CN101791365A - Discorea nipponica makino extractive and application thereof in preparing medicaments for resisting free radicals and reducing blood fat - Google Patents
Discorea nipponica makino extractive and application thereof in preparing medicaments for resisting free radicals and reducing blood fat Download PDFInfo
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Abstract
The invention provides a discorea nipponica makino extractive and application thereof in preparing medicaments for resisting free radicals and reducing blood fat. The discorea nipponica makino extractive is obtained by extracting discorea nipponica makino medicinal materials with an alcoholic solution and then extracting with chloroform and normal butanol. The discorea nipponica makino extractive comprises steroidal compounds, phytosterol and benzylacid, and has better activity for reducing fat, resisting coagulation and resisting free radicals. The extractive can be further separated and purified by adopting silicagel column chromatography, oxide dispersion strengthened (ODS) meslow column chromatography and semi-preparation high performance liquid chromatography (HPLC). The steroidal compounds comprise diosgenin, dioscin, prosapogenin and trillin which all have better activity for reducing fat, resisting coagulation and resisting free radicals and are effective components of discorea nipponica makino for reducing blood fat, wherein the effects of the dioscin and the prosapogenin are more remarkable.
Description
Technical field
The present invention relates to Chinese medicine extract and application thereof, relate in particular to Discorea nipponica makino extractive and the application in preparation free radical resisting, blood lipid-lowering medicine thereof.
Background technology
The tradition Chinese medicine is one of rarity in the magnificent cultural treasure-house of motherland, for the prosperity of the Chinese nation has been made huge contribution.Chinese medicine is the natural materials in order to prevent and cure diseases under instruction of Chinese Medicine theory, and Chinese medicine and pharmacy is gone through the development in thousands of years, and process successive dynasties medicine scholar's is constantly additional, perfect, has formed unique theoretical system in secular practice.To the deepening continuously of Chinese medicine research, it more and more is subject to people's attention with its definite curative effect and lower toxicity along with both at home and abroad.Along with China's entry into the WTO, the Chinese medicine development is faced with unprecedented opportunities.For making Chinese medicine in international medical market competition, obtain bigger development, set up and improve Chinese medicine series standard criterion, for the modernization of Chinese medicine provides safeguard very urgent.
Along with the development of society, human diseases spectrum quietly changes, and medical model changes prevention, health care, treatment, rehabilitation into by simple disease treatment and combines, and various alternative medicines and traditional medicine are just being brought into play increasing effect.Cardiovascular and cerebrovascular disease is called as human health " first killer ", add up according to The World Health Organization (WHO), the whole world all has 1,500 ten thousand people to die from cardiovascular and cerebrovascular disease every year, in recent years statistical data shows, the population of China cardiovascular and cerebrovascular disease rises year by year, state such as total number of persons has been much higher than U.S., adds, method, day, Switzerland.Crowd's hypertension prevalence is about 14% more than 15 years old, increases by one times before 30 years.The whole nation annual because of the number of cardiovascular and cerebrovascular disease death about 2,600,000, on average per hour dead 300 people.Treatment coronary heart disease, anginal Western medicine commonly used are nitrate esters medicine, beta-blocker, calcium antagonist and medicament for resisting platelet aggregation.With the nitroglycerin is the direct blood vessel dilating smooth muscle of nitrate esters medicine energy of representative, and the expansion peripheral vessels reduces the blood flow of venous return to heart, thereby alleviates the heart burden; Simultaneously also can coronary artery dilator, directly improve the ischemic region blood supply of cardiac muscle, cause angina pectoris to be alleviated.But said medicine simultaneously also has many untoward reaction, and for example: headache, flushing, tinnitus, dizzy, blood pressure drops, heart beating are overrun etc., and life-time service can produce drug resistance.Chinese medicine has unique advantage in treatment aspect the thrombotic disease, and this point is confirmed by for many years clinical trial and experimentation.
Dioscorea nipponica Mak. Ningpo Yam Rhizome (Dioscorea nipponica Mak.) is the Dioscoreaceae yam, perennial voluble herb.Rhizome is horizontal to be walked, cylindrical, yellowish-brown.Stem is left-handed, reaches 5 meters, and being close to does not have hair.Leaf tool long handle, alternate, avette or width egg shape, long 5～12cm, common 5～7 split, the base portion heart.The top sliver has long point, 9 of veins, and base goes out, and offshoot is netted.Brightly yellowish green, unisexuality, dioecism; The inflorescence axil is given birth to, and is sagging; The multiple spike of staminate inflorescence, the female inflorescence spike; Male flower is little, and is bell, tapel 6, and stamen 6 and is born on the perianth tube; Female perianth 6, the square circle, stigma 3 splits, and sliver 2 splits again.The capsule shape of falling ovum ellipse, tool 3 wings.Seed tool rectangle wing.6～August of florescence, really 8～October of phase.Medical material is the dry rhizome of Dioscorea nipponica Mak. Ningpo Yam Rhizome, claim again Rhizoma Dioscoreae Nipponicae, CHUANDILONG, Dioscorea nipponica Mak. Ningpo Yam Rhizome, Dioscorea nipponica bone, Os Draconis, dioscorea japonica, Canis familiaris L. Rhizoma Dioscoreae, chicken bone, mountain Radix Dichroae, Radix Dichroae, wear mountain bone, fiery rattan root, Rhizoma Dioscoreae Hypoglaucae, HUANGJIANG, native mountain potato, bamboo root potato, ferrum root potato, male Rhizoma Zingiberis Recens, yellow whip, mountain flower, JINGANGGU etc.Be elongated cylindrical, long 10～20cm, the about 1.5cm of diameter, the most irregular branches of tool, the surface soil yellow has the fibrous root residue of most thin longitudinal grins and projection, and holotype is Cornu Cervi slightly seemingly.Matter is hard, and section is faint yellow, mealiness, and visible most fine-meshed vascular bundles are dispersed in.Feeble QI, bitter in the mouth.Slightly long with rhizome, khaki, the hard person of matter is for well.
Dioscorea nipponica Mak. Ningpo Yam Rhizome often is born in the hill sylvan life, and happiness is born under hillside shrubbery and the sparse woods or in the limes marginis crack of stone of thicker soil, it has a very wide distribution, and adaptability is strong, and the cultivation breeding is easier, but still is in wild state at present, and artificial culture also is in the exploratory stage.Mainly be distributed in ground such as the Inner Mongol, Heilungkiang, Jilin, Liaoning, Hebei, Henan, Shaanxi, Shanxi, Gansu, Sichuan, Guizhou, Hubei, Hunan, Shandong, Anhui, Jiangsu, Zhejiang and Jiangxi.
Dioscorea nipponica Mak. Ningpo Yam Rhizome has antiinflammatory, eliminates the phlegm, relievings asthma, increases coronary flow, blood fat reducing, improves cardiovascular function and pharmacologically actives such as antibiotic and antiviral, but decreased heart rate also, strengthen myocardial contraction, increase urine amount every day, improve coronary circulation, reduce arteriotony, be particularly useful for slight arteriosclerosis.
Summary of the invention
One aspect of the present invention provides a kind of Discorea nipponica makino extractive, this extract be with the Dioscorea nipponica Mak. Ningpo Yam Rhizome medical material after alcoholic solution extracts, the extract that obtains with chloroform and n-butanol extraction is comprising steroidal compounds, plant sterol and benzoic acid.Preferred 60～70% alcoholic solution of alcoholic solution, more preferably 65% alcoholic solution.
This extract can further adopt silica gel column chromatography, ODS mesolow column chromatography and half preparative high-performance liquid chromatographic separation and purification.
Steroidal compounds in the extract comprises: diosgenin, dioscin, protodioscin and cryptogenin.
Plant sterol in the extract is a cupreol.
The present invention provides the application of Discorea nipponica makino extractive in preparation free radical resisting, blood lipid-lowering medicine on the other hand.
The present invention selects the hyperlipidemia animal model, activity with content, serum superoxide dismutases (SOD) and the lipid peroxidation (LPO) of bleeding time, clotting time, T-CHOL (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), HDL-C (HDL-C) is an index, Discorea nipponica makino extractive is reached the main steroidal compounds that wherein comprises, promptly the free radical resisting of diosgenin, dioscin, protodioscin and cryptogenin, effect for reducing blood fat are studied.
The chloroform of Dioscorea nipponica Mak. Ningpo Yam Rhizome 65% ethanol extraction provided by the invention and n-butanol extraction partly have blood fat reducing preferably, anticoagulant, free radical resisting activity.Further adopt multiple separating and purifying technologies such as silica gel column chromatography, ODS mesolow column chromatography and half preparative high-performance liquid chromatographic, separate 4 steroidal compounds that obtain, diosgenin (diosgenin),, dioscin (dioscin), protodioscin (protodioscin) and cryptogenin (trillin) all have blood fat reducing preferably, anticoagulant, free radical resisting activity, is the effective ingredient of Dioscorea nipponica Mak. Ningpo Yam Rhizome effect for reducing blood fat.Wherein dioscin and protodioscin effect are more remarkable.
Description of drawings
Fig. 1 is the extraction separation flow chart of Dioscorea nipponica Mak. Ningpo Yam Rhizome.
The specific embodiment
Introduce the present invention in detail below in conjunction with drawings and the specific embodiments; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also are considered as protection scope of the present invention.
One, experiment material
1, instrument:
RE-52A rotary evaporator Shanghai Yarong Biochemical Instrument Plant
876-1 type vacuum drying oven Shang Hai Jin Min scientific instrument company limited
Haikang China biochemical instrument maker on the ZF7B ultraviolet analysis instrument for three purposed
Jc18-1 infrared lamp Shanghai Xinhua Lamp Factory
DILW type electronic thermostatic water-bath Beijing bright forever Medical Instruments factory
The 101-3-BS electric heating constant temperature forced air drying Shanghai medical apparatus and instruments factory of making a leapleap forward
Case
BP210S electronic balance Germany Satorius company
Beckman DU640 uv-spectrophotometric day island proper Tianjin company
Meter
BT-224 automatic biochemistry analyzer Italy scientific instrument company
Mesolow column chromatography instrument day island proper Tianjin company
Centrifuge TGL-16C goes up Hai'an booth Instr Ltd.
Japan Yanaco micro melting point apparatus Japan Yanaco company
Bruker Tensor37 type infrared spectrometer Switzerland Bruker company
Bruker AV-400 type nuclear magnetic resonance analyser Switzerland Bruker company
Tianjin, island LC-8A type prepares liquid phase day island proper Tianjin company
Micromass Quattro type second order ms U.S. Waters company
Instrument
2, reagent and reagent
Rich Dihua, petroleum ether (60～90 ℃, analytical pure) Tianjin worker's company limited
Ethyl acetate (analytical pure) Tianjin Da Mao chemical reagent factory
Dehydrated alcohol (analytical pure) Guangzhou Chemical Reagent Factory
Rich Dihua, chloroform (analytical pure) Tianjin worker's company limited
Concentrated sulphuric acid (analytical pure) Tianjin Da Mao chemical reagent factory
Silica gel (200～300 order) Haiyang Chemical Plant, Qingdao
Silica GF254 (60 order) Haiyang Chemical Plant, Qingdao
The German MERCK of ODS (40 order) company
Malonaldehyde (MDA) test kit
Bio-engineering research institute is built up in Nanjing
Thiobarbituricacid (TBA colorimetric) examination
Bio-engineering research institute is built up in Nanjing
The agent box
Bio-engineering research institute is built up in xanthine oxidase (hydroxylamine assay) test kit Nanjing
Propylthiouracil (lot number 050401) Guangzhou Kanghe Pharmaceutical Co., Ltd.
Cholesterol (lot number 030814) Beijing chemical reagents corporation
DIAOXINXUE KANG JIAONANG (lot number Chengdu Diao Pharmaceutical Group Co., Ltd
0507060)
3, animal
Common cleaning level SD male rat, body weight 200 ± 20g, Guangdong Province's Experimental Animal Center provides.
The quality certification number: SCXK (Guangdong) the word 2005A011 that checks and affirm in 2003-0002 Guangdong
Two, the preparation of extract
As shown in Figure 1, be the extraction separation flow chart of Dioscorea nipponica Mak. Ningpo Yam Rhizome of the present invention.Get exsiccant Dioscorea nipponica Mak. Ningpo Yam Rhizome medical material 5kg, cross 40 mesh sieves after crushed, add 65% ethanol of 10 times of amounts, heating and refluxing extraction 2 times, each 2h filters, and merges ethanol extract, and decompression recycling ethanol gets ethanol extraction extractum 630g.Add 2L water with the extractum suspendible after, successively with chloroform, the extraction of n-butyl alcohol equal-volume 3 times, combining extraction liquid, behind the decompression and solvent recovery, chloroform layer extractum (60g), n-butanol layer extractum (160g).
Get chloroform layer extractum 10g, (f 5cm * 38cm) was with petroleum ether-ethyl acetate system gradient (100: 1 through silica gel (200-300 order) post; 95: 5; 90: 10; 80: 20; 70: 30) eluting carries out column chromatography, obtains 20 fractions (Fr.1-Fr.20).Fr.5 and Fr.8 are carried out silica gel column chromatography repeatedly,, obtain diosgenin (chemical compound 1) and cupreol (chemical compound 2) respectively with petroleum ether-ethyl acetate gradient elution, separation and purification.
Get n-butyl alcohol part extractum 10g, (f5cm * 38cm) was with chloroform-methanol-water system gradient (10: 1 through silica gel (200-300 order) post; 8: 2: 0.2; 7: 3: 0.5; 6: 4: 0.8) eluting carries out column chromatography, obtain 30 fractions (Fr.1～Fr.30), with Fr.15 and Fr.20 ethyl acetate-methanol (6: 4) recrystallization, obtain dioscin (chemical compound 3) and benzoic acid (chemical compound 4) respectively, other fractions are through the open column chromatography of ODS, methanol-water (6: 4,7: 3,8: 2) behind the gradient elution, fraction continues separation and purification through the preparation liquid phase, obtains protodioscin (chemical compound 5) and cryptogenin (chemical compound 6).
Three, the affirmation of main chemical compositions in the Discorea nipponica makino extractive
Chemical compound 1: diosgenin (diosgenin)
White, needle-shaped crystals (petroleum ether-ethyl acetate), C
27H
42O
3, the Liebermann-Burchard reaction is positive, and with A reagent (Anisaldehyde, p-tolyl aldehyde) reaction displaing yellow, does not develop the color with E reagent (Ehrlish, Paradimethylaminobenzaldehyde) reaction, infers that this chemical compound belongs to the Spirostance chemical compound.IR
KBr MaxCm
-1: 3452 (OH), 980,919,898,865 (919＜898cm
-1, the characteristic absorption of 25R spirostane); ESI-MS m/z:415[M+H]
+, 437[M+Na]
+ 13The C-NMR spectrogram and
1The H-NMR spectral data is consistent with the dioscin metadata that document is reported.Through carrying out three kinds of solvent systems thin layer altogether, spot colors and R with the diosgenin reference substance
fBe worth in full accordly, so chemical compound 1 is accredited as diosgenin, structure is as follows:
Chemical compound 2: cupreol (β-sitosterol)
White needle (chloroform), C
29H
50O, mp.137～138 ℃.IR
KBr maxcm
-1：3426(OH)，2936、1464、1380(CH
3)，1661(C＝C)；EI-MS?m/z：414(M)，396(M-H
2O)，381(M-H
2O-CH
3)。
13The C-NMR spectrogram and
1The H-NMR spectral data is consistent with the cupreol data that document is reported.Through carrying out three kinds of solvent systems thin layer altogether, R with the cupreol reference substance
fBe worth in full accordly, so chemical compound 2 is accredited as cupreol, its structure is as follows:
Chemical compound 3: dioscin (dioscin)
White needle (ethyl acetate-methanol), C
45H
72O
16, mp276～278 ℃, Liebermann-Burchard and Molish reaction are positive, and with A reagent reacting displaing yellow, do not develop the color with the E reagent reacting, infer that this chemical compound belongs to the spirostanol saponin compounds.Acid hydrolysis products and diosgenin reference substance carry out three kinds of solvent systems thin layer altogether, spot colors and R
fBe worth in full accordly, determine that aglycon is a diosgenin.IR
KBr MaxCm
-1: 3411 (OH), 980,919,899,865 (919＜899cm
-1, the characteristic absorption of 25R spirostane); ESI-MSm/z:869[M+H]
+, 891[M+Na]
+, 723[M-Rha]
+, consistent with bibliographical information.
13The C-NMR data and
1The H-NMR data are consistent with the dioscin data that document is reported.Through carrying out three kinds of solvent systems thin layer altogether, R with the dioscin reference substance
fBe worth in full accordly, so chemical compound 3 is accredited as dioscin, structure is as follows:
Chemical compound 4: benzoic acid (benzoic acid)
The white needle, C
7H
6O
2, 122～124 ℃ of mp are positive with the iron chloride reaction.IR
KBr MaxCm
-1: 3100-2600 (OH), 1680 (C=O), 1600,1580 (C=C) are consistent with bibliographical information.ESI-MS?m/z：121[M-H]
-，77[M-H-CO
2]
-。
13C-NMR and
1The H-NMR data are consistent with the benzoic acid data that document is reported.Through carrying out three kinds of solvent systems thin layer altogether, R with the benzoic acid reference substance
fBe worth in full accordly, so chemical compound 4 is accredited as benzoic acid, its structure is as follows:
Chemical compound 5: protodioscin (protodioscin)
White powder, C
51H
84O
22, 187～189 ℃ of mp, Liebermann-Burchard and Molish reaction are positive, vanillin reaction displaing yellow.ESI-MS?m/z：1031[M-H
2O]
+，1071[M+Na]
+，1087[M+K]
+，869[M-Glc]
+。
13C-NMR and
1The H-NMR data are consistent with the protodioscin data that document is reported.So chemical compound 5 is accredited as protodioscin.
Chemical compound 6: cryptogenin (trillin)
Colourless needle (chloroform-methanol), 239～242 ℃ of mp, Liebermann-Burchard and Molish reaction are positive, and with A reagent reacting displaing yellow, do not develop the color with the E reagent reacting, infer that this chemical compound belongs to the spirostanol saponin compounds.IR
KBr MaxCm
-1: 3472 (OH), 980,915,899,865 (915＜899cm
-1, the characteristic absorption of 25R spirostane); ESI-MS m/z:577[M+H]
+, 271[M+H-C
8H
15O
2-C
6H
11O
5]
+, 253[M+H-C
8H
15O
2-C
6H
11O
5-H
2O]
+ 13C-NMR and
1The H-NMR data are consistent with the cryptogenin data that document is reported.Through carrying out three kinds of solvent systems thin layer altogether, spot colors and R with the cryptogenin reference substance
fBe worth in full accord, so chemical compound 6 is accredited as cryptogenin.
Four, the free radical resisting of Discorea nipponica makino extractive, hypolipidemic activity
1, the preparation of sample solution
The present invention has carried out system thinking to extracting solvent.Adopt behind water, Different concentrations of alcohol and the decocting method such as different concentration ethanol precipitation to prepare Discorea nipponica makino extractive, observe of the influence of the prepared extract of Different Extraction Method, thereby determine the optimum extraction solvent high fat rat fat level, blood viscosity and body free radical.
Prepare following each medicinal liquid successively by table 1:
Dioscorea nipponica Mak. Ningpo Yam Rhizome decocting liquid: it is an amount of to get the Dioscorea nipponica Mak. Ningpo Yam Rhizome medicinal powder, adds 10 times of water, decocts 2 times, and each 2h merges 2 times decocting liquid, is concentrated into 1.5gmL
-1, standby.
The Dioscorea nipponica Mak. Ningpo Yam Rhizome alcohol extract: it is an amount of to get the Dioscorea nipponica Mak. Ningpo Yam Rhizome medicinal powder, and 95%, 65%, the 45% and 15% ethanol liquid that adds 10 times of amounts decocts 2 times, and each 2h merges 2 times ethanol liquid, and pressure reducing and steaming ethanol, residue add the suitable quantity of water dissolving, and concentration is the same, and is standby.
Dioscorea nipponica Mak. Ningpo Yam Rhizome water decoction-alcohol sedimentation liquid: it is an amount of to get the Dioscorea nipponica Mak. Ningpo Yam Rhizome medicinal powder, adds 10 times of water gagings, decocts 2 times, each 2h merges 2 times decocting liquid, adds an amount of dehydrated alcohol again, make it contain the alcohol amount and reach 95%, 65%, 45% and 15%, refrigerator takes out after leaving standstill 18h, sucking filtration is got subsequent filtrate and boil off ethanol on Rotary Evaporators, and residue adds the suitable quantity of water dissolving, concentration is the same, and is standby.
The extracting method of table 1, Dioscorea nipponica Mak. Ningpo Yam Rhizome
| Numbering | Extracting method |
| ??1 | Decocting liquid |
| ??2 | 95% alcohol extract |
| ??3 | 65% alcohol extract |
| ??4 | 45% alcohol extract |
| ??5 | 15% alcohol extract |
| ??6 | 95% alcohol deposit fluid |
| ??7 | 65% alcohol deposit fluid |
| ??8 | 45% alcohol deposit fluid |
| ??9 | 15% alcohol deposit fluid |
2, the foundation of hyperlipidemia animal model
(1) pharmacology index choice
Selection causes the rat high blood lipid model with high lipid food (containing Adeps Sus domestica, cholesterol 3%, Fel Sus domestica salt 0.5% and methylthiouracil 0.2%) feeding animals, is the pharmacology index with bleeding time, clotting time, blood lipid level and free radical resisting activity.
(2) method for building up of model
27 ℃ of room temperatures, under the condition of humidity 60%, after one week of animal feeding, be divided into blank group, high blood lipid model group, solvent matched group (DMSO aqueous solution), DIAOXINXUE KANG group (0.5mg total steroidal saponin/kg), 1～No. 9 medicine group (decocting liquid of Dioscorea nipponica Mak. Ningpo Yam Rhizome different solvents extract group at random, 95%, 65%, 45%, 15% alcohol extract, 95%, 65%, 45%, 15% alcohol deposit fluid).Except that solvent matched group 8, all the other respectively organize every group of 10 animals.Except that the blank group is fed normal diet, all the other each groups all give high lipid food, raise after 14 days continuously, inspection fasting the previous day be can't help drinking more than 8 hours, every animal all detects blood fat through the eyeball blood sampling, affirmation modeling success (blood lipid level before the administration, some of the rats that process check rejecting blood fat does not raise).Continue to give high fat diet subsequently.
(3) medication and dosage
Fed with high beginning in about the 15th day administration is tested 1～No. 9 medicine group respectively through irritating the administration of appetite clothes, irritates the stomach amount for being equivalent to crude drug 3gkg
-1, once a day, successive administration altogether around 28 days, (after the last administration, fasting can't help drinking 8h) surveys rat bleeding time, clotting time.All animals is taken a blood sample through eye socket, gets serum, detects blood fat (blood fat after the administration) and lipid peroxidase level.
(4) detect index
A, hemorrhage, clotting time
Bleeding time, clotting time can detect the flowability and the coagulability of blood, assay method: the bleeding time is used the docking method, and clotting time is controlled room temperature at 27 ℃ with capillary glass-tube method and slide method, and humidity is in about 60%.
B, blood fat
Blood drawing detects T-CHOL (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and HDL-C (HDL-C) content.
C, free radical resisting activity
Get veins of upper extremity blood on an empty stomach, centrifuging and taking serum, cryopreservation detects activity of SOD in serum and LPO content.Active xanthine oxidase (hydroxylamine assay) method that adopts of SOD, LPO (malonaldehyde MDA) detects and adopts thiobarbituricacid (TBA colorimetric) method, utilize Nanjing build up bio-engineering research test kit measure.
(5) result judges
Adopt computer statistics software SPSS version 10.0 to carry out variance analysis F, q and t check, P＜0.05 is judged to has significant difference.
3, experimental technique
27 ℃ of room temperatures, under the condition of humidity 60%, the SD rat gives the high lipid food diet after 14 days continuously, randomly draw every group 30% animal eyeball after vascular plexus get blood, detect T-CHOL (TC), triglyceride blood lipid levels such as (TG).As a result, with the blank group relatively, the high blood lipid model success rate reaches 99%, prove that the modeling type is successful, reject indivedual blood lipid levels animal on the low side after, carry out fat-reducing medicament and test.
(1) to the influence of rat bleeding time, clotting time
Comprehensive three kinds of method of inspection index results, and with hyperlipidemia model group and solvent matched group relatively, respectively be subjected to reagent all can reduce blood viscosity, make hemorrhage time lengthening (P＜0.05), but compare with positive drug DIAOXINXUE KANG group, each test group there is no significant difference (P＞0.05).Remove 95% ethanol extract, 95% pure hypostasis, 15% precipitate with ethanol beyond the region of objective existence, all the other administration group effects are all better.The results are shown in Table 2
Table 2, to the influence of rat bleeding time, clotting time
* compare P＜0.05 with the blank group,
# and positive controls be P＜0.05 relatively
(2) to the influence of rat fat level
After the SD rat gave the high lipid food diet continuously, untreated hyperlipidemia model group blood lipid level significantly raise, and showed that the bleeding time of blood obviously shortens than the blank group; Meanwhile, after the treatment through a course of treatment, be subjected to each group of reagent all in hypercholesterolemia reducing (TC), triglyceride (TG) level in various degree.Low density lipoprotein, LDL (LDL-C) and high density lipoprotein (HDL-C) content and HDL-C/LDL-C ratio, solvent matched group and model group do not have significant difference; Compare with hyperlipidemia model group and solvent matched group, remove 95% pure hypostasis, 65% precipitate with ethanol beyond the region of objective existence, but respectively be subjected to all hypercholesterolemia reducing (TC) levels (P＜0.05) of reagent, but little to triglyceride (TG) level affects.To the lipoprotein especially influence of HDL/LDL ratio, from this experimental result, the DIAOXINXUE KANG group is to promoting the HDL level, it is general to improve HDL/LDL ratio effect, but be subjected to 95% ethanol extract in the reagent, 65% ethanol extract, 15% ethanol extract, 15% pure hypostasis better for the horizontal effect of rising HDL, can obviously improve HDL/LDL ratio, wherein 65% ethanol extract and 15% ethanol extract effect are better than DIAOXINXUE KANG group (P＜0.05), and the rising of the level of HDL is very big for the hypercholesterolemia reducing effect, is one of reason that T-CHOL reduces in these the four groups experiments.The results are shown in Table 3, table 4.
Table 3, to the influence of rat T-CHOL, triglyceride levels
* compare P＜0.05 with the blank group,
Table 4, to the influence of rat high density lipoprotein and low-density lipoprotein white level
* compare P＜0.05 with the blank group,
# and positive controls be P＜0.05 relatively
(3) to the influence of free radicals in rats level
The SOD level has the effect that free radical resisting generates, and stable for keeping organismic internal environment, arteriosclerosis, anti-apoptotic have important function, and LPO is one of leading indicator of free radical generation.Be subjected to reagent in the performance blood fat reducing, serum superoxide dismutases (SOD) and lipid peroxidation (LPO) also there is in various degree influence, wherein decocting liquid, 95% ethanol extract, 65% ethanol extract, 15% ethanol extract, 15% pure hypostasis are subjected to reagent to improving serum superoxide dismutases (SOD) level (P＜0.05), it is comparatively remarkable to reduce lipid peroxidation (LPO) level (P＜0.05) effect simultaneously, sees Table 5.
Table 5, to the influence of rat blood serum superoxide dismutase (SOD) and lipid peroxidation (LPO) level
* compare P＜0.05 with the blank group,
Comprehensive relatively each administration group is to hyperlipemia rat bleeding time, clotting time, blood lipid level, can draw after the active influence of free radical resisting: 65% ethanol extraction is all comparatively remarkable to the every index influence that detects, so the present invention determines that 65% ethanol is the optimum extraction solvent.
4, the determination of activity of effective site
The preparation of sample solution: take by weighing required Dioscorea nipponica Mak. Ningpo Yam Rhizome medical material, pulverized 40 mesh sieves, with 65% ethanol (10 times of amounts) reflux, extract, 2 times, each 2h filters, and filtrate merges, and rotary evaporation is flung to ethanol, filter spissated aqueous solution.With chloroform extraction 3 times, press chloroform at every turn: water=extraction in 1: 1, combining extraction liquid volatilizes, be dissolved in water, dilute the aqueous solution (A medicine group) of debita spissitudo.Water layer with n-butanol extraction 3 times, press n-butyl alcohol at every turn: water=extract at 1: 1, combining extraction liquid volatilizes, be dissolved in water, dilute the aqueous solution (B medicine group) of debita spissitudo.The water layer of last gained is evaporated to debita spissitudo (C medicine group).
Free radical resisting, the experiment of blood fat reducing drug effect: the preparation of experiment material, hyperlipidemia animal model, detection index and experimental technique are all with aforementioned.
At random rat is divided into blank group, hyperlipidemia model group, solvent matched group, DIAOXINXUE KANG group, A medicine group, B medicine group and C medicine group.
(1) to the influence of rat bleeding time, clotting time
Comprehensive three kinds of method of inspection index results compare with hyperlipidemia model group and solvent matched group, respectively are subjected to reagent all can prolong the bleeding time, reduce blood viscosity (P＜0.05); But compare with positive drug DIAOXINXUE KANG group, each test group there is no significant difference (P＞0.05).Wherein chloroform layer and n-butanol layer effect are more remarkable, and three kinds of detection method gained results all are better than the DIAOXINXUE KANG group.The results are shown in Table 6.
Table 6, to the influence of rat bleeding time, clotting time
* compare P＜0.05 with the blank group,
# and positive controls be P＜0.05 relatively
(2) to the influence of rat fat level
After the treatment through a course of treatment, be subjected to each group of reagent all in varying level hypercholesterolemia reducing (TC), triglyceride (TG) level.Compare with hyperlipidemia model group and solvent matched group, but respectively be subjected to reagent group all hypercholesterolemia reducing (TC) level and triglyceride (TG) level (P＜0.05).To the lipoprotein especially influence of HDL/LDL ratio, better with chloroform layer and n-butanol layer effect.The results are shown in Table 7, table 8.
Table 7, to the influence of rat T-CHOL (TC) level and triglyceride (TG) level
* compare P＜0.05 with the blank group,
Compare P＜0.05 with the hyperlipidemia model group,
Table 8, to the influence of rat high density lipoprotein (HDL) and low density lipoprotein, LDL (LDL) level
* compare P＜0.05 with the blank group,
# and positive controls be P＜0.05 relatively
(3) to the influence of free radicals in rats level
Chloroform layer and n-butanol layer are to improving serum superoxide dismutases (SOD) level (P＜0.05) as can be known from experimental result, and it is comparatively remarkable to reduce lipid peroxidation (LPO) level (P＜0.05) effect simultaneously.See Table 9.
Table 9, to the influence of rat blood serum superoxide dismutase (SOD) and lipid peroxidation (LPO) level
* compare P＜0.05 with the blank group,
Above-mentioned experimental result shows, the rat high blood lipid model that high lipid food (containing Adeps Sus domestica, cholesterol 3%, Fel Sus domestica salt 0.5% and methylthiouracil 0.2%) feeding animals is caused, the chloroform layer of Dioscorea nipponica Mak. Ningpo Yam Rhizome 65% ethanol extract and n-butanol layer all have appreciable impact to go out clotting time, blood lipid level and the free radical level of hyperlipemia rat, illustrate that the active component of the blood fat reducing that contains in the chloroform layer of Dioscorea nipponica Mak. Ningpo Yam Rhizome extracting solution and n-butanol layer, anticoagulant, free radical resisting is more.
Five, free radical resisting, the hypolipidemic activity of main steroidal compounds in the Discorea nipponica makino extractive
1, the preparation of sample solution
4 steroidal compounds dioscins (dioscin), protodioscin (protodioscin), cryptogenin (trillin) and diosgenin (diogenin) that above-mentioned separation obtains are used dimethyl sulfoxide (DMSO) dissolving respectively, and adding distil water is diluted to 1.0mgmL
-1, place 4 ℃ of refrigerators to preserve.
2, experimental technique
27 ℃ of room temperatures, under the condition of humidity 60%, with rat feeding after one week, be divided into blank group, high blood lipid model group, solvent matched group (dimethyl sulphoxide solution), DIAOXINXUE KANG group (0.5mg total steroidal saponin/kg), experiment No. 1 medicine group (dioscin solution), No. 2 medicine groups (protodioscin solution), No. 3 medicine groups (cryptogenin solution) and No. 4 medicine groups (diosgenin solution) at random, remove 8 of solvent matched groups, all the other respectively organize every group of 10 animals, and experimental technique thereafter is ditto described.
Rat to after the conclusive evidence modeling success begins administration after raising about the 15th day, intraperitoneal injection, (injection volume 0.5mL/kg body weight) once a day, successive administration 28 days (after the last administration, fasting can't help drinking 8h) is surveyed rat bleeding time, clotting time.All animals is taken a blood sample through eye socket, gets serum, detects blood fat (blood fat after the administration) and lipid peroxidase level.
3, experimental result
(1) each monomeric compound is to the influence of rat bleeding time, clotting time
Comprehensive three test ratings compare with hyperlipidemia model group and solvent matched group, respectively are subjected to reagent all can prolong the bleeding time, reduce blood viscosity (P＜0.05); Compare with positive drug DIAOXINXUE KANG group, each test group there is no significant difference (P＞0.05).With protodioscin solution and dioscin solution most pronounced effects, bleeding time, two kinds of detection method indexs of clotting time all near or be better than the DIAOXINXUE KANG group.Experimental result is all used means standard deviation
Expression, the t inspection statistics is handled, and the results are shown in Table 10.
Table 10, monomeric compound are to the influence of rat bleeding time, clotting time
* compare P＜0.05 with the blank group,
Compare P＜0.05 with the hyperlipidemia model group,
# and positive drug group be P＜0.05 relatively
The result shows, after the treatment through a course of treatment, compares with the hyperlipidemia model group, respectively is subjected to reagent all can prolong the bleeding time, reduces blood viscosity (P＜0.05).With positive drug DIAOXINXUE KANG group relatively, dioscin solution is close with protodioscin solution effect or slightly be better than DIAOXINXUE KANG group, all a little higher than DIAOXINXUE KANG group of two kinds of detection method indexs of bleeding time and clotting time.
(2) each monomeric compound is to the influence of rat fat level
After the SD rat gave the high lipid food diet continuously, untreated hyperlipidemia model group blood lipid level significantly raise, and the bleeding time that shows blood is than the apparent in view shortening of blank group; Meanwhile, after the treatment through a course of treatment, be subjected to each group of reagent all in varying level hypercholesterolemia reducing (TC), triglyceride (TG) level.To low density lipoprotein, LDL (LDL-C), high density lipoprotein (HDL-C) content and HDL-C/LDL-C ratio, solvent and blank group and model group do not have significant difference; Compare with hyperlipidemia model group and solvent matched group, but respectively be subjected to reagent all hypercholesterolemia reducing (TC) level and triglyceride (TG) level (P＜0.05).To the lipoprotein especially influence of HDL/LDL ratio, better with cryptogenin solution and diosgenin solution, effect is better than DIAOXINXUE KANG group (P＜0.05).From this experimental result, be subjected in the reagent protodioscin solution and diosgenin solution best, and that the level of HDL raise is very big for the hypercholesterolemia reducing effect for the horizontal effect of rising HDL, be one of reason that T-CHOL reduces in these the four groups experiments.The results are shown in Table 11, table 12.
Table 11, monomeric compound are to the influence of rat T-CHOL (TC), triglyceride (TG) level
* compare P＜0.05 with the blank group,
Table 12, monomeric compound are to the influence of rat high density lipoprotein (HDL) and low density lipoprotein, LDL (LDL) level
* compare P＜0.05 with the blank group,
# and positive drug group be P＜0.05 relatively
The result shows, after the treatment through a course of treatment, is subjected to each group of reagent all in varying level hypercholesterolemia reducing (TC), triglyceride (TG) level.To low density lipoprotein, LDL (LDL), high density lipoprotein (HDL) content and HDL/LDL ratio, solvent matched group (DMSO and distilled water) does not have significant difference with model group, compare with the hyperlipidemia model group, but respectively be subjected to reagent all hypercholesterolemia reducing (TC) and triglyceride (TG) level (P＜0.05).Influence to high and low density lipoprotein, especially HDL/LDL ratio, be subjected to that cryptogenin solution and diosgenin solution can obviously improve HDL/LDL ratio for the horizontal most pronounced effects of rising HDL in the reagent, effect is better than DIAOXINXUE KANG group (P＜0.05).Though it is dioscin solution and protodioscin solution also can promote the HDL level, not obvious to improving the effect of HDL/LDL ratio.
(3) each monomeric compound is to the influence of free radicals in rats level
Four groups are subjected to reagent solution to improving SOD level (P＜0.05), and reducing LPO level (P＜0.05) simultaneously all has certain effect, and wherein dioscin solution, protodioscin solution and cryptogenin solution are comparatively remarkable.See Table 13.
Table 13, monomeric compound are to the influence of rat blood serum superoxide dismutase (SOD) and lipid peroxidation (LPO) level
* compare P＜0.05 with the blank group,
The result shows, be subjected to reagent that serum superoxide dismutases (SOD) and lipid peroxidation (LPO) are also had in various degree influence, wherein dioscin solution, protodioscin solution and cryptogenin solution are to improving serum superoxide dismutases (SOD) level (P＜0.05), it is comparatively remarkable to reduce lipid peroxidation (LPO) level (P＜0.05) effect simultaneously, substantially parallel with the effect of its blood fat reducing, reduction blood viscosity.
Above-mentioned experimental result shows, 4 kinds of steroidal compounds through lumbar injection have significantly effect to reducing the hyperlipemia rat blood lipid level, reduce blood viscosity and removing the body free radical, be the effective ingredient of Dioscorea nipponica Mak. Ningpo Yam Rhizome effect for reducing blood fat, wherein dioscin and protodioscin most pronounced effects.
Claims (7)
1. a Discorea nipponica makino extractive is characterized in that, described extract be with the Dioscorea nipponica Mak. Ningpo Yam Rhizome medical material after alcoholic solution extracts, the mixture that obtains with chloroform and n-butanol extraction is comprising steroidal compounds, plant sterol and benzoic acid.
2. extract as claimed in claim 1 is characterized in that, described alcoholic solution is 60%～70% alcoholic solution.
3. extract as claimed in claim 2 is characterized in that, described alcoholic solution is 65% alcoholic solution.
4. extract as claimed in claim 1 is characterized in that, this extract can further adopt silica gel column chromatography, ODS mesolow column chromatography and half preparative high-performance liquid chromatographic separation and purification.
5. extract as claimed in claim 1 is characterized in that, the steroidal compounds in the described extract comprises: diosgenin, dioscin, protodioscin and cryptogenin.
6. extract as claimed in claim 1 is characterized in that, the plant sterol in the described extract is a cupreol.
7. the application of Discorea nipponica makino extractive as claimed in claim 1 in preparation free radical resisting, blood lipid-lowering medicine.
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