present invention.
Laboratory animal: Kunming mouse, body weight 18-22g; Livid purple blue rabbit, body weight 1.8-2.5kg; The SD male rat, body weight 170-220g is provided by animal housing of China Medicine University, the quality certification number, Soviet Union's kinoplaszm 97004.
Medicine name: Caulis Akebiae total saponins sample 1 (containing akebin D5%), Caulis Akebiae total saponins sample 2 (containing akebin D55%), Caulis Akebiae total saponins sample 3 (containing akebin D95%), the unit of providing: Zhejiang Deyer Pharmaceutical Co., Ltd. provides, lot number: 980813
Powder for fracture and trauma: Chinese Jiamusi pharmaceutical factory of traditional Chinese medicine produces, and blackly defends the accurate word (1982) of medicine No. 301173.
According to phosphine: the honest day fine pharmaceutical Co. Ltd in Lianyun Harbour produces.
Radix Salviae Miltiorrhizae Tabellae: Shanghai No.3 Traditional Chinese Medicine Pharmaceutical Factory produces, and ZZ-3885-defends in Shanghai the accurate word (1995) of medicine No. 042067.
The ketoprofen capsule: pharmaceutical factory (Jiangsu) produces in the China Soviet Union, the accurate word of Su Wei medicine (1987) and No. 355202.
Compound method: Caulis Akebiae total saponins sample 1, sample 2, sample 3 granules and positive drug are ground into fine powder, are mixed with the suspension of desired concn with 0.5%CMC-Na.
Reagent: serum inorganic phosphorus is measured test kit: the glad biotechnology research of Shanghai section is produced.Alkali phosphatase reagent: Changzhou Medical House reagent station, lot number: 971104.Retinoic acid: Shanghai No.6 Pharmaceutical Factory produces.
Instrument: the close analyzer of dual intensity x ray bone, LUNAR DPX-L type U.S. LUNAR company produces
1. Caulis Akebiae total saponins (containing 5-95% akebin D) is to the influence of mice self ecchymosis
Referring to: Wang Nan, Jia Junsheng, Cao Difang etc., the benefiting qi and removing blood stasis method is treated the experimental research of subcutaneous and intracerebral hematoma, Shanghai Chinese medicine, 1985, (1): 45-48.
Get 140 of body weight 18-22g male mices, be divided into 14 groups at random, negative control group (giving 0.5%CMC-Na0.4ml) by body weight; Positive controls (giving Radix Salviae Miltiorrhizae Tabellae 0.3g/kg); Caulis Akebiae total saponins sample 1 height (4.4g/kg), low (2.2g/kg) 2 dosage; Caulis Akebiae total saponins sample 2 height (0.8g/kg), low (0.4g/kg) 2 dosage; Caulis Akebiae total saponins sample 3 height (0.46g/kg), low (0.23g/kg) 2 dosage.On-off total saponin as well as sample 4 height (4.4g/kg), low (2.2g/kg) 2 dosage; On-off total saponin as well as sample 5 height (0.8g/kg), low (0.4g/kg) 2 dosage; On-off total saponin as well as sample 6 height (0.46g/kg), low (0.23g/kg) 2 dosage.Make the ecchymosis model before the administration; Every Mus one branch hole posterior vein is got blood 0.5ml, adds the CaCl2 solution 0.1ml mixing of the 0.1M contain penicillin 100,000 units, and it is subcutaneous to inject self nape portion immediately, causes ecchymosis.Pressed last method successive administration 7 after the moulding, every morning, 8:00-10:00 was administered once.The administration volume is 0.4ml/20g.Put to death in 8th, and separated the ecchymosis enclosed mass, scales/electronic balance weighing, and data are organized a t check compare, the results are shown in Table 1.
Table 1 Caulis Akebiae total saponins and on-off total saponin as well as gastric infusion are to influence (X ± S) (n=10) of mice self ecchymosis
| Group | Dosage (g/kg) | Subcutaneous residual quantity clot weight (mg) |
| Negative control | - | 76.55±20.44 |
| Radix Salviae Miltiorrhizae Tabellae | 0.3 | 27.88±7.07 *** |
| Caulis Akebiae total saponins sample 1 | 4.4 | 26.89±5.01 *** |
| 2.2 | 27.06±6.15 *** | |
| Caulis Akebiae total saponins sample 2 | 0.8 | 20.34±3.56 *** |
| 0.4 | 26.63±6.02 *** | |
| Caulis Akebiae total saponins sample 3 | 0.46 | 20.22±1.66 *** |
| 0.23 | 25.97±3.13 *** | |
| On-off total saponin as well as sample 4 | 4.4 | 25.93±4.09 *** |
| 2.2 | 26.78±4.12 *** | |
| On-off total saponin as well as sample 5 | 0.8 | 19.91±3.77 *** |
| 0.4 | 25.71±5.92 *** | |
| On-off total saponin as well as sample 6 | 0.46 | 19.25±3.11 *** |
| 0.23 | 24.79±4.12 *** |
* *Compare with negative control group P＜0.01
By table 1 as seen, Caulis Akebiae total saponins sample 1 (containing akebin D5%), Caulis Akebiae total saponins sample 2 (containing akebin D55%), high and low 2 dosage groups of Caulis Akebiae total saponins sample 3 (containing akebin D95%), on-off total saponin as well as sample 4 (containing akebin D5%), on-off total saponin as well as sample 5 (containing akebin D55%), high and low 2 dosage groups of on-off total saponin as well as sample 6 (containing akebin D95%) and Radix Salviae Miltiorrhizae Tabellae group can make the weight of mice self ecchymosis piece obviously alleviate (P＜0.01).
2, the influence of Caulis Akebiae total saponins (containing 5～95% akebin D) xylol induced mice auricle edema
Get 80 of male mices, body weight 18-22g is divided into 8 groups at random by body weight, negative control group (giving 0.5%CMC-Na); Positive controls (giving ketoprofen 0.18g/kg); All the other 6 groups give Caulis Akebiae total saponins sample 1 height (4.4g/kg), low (2.2g/kg) 2 dosage respectively; Caulis Akebiae total saponins sample 2 height (0.8g/kg), low (0.4g/kg) 2 dosage; Caulis Akebiae total saponins sample 3 height (0.46g/kg), low (0.23g/kg) 2 dosage.Press above-mentioned dosage successive administration 7 days, and the results are shown in Table 2.
Influence (X ± S) (n=10) of table 2 Caulis Akebiae total saponins gastric infusion xylol induced mice auricle edema
| Group | Dosage (g/kg) | Swelling degree (mg) |
| Negative control | - | 20.6±4.24 |
| Ketoprofen | 0.18 | 6.91±1.22 *** |
| Caulis Akebiae total saponins sample 1 | 4.4 | 13.69±1.75 *** |
| 2.2 | 15.44±2.24 *** | |
| Caulis Akebiae total saponins sample 2 | 0.8 | 8.64±2.40 *** |
| 0.4 | 14.09±2.49 *** | |
| Caulis Akebiae total saponins sample 3 | 0.46 | 8.89±1.48 *** |
| 0.23 | 13.84±2.01 *** |
* *Compare with negative control group P＜0.01
By table 2 as seen, Caulis Akebiae total saponins sample 1 (containing akebin D5%), Caulis Akebiae total saponins sample 2 (containing akebin D55%), high and low 2 dosage groups of Caulis Akebiae total saponins sample 3 (containing akebin D95%) and ketoprofen group xylol induced mice auricle edema have significant inhibitory effect (P＜0.01).
3, the influence of Caulis Akebiae total saponins (containing 5～95% akebin D) Dichlorodiphenyl Acetate induced mice pain.
Get 80 of mices, body weight 18-22g is divided into 8 groups at random by body weight, and administering mode and dosage the results are shown in Table 3 with 2.
Influence (X ± S) (n=10) of table 3 Caulis Akebiae total saponins gastric infusion Dichlorodiphenyl Acetate induced mice pain
| Group | Dosage (mg/kg) | Turn round body time of occurrence (S) | Turn round body number of times (inferior) |
| Negative control | - | 122.5±61.82 | 28.5±6.82 |
| Ketoprofen | 0.18 | 254.3±97.32 *** | 11.5±5.64 *** |
| Caulis Akebiae total saponins sample 1 | 4.4 | 237.3±62.1 ** | 21.68±7.72 ** |
| 2.2 | 209/5±52.9 ** | 24.57±6.11 ** | |
| Caulis Akebiae total saponins sample 2 | 0.8 | 302.4±80.38 *** | 18.0±6.78 *** |
| 0.4 | 202.4±59.73 ** | 22.2±5.05 ** | |
| Caulis Akebiae total saponins sample 3 | 0.46 | 314.1±47.6 *** | 17.1±4.40 *** |
| 0.23 | 208.7±67.9 ** | 19.87±8.91 *** |
*P＜0.05
* *Compare with negative control group P＜0.01
By table 3 as seen, Caulis Akebiae total saponins sample 1 (containing akebin D5%), Caulis Akebiae total saponins sample 2 (containing akebin D55%), high and low 2 dosage group Dichlorodiphenyl Acetate induced mice pain of Caulis Akebiae total saponins sample 3 (containing akebin D95%) have obvious inhibitory action (P＜0.01, P＜0.05), mouse writhing number of times and turn round the comparison of body time of occurrence and negative control group first there were significant differences, the ketoprofen group also has significant analgesia role (P＜0.01) to this model.
4, Caulis Akebiae total saponins (containing 5～95% akebin D) is to the therapeutical effect of rabbit fracture of radius
Get 128 of body weight 1.8-2.5kg rabbit, male and female half and half, the experiment beginning is the preparation fracture model earlier, carry out the intravenous anesthesia of rabbit ear edge with 1.5% pentobarbital sodium 2ml/kg (30mg/kg), under aseptic condition, cause the wide standard transverse fracture of bilateral radius sclerotin complete collyriculum 3mm in pronator ters stop far-end with special hacksaw, the operative incision position splashes into 0.1ml (40,000 u) penicillin and 0.1ml (120mg) streptomycin, skin suture then.Negative control group gives normal diet, and positive controls gives powder for fracture and trauma 0.3g/kg, and other 6 groups give Caulis Akebiae total saponins sample 1 height (2.2g/kg), low (1.1g/kg) 2 dosage respectively; Caulis Akebiae total saponins sample 2 height (0.2g/kg), low (0.1g/kg) 2 dosage; Caulis Akebiae total saponins sample 3 height (0.116g/kg), low (0.058g/kg) 2 dosage.Administration every day 1 time, the sacrificed by exsanguination animal in batches in administration 21 days and 35 days, get blood and anatomical isolation bilateral radius and detect following several indexs:
4.1 the mensuration of serum alkaline phosphatase
Serum alkaline phosphatase is measured in the femoral venous catheter blood-letting, the results are shown in Table 4.
Table 4 Caulis Akebiae total saponins is to influence (X ± S) (n=8) of fracture rabbit anteserum alkali phosphatase
| Group | Dosage (mg/kg) | Serum alkaline phosphatase (U/L) | |
| 21 days | 35 days | ||
| Negative control | - | 96.7±35.3 | 100.5±13.1 |
| Powder for fracture and trauma | 0.3 | 134.3±17.8 ** | 133.4±4.9 *** |
| Caulis Akebiae total saponins sample 1 | 2.2 | 137.1±13.1 ** | 137.0±15.5 *** |
| 1.1 | 130.9±16.8 ** | 130.1±17.1 *** | |
| Caulis Akebiae total saponins sample 2 | 0.2 | 136.9±15.8 ** | 137.4±8.4 *** |
| 0.1 | 131.7±10.4 ** | 131.0±6.2 *** | |
| Caulis Akebiae total saponins sample 3 | 0.116 | 136.4±11.7 ** | 136.8±7.5 *** |
| 0.058 | 132.9±16.3 ** | 133.0±8.1 *** | |
*P＜0.05
* *Compare with negative control group P＜0.01
By table 4 as seen, Caulis Akebiae total saponins sample 1 (containing akebin D5%), Caulis Akebiae total saponins sample 2 (containing akebin D55%), there is obvious rising effect (P＜0.01, P＜0.05) the high and low dose group of Caulis Akebiae total saponins sample 3 (containing akebin D95%) and powder for fracture and trauma group 21 days and 35 days after administration to fracture rabbit anteserum alkaline phosphatase concentration.
4.2 bone weight coefficient and bone ash Determination on content
Get the left side radius, the mensuration of weighing bone weight coefficient, the g/100g body weight is represented; Get the dry and carbonization of left side radius and be placed in 800 ℃ of muffle furnaces ashing and made bone ash in 8 hours, measure bone ash content, heavily represent, the results are shown in Table 5 with the g/g bone.
Table 5 Caulis Akebiae total saponins is to influence (X ± S) (n=8) of fracture rabbit radius bone weight coefficient and bone ash content
| Group | Dosage g/kg | Bone weight coefficient (g/100g body weight) | Bone ash content (the g/g bone is heavy) | ||
| 21 days | 35 days | 21 days | 35 days | ||
| Negative control | - | 0.072±0.004 | 0.073±0.011 | 0.354±0.032 | 0.357±0.015 |
| Powder for fracture and trauma | 0.3 | 0.092±0.021 ** | 0.097±0.016 *** | 0.410±0.036 *** | 0.418±0.026 *** |
| Caulis Akebiae total saponins sample 1 | 2.2 | 0.092±0.012 *** | 0.099±0.010 *** | 0.418±0.031 *** | 0.429±0.019 *** |
| 1.1 | 0.086±0.016 ** | 0.091±0.020 *** | 0.407±0.029 *** | 0.410±0.041 *** | |
| Caulis Akebiae total saponins sample 2 | 0.2 | 0.092±0.013 *** | 0.100±0.016 *** | 0.426±0.036 *** | 0.430±0.015 *** |
| 0.1 | 0.088±0.015 ** | 0.097±0.011 *** | 0.410±0.020 *** | 0.411±0.021 *** | |
| Caulis Akebiae total saponins sample 3 | 0.116 | 0.092±0.019 *** | 0.101±0.016 *** | 0.427±0.027 *** | 0.431±0.022 *** |
| 0.058 | 0.087±0.014 ** | 0.096±0.014 *** | 0.412±0.024 *** | 0.413±0.029 *** | |
*P＜0.05
* *P＜0.01 and corresponding negative control group comparison in period
By table 5 as seen, Caulis Akebiae total saponins sample 1 (containing akebin D5%), Caulis Akebiae total saponins sample 2 (containing akebin D55%), the high and low dose group of Caulis Akebiae total saponins sample 3 (containing akebin D95%) and powder for fracture and trauma group can make bone weight coefficient and bone ash content obviously raise (P＜0.01, P＜0.05) in 21 days and 35 days in administration.
4.3 the mensuration of bone mineral density
Get the left side radius, measure the fracture site bone mineral density, the results are shown in Table 6 with dual-energy x-ray bone densitometry instrument.
Table 6 Caulis Akebiae total saponins is to fracture rabbit radius bone mineral density influence (X ± S) (n=8)
| Group | Dosage (g/kg) | Bone mineral density (g/cm 2) | |
| 21 days | 35 days | ||
| Negative control | - | 0.109±0.066 | 0.088±0.060 |
| Powder for fracture and trauma | 0.3 | 0.196±0.072 ** | 0.151±0.055 ** |
| Caulis Akebiae total saponins sample 1 | 2.2 | 0.255±0.043 *** | 0.168±0.054 ** |
| 1.1 | 0.166±0.072 ** | 0.152±0.041 ** | |
| Caulis Akebiae total saponins sample 2 | 0.2 | 0.277±0.062 *** | 0.163±0.066 ** |
| 0.1 | 0.178±0.056 ** | 0.156±0.049 ** | |
| Caulis Akebiae total saponins sample 3 | 0.116 | 0.265±0.047 *** | 0.164±0.021 ** |
| 0.058 | 0.171±0.053 ** | 0.158±0.063 ** | |
*P＜0.05
* *P＜0.01 and corresponding negative control group comparison in period
By table 6 as seen, Caulis Akebiae total saponins sample 1 (containing akebin D5%), Caulis Akebiae total saponins sample 2 (containing akebin D55%), the high and low dose group of Caulis Akebiae total saponins sample 3 (containing akebin D95%) and powder for fracture and trauma group can make rabbit fracture of radius position bone mineral density obviously increase (P＜0.01, P＜0.05) in 21 days and 35 days after administration.
4.4 X-ray film is measured
Get the left side radius and carry out X-ray film mensuration, comprise 3 indexs: 1. situation (fuzzy animal example number occurring) appears in the fracture site edge blurry, and the result is carried out the X2 check; 2. periosteal reaction: promptly measure the dermoskeleton film thickness, method is optional 3 points in the most obvious place of periosteal reaction outside the fracture site both sides, measures that it is thick wide, averages; 3. callus density: adopt 3 grades of point-scores, shade is light fuzzy, can identification person be I reluctantly; Shade is more shallow, and obviously visible person is the II level; Shade is darker, even obvious calcification person is arranged for the III level, and the gained result analyzes (referring to Chai Benpu, Tang Xueming, the ultrastructural studies of experimental union of fracture, Chinese journal of orthopedics, 1982,2 (2): 117) with Riddit.Above-mentioned three results see Table 7 respectively, table 8 and table 9.
Table 7 Caulis Akebiae total saponins fractures to Os Leporis seu Oryctolagi, and end margin is fuzzy influence to occur
| Group | Dosage (g/kg) | Number of animals (only) | The routine number (only) that fracture site is fuzzy | |
| 21 days | 35 days | |||
| Negative control | - | 8 | 1 | 1 |
| Powder for fracture and trauma | 0.3 | 8 | 6 ** | 8 *** |
| Caulis Akebiae total saponins sample 1 | 2.2 | 8 | 6 ** | 8 *** |
| 1.1 | 8 | 5 ** | 7 *** | |
| Caulis Akebiae total saponins sample 2 | 0.2 | 8 | 6 ** | 8 *** |
| 0.1 | 8 | 5 ** | 7 *** | |
| Caulis Akebiae total saponins sample 3 | 0.116 | 8 | 7 *** | 8 *** |
| 0.0.58 | 8 | 5 ** | 8 *** | |
*P＜0.05
* *P＜0.01 and corresponding negative control group comparison in period
Table 8 Caulis Akebiae total saponins is to influence (X ± S) (n=8) of rabbit fractured bones periosteum thickness
| Group | Dosage (g/kg) | Periosteum thickness | |
| 21 days | 35 days | ||
| Negative control | - | 1.63±1.09 | 2.35±1.04 |
| Powder for fracture and trauma | 0.3 | 1.74±1.55 | 0.98±1.25 ** |
| Caulis Akebiae total saponins sample 1 | 2.2 | 2.77±1.01 ** | 0.79±1.01 **△△△ |
| 1.1 | 2.63±1.38 ** | 0.92±0.87 **△△ | |
| Caulis Akebiae total saponins sample 2 | 0.2 | 2.85±1.32 ** | 0.77±0.99 **△△△ |
| 0.1 | 2.66±1.39 ** | 0.94±1.06 **△△ | |
| Caulis Akebiae total saponins sample 3 | 0.116 | 2.87±1.42 ** | 0.76±0.83 **△△△ |
| 0.058 | 2.70±1.21 ** | 0.91±1.03 **△△ | |
*P＜0.05 and corresponding negative control group comparison in period,
△ △P＜0.05,
△ △ △P＜0.01 is with comparison in 21 days on the same group.
*P＜0.05
* *P＜0.01 and corresponding negative control group comparison in period
Table 9 Caulis Akebiae total saponins is to the influence of fracture Os Leporis seu Oryctolagi crust density
| Group | Dosage (g/kg) | Number of animals (only) | Callus density number of animals at different levels (only) | |||||
| 21 days | 35 days | |||||||
| I | II | III | I | II | III | |||
| Negative control | - | 8 | 8 | 0 | 0 *** | 5 | 3 | 0 |
| Powder for fracture and trauma | 0.3 | 8 | 0 | 4 | 4 *** | 0 | 2 | 6 *** |
| Caulis Akebiae total saponins sample 1 | 2.2 | 8 | 0 | 4 | 4 *** | 0 | 0 | 8 *** |
| 1.1 | 8 | 0 | 5 | 3 *** | 0 | 2 | 6 *** | |
| Caulis Akebiae total saponins sample 2 | 0.2 | 8 | 0 | 4 | 4 *** | 0 | 0 | 8 *** |
| 0.1 | 8 | 0 | 5 | 3 *** | 0 | 2 | 6 *** | |
| Caulis Akebiae total saponins sample 3 | 0.116 | 8 | 0 | 3 | 5 *** | 0 | 0 | 8 *** |
| 0.058 | 8 | 0 | 4 | 4 *** | 0 | 1 | 7 *** | |
* *Compare with negative control group P＜0.01
By table 7,8 and 9 as seen, Caulis Akebiae total saponins sample 1 (containing akebin D5%), Caulis Akebiae total saponins sample 2 (containing akebin D55%), the height of Caulis Akebiae total saponins sample 3 (containing akebin D95%), low dose group and fracture are frustrated fracture site blurs when organizing after administration 21 days and 35 days the routine number of loosing obviously more than negative control group (P＜0.01, P＜0.05), the periosteum thickness of 21 days each administration groups is thick than positive controls after the administration, but compare there was no significant difference with the latter, the periosteum thickness of 35 days each administration groups of administration obviously is thinner than negative control group (P＜0.05), and 35 days periosteum thickness obviously is thinner than after the administration 21 days periosteum thickness after the administration of Caulis Akebiae total saponins, show that administration group callus is absorbed, fracture is repaired substantially; In addition, the callus density of each administration group is obviously greater than negative control group (P＜0.01).Above-mentioned X-ray film result shows that each administration group speed of fracture union is obviously faster than negative control group.
4.5 fracture resistence force test
Press document (Qiu Tong, Lin Weiqi, Wang Shen, the influence that " synthetism Yilidan Chinese patent medicine " shows rabbit ulnar fracture X-ray film, Gansu college of traditional Chinese medicine journal, 1992,6,9 (2): 35-36) method design fracture resistence force assay device.As shown in Figure 1, when the rabbit radius was done the stress destruction test, assay device can be similar to the simple beam that is subjected to concentrfated load, and the weight of rabbit radius can be ignored.Fig. 1 is a rabbit radius fracture resistence force assay device, and wherein: 1 is rabbit radius long 1; P is load (loading gradually with the method for hanging counterweight in the test); A is the distance of load p apart from rabbit radius left end point; B is that load p is apart from rabbit radius right endpoint distance; B is a load p application point, also is the rabbit radius place that fractures; A, C are radius two end supports point.Set up coordinate, then rabbit radius bending moment such as Fig. 2.By Fig. 2 bending moment figure as can be known, maximum bending moment place is the displacement that the rabbit radius fractures and locates.
We measured after respectively at administration in 21 days and 35 days, the results are shown in Table 10.
Table 10 Caulis Akebiae total saponins is to influence (X ± S) (n=8) of rabbit fractured bones bone fracture resistence force
| Group | Dosage (g/kg) | The maximum bending moment (Nm) of fracturing | |
| 21 days | 35 days | ||
| Negative control | - | 0.197±0.021 | 0.240±0.021 |
| Powder for fracture and trauma | 0.3 | 0.371±0.051 *** | 0.478±0.032 ***△△△ |
| Caulis Akebiae total saponins sample 1 | 2.2 | 0.476±0.054 *** | 0.609±0.017 ***△△△ |
| 1.1 | 0.407±0.048 *** | 0.581±0.041 ***△△△ | |
| Caulis Akebiae total saponins sample 2 | 0.2 | 0.488±0.044 *** | 0.611±0.094 ***△△△ |
| 0.1 | 0.404±0.032 *** | 0.586±0.054 ***△△△ | |
| Caulis Akebiae total saponins sample 3 | 0.116 | 0.491±0.047 *** | 0.616±0.042 ***△△△ |
| 0.058 | 0.412±0.056 *** | 0.579±0.023 ***△△△ | |
* *P＜0.01 and corresponding negative control group comparison in period,
△ △ △P＜0.05,
△ △ △P＜0.01 is with comparison in 21 days on the same group.
By table 10 as seen, administration is Caulis Akebiae total saponins sample 1 (containing akebin D5%) in the time of 21 days, Caulis Akebiae total saponins sample 2 (containing akebin D55%), the maximum bending moment of the high and low dose group of Caulis Akebiae total saponins sample 3 (containing akebin D95%) and the fractured bones of powder for fracture and trauma group is obviously greater than negative control group (P＜0.01).The maximum bending moment of administration each administration group fractured bones in the time of 35 days is obviously greater than negative control group (P＜0.01).In addition, the fractured bones fracture resistence force effect of each administration group of 35 days of administration significantly is better than 21 days on the same group (P＜0.01, P＜0.05).
4.6 the mensuration of bone calcium, bone phosphorus
Make bone ash by the 6-4.2 method, get quantitative bone ash Tianjin, island AA-670 flame atom spectrum measuring calcium content of bone; In addition a certain amount of bone ash is dissolved in 6NHCL, measures bone phosphorus with serum inorganic phosphorus determination test box.The gained result is converted to every gram bone heavily contains what milligram bone calcium or bone phosphorus, then it is organized a t check, the results are shown in Table 11.
Table 11 Caulis Akebiae total saponins is to influence (X ± S) (n=8) of fracture Os Leporis seu Oryctolagi calcium and bone phosphorus
| Group | Dosage (g/kg) | Bone calcium (the mg/g bone is heavy) | Bone phosphorus (the mg/g bone is heavy) | ||
| 21 days | 35 days | 21 days | 35 days | ||
| Negative control | - | 112.5±11.9 | 128.9±8.4 | 1.406±0.028 | 1.41±0.014 |
| Powder for fracture and trauma | 0.3 | 140.6±21.7 *** | 148.5± 12.6 *** | 1.604±0.167 ** | 1.640±0.073 *** |
| Caulis Akebiae total saponins sample 1 | 2.2 | 136.9±12.3 *** | 148.2±8.0 *** | 1.67±0.143 *** | 1.727±0.091 *** |
| 1.1 | 120.1±7.9 | 137.6±7.3 | 1.53±0.211 ** | 1.688±0.028 *** | |
| Caulis Akebiae total saponins sample 2 | 0.2 | 137.0±14.0 *** | 148.9±6.4 *** | 1.68±0.079 *** | 1.725±0.078 *** |
| 0.1 | 119.2±4.9 | 139.1±10.39 | 1.51±0.159 ** | 1.675±0.100 *** | |
| Caulis Akebiae total saponins sample 3 | 0.116 | 138.2±11.6 *** | 150.1± 11.8 *** | 1.69±0.108 *** | 1.766±0.126 *** |
| 0.058 | 119.9±14.2 | 139.3±7.5 | 1.53±0.105 ** | 1.689±0.098 *** | |
*P＜0.05
* *P＜0.01 and corresponding negative control group comparison in period
By table 11 as seen, Caulis Akebiae total saponins sample 1 (containing akebin D5%), Caulis Akebiae total saponins sample 2 (containing akebin D55%), the high and low dose group of Caulis Akebiae total saponins sample 3 (containing akebin D95%) and powder for fracture and trauma 21 days and 35 days after administration have remarkable rising effect (P＜0.01, P＜0.05) to fractured bones bone phosphorus content; This product high dose group and powder for fracture and trauma group can make after the administration fractured bones bone calcium of 21 days and 35 days obviously raise (P＜0.01), and low dose group also has the rising effect to it, but with negative control group than no significant difference.
4.7 fracture of radius position histopathologic examination
Ear right side radius specimen is fixed through 10% formalin, decalcification, and right wax section, HE dyeing is observed and is respectively organized the pathological tissue change of fracture of radius position.The results are shown in Table 12.
Table 12 Caulis Akebiae total saponins is to influence (X ± S) (n=8) of position, Os Leporis seu Oryctolagi folding part bone trabecula width
| Group | Dosage (g/kg) | Bone trabecula width (mm) | |
| 21 days | 35 days | ||
| Negative control | - | 0.017±0.002 | 0.019±0.004 |
| Powder for fracture and trauma | 0.3 | 0.021±0.006 ** | 0.021±0.006 ** |
| Caulis Akebiae total saponins sample 1 | 2.2 | 0.028±0.008 *** | 0.029±0.004 *** |
| 1.1 | 0.017±0.004 | 0.021±0.011 ** | |
| Caulis Akebiae total saponins sample 2 | 0.2 | 0.029±0.011 *** | 0.029±0.012 *** |
| 0.1 | 0.018±0.008 | 0.020±0.004 ** | |
| Caulis Akebiae total saponins sample 3 | 0.116 | 0.030±0.009 *** | 0.031±0.006 *** |
| 0.058 | 0.019±0.012 | 0.020±0.014 ** |
*P＜0.05,
* *P＜0.01 is with corresponding negative control group comparison in period
By table 12 as seen, Caulis Akebiae total saponins sample 1 (containing akebin D5%), Caulis Akebiae total saponins sample 2 (containing akebin D55%), the high dose group of Caulis Akebiae total saponins sample 3 (containing akebin D95%) and powder for fracture and trauma group can make fracture site bone trabecula width significantly increase (P＜0.01, P＜0.05) in 21 days after administration; High dose group also can make the fracture site bone trabecula obviously widen (P＜0.05) in 35 days after administration, and the bone trabecula surrounding bone hyperplasia of each administration group is active, and visible more osteoblast, and is the most obvious with high dose group especially.Pathological examination report shows, latter's bone trabecula is broadening but also arrangement very intensive not only.
5. Caulis Akebiae total saponins (containing 5～95% akebin D) is to the influence of osteoporosis rat due to the retinoic acid
Get 210 of rats, be divided into 21 groups at random by body weight, one group as the normal control group, give 0.5%CMC-Na, all the other 20 groups of rats all give retinoic acid 70mg/kg, with the preparation osteoporosis model, in these 20 groups of rats, 1 group gives 0.5%CMC-Na as model control group, and 1 group gives according to phosphine as positive controls (40mg/kg), all the other 18 groups give respectively Caulis Akebiae total saponins sample 1 height (2.2g/kg), in (1.1g/kg), low (0.55g/kg) 3 dosage; Caulis Akebiae total saponins sample 2 height (0.4g/kg), in (0.2g/kg), low (0.1g/kg) 3 dosage; Caulis Akebiae total saponins sample 3 height (0.23g/kg), in (0.115g/kg), low (0.058g/kg) 3 dosage and on-off total saponin as well as sample 4 height (2.2g/kg), in (1.1g/kg), low (0.55g/kg) 3 dosage; On-off total saponin as well as sample 5 height (0.4g/kg), in (0.2g/kg), low (0.1g/kg) 3 dosage; On-off total saponin as well as sample 6 height (0.23g/kg), in (0.115g/kg), low (0.058g/kg) 3 dosage.Animal is irritated stomach retinoic acid and medicine after 2 weeks simultaneously, withdraws retinoic acid, continues 2 weeks of administration, after drug withdrawal, rat put to death in second day, dissect the bilateral femur, the left side femur is used to measure bone weight coefficient, bone mineral density, bone ash content and bone calcium, bone phosphorus, and the right side femur is used to do the pathology histology.Every index determining method the results are shown in Table 13-16 with 4.
Table 13 Caulis Akebiae total saponins and on-off total saponin as well as are to influence (X ± S) (n=10) of osteoporosis rat bone weight coefficient and bone ash content
| Group | Dosage (g/kg) | Bone weight coefficient (g/100g body weight) | Bone ash content (the g/g bone is heavy) |
| Normal control | - | 0.346±0.037 | 0.276±0.012 |
| The model contrast | - | 0.272±0.018 △△△ | 0.207±0.013 △△△ |
| According to phosphine | 0.04 | 0.336±0.026 *** | 0.257±0.018 *** |
| Caulis Akebiae total saponins sample 1 | 2.2 | 0.343±0.015 *** | 0.258±0.019 *** |
| 1.1 | 0.335±0.031 *** | 0.253±0.027 *** | |
| 0.55 | 0.329±0.019 *** | 0.243±0.030 *** | |
| Caulis Akebiae total saponins sample 2 | 0.4 | 0.342±0.025 *** | 0.259±0.022 *** |
| 0.2 | 0.334±0.013 *** | 0.252±0.018 *** | |
| 0.1 | 0.329±0.021 *** | 0.244±0.017 *** | |
| Caulis Akebiae total saponins sample 3 | 0.232 | 0.346±0.037 *** | 0.260±0.015 *** |
| 0.116 | 0.336±0.031 *** | 0.253±0.033 *** | |
| 0.058 | 0.330±0.026 *** | 0.244±0.011 *** | |
| On-off total saponin as well as sample 4 | 2.2 | 0.341±0.022 *** | 0.253±0.028 *** |
| 1.1 | 0.332±0.041 *** | 0.254±0.017 *** | |
| 0.55 | 0.327±0.021 *** | 0.245±0.047 *** | |
| On-off total saponin as well as sample 5 | 0.4 | 0.347±0.013 *** | 0.260±0.019 *** |
| 0.2 | 0.339±0.045 *** | 0.254±0.036 *** | |
| 0.1 | 0.330±0.027 *** | 0.242±0.052 *** | |
| On-off total saponin as well as sample 6 | 0.232 | 0.344±0.071 *** | 0.261±0.045 *** |
| 0.116 | 0.341±0.044 *** | 0.257±0.016 *** | |
| 0.058 | 0.336±0.021 *** | 0.249±0.038 *** |
△ △ △Compare with the normal control group P＜0.01,
* *Compare with model control group P＜0.01
Table 14 Caulis Akebiae total saponins and on-off total saponin as well as are to influence (X ± S) (n=10) of osteoporosis rat femur density
| Group | Dosage (g/kg) | Bone mineral density (g/cm 2) | |||
| #1 | #2 | #3 | #4 | ||
| Normal control | - | 0.062±0.014 | 0.064±0.012 | 0.099±0.039 | 0.124±0.017 |
| The model contrast | - | 0.031±0.012 △△△ | 0.028±0.017 △△△ | 0.046±0.027 △△△ | 0.017±0.027 △△△ |
| According to phosphine | 0.04 | 0.057±0.010 *** | 0.055±0.017 *** | 0.078±0.024 ** | 0.114±0.024 *** |
| 2.2 | 0.061±0.015 *** | 0.061±0.019 *** | 0.085±0.037 *** | 0.115±0.039 *** | |
| Caulis Akebiae total saponins sample 1 | 2.2 | 0.061±0.015 *** | 0.061±0.019 *** | 0.085±0.037 *** | 0.115±0.039 *** |
| 0.55 | 0.069±0.029 *** | 0,069±0.022 *** | 0.085±0.025 *** | 0.109±0.026 *** | |
| Caulis Akebiae total saponins sample 2 | 0.4 | 0.062±0.025 *** | 0.061±0.021 *** | 0.087±0.028 *** | 0.116±0.036 *** |
| 0.2 | 0.062±0.024 *** | 0.060±0.020 *** | 0.080±0.012 *** | 0.113±0.007 *** | |
| 0.1 | 0.060±0.023 *** | 0.053±0.020 *** | 0.078±0.018 *** | 0.106±0.028 *** | |
| Caulis Akebiae total saponins sample 3 | 0.232 | 0.063±0.017 *** | 0.062±0.029 *** | 0.08±0.036 *** | 0.118±0.014 *** |
| 0.116 | 0.063±0.031 *** | 0.061±0.030 *** | 0.088±0.018 *** | 0.115±0.025 *** | |
| 0.058 | 0.061±0.026 *** | 0.060±0.011 *** | 0.083±0.034 *** | 0.110±0.017 *** | |
| On-off total saponin as well as sample 4 | 2.2 | 0.059±0.023 *** | 0.060±0.034 *** | 0.086±0.029 *** | 0.109±0.0179 *** |
| 1.1 | 0.058±0.037 *** | 0.059±0.044 *** | 0.083±0.025 *** | 0.110±0.051 *** | |
| 0.55 | 0.057±0.032 *** | 0.058±0.071 *** | 0.080±0.041 *** | 0.106±0.076 *** | |
| On-off total saponin as well as sample 5 | 0.4 | 0.061±0.035 *** | 0.060±0.028 *** | 0.088±0.032 *** | 0.117±0.028 *** |
| 0.2 | 0.060±0.011 *** | 0.058±0.033 *** | 0.083±0.041 *** | 0.114±0.030 *** | |
| 0.1 | 0.058±0.039 *** | 0.055±0.024u | 0.075±0.025 *** | 0.101±0.020 *** | |
| On-off total saponin as well as sample 6 | 0.232 | 0.064±0.070 *** | 0.063±0.044 *** | 0.087±0.046 *** | 0.117±0.023 *** |
| 0.116 | 0.062±0.013 *** | 0.062±0.022 *** | 0.085±0.073 *** | 0.116±0.018 *** | |
| 0.058 | 0.060±0.025 *** | 0.060±0.043 *** | 0.081±0.051 *** | 0.112±0.065 *** |
*P＜0.05,
* *Compare with model control group P＜0.01;
△ △ △Compare with the normal control group P＜0.01
# annotates; The 1-femoral head; 2-greater trochanter portion; The 3-femoral neck; Portion between the 4-rotor
Table 15 Caulis Akebiae total saponins and on-off total saponin as well as are to influence (X ± S) (n=10) of osteoporosis rat bone calcium, bone phosphorus
| Group | Dosage (g/kg) | Bone calcium (the mg/g bone is heavy) | Bone phosphorus (the mg/g bone is heavy) |
| Normal control | - | 94.0±4.5 | 2.54±0.13 |
| The model contrast | - | 62.2±6.6 △△△ | 2.16±0.16 △△△ |
| According to phosphine | 0.04 | 82.6±7.3 *** | 2.42±0.09 *** |
| Caulis Akebiae total saponins sample 1 | 2.2 | 85.7±6.5 *** | 2.43±0.12 *** |
| 1.1 | 75.9±4.9 *** | 2.38±0.08 *** | |
| 0.55 | 68.8±5.6 *** | 2.37±0.12 *** | |
| Caulis Akebiae total saponins sample 2 | 0.4 | 86.4±7.3 *** | 2.44±0.10 *** |
| 0.2 | 76.0±4.3 *** | 2.38±0.11 *** | |
| 0.1 | 67.0±5.3 *** | 2.36±0.09 *** | |
| Caulis Akebiae total saponins sample 3 | 0.232 | 87.7±6.1 *** | 2.46±0.07 *** |
| 0.116 | 77.4±4.8 *** | 2.40±0.10 *** | |
| 0.058 | 67.4±3.7 *** | 2.37±0.09 *** | |
| On-off total saponin as well as sample 4 | 2.2 | 86.2±7.1 *** | 2.47±0.34 *** |
| 1.1 | 77.1±5.4 *** | 2.41±0.29 *** | |
| 0.55 | 69.4±6.1 *** | 2.40±0.38 *** |
| On-off total saponin as well as sample 5 | 0.4 | 87.1±3.7 *** | 2.48±0.36 *** |
| 0.2 | 77.9±2.4 *** | 2.42±0.53 *** | |
| 0.1 | 68.3±4.4 *** | 2.40±0.35 *** | |
| On-off total saponin as well as sample 6 | 0.232 | 88.5±6.0 *** | 2.51±0.18 *** |
| 0.116 | 80.1±7.6 *** | 2.46±0.24 *** | |
| 0.058 | 73.5±2.1 *** | 2.42±0.38 ** |
△ △ △Compare with the normal control group P＜0.01,
* *Compare with model control group P＜0.01
Table 16 Caulis Akebiae total saponins and on-off total saponin as well as are to influence (X ± S) (n=10) of the capital bone trabecula width of osteoporosis rat
| Group | Dosage (g/kg) | Bone trabecula width (mm) |
| Normal control | - | 0.018±0.005 |
| The model contrast | - | 0.009±0.003 △△△ |
| According to phosphine | 0.04 | 0.024±0.003 ***△△△ |
| Caulis Akebiae total saponins sample 1 | 2.2 | 0.023±0.006 ***△△△ |
| 1.1 | 0.016±0.003 *** | |
| 0.55 | 0.014±0.008 ** | |
| Caulis Akebiae total saponins sample 2 | 0.4 | 0.025±0.007 ***△△△ |
| 0.2 | 0.016±0.005 *** | |
| 0.1 | 0.015±0.004 *** | |
| Caulis Akebiae total saponins sample 3 | 0.232 | 0.025±0.005 ***△△△ |
| 0.116 | 0.019±0.007 *** | |
| 0.058 | 0.015±0.002 *** | |
| On-off total saponin as well as sample 4 | 2.2 | 0.021±0.009 ***△△△ |
| 1.1 | 0.015±0.004 *** | |
| 0.55 | 0.014±0.007 ** | |
| On-off total saponin as well as sample 5 | 0.4 | 0.024±0.004 ***△△△ |
| 0.2 | 0.020±0.007 *** | |
| 0.1 | 0.017±0.005 *** | |
| On-off total saponin as well as sample 6 | 0.232 | 0.020±0.006 *** |
| 0.116 | 0.018±0.004 *** | |
| 0.058 | 0.016±0.003 *** |
* *P＜0.05,
* *Compare with model control group P＜0.01,
△ △ △Compare with the normal control group P＜0.01
By table 13-16 as seen, Caulis Akebiae total saponins sample 1 (containing akebin D5%), Caulis Akebiae total saponins sample 2 (containing akebin D55%), the height of Caulis Akebiae total saponins sample 3 (containing akebin D95%), in, low dose group, on-off total saponin as well as sample 4 (containing akebin D5%), on-off total saponin as well as sample 5 (containing akebin D55%), the height of on-off total saponin as well as sample 6 (containing akebin D95%), in, low dose group reaches the bone weight coefficient that can make osteoporosis rat according to the phosphine group, bone ash content, bone mineral density, the bone trabecula width, the bone phosphorus content significantly increases (P＜0.01, P＜0.05), this product height, in, low dose group and can make calcium content of bone significantly raise (P＜0.01) according to the phosphine group.Each administration group all can make the interior osteocyte of femur and destroy hyperplasia active.Caulis Akebiae total saponins (containing 5～95% akebin D) has stronger promoting healing effect to rabbit bilateral radius fracture, and can obviously alleviate the lesion degree of osteoporosis rat due to the retinoic acid.
6, akebin D monomer (carry from Radix Dipsaci) is to the influence experiment of external osteocyte growth
Table 17, medicine are to the influence result of osteoblast differentiation and propagation
| Group | Molecular weight | Solvent (ml) | Medicine original content (mol/L) | Medicine ultimate density (mol/L) | OD 405Nm (MTT result) |
| Akebin D | 952 | Methanol 0.03 MEM 0.5 | 2.526*10 -3 | 2.52*10 -5 2.52*10 -6 2.52*10 -7 | 0.943±0.070*** 0.952±0.067*** 0.910±0.224* |
| Diethylstilbestrol | 268. 36 | Ethanol 0.5 | 3.632*10 -3 | 3.632*10 -6 3.632*10 -7 3.632*10 -8 | 0.824±13333* 0.981±0.164** 0.819±0.070** |
| According to phosphine | 249. 99 | H 2O 0.5 | 3.064*10 -2 | 3.064*10 -5 3.064*10 -6 3.064*10 -7 | 1.008±0.104*** 0.945±0.239* 0.969±0.176** |
| Ethanol | - | 1％ | - | 0.767±0.105 | |
| Culture medium | - | - | - | 0.756±0.050 |
* *P＜0.001,
*P＜0.01,
*P＜0.05 vs contrast
Akebin D monomer (carry from Radix Dipsaci) is to the significant facilitation that has of external osteocyte growth.
We are divided into the effect experiment of Caulis Akebiae total saponins (containing 5～95% akebin D) and on-off total saponin as well as (containing 5～95% akebin D) 6 administration groups and carry out, and are called sample 1, sample 2, sample 3, sample 4, sample 5, sample 6.
Akebin D content in each sample: the akebin D content difference 5%, 55%, 95% in sample 1 and sample 4, sample 2 and sample 5, sample 3 and the sample 6.
The dosage of each sample different animals:
The dosage of rabbit is respectively: sample 1 and sample 4 height (2.2g/kg), low (1.1g/kg) 2 dosage; Sample 2 and sample 5 height (0.2g/kg), low (0.1g/kg) 2 dosage; Sample 3 and sample 6 height (0.116g/kg), low (0.058g/kg) 2 dosage;
The dosage of rat is: sample 1 and sample 4 height (4.4g/kg), in (2.2g/kg), low (1.1g/kg) 3 dosage; Sample 2 and sample 5 height (0.4g/kg), in (0.2g/kg), low (0.1g/kg) 3 dosage; Sample 3 and sample 6 height (0.23g/kg), in (0.115g/kg), low (0.058g/kg) 3 dosage;
The dosage of mice is: sample 1 and sample 4 height (4.4g/kg), low (2.2g/kg) 2 dosage; Sample 2 and sample 5 height (0.8g/kg), low (0.4g/kg) 2 dosage; Sample 3 and sample 6 height (0.46g/kg), low (0.23g/kg) 2 dosage.
Be expressed as follows after the cubage of the metering of each administration group by akebin D in the sample: the dosage of rabbit: sample 1 and sample 4 height (0.11g/kg), low (0.055g/kg) 2 dosage; Sample 2 and sample 5 height (0.11g/kg), low (0.055g/kg) 2 dosage; Sample 3 and sample 6 height (0.1102g/kg), low (0.0551g/kg) 2 dosage.
The dosage of rat: sample 1 and sample 4 height (0.22g/kg), in (0.11g/kg), low (0.055g/kg) 3 dosage; Sample 2 and sample 5 height (0.22g/kg), in (0.11g/kg), low (0.055g/kg) 3 dosage; Sample 3 and sample 6 height (0.2185g/kg), (0.1093g/kg), low (0.0551g/kg) 3 dosage.
The dosage of mice: sample 1 and sample 4 height (0.22g/kg), low (0.11g/kg) 2 dosage; Sample 2 and sample 5 height (0.44g/kg), low (0.22g/kg) 2 dosage; Sample 3 and sample 6 height (0.437g/kg), low (0.2185g/kg) 2 dosage.
Above-mentioned experimental result (5 kinds of experimental models, 16 experiments detect indexs) show: different Caulis Akebiae total saponins samples do not have dependency with different on-off total saponin as well as samples (the percentage amounts difference that contains akebin D) effect experiment result and total saponins amount, and have the dose-effect dependency with the content of akebin D in the total saponins sample.Akebin D monomer (carry from Radix Dipsaci) is to the significant facilitation that has of external osteocyte growth.The content of drug effect and akebin D has this experimental result of dose-effect dependency and shows: the active component of treatment fracture and osteoporosis is akebin D.
Claims (7)
1, akebin D and the Caulis Akebiae total saponins that contains akebin D 〉=5% are prevented and treated osteoporosis and/or are promoted purposes in the bone injury healing medicine in preparation.
2, the purposes in the described medicine of claim 1, it is characterized in that: the Caulis Akebiae total saponins contains akebin D5-95%.
3, the purposes in the described medicine of claim 1, it is characterized in that: described bone injury comprises fracture, bone splits.
4, the purposes in the described medicine of claim 1 is characterized in that: described medicine is to contain akebin D and the pharmaceutically acceptable component and/or the carrier for the treatment of effective dose.
5, the purposes in the described medicine of claim 1 is characterized in that: described medicine is to contain the Caulis Akebiae total saponins that contains akebin D and the pharmaceutically acceptable component and/or the carrier for the treatment of effective dose.
6, the purposes in claim 4 or the 5 described medicines is characterized in that: pharmaceutically acceptable component is for uniting the chemical compound or the compositions of preventing and treating osteopathia with akebin D or the Caulis Akebiae total saponins that contains akebin D.
7, the purposes in claim 4 or the 5 described medicines is characterized in that: described medicine is to contain akebin D or contain the Caulis Akebiae total saponins of akebin D and oral formulations, parenteral preparation or the external preparation of pharmaceutically acceptable component and/or carrier.
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| CN 03158233 CN1209116C (en) | 2003-09-16 | 2003-09-16 | Application of akebia saponin D in preparation of medicine for proventing and treating osteo pathic |
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| CN 03158233 CN1209116C (en) | 2003-09-16 | 2003-09-16 | Application of akebia saponin D in preparation of medicine for proventing and treating osteo pathic |
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| JP5374054B2 (en) * | 2008-02-19 | 2013-12-25 | 田村薬品工業株式会社 | New saponin compounds containing material and their uses |
| CN101366721B (en) * | 2008-08-25 | 2010-12-15 | 马仁强 | Bulk medicament for treating orthopedic disorders and preparation method thereof |
| CN102462688A (en) * | 2010-11-04 | 2012-05-23 | 中国人民解放军第二炮兵总医院 | Application of akebiasaponin D in preparing medicines for treating and preventing fatty liver and related diseases |
| 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 |
| 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 |
| 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 |
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