CN100500193C

CN100500193C - Extract from decoction of rehmannia including six elements, combination of medication, and medical use

Info

Publication number: CN100500193C
Application number: CNB200410058600XA
Authority: CN
Inventors: 赵毅民; 乔善义; 张永祥; 周文霞; 马渊; 齐春会; 董剑英; 孙磊; 程军平; 赵修南; 童静; 任凤霞; 熊善丽; 郭继芬
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2003-08-19
Filing date: 2004-08-19
Publication date: 2009-06-17
Anticipated expiration: 2024-08-19
Also published as: CN1626228A

Abstract

An extractive from decoction of rehmannia including six elements, comprises 11.4-17 weight percents amylase, and/or 1.85-2.27 weight percents total glucosides (Monod glucoside, Loganin, Paeoniflorin) and/or 23.5-35.5 weight percents manninotriose according to weight of the decoction of rehmannia including six elements, wherein the rehmannia including six elements is compounded with rehmanniae vaporata, cornus, yam, rhizoma alismatis, cortex moutan, Tuckahoe in a ratio of 8:2:4:3:3:3 in weight.

Description

LIUWEI DIHUANG TANG extract, its pharmaceutical composition and medical usage thereof

Technical field

The present invention relates to LIUWEI DIHUANG TANG extract (being called for short LW-ABC), its extracting method, the pharmaceutical composition that contains this extract and medical usage thereof, described medical usage comprises the purposes of treatment or adjuvant treatment of diseases, and related disease comprises tumor, tumor chemistry or radiocurable side effect, leukopenia, aging immunologic hypofunction, infection etc.; Autoimmune disease such as systemic lupus erythematosus (sle), nephrotic syndrome, nephritis, bronchitis and bronchial asthma, prostatitis, aplastic anemia, essential thrombopenia, myasthenia gravis reach the immunologic function disorders that the patient showed such as Senile disease; And with the syndrome of deficiency of kidney yin reproductive endocrine function low or disorders comprises climacteric syndrome, ovarian hypofunction, infertility, habitual abortion, autonomic nervous dysfunction, chloasma, gynaecomastia, senile sexual disorder, climacteric osteoporosis, senile osteoporosis etc.

Technical background

LIUWEI DIHUANG TANG is by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria Six-element Chinese medicine are formed with the part by weight compatibility of 8:4:4:3:3:3, be mainly used in the treatment soreness of the waist and knees, have a dizzy spell, Hiccough and deaf, seminal emission, quench one's thirst, night sweat, osteopyrexia and fever, tooth shakes; the card of deficiency of kidney yin such as dribbling urination; widely use in tcm clinical practice; and in secular clinical practice, constantly developed; range of application constantly enlarges. in modern clinical treatment, still be widely used in having hundreds of treatment of diseases or the auxiliary treatment of syndrome of deficiency of kidney yin performance; comprise tumor; autoimmune disease such as systemic lupus erythematosus (sle); nephrotic syndrome; nephritis leukopenia bronchitis and bronchial asthma; prostatitis; aplastic anemia essential thrombopenia; myasthenia gravis; climacteric syndrome; prostate hyperplasia; infertility; hypertensive state of pregnancy neurasthenia; parkinson; hypertension; coronary heart disease; arrhythmia; pyelonephritis; diabetes etc. has obtained excellent curative.But should just no matter as decoction, pill, capsule or other dosage form, all there be obviously deficiency in actual applications at present, mainly showed the following aspects: the one, dosage is big, and the medication inconvenience is difficult to make the convenient pharmaceutical dosage form that uses; The 2nd, effective ingredient is indeterminate, not with effective ingredient as quality control index, be difficult to guarantee the stable and controlled of product quality; The 3rd, when a variety of causes causes active constituent content in the medical material big variation to occur, do not have effective means to guarantee the stable of active constituent content in the product, thereby can't guarantee the effectiveness of clinical application.Therefore, improve traditional LIUWEI DIHUANG TANG, determine its effective ingredient and content range, thereby the LIUWEI DIHUANG TANG of making stable effective ingredients has important practical significance.

Summary of the invention

The inventor after deliberation, have now found that by traditional LIUWEI DIHUANG TANG is scientifically handled, when keeping the various drug effects of former LIUWEI DIHUANG TANG, volume in the time of can reducing the LIUWEI DIHUANG TANG use greatly, obviously improve active constituent content in the LIUWEI DIHUANG TANG, determine the distribution and the content of effective ingredient in the LIUWEI DIHUANG TANG, thereby guarantee LIUWEI DIHUANG TANG safety effectively and use effectively.LIUWEI DIHUANG TANG extract of the present invention has good usability, and dissolubility is 〉=1.5g/ml in the water at normal temperatures.

Therefore, first aspect present invention relates to LIUWEI DIHUANG TANG extract, it comprises in LIUWEI DIHUANG TANG extract weight, 11.4-17 the polysaccharide of weight %, and/or the mannotriose of the total glycosides of 1.85-2.27% weight % (morroniside, meliatin, peoniflorin) and/or 23.5-35.5 weight %, wherein LIUWEI DIHUANG TANG is made up of with the part by weight compatibility of 8:4:4:3:3:3 Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria Six-element Chinese medicine.

The present invention relates to pharmaceutical composition on the other hand, it comprises the polysaccharide that contains 11.4-17 weight %, and/or the LIUWEI DIHUANG TANG extract and the pharmaceutical carrier of the mannotriose of the total glycosides of 1.85-2.27% weight % (morroniside, meliatin, peoniflorin) and/or 23.5-35.5 weight %, wherein LIUWEI DIHUANG TANG is made up of with the part by weight compatibility of 8:4:4:3:3:3 Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria Six-element Chinese medicine.

Further aspect of the present invention relates to the six drugs containing rehmanniae preparation method of extract, and it comprises:

I) will be concentrated into extractum then by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria by the mixture boiling water steaming and decocting twice that 8:4:4:3:3:3 forms;

Ii) follow to stir in extractum to add ethanol to concentration of alcohol 30% (v/v) placement, centrifugal, flushing is merged into supernatant with supernatant and washing liquid;

Iii) concentrated supernatant is followed to stir to add 95% ethanol to concentration of alcohol 60% (v/v) toward concentrated solution in, placement, separate position A and supernatant;

Iv) iii) middle supernatant is carried out column chromatography, water and polar organic solvent eluting get position B after the organic solvent eluent concentrates;

V) iv) middle water elution liquid concentrates the back and goes up activated-charcoal column, uses ethanol elution, gets position C after ethanol elution concentrates;

Vi) will iii) middle position A, iv) middle position B and v) middle position C mixing.

According to the present invention, LIUWEI DIHUANG TANG extract can obtain by following technological process:

Specifically, by the proportioning of classical each single medicinal material of LIUWEI DIHUANG TANG, form crude drug.Add boiling water and decoct, filter, get extracting solution.Extracting solution is concentrated into proper volume, adds alcohol to final concentration 30%, stirs, and placement is spent the night.Centrifugal treating (2500 rev/mins), precipitation is abandoned.The supernatant merging is concentrated into an amount of, and determining alcohol is adjusted to 60%, stirs, and places, with the method centrifugal treating.Precipitation is carried out secondary alcohol precipitation again with method and is handled.The gained precipitation concentrates except that behind the alcohol with water dissolution, and lyophilization is position A.Three times supernatant merges, and alcohol is removed in the rotating thin film evaporation, carries out macroporous adsorbent resin column chromatography, and water and polar organic solvent be eluting successively, and polar organic solvent eluting position concentrates removes the back lyophilizing of desolvating, and is position B.Activated carbon column chromatography is carried out after concentrating in the water elution position, and water and ethanol is eluting successively, and pure eluting position concentrates removes alcohol back lyophilizing, is position C.A, B and three positions of C are merged, be LIUWEI DIHUANG TANG extract LW-ABC.

Big pore adsorption resin is the low pole macroporous adsorbent resin of all models in the above-mentioned LW-ABC preparation method; The ethanol precipitation twice used alcohol that forms sediment is C1-4 lower alkyl alcohol (as: methanol, ethanol etc.), and concentration range is respectively 10%-40% and 50%-95%; The used polar organic solvent of macroporous adsorption resin chromatography eluting is the polar organic solvent (as: methanol, ethanol, propanol and acetone etc.) that all macroporous resins can allow use, and concentration range is 10%-95%.The used alcohol of activated carbon chromatography eluting is methanol and ethanol, and eluting concentration is 20%-95%.

The invention still further relates to LIUWEI DIHUANG TANG extract and be used for prevention or treatment tumor in preparation, side effect in tumor chemistry or the radiation therapy process, leukopenia, the aging immunologic hypofunction, infect, systemic lupus erythematosus (sle), nephrotic syndrome, nephritis, bronchitis and bronchial asthma, prostatitis, aplastic anemia, essential thrombopenia, myasthenia gravis, senile immunologic hypofunction or disorder, climacteric syndrome, ovarian hypofunction, habitual abortion, infertility, chloasma, gynaecomastia, climacteric osteoporosis, senile osteoporosis, purposes in the product of autonomic nervous dysfunction, wherein LIUWEI DIHUANG TANG extract comprises in LIUWEI DIHUANG TANG extract weight, 11.4-17 the polysaccharide of weight %, and/or the total glycosides (morroniside of 1.85-2.27% weight %, meliatin, peoniflorin) and/or the mannotriose of 23.5-35.5 weight %, wherein LIUWEI DIHUANG TANG is by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria Six-element Chinese medicine are formed with the part by weight compatibility of 8:4:4:3:3:3.

According to the present invention, the content of total glycosides is morroniside content in the LIUWEI DIHUANG TANG of the present invention, meliatin content and content of paeoniflorin sum.

According to the present invention, extract A (position A) is to contain the LIUWEI DIHUANG TANG extract that polysaccharide is a feature.

According to the present invention, extract B (position B) is to contain the LIUWEI DIHUANG TANG extract that glycoside is a feature.

According to the present invention, extract C (position C) is to contain the LIUWEI DIHUANG TANG extract that mannotriose is a feature.

Description of drawings

Fig. 1-1 criticizes the stricture of vagina collection of illustrative plates for the total ion current of LIUWEI DIHUANG TANG extract

Fig. 1-2 is the HPLC-DAD finger printing of LIUWEI DIHUANG TANG extract

Fig. 2 is a morroniside reference substance chromatograph

Fig. 3 is a meliatin reference substance chromatograph

Fig. 4 is the peoniflorin reference substance

Fig. 5 is the sample high performance liquid chromatography

Fig. 6 is a sample total ion current chromatograph

Fig. 7 is morroniside negative ion mass spectrum figure in the sample

Fig. 8 is the plain negative ion mass spectrum figure of malleable iron in the sample

Fig. 9 is peoniflorin negative ion mass spectrum figure in the sample

Figure 10 reveals three saccharide chromatograms for glycosides

Figure 11 is an oligosaccharide sample segment chromatogram

Figure 12 is the influence of LW-ABC to the SAMP8 oestrous cycle

Figure 13 is the influence of LW-ABC to SAMP8 serum estradiol level

Figure 14 is the influence of LW-ABC to SAMP8 hypophysis LH content

Figure 15 is the influence of LW-ABC to SAMP8 hypothalamus GnRH content

Figure 16 is LW-ABC handles (deficiency of the kidney yin) mouse ovarian weight to corticosterone influence

Figure 17 is LW-ABC handles (deficiency of the kidney yin) mice serum glycol level to cortex influence

Figure 18 is LW-ABC handles (deficiency of the kidney yin) mice hypophysis LH content to cortex influence

According to the present invention, LIUWEI DIHUANG TANG extract of the present invention is qualitative discriminating as follows:

(1) alphanaphthol reaction (Molish reaction)

LIUWEI DIHUANG TANG extract adds three of 10% alpha-Naphthol ethanolic solutions with an amount of water-soluble solution, Slowly add the concentrated sulfuric acid along test tube wall then, make formation two-layer up and down, after a while at the interface two-layer As seen the purple ring represents to have oligosaccharides, glycoside and polysaccharide composition to exist.

(2) qualitative examination of the iridoid glycoside take loganin as representative

LIUWEI DIHUANG TANG extract leaches with an amount of 95% ethanol, and leachate is liquid to be measured. With horse The ethanolic solution of money element and morroniside is product in contrast. and liquid to be measured and contrast liquid difference point sample are in silicon On glue G lamellae and the silica gel G-0.3%CMC-Na lamellae, respectively with solvent A (just Butanols/ethanol/water, 15:3:3) (chloroform/methyl alcohol 16:4) launches, and divides with solvent B Do not use the colour developing of 10% vanillic aldehyde solution (then spray again 72% sulfuric acid) and Godin reagent, respectively Dried by the fire 5 minutes at 105 ℃ and 80-90 ℃, liquid to be measured has identical colour developing spot with contrast liquid The point.

(3) the HPLC finger-print of LIUWEI DIHUANG TANG extract:

Liquid phase chromatogram condition

Zorbax Eclipse XDB C-8 post (150mm * 4.6mm, 5 μ m), flow rate of mobile phase 1.00ml/min detects wavelength 254nm, 7 ℃ of column temperatures, CH₃CN-H ₂O binary ladder Degree wash-out (0min:2:98; 50min:24:76,60min:56:44), sample size 20 Wash-out is complete in the μ l, all components 60min.

Result such as Fig. 1-1, shown in Fig. 1-2:

According to the present invention, LIUWEI DIHUANG TANG extract of the present invention is quantitatively differentiated as follows:

One, the assay of methods of glycosides in the extract

Instrument and reagent

Agilent1100 high efficiency liquid phase system contains quaternary gradient pump, DAD detector, column temperature Case and chem workstation. The API of U.S. ABI company 3000 liquid chromatograph-mass spectrometers are joined Turbo Ionspray ionization source and Analyst 1.1 data handling systems are arranged.

Morroniside, loganin reference substance are determined structure through spectrum, the HPLC face for this chamber self-control Long-pending normalization method records purity more than 98.0%. The Paeoniflorin reference substance is available from Chinese medicine biology Goods calibrating institute. cultivated land, Fructus Corni, Chinese yam, Poria cocos, rhizoma alismatis, moutan bark are all available from Beijing Medicinal material distribution center of Tongrentang. Methyl alcohol is chromatographically pure, and water is deionized water.

Experimental technique

1. chromatographic condition: Diamonsil C₁₈(4.6mm * 250mm, 5 μ m) chromatographic column, mobile phase is methyl alcohol-water (33:67), flow velocity 1.0mL.min^-1, 30 ℃ of column temperatures, DAD inspection Survey wavelength 236nm, sample size 20 μ L. Theoretical cam curve is pressed the loganin peak and is calculated greater than 3000, With this understanding, morroniside, loganin, Paeoniflorin all can reach good base with other components Line separates.

2. mass spectrum condition: anion detects, and sprays voltage and is-4000V DP voltage-70V, FP Voltage-290V, 300 ℃ of TEM, NEB are that 8, CUR is 11.

3. the preparation of reference substance solution: precision takes by weighing morroniside, loganin, Paeoniflorin reference substance Each 10mg places in the 10mL volumetric flask, is settled to scale with methyl alcohol, shakes up. Respectively therefrom Draw 75,100,150,300,600 μ L, thin up becomes 10mL, be prepared into 5 parts right According to product solution.

4. the preparation of sample solution: get this extract 8.92g, add 80 milliliters of 60% ethanol, Fully stir, place, (2500 rev/mins, 25min), precipitation is used 60% second again in centrifugal processing The alcohol precipitation, precipitation is abandoned. Merge supernatant twice, through the evaporation of rotation film, remove ethanol, advance The row macroporous adsorbent resin column chromatography, water and 30% ethanol is wash-out successively, 30% ethanol elution section The position concentrates except freeze-drying behind the alcohol, is the glucoside position. and get and add in right amount water to be made into concentration be 40mg/mL, Totally 3 parts, it is rear by above-mentioned chromatographic condition sample introduction to filter (filter membrane of 0.45 μ m), calculates the Monot The content of glycosides, loganin, Paeoniflorin.

The result

Under above-mentioned chromatographic condition, respectively 3 reference substances are carried out HPLC-DAD and HPLC-MS analysis.Morroniside, meliatin, peoniflorin retention time are respectively 6.0,11.0, and 12.0min, Electrospray Mass Spectrometry detect the conclusive evidence mass number and conform to value of calculation; Again sample is carried out HPLC-DAD and HPLC-MS analysis, the chromatographic retention of main component all conforms to reference substance with mass spectrometric data, the results are shown in Figure 2-9.

The do not fall standard curve equation of glycosides of result is A=2772.8C-13.28, r=0.9998, the range of linearity 7.4～60mg.L ^-1The standard curve equation of meliatin is A=2676.3C-4.14, r=0.9996, the range of linearity 7.7～62mg.L ^-1The standard curve equation of peoniflorin is A=2541.3C-42.49, r=0.9991, the range of linearity 8.5～68mg.L ^-1

Morroniside, meliatin, peoniflorin average recovery result of the test in the table 1 six drugs containing rehmanniae decocting liquid

The RP-HPLC analysis result shows that the relative retention time of main component morroniside (a), meliatin (b), peoniflorin (c) is respectively 1.0,1.75～1.85,1.90～2.10 in the extract.HPLC-MS the analysis showed that, the main component a in the sample, b and c have the quasi-molecular ion of [M-H] respectively (a is: 405 and 243 with [M-glc] fragment peak in (-) ESI-MS second order ms; B is: 389 and 227; C is: 479 and 317), see Fig. 6～Fig. 9.

Sample size is measured: morroniside, meliatin, paeoniflorin content are respectively 6.2-7.6%, 3.3-4.1%, 3.0-3.6% in the sample, and three parts add and are 12.5-15.3%.Conversion adds and is 1.85-2.27% for three parts in LIUWEI DIHUANG TANG extract.

Two, the assay of oligosaccharide (mannotriose) in the extract

Instrument and reagent

Mannotriose is this prepared in laboratory, is defined as mannotriose through spectrum, HPLC-MS, measures its purity by area normalization method and reaches more than 98%; Acetonitrile is a chromatographically pure; Water is deionized water.

Instrument: Waters 600 pumps, Waters 410 differential detectors.

Experimental technique

1, chromatographic condition Intersil nh 2 column (4.6mm * 250mm); Column temperature is a room temperature; Mobile phase be acetonitrile-water (67:33, v/v); Flow velocity is 1.0mL/mL; The 20uL injection valve; The differential detector.

2, the preparation of standard curve takes by weighing mannotriose reference substance 0.0170g, add water and be made into 5mL solution, shake up the back and cross the filter membrane of 0.45 μ m, therefrom draw 3,2.5 respectively then, 1.5, being made into the 5mL aqueous solution 1.25mL add water, shaking up, is 5 parts of reference substance solution altogether. analyze by above-mentioned chromatographic condition sample introduction, chromatographic peak integral area and reference substance concentration are done regression analysis, investigate the range of linearity.

3, precision experiment is by above-mentioned chromatographic condition, with same reference substance solution continuous sample introduction 6 times, calculates RSD value, the measure of precision of investigation instrument.

4, stability experiment is pressed above-mentioned chromatographic condition, with same sample solution sample introduction, and 0,30min, 1h, 2h, 4h, 8h measures, and investigates the stability of sample solution.

5, sample determination is got this extract 8.92g, adds 80 milliliters of 60% ethanol, fully stirs, and places.(2500 rev/mins of centrifugal treating, 25min), precipitation reuse 60% ethanol precipitation, precipitation is abandoned. and merge supernatant twice, evaporate through rotating thin film, remove ethanol, carry out macroporous adsorbent resin column chromatography, activated carbon column chromatography is carried out after concentrating in the water elution position, and water and 30% ethanol is eluting successively, 30% ethanol elution position concentrates removes alcohol back lyophilizing, is the oligosaccharide position.Take by weighing by 3 parts of 0.0757g of oligosaccharide sample, 0.0755g, 0.0835g places the 25mL volumetric flask, adds water to scale, shakes up, and filters the back by above-mentioned chromatographic condition sample introduction, according to the content of the calculated by peak area oligosaccharide that records.

6, repeated experiment prepares 3 duplicate samples simultaneously by technology under same condition, takes by weighing a certain amount of aqueous solution that concentration is 3mg/mL that is made into, and analyzes at above-mentioned chromatographic condition sample introduction.

The result

Under above-mentioned chromatographic condition, mannotriose reference substance aqueous solution is favorable linearity in 0.85mg/mL～3.5mg/mL concentration range, and the standard curve equation is that Y=6.53 * 10-8A-0.023[Y is concentration mg/mL, and A is a peak area].The precision experimental result shows that it is good that this tests used instrument precision; The sample aqueous solution room temperature is measured in following 8 hours, and the result is very stable; Repeated experiment result (seeing Table 2) shows, utilizes behind the water decoction-alcohol sedimentation pure molten part to cross macroporous resin and obtains the water elution part, obtains the extraction and separation process good reproducibility of active oligosaccharide again through the carbon column chromatography.

Analysis result shows that main component is a mannotriose in the oligosaccharide part in the sample, also has a spot of stachyose and disaccharide.The relative retention time of disaccharide, mannotriose and stachyose is respectively 1,1.40～1.53,1.55～1.70 (seeing Figure 10,11).In the C of position, mannotriose on average accounts for 36.8-56.8%, converts to mannotriose in LIUWEI DIHUANG TANG extract and on average accounts for 23.5-35.5%.

Table 2 HPLC measures oligosaccharide sample repeated experiment

Three, content Determination of Polysaccharide in the extract:

Instrument and reagent:

Reagent a: xenol (AR.): being configured to mass ratio with 0.5% sodium hydroxide is 0.15%, and refrigerator is preserved in January effective; Sodium tetraborate (AR.)-sulphuric acid test solution: the concentrated sulfuric acid solution of 0.0125mol/mL sodium tetraborate; Galacturonic acid (Sigma) titer: 80 ℃ are configured to 60 μ g/mL after being dried to constant weight, and refrigerator is preserved standby.Sulphuric acid is analytical pure; Phenol is analytical pure, heavily steams the back refrigerator and preserves, and is configured to 5% aqueous solution with before adding water; Glucose is an analytical pure, and 105 ℃ are dried to constant weight.

Instrument: cintra-20 (Australia) uv-spectrophotometric instrument

Experimental technique

1. an xenol method

1.1 the accurate Gala A titer 0,0.3,0.6 of drawing of the preparation of standard curve, 0.9,1.2,1.5mL places the volumetric flask of 6 25mL respectively, the next choice adds water to 1.5mL, shakes up the ice bath cooling, add sodium tetraborate-concentrated sulphuric acid 9.0mL and shake up, boiling water bath 5min takes out the ice bath cooling, hydroxyl biphenyl solution 0.15mL between adding, shake up, place half an hour after, measure absorption value down in 525nm.

1.2 the accurate absorption of precision experiment GalaA standard solution 0.9mL6 part, surplus operation is the same, investigates the precision of instrument under similarity condition.

1.3 the preparation of sample: get this extract 8.83g, add 80 milliliters of 60% ethanol, fully stir, place, centrifugal treating (2500 rev/mins 25min), precipitate reuse 60% ethanol precipitation once, supernatant discarded.Behind the precipitation water dissolution, lyophilization is standby.

1.4 stability experiment takes by weighing sample and disposes certain density solution, draws 1.0mL, surplusly presses first operation down, 0,20min, 40min, 1h, 3h, 5h, 8h measure absorbance, investigate sample and with the stability of reaction reagent generation colored substance.

1.5 the sample repeated experiment prepares 3 parts in sample simultaneously by technique processing method under similarity condition, press configuration solution under the sample determination item, draws 1.0mL, absorbance is measured in the same operation, investigates the stability of this separation method.

1.6 sample determination takes by weighing 6 parts in same sample, being configured to concentration is 0.4mg/mL, draw 1.0mL, it is divided into two groups, by first operation, first group adds developer, second group of 0.5% sodium hydroxide solution that adds isodose, first group record absorbance and deduct second group after, calculate the content of 60% precipitate with ethanol acidic polysaccharose.

1.7 the average recovery precision takes by weighing five parts in sample, every part of precision adds galacturonic acid standard solution 1.0,2.0,3.0 successively, 4.0 5.0mL adds the water standardize solution, after shaking up, draw 1.0mL by first operation down, measure absorbance, calculate recovery rate is investigated the stability of this quantitative approach.

2. phenolsulfuric acid method

2.1 the preparation precision of standard curve takes by weighing the glucose reference substance that is dried to constant through 105 ℃, being made into concentration is the 0.3mg/mL reserve liquid.Draw reserve liquid 1.0,2.0 respectively, 3.0,4.0,5.0,6.0 place the volumetric flask of 50mL, add water and are settled to scale and shake up, and are made into titer.Draw each 2.0mL of standard solution respectively and place the 25mL measuring bottle, add 5% (g/v) phenol solution 1.8mL, mixing, add concentrated sulphuric acid 7.5mL (10～20s) rapidly, mixing is put and is heated 20min in the boiling water bath, takes out in the psychrolusia of back and cools off 30min, be blank with the same operation repetitive of 2.0mL distilled water in addition, measure absorbance down in 490nm.

2.2 the preparation precision of calibration trace takes by weighing the galacturonic acid 0.0595g of 80 ℃ of oven dry, adds water and is made into 100mL solution, after shaking up, therefrom take out 1.0,2.0,3.0,4.0,5.0 6.0mL adds water to scale in 6 50mL volumetric flasks, after shaking up, therefrom get 2.0mL, place the 25mL volumetric flask, surplus operation is with first.

2.3 accurate totally 6 parts of the same standard solution 2.0mL that draw of precision experiment by first operation down, measure absorption value A, investigate the precision of instrument under similarity condition.

2.4 the accurate sample solution 2.0mL that draws of stability experiment, by first operation down, 0,10min, 30min, 1.0h, 1.5h, 2.5h, the 4.0h time point is surveyed absorption value, investigates stability of sample.

2.5 sample determination takes by weighing 3 parts in same sample, being configured to concentration is 0.04mg/mL, draws sample solution 2.0mL, places the 25mL volumetric flask, and surplus operation is measured absorbance by first, calculates the content of 60% precipitate with ethanol neutral polysaccharide.

2.6 the sample repeated experiment prepares 3 parts in sample simultaneously by technique processing method under similarity condition, press configuration solution under the sample determination item, draws 2.0mL and places the 25mL volumetric flask, absorbance is measured in surplus the same operation, investigates the repeatability of this separation method.

2.7 average recovery experiment precision takes by weighing 5 parts in sample, every part of accurate successively glucose standard solution 1.0,2.0,3.0 that adds, 4.0 5.0mL adds the water standardize solution, after shaking up, draw 2.0mL by first operation down, measure absorbance, calculate recovery rate is investigated the stability of this quantitative approach.

Experimental result:

Adopt an xenol method and the coupling of phenolsulfuric acid method, the content of the polysaccharide in the working sample macromole position after treatment.The xenol method is measured the content of alduronic acid between using earlier, and is accurately reliable; Measure the content of sugar then with the phenolsulfuric acid method, simultaneously alduronic acid is made reference substance, do calibration trace by this method, the glucuronic acid content that the first step is recorded is brought calibration trace into and is proofreaied and correct, obtain calculating absorbance. after from the absorbance that this method records, deducting value of calculation, bring the content that calculates neutral sugar in this method standard curve into, neutral sugar and acid sugar aldehydic acid add and promptly get the amount of total polysaccharides, convert out the content of total polysaccharides in sample according to the weight of raw sample again. measurement result shows that the content of total polysaccharides is between the 11.4-17% scope in the extract.

Table 3. an xenol method repeated experiment result

Table 4 phenolsulfuric acid method repeated experiment result

Further, the active polysaccharide position in the LIUWEI DIHUANG TANG mainly is made of acid heteroglycan.The active polysaccharide position after the hydrolysis, to launch on the cellulose thin-layer chromatography plate, with the monosaccharide standard control, can clearly detect rhamnose, arabinose, glucose, galactose and galacturonic acid with sample after the hydrolysis under acid condition.

The polysaccharide acid hydrolysis is analyzed sugar and is formed:

Get 5mg polysaccharide sample and add 2M trifluoroacetic acid 3ml, tube sealing was 100 ℃ of hydrolysis 10 hours, after a large amount of trifluoroacetic acids is removed in 40 ℃ of distilling under reduced pressure of hydrolyzed solution, divide five times and add 3ml methanol, azeotropic distillation, remove residual trifluoroacetic acid, residue is dissolved in the 0.3ml water and obtains testing sample.

With testing sample and standard monosaccharide aqueous solution point sample on water saturated cellulose thin-layer chromatography plate, use ethyl acetate: pyridine: glacial acetic acid: water (5:5:1:3) mixed solution is developing solvent, launched eight hours, after launching 16 centimetres approximately, take out, dry up developing solvent, spray is with the developer aniline-phthalic acid, 100 ℃ were dried by the fire 5 minutes. with Rf value and the spot colors and the comparison of standard monosaccharide of polysaccharide sample colour developing speckle, can determine that the monosaccharide of sample polysaccharide is formed.

Can obtain the feature thin-layer chromatogram at the active polysaccharide position in the LIUWEI DIHUANG TANG as stated above.Analysis result shows that Liuwei Dihuang polysaccharides mainly is made of rhamnose, arabinose, glucose, galactose and galacturonic acid, and contains micro-xylose and mannose.

According to the present invention, LIUWEI DIHUANG TANG extract LW-ABC of the present invention has obvious improvement or regulates the activity of immunologic function, to with syndrome of deficiency of kidney yin and have various diseases and the reproductive endocrine function low or disorderly various diseases, particularly tumor of immunologic hypofunction, in tumor chemistry or the radiation therapy process, leukopenia, the aging immunologic hypofunction, infect, systemic lupus erythematosus (sle), nephrotic syndrome, nephritis, bronchitis and bronchial asthma, prostatitis, aplastic anemia, essential thrombopenia, myasthenia gravis, senile immunologic hypofunction or disorder, climacteric syndrome, ovarian hypofunction, habitual abortion, infertility, chloasma, gynaecomastia, climacteric osteoporosis, senile osteoporosis, diseases such as autonomic nervous dysfunction have treatment or auxiliary treatment effect.

According to the present invention, LIUWEI DIHUANG TANG extract of the present invention is by the polysaccharide position, and glycoside position and oligosaccharide class position three parts are formed, and the content range that can measure composition is: total polysaccharides 11.4-17%, total glycoside 1.85-2.27%, oligosaccharide 23.5-35.5%.

According to the present invention, the invention still further relates to pharmaceutical composition, comprise LIUWEI DIHUANG TANG extract of the present invention and one or more pharmaceutical carriers or excipient as active component.

According to the present invention, the invention still further relates to LIUWEI DIHUANG TANG extract and be used to prepare treatment with syndrome of deficiency of kidney yin and have the various diseases and the low or disorderly various diseases of reproductive endocrine function of immunologic hypofunction, as: tumor, side effect when tumor chemistry or radiotherapy, leukopenia, the aging immunologic hypofunction, infect, systemic lupus erythematosus (sle), nephrotic syndrome, nephritis, bronchitis and bronchial asthma, prostatitis, aplastic anemia, essential thrombopenia, myasthenia gravis, senile immunologic hypofunction or disorder, climacteric syndrome, ovarian hypofunction, habitual abortion, infertility, chloasma, gynaecomastia, climacteric osteoporosis, senile osteoporosis, the purposes of the medicine that autonomic nervous dysfunction etc. are sick.

According to the present invention, LIUWEI DIHUANG TANG extract of the present invention can be used separately or with pharmaceutical compositions, and its administering mode can be decided as the case may be, can pass through oral, non-intestinal or topical, form of administration can be a tablet for example, pill, capsule, unguentum, oral liquid etc.

The specific embodiment

Embodiment

The following examples and biological activity test are to further describe of the present invention, but do not mean that any limitation of the invention.

Embodiment 1: the preparation of LIUWEI DIHUANG TANG extract LW-ABC

Proportioning by each single medicinal material of LIUWEI DIHUANG TANG, be Radix Rehmanniae Preparata: Fructus Corni: Rhizoma Dioscoreae: Rhizoma Alismatis: Cortex Moutan: Poria (8:4:4:3:3:3) or to the part flavour of a drug add, after the decrement, form crude drug totally 1500 grams, the boiling water that adds six times of crude drug weight decocts twice, each two hours, filter with absorbent cotton with gauze (six layers), be evaporated to crude drug (weight) and compare 1:1 with extractum (volume).

Extractum is followed to stir and is added ethanol to ethanol final concentration 30% (v/v), stirs, and placement is spent the night.Centrifugal treating (2500 rev/mins, 25min).Precipitation is with 30% alcohol flushing 4 times, and centrifugal, precipitation is abandoned.The supernatant merging is concentrated into 1000 milliliters, with 95% ethanol the ethanol final concentration is adjusted to 60%, stirs, and placement is spent the night, with the method centrifugal treating.Precipitation is carried out secondary alcohol precipitation again with 60% ethanol and is handled.The gained precipitation concentrates except that behind the alcohol with water dissolution, and lyophilization obtains pale powder (position A, 51.92 grams).Three times supernatant merges, the rotating thin film evaporation, remove ethanol, carry out the HP-20 macroporous adsorbent resin column chromatography, resin column bed volume (milliliter) and applied sample amount (gram) be than being 10:1, water and 30% ethanol eluting successively then, and 30% ethanol elution position concentrates removes lyophilizing behind the alcohol, obtain pale brown toner end (position B, 38.6 grams).Activated carbon column chromatography is carried out after concentrating in the water elution position, and activated-charcoal column bed volume (milliliter) and applied sample amount (gram) are than (20:1), and water and 30% ethanol is eluting successively, and 30% ethanol elution position concentrates removes alcohol back lyophilizing, obtains white powder (position C, 151 restrain).A, B and three positions of C are merged, form extract.

It has aforesaid qualitative reaction feature.

With aforesaid quantitative approach record extract obtained in: polysaccharide 14.2 weight %, total glycoside 2.06 weight %, oligosaccharide 29.5 weight %.

Embodiment 2: the biological activity test of embodiment 1 LIUWEI DIHUANG TANG extract LW-ABC

One, LW-ABC is to the effect of immunologic function

1.LW-ABC to " deficiency of the kidney yin " effect of immunologic function

The preparation of " deficiency of the kidney yin " mouse model is carried out as follows: give Balb/c mouse subcutaneous injection corticosterone suspension, 25mg/kg, 2 times/day, continuous 7 days.Began to irritate stomach the same day and gave LIUWEI DIHUANG TANG or LIUWEI DIHUANG TANG active component prescription LW-ABC in making to touch, 1 time/day, continuous 7 days, after the last administration, experimentized in 4 hours.Sacrificed by decapitation after mice is weighed, take out thymus and spleen respectively, weigh, calculate thymus and spleen index. during the lymphproliferation response experiment with the mice sacrificed by decapitation, the aseptic spleen of getting, the preparation splenocyte suspension, adjusting cell concentration with complete RPMI-1640 culture fluid is 5 * 106 cells/ml, that gets 50 μ l variable concentrations is subjected to the reagent thing, 50 μ l Con A (final concentration is 0.5ug/ml) and 100 μ l cell suspension add in 96 orifice plates successively, matched group with RPMI-1640 replaced C on A or (with) medicine, put 37 ℃, 5%CO2 was incubated incubation 56 hours, and every hole adds 20 μ l 3H-TdR (10 μ Ci/ml), continues to be cultured to 72 hours, with multiple tracks cell harvesting instrument collecting cell on glass fiber filter paper, after 80 ℃ of oven dry, glass fiber filter paper is placed in the scintillation solution, survey the cpm value with liquid scintillation counter.Every sample 4 parallel holes, the result is with average cpm value representation.The result shows, compares with matched group, and model mice thymus and index and spleen index obviously descend, and the splenocyte lymphproliferation response obviously descends; Orally give LW-ABC has obvious improvement effect (table 5) to the reduction of model mice thymus index and splenocyte breeder reaction ability.

Table 5.LW-ABC is to " deficiency of the kidney yin " effect of immunologic function

Annotate: the kidney yin method of touching of fabricateing: 25mg/kg corticosterone suspension sc, 2/ day; Make to touch and began gastric infusion the same day, 1 time/day, continuous 7 days, began experiment after the last administration in 4 hours; LW: LIUWEI DIHUANG TANG is the side entirely. ^*P＜0.05, ^*Compare with matched group p＜0.01; #p＜0.05, compare with the deficiency of the kidney yin group ##p＜0.01; Mean ± SD, n=10.

2.LW-ABC influence to caused by cyclophosphamide mouse boosting cell antibody formation reaction

Balb/c mice, gastric infusion 7 days, 1 time/day are used in experiment.In administration every mouse peritoneal injection sheep red blood cell (SRBC) (SRBC) suspension 0.2ml (5 * 107 erythrocyte) in the time of the 3rd day/only, the immune same day, except that the normal control group, each organizes mouse peritoneal injection cyclophosphamide, 15mg/kg, and next day to experimentize after with the SRBC immunity with of dosage duplicate injection on the 4th day, mice sacrificed by decapitation during experiment, get spleen, the preparation splenocyte suspension, the adjustment cell concentration is 2.5 * 106/ml.Extracting spleen cell suspension 400 μ l mix with 50%SRBC (50 μ l) and 50% complement (by the fresh guinea pig serum 50 μ l of 1:1 dilution), getting 100 μ l adds in the CunninghamShi cell, paraffin sealing is placed in 37 ℃ of incubators and hatched 1 hour, hemolysis plaque (PFC) forms number in the numeration cell, and the result represents with PFC/106 splenocyte.The result shows that cyclophosphamide is handled mouse boosting cell antibody formation reaction ability and obviously descended than matched group, and orally give LIUWEI DIHUANG TANG and LW-ABC all have obvious improvement effect (table 6).

Table 6.LW-ABC is to the influence of caused by cyclophosphamide immunologic hypofunction mouse antibodies reaction of formation

Annotate: LW: LIUWEI DIHUANG TANG. ^*Compare with the normal control group P＜0.01; #P＜0.05, compare with the Cy model group ##P＜0.01.

3.LW-ABC influence to the breeder reaction of tumor-bearing mice splenocyte

The preparation of transplanted tumor mouse model is carried out as follows: get the S180 ascites mice of going down to posterity, and the aseptic ascites of getting, with the normal saline dilution, the adjustment cell concentration is 2 * 105/ml.Give the right front axil subcutaneous injection of Balb/c mice S180 cell suspension, 0.2ml/ mice.Mice is 24 hours orally give LIUWEI DIHUANG TANG or LW-ABC after implanted tumor cells, continuous 12 days, experimentize, the mouse boosting cell breeder reaction is undertaken by preceding method. and the result shows, the orally give LIUWEI DIHUANG TANG has obvious improvement effect to the breeder reaction of the inductive tumor-bearing mice splenocyte of Con A, but inductive splenocyte breeder reaction does not have obvious effect to LPS; Orally give LW-ABC all has obvious improvement effect (table 7) to Con A and the inductive splenocyte breeder reaction of LPS.

Table 7.LW-ABC is to lotus S ₁₈₀The influence of sarcoma mouse boosting cell breeder reaction

Annotate: ^*P＜0.05, ^*Compare with lotus tumor matched group P＜0.01.

The main foundation that changes the simulation nephrasthenia syndrome with the animal adrenal gland cortex hormone function is that clinical insufficiency of kidney-YANG patient shows as hypoadrenocorticism, deficiency of the kidney yin patient clinical manifestation has hyperinterrenal phenomenon, and adrenocortical hormone also obviously increases in aging course.In addition, clinical in treatment needs to use the adrenocortical hormone patients to show as deficiency of the kidney yin for a long time in a large number, hormone shows as insufficiency of kidney-YANG after stopping using. according to above result, designed the method for application adrenocortical hormone preparation " nephrasthenia syndrome " animal model.It is the modeling method of present widely used animal nephrasthenia syndrome that animal gives the modeling method that adrenal gland's glucocorticoid prepares nephrasthenia syndrome.In big quantity research, the animal applications 17-hydroxy-11-dehydrocorticosterone phase is " deficiency of the kidney yin ", and the back of stopping using is " insufficiency of kidney-YANG ".The exogenous corticosterone that this institute adopts is handled the method that mice was made " deficiency of the kidney yin " model in 7 days, obtains scholar's approval substantially, and has been used by lot of documents.Above-mentioned experimental result shows, the compatible composition LW-ABC of orally give LIUWEI DIHUANG TANG extract is to " deficiency of the kidney yin " model mice, cyclophosphamide processing mice and the equal tool of immunologic hypofunction that tumor-bearing mice showed improve significantly, the effect of the full side of LIUWEI DIHUANG TANG of dosage reckonings such as its action intensity is not less than, the drug effect of the full side of LIUWEI DIHUANG TANG that shown the LW-ABC fundamental reaction, prompting LW-ABC all has obvious improvement or regulating action for immunologic hypofunction that multiple reason caused or dysequilibrium with syndrome of deficiency of kidney yin, clinical treatment of diseases or the auxiliary treatment that can be used for immunologic hypofunction due to a variety of causes of syndrome of deficiency of kidney yin or the disorderly performance of balance.

Two, LW-ABC is to the effect of reproductive endocrine system

1.LW-ABC effect to quick aging model mice (SAM) hypothalamic pituitary gonadal axis function

(1) LW-ABC is to the influence of SAMP8 oestrous cycle

The mensuration of oestrous cycle is carried out as follows: soak normal saline with the disinfectant cotton swab and insert the about 0.5cm of mouse vagina, rotating the back gently takes out, vaginal exfoliated on the cotton swab evenly is applied on the clean microscope slide, treat behind the smear natural air drying with the alkaline methylene blue solution 5min that dyes, slowly wash with distilled water then, in microscopically observation of cell form, determine the oestrous cycle behind the natural air drying.

The determination methods of oestrous cycle

Select for use 9-12 monthly age SAM quick aging subbreed SAMP8 to be used for experiment, being contrast with monthly age anti-quick aging subbreed SAMR1. set up the full side's matched group of LIUWEI DIHUANG TANG during experiment, dosage is 10g/kg.The result shows, and compares with monthly age SAMR1, and SAMP8 oestrous cycle and diestrus obviously prolong, and shortening is arranged rutting period, and LIUWEI DIHUANG TANG can obviously shorten model mice oestrous cycle and diestrus, prolongs its rutting period; Orally give LW-ABC also can obviously shorten model mice oestrous cycle and diestrus, dosage obvious lengthening model mice rutting period (seeing Figure 12) during for 3.2g/kg.

(2) LW-ABC is to the influence of SAMP8 serum E2 level

Application of radiation immunoassay detection kit (Tianjin consonance biotechnology research institute product) detects mice serum E2 level. and specific operation process is undertaken by the test kit explanation, (μ l) is as shown in the table for operation sequence and application of sample amount, and standard substance, NSB, T are two-tube.

Mice serum E2 detecting operation program

Experimental result shows, and with monthly age SAMR1 relatively, SAMP8 serum E2 level obviously reduces; The orally give LIUWEI DIHUANG TANG has obvious improvement effect to the decline of SAMP8 serum E2 level, and oral LW-ABC is to the reduction of the SAMP8 serum E2 level effect (Figure 13) that also has clear improvement.

(3) LW-ABC is to the influence of SAMP8 hypophysis LH level

Use time-resolved fluoroimmunoassay and detect mice hypophysis LH content.The sacrificed by decapitation mice, get place's hypophysis, place liquid nitrogen rapidly, from liquid nitrogen, take out hypophysis during detection, treat that liquid nitrogen evaporates into to weigh after stable, move to then in the centrifuge tube that fills 400 μ l lysates (aqueous solution of Na3PO4 10mM, NaCl 0.14M, BSA 1%), centrifuge tube is placed ice bath, prepare homogenate with ultrasonic method, 4 ℃ of centrifugalize supernatants are used for the mensuration of LH level. and the hypophysis sample is pressed the 1:29 dilution proportion before measuring.Use 96 hole assay plate and measure, with cleaning mixture washing test hole, add 50 μ l hLH standard substance or blood serum sample and 200 μ l tracer working solutions then successively before the application of sample.Fast concussion 2 minutes on agitator, 4-10 ℃ of standing over night, bradyseism 1 hour under room temperature then, measure hole 6 times with the cleaning mixture washing, every hole adds 200 μ l and strengthens liquid, slowly shakes 5 minutes on oscillator, differentiates luminoscope with the time after 10-15 minute and measures fluorescence.According to standard curve, the content of LH in the calculation sample.The result shows, and compares with monthly age SAMR1 matched group, and SAMP8 hypophysis LH content obviously raises.The orally give LIUWEI DIHUANG TANG can obviously reduce the content of SAMP8 hypophysis LH; Orally give LW-ABC also has the effect (seeing Figure 14) of obvious reduction to the level rising of SAMP8 hypophysis LH.

(4) LW-ABC is to the influence of SAMP8 hypothalamus GnRH content

The detection of mouse hypothalamus gonadotropin releasing hormone (GnRH) adopts the radioimmunoassay kit of naval's radioimmunoassay technique institute production to detect the .SAMP8 sacrificed by decapitation, separates and takes out hypothalamus, and it is standby to place liquid nitrogen to preserve.From liquid nitrogen, take out hypothalamus during mensuration, treat liquid nitrogen evaporate into stable after, weigh, place and fill the centrifuge tube that 300 μ l boil normal saline, continue to boil 3min, add 150 μ l glacial acetic acid then, place ice bath, ultrasonic method prepares homogenate, adds among the 150 μ l NaOH and glacial acetic acid, add 25 μ l aprotiniies then, mixing, 4 ℃ of centrifugalize supernatants, it is standby to be placed in-70 ℃ of preservations after packing. undertaken by the test kit explanation during test, (μ l) is as shown in the table for operation sequence and application of sample amount, and T, NSB, standard substance are two-tube.

Mouse hypothalamus GnRH assay operation sequence

The result shows, compare with the SAMR1 matched group, SAMP8 hypothalamus GnRH content obviously raises. and the orally give LIUWEI DIHUANG TANG can obviously reduce SAMP8 hypothalamus GnRH content, and orally give LW-ABC also has obvious reduction effect (seeing Figure 15) to the rising of SAMP8 hypothalamus GnRH content.

2.LW-ABC effect to " deficiency of the kidney yin " mouse hypothalamus-hypophysis-hypothalamic pituitary ovarium axis dysfunction

(1) LW-ABC is to the influence of " deficiency of the kidney yin " mouse ovarian weight

The sacrificed by decapitation mice is got ovary and weighs. and the result shows, handles after 5 days mouse ovarian weight than matched group obviously down with corticosterone.Reduction has obvious improvement effect to orally give LW to corticosterone induced mice ovary weight, and orally give LW-ABC also can obviously improve the ovary weight (Figure 16) of model mice.

(2) LW-ABC is to the influence of " deficiency of the kidney yin " mice serum E2 level

Application of radiation immune analysis method (as described above) is measured mice serum E2 level.The result shows that " deficiency of the kidney yin " mice serum E2 level obviously descends than matched group.The orally give LIUWEI DIHUANG TANG has obvious improvement effect to the decline of model mice serum E2 level, and reduction also has obvious improvement effect (Figure 17) to orally give LW-ABC to model mice serum E2 level.

(3) LW-ABC is to the influence of " deficiency of the kidney yin " mice hypophysis LH content

Use time-resolved fluoroimmunoassay (as described above) and detect mice hypophysis LH content.The result shows that the normal matched group of the content of model mice hypophysis LH obviously descends, and oral LIUWEI DIHUANG TANG and LW-ABC all can obviously improve the content (Figure 18) of model mice hypophysis LH.

3.LW-ABC influence to " deficiency of the kidney yin " mouse testis weight and level of serum testosterone

" deficiency of the kidney yin " model mice preparation method is with aforementioned. sacrificed by decapitation after mice is weighed, and get testis and weigh, calculate testis index.Serum testosterone uses the Beckman full-automatic microsome chemical illumination immunity analysis instrument of Ku Erte Access and the matched reagent box is measured, and the detection sensitivity of this instrument is 0.5ng/ml.Place 0.2ml to measure pipe respectively test serum during mensuration, put into the instrument test frame, set the Instrument measuring program, automatically measure by instrument, and calculating serum testosterone content. the result shows, the model mice level of serum testosterone obviously reduces than matched group, and the orally give LIUWEI DIHUANG TANG does not have obvious improvement effect to the decline of model mice level of serum testosterone, and orally give LW-ABC has obvious improvement effect (table 8) to the decline of model mice level of serum testosterone.

Table 8.LW-ABC is to the influence of " deficiency of the kidney yin " mouse testis weight and serum corticosterone level

Annotate: ^*P＜0.05, ^*Compare with matched group p＜0.01; #p＜0.05, compare with the deficiency of the kidney yin group ##p＜0.01; Mean ± SD, n=10.

Quick aging model mice (senescence-accelerated mouse, SAM) be to teach in the mice of cultivating a kind of quick aging successfully by Kyoto Univ Japan bamboo field, (the senescence-accelerated mouse-prone of P system that comprises quick aging, SAMP) and (the senescence-accelerated mouse-resistant of the R of anti-quick aging system, SAMR), the characteristics of P system be occur age fast with increasing, each system of carrying out property whole body aging, R system then has the physiological feature of normal mouse, is the contrast of P system.SAMP8 is one of quick aging subbreed, being characterized in increasing its maincenter learning and memory function and immunologic function carrying out property decline fast in age. the kind Mus of SAMR1 and SAMP8 subbreed has been introduced in my chamber from Kyoto Univ Japan's bamboo field professor's laboratory from 1994, in me institute's laboratory animal laboratory breeding and raising, and be applied to experimentation.

We show former studies, SAMP8 is with increasing the dysequilibrium that occurs hypothalamic-pituitary-adrenal (HPA) and gonad (HPG) s function age fast, quite similar with the variation of mankind aging's property hpa axis and HPG axle, show as carrying out property of serum corticoid level rising in increasing rheological processes; Female SAMP8 shortens rutting period, interval, prolong, serum E2 level descends, and hypophysis LH level and hypothalamus GnRH level raise, and has similar performance with human syndrome of deficiency of kidney yin, aging, Woman climacteric and the HPG s function is low or dysequilibrium is relevant numerous disease.The prolongation of oestrous cycle or irregular be the low direct performance of reproductive endocrine function in the aging course, therefore, the early stage decline fast of sexual function also is one of old and feeble fast important behaviour of SAMP8, its hypophysis LH level to increase that rheological properties raises be ovarian hypofunction, cause estrogen level to reduce, thereby make due to hypothalamus-hypophysis negative feedback inhibition weakens.

This experimental result shows, orally give LW-ABC lowly has obvious improvement or regulating action to the HPG s function that SAMP8 showed, its effect is similar with the full side of LIUWEI DIHUANG TANG, shows that LW-ABC has obvious improvement or regulating action .LW-ABC to the hypofunction of aging HPG axle or balance disorder the reduction of " deficiency of the kidney yin " model mice ovary weight reduction due to the adrenocortical hormone, serum E2 and hypophysis LH level is also had obvious improvement effect; Decline to male " deficiency of the kidney yin " mice serum testosterone levels also has obvious improvement effect, and low or dysequilibrium has obvious improvement or regulating action to " deficiency of the kidney yin " HPG s function that mice showed to show LW-ABC.The The above results prompting, LW-ABC to HPG s function due to old and feeble, the deficiency of the kidney yin low or balance disorderly all have improve or regulating action, clinically can be used for aging and with the low disease of HPG s function of syndrome of deficiency of kidney yin such as climacteric syndrome, ovarian hypofunction, infertility, habitual abortion, autonomic nervous dysfunction, chloasma, gynaecomastia, senile deterioration, climacteric osteoporosis, prevention such as senile osteoporosis and treatment.

Claims

1. LIUWEI DIHUANG TANG extract, it comprises in LIUWEI DIHUANG TANG extract weight, 11.4-17 the polysaccharide of weight %, 1.85-2.27 total glycosides that weight % is made up of morroniside, meliatin and peoniflorin and the mannotriose of 23.5-35.5 weight %, wherein LIUWEI DIHUANG TANG is made up of with the part by weight compatibility of 8:4:4:3:3:3 Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria Six-element Chinese medicine, and wherein LIUWEI DIHUANG TANG extract is following makes:

Ii) follow to stir in extractum to add ethanol to concentration of alcohol 30v/v% placement, centrifugal, flushing is merged into supernatant with supernatant and washing liquid;

Iii) concentrated supernatant is followed to stir to add 95v/v% ethanol to concentration of alcohol 60v/v% toward concentrated solution in, placement, separate position A and supernatant;

Iv) iii) middle supernatant is carried out column chromatography on macroporous adsorbent resin, water and polar organic solvent eluting get position B after the organic solvent eluent concentrates;

2. pharmaceutical composition, it comprises LIUWEI DIHUANG TANG extract and pharmaceutical carrier in the claim 1, wherein LIUWEI DIHUANG TANG is made up of with the part by weight compatibility of 8:4:4:3:3:3 Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria Six-element Chinese medicine.

3. the preparation method of LIUWEI DIHUANG TANG extract, it comprises:

4. the described method of claim 3, wherein big pore adsorption resin is the low pole macroporous adsorbent resin of all models; The ethanol precipitation twice used alcohol that forms sediment is C _1-4Lower alkyl alcohol, concentration range are respectively 10%-40% and 50%-95%; The used polar organic solvent of macroporous adsorption resin chromatography eluting is the polar organic solvent that all macroporous resins can allow use, and concentration range is 10%-95%; The used alcohol of activated carbon chromatography eluting is methanol and ethanol, and determining alcohol is 20%-95%.

5. according to the LIUWEI DIHUANG TANG extract of claim 1, wherein said extract is to be used for the treatment of tumor, side effect in tumor chemistry or the radiation therapy process, leukopenia, the aging immunologic hypofunction, infect, systemic lupus erythematosus (sle), nephrotic syndrome, nephritis, bronchitis and bronchial asthma, prostatitis, aplastic anemia, thrombocytopenic purpura, myasthenia gravis, senile immunologic hypofunction or disorder, climacteric syndrome, ovarian hypofunction, habitual abortion, infertility, chloasma, gynaecomastia, climacteric osteoporosis, the extract of senile osteoporosis or autonomic nervous dysfunction.

6. the pharmaceutical composition of claim 2, wherein said compositions are the treatment tumors, side effect in tumor chemistry or the radiation therapy process, leukopenia, the aging immunologic hypofunction, infect, systemic lupus erythematosus (sle), nephrotic syndrome, nephritis, bronchitis and bronchial asthma, prostatitis, aplastic anemia, thrombocytopenic purpura, myasthenia gravis, senile immunologic hypofunction or disorder, climacteric syndrome, ovarian hypofunction, habitual abortion, infertility, chloasma, gynaecomastia, climacteric osteoporosis, the medical compounds of senile osteoporosis or autonomic nervous dysfunction.

7. the extract of claim 1 is in preparation treatment tumor, side effect in tumor chemistry or the radiation therapy process, leukopenia, the aging immunologic hypofunction, infect, systemic lupus erythematosus (sle), nephrotic syndrome, nephritis, bronchitis and bronchial asthma, prostatitis, aplastic anemia, thrombocytopenic purpura, myasthenia gravis, senile immunologic hypofunction or disorder, climacteric syndrome, ovarian hypofunction, habitual abortion, infertility, chloasma, gynaecomastia, climacteric osteoporosis, purposes in the product of senile osteoporosis or autonomic nervous dysfunction.