CN1663593A - Glucoside active site LWD-b in Liuwei Dihuang decoction, its preparation and medical usage - Google Patents
Glucoside active site LWD-b in Liuwei Dihuang decoction, its preparation and medical usage Download PDFInfo
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- CN1663593A CN1663593A CN 200410058598 CN200410058598A CN1663593A CN 1663593 A CN1663593 A CN 1663593A CN 200410058598 CN200410058598 CN 200410058598 CN 200410058598 A CN200410058598 A CN 200410058598A CN 1663593 A CN1663593 A CN 1663593A
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- 210000003462 vein Anatomy 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
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Abstract
A Liuwei Dihuang decoction (Glucoside active site LWD-b) extract includes, by weight of extract, 12.5-15.3 weight % total glycosides (morroniside, loganin, paeoniflorin), wherein the Liuwei Dihuang decoction is prepared by rehmannia, dogberry, Chinese potato, rhizoma alismatis, cortex moutan and tuckahoe with a weight proportion of 8:4:4:3:3:3.
Description
Technical field
The present invention relates to LIUWEI DIHUANG TANG extract (glucosides class active site LWD-b), its preparation method, and be used for the treatment of or auxiliary treatment with the various diseases of syndrome of deficiency of kidney yin and immune dysfunction, comprise 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, thrombocytopenic purpura, myasthenia gravis reach the immunologic function disorders that the patient showed such as Senile disease; And reproduction, hormonal system hypofunction or the disorderly disease that causes, comprise climacteric syndrome, ovarian hypofunction, infertility, habitual abortion, autonomic nervous dysfunction, chloasma, gynaecomastia, senile sexual disorder, climacteric osteoporosis, purposes such as senile osteoporosis.
Technical background
LIUWEI DIHUANG TANG is that the classics of the traditional Chinese medical science " nourishing kidney yin " are represented name side, and the shenqi pill of being got in " Medical Treasures of the Golden Chamber " by the well-known doctor Qian Yi of the Song dynasty goes to osmanthus attached, becomes " warming and recuperating the kidney-YANG " into " nourishing kidney yin ", initiates in key to Therapeutics of Children's Diseases.This side by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria Six-element Chinese medicine with 8: 4: 4: 3: 3: 3 part by weight compatibility is formed, and is mainly used in the treatment soreness of the waist and knees, has a dizzy spell, Hiccough and deaf, night sweat is quenched one's thirst in seminal emission, osteopyrexia and fever, tooth shakes, and the card of deficiency of kidney yin such as dribbling urination is widely used 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, the aplastic anemia thrombocytopenic purpura, myasthenia gravis, climacteric syndrome, prostate hyperplasia, infertility, the hypertensive state of pregnancy neurasthenia, parkinson, hypertension, coronary heart disease, arrhythmia, pyelonephritis, diabetes etc. have 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, medication inconvenience, the pharmaceutical dosage form that is difficult to make modern convenient use; 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.
Summary of the invention
The inventor has now found that the glucosides class active site of beat all discovery LIUWEI DIHUANG TANG still has good various pharmaceutically actives by traditional LIUWEI DIHUANG TANG is scientifically handled after deliberation, its at normal temperatures in the water dissolubility be 〉=1.5g/ml.
Therefore, first aspect present invention relates to LIUWEI DIHUANG TANG (glucosides class active site LWD-b) extract, it comprises, in extract weight, 12.5-15.3 the total glycosides of weight % (morroniside, meliatin, peoniflorin), wherein LIUWEI DIHUANG TANG by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria Six-element Chinese medicine with 8: 4: 4: 3: 3: 3 part by weight compatibility is formed.
The present invention relates to pharmaceutical composition on the other hand, and it comprises total glycosides (morroniside, meliatin, peoniflorin) LIUWEI DIHUANG TANG extract and the pharmaceutical carrier that contains 12.5-15.3% weight %.
Further aspect of the present invention relates to the method that preparation contains the extract of 12.5-15.3 weight % total glycosides, and it comprises:
I) will be by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria by 8: 4: 4: the mixture of forming at 3: 3: 3 be concentrated into extractum then with boiling water steaming and decocting twice;
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 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.
The extract according to the present invention (glucosides class active site LWD-b) can obtain by following technological process:
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, and it is about 30% to final concentration to add alcohol, stirs, and placement is spent the night.Centrifugal treating (2500 rev/mins) discards precipitation.The merging supernatant is concentrated in right amount, determining alcohol is adjusted to be about 60%, stirs, and places, and centrifugal treating keeps supernatant.After precipitation is redissolved with suitable quantity of water, carry out the ethanol precipitation twice shallow lake again with method and handle.Three times supernatant merges, concentrate and remove alcohol, the gained extracting solution carries out macroporous adsorbent resin column chromatography, successively water and polar organic solvent eluting, polar organic solvent eluting position concentrates removes the back lyophilizing of desolvating, and is the glucosides class active site LWD-b of LIUWEI DIHUANG TANG.Technological process is as follows:
Big pore adsorption resin can be the low pole macroporous adsorbent resin of all models; The used alcohol of two step alcohol precipitations can be C
1-4Lower alkyl alcohol, as: methanol, ethanol etc., concentration range is respectively 10%-40% and 50%-95%; The used polar organic solvent of macroporous adsorption resin chromatography eluting is 10%-95% for polar organic solvent (as: methanol, ethanol, propanol and the acetone etc.) concentration range that all macroporous resins can allow to use.
According to the present invention, total glycosides comprises morroniside among the present invention, meliatin and peoniflorin.
According to the present invention, 12.5-15.3 weight % total glycosides is based on the percentage ratio that extract weight calculates in the extract of the present invention.
According to the present invention, position B is glucosides class active site LWD-b or LWD-b in extract of the present invention or the LIUWEI DIHUANG TANG among the present invention.
Description of drawings
Fig. 1-1 criticizes the stricture of vagina collection of illustrative plates for the total ion current at LIUWEI DIHUANG TANG extract LWD-b position
Fig. 1-2 is the HPLC-DAD finger printing at LIUWEI DIHUANG TANG extract LWD-b position
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 is the influence of oral LWD-b to normal mouse boosting cell breeder reaction
Figure 11 is the influence of LWD-b to the breeder reaction of lotus S180 sarcoma mouse boosting cell
Figure 12 is the influence of LWD-b to the breeder reaction of CJ sensitized mice splenocyte.
According to the present invention, LIUWEI DIHUANG TANG glucosides class active site LWD-b of the present invention is qualitative identification as follows:
(1) alphanaphthol reaction (Molish reaction)
LWD-b dissolves with suitable quantity of water, adds three of 10% alpha-Naphthol alcoholic solution, slowly adds concentrated sulphuric acid along test tube wall then, makes formation two-layer up and down, and at two-layer visible at the interface purple ring, expression has the glycoside composition to exist after a while.
(2) be the qualitative examination of the iridoid glycoside constituents of representative with the meliatin among the LWD-b
LIUWEI DIHUANG TANG extract LWD-b leaches with an amount of 95% ethanol, and leachate is a liquid to be measured.With the alcoholic solution of meliatin and morroniside product in contrast.Liquid to be measured and contrast liquid difference point sample are on silica gel g thin-layer plate and silica gel G-0.3%CMC-Na lamellae, respectively with developing solvent A (n-butyl alcohol/ethanol/water, 15: 3: 3) and developing solvent B (chloroform/methanol, 16: 4) launch, use 10% vanillin solution (then spray 72% sulphuric acid again) and Godin reagent colour development respectively, dried by the fire 5 minutes at 105 ℃ and 80-90 ℃ respectively, liquid to be measured has identical colour developing speckle with contrast liquid.
(3) the HPLC finger printing of LIUWEI DIHUANG TANG extract LWD-b:
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, CH
3CN-H
2O binary gradient elution (0min:2: 98; 50min:24: 76,60min:56: 44), eluting finishes in the sample size 20 μ l, all components 60min.
The results are shown in accompanying drawing 1-1 and 1-2:
According to the present invention, LIUWEI DIHUANG TANG glucosides class active site LWD-b of the present invention is quantitative identification as follows:
Instrument and reagent
Agilent 1100 high performance liquid chromatogram systems contain quaternary gradient pump, DAD detector, column oven and chem workstation.The API of American AB I company 3000 liquid chromatograph-mass spectrometers are furnished with Turbo Ionspray ionization source and Analyst 1.1 data handling systems.
Morroniside, meliatin reference substance are determined structure for this chamber self-control through spectrum, and the HPLC area normalization method records purity more than 98.0%.The peoniflorin reference substance is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Poria, Rhizoma Alismatis, Cortex Moutan are all available from medical material distribution center of Beijing Tongrentang.Methanol is chromatographically pure, and water is deionized water.
Experimental technique
1. chromatographic condition: Diamonsil C
18(4.6mm * 250mm, 5 μ m) chromatographic column, mobile phase is methanol-water (33: 67), flow velocity 1.0mL.min
-1, 30 ℃ of column temperatures, DAD detects wavelength 236nm, sample size 20 μ L.Theoretical cam curve is pressed the meliatin peak and is calculated greater than 3000, and with this understanding, morroniside, meliatin, peoniflorin all can reach good baseline separation with other components.
2. mass spectrum condition: anion detects, and injection electric is-4000V, DP voltage-70V, and FP voltage-290V, 300 ℃ of TEM, NEB are 8, CUR is 11.
3. the preparation of reference substance solution: precision takes by weighing morroniside, meliatin, each 10mg of peoniflorin reference substance, places in the 10mL volumetric flask,, shakes up to scale with methanol constant volume.Therefrom draw 75,100,150,300,600 μ L respectively, thin up becomes 10mL, is prepared into 5 parts of reference substance solution.
4. the preparation of sample solution: get this extract and add water in right amount to be made into concentration be 40mg/mL, totally 3 parts, filter (filter membrane of 0.45 μ m) back by above-mentioned chromatographic condition sample introduction, calculate morroniside, meliatin, content of 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
| Reference substance | Sample weighting amount/g | Addition/mg | The amount of recording/mg | The response rate/% | Average recovery rate/% |
| Morroniside | 0.2512 | ????2.5 | ????4.53 | ????100.8 | ????98.8 |
| 0.2508 | ????2.6 | ????4.68 | ????102.8 | ||
| 0.2510 | ????2.3 | ????4.22 | ????96.2 | ||
| 0.2506 | ????2.2 | ????4.10 | ????95.2 | ||
| 0.2509 | ????2.2 | ????4.18 | ????98.8 | ||
| Meliatin | 0.2512 | ????1.6 | ????2.64 | ????102.2 | ????98.3 |
| 0.2508 | ????1.3 | ????2.26 | ????96.7 | ||
| 0.2510 | ????1.4 | ????2.39 | ????99.0 | ||
| 0.2506 | ????1.2 | ????2.15 | ????95.7 | ||
| 0.2509 | ????1.2 | ????2.18 | ????98.0 | ||
| Peoniflorin | 0.2512 | ????1.5 | ????2.69 | ????98.9 | ????99.6 |
| 0.2508 | ????1.6 | ????2.85 | ????102.9 | ||
| 0.2510 | ????1.4 | ????2.56 | ????96.7 | ||
| 0.2506 | ????1.5 | ????2.72 | ????101.1 | ||
| 0.2509 | ????1.4 | ????2.58 | ????98.3 |
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.
The sample size measurement result shows, morroniside, meliatin, paeoniflorin content scope are respectively 6.2-7.6%, 3.3-4.1%, 3.0-3.6% in the sample, three parts add and, the content range in LIUWEI DIHUANG TANG glucosides class active site LWD-b is at 12.5-15.3%.
According to the present invention, first aspect present invention relates to LIUWEI DIHUANG TANG glucosides class active site LWD-b, this active site 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 immune dysfunction, 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, senile osteoporosis, diseases such as autonomic nervous dysfunction have treatment or auxiliary treatment effect.
According to the present invention, the main active among the LIUWEI DIHUANG TANG glucosides class active site LWD-b of the present invention is morroniside, meliatin and peoniflorin, three parts add and, the content range in LWD-b is between 12.5-15.3%.
According to the present invention, the invention still further relates to the pharmaceutical composition that contains LWD-b, comprise LWD-b 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 LWD-b 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, 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, senile osteoporosis, the purposes of the medicine that autonomic nervous dysfunction etc. are sick.
According to the present invention, LWD-b of the present invention can be separately or is used with pharmaceutical compositions, and its administering mode can be decided as the case may be, can pass through oral, non-intestinal or topical, and form of administration can be a tablet for example, capsule, electuary, pill, unguentum 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 glucosides class active site LWD-b
Each single medicinal material of LIUWEI DIHUANG TANG is by proportioning commonly used, as 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 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 extractum (volume) than 1: 1.
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.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) are than being 10: 1, water and 30% ethanol eluting successively then, 30% ethanol elution position concentrates removes alcohol back lyophilizing, obtains pale brown toner end and is LWD-b (38.6 gram).It has aforesaid qualitative reaction feature.
Measuring the extract obtained middle total glycosides content of present embodiment by aforementioned institute to method is 13.9%.
Embodiment 2: the biological activity test of embodiment 1 LIUWEI DIHUANG TANG extract LWD-b
One, LWD-b is to the effect of immunologic function
1. oral LWD-b is to the influence of normal mouse boosting cell breeder reaction
Female Balb/c mice is irritated stomach and gives LWD-b, and 1 time/day, continuous 7 days.Sacrificed by decapitation mice on the 8th, the aseptic spleen of getting, the preparation splenocyte suspension, adjusting cell concentration with complete RPMI-1640 culture fluid is 5 * 10
6Cell/ml gets 100 μ l Con A (final concentration is 0.5ug/ml) and 100 μ l cell suspension and adds successively in 96 orifice plates, and matched group is put 37 ℃, 5%CO with RPMI-1640 replaced C on A
2Incubated incubation 56 hours, every hole adds 20 μ l
3H-TdR (10 μ Ci/ml) continues to be cultured to 72 hours,, after 80 ℃ of oven dry, glass fiber filter paper is placed in the scintillation solution on glass fiber filter paper with multiple tracks cell harvesting instrument collecting cell, surveys the cpm value with liquid scintillation counter.Every sample is established 5 parallel holes, and the result is with average cpm value representation.The result shows, orally give LWD-b (0.07,0.14g/kg) all has obvious facilitation (the results are shown in Figure 10) to spontaneous breeder reaction of splenocyte and the inductive breeder reaction of ConA.
2. oral LWD-b is to the influence of caused by cyclophosphamide mouse boosting cell antibody formation reaction
The Balb/c mice, female, gastric infusion 9 days, 1 time/day.In administration every mouse peritoneal injection sheep red blood cell (SRBC) (SRBC) suspension 0.2ml (5 * 107 erythrocyte) in the time of the 5th day/only, on the immune same day, except that the normal control group, each organizes mouse peritoneal injection cyclophosphamide (Cy), 15mg/kg, next day is with the dosage duplicate injection once.To experimentize in the 4th day after the SRBC immunity, the mice sacrificed by decapitation is got spleen during experiment, the preparation splenocyte suspension, and 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 dilution in 1: 1), 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 Cy handles mouse boosting cell antibody formation reaction ability and obviously descends than matched group, and orally give LWD-b has obvious improvement effect (table 2).
Table 2.LWD-b is to the influence of caused by cyclophosphamide immunologic hypofunction mouse antibodies reaction of formation
| Group | Dosage (g/kg) | ????PFC/10 6Splenocyte |
| The normal control group | ???/ | ????997±274 |
| Model control group | ??15mg | ????364±109 ** |
| Model+LW | ??10 | ????605±120## |
| Model+LWD-b | ??0.07 | ????433±170 |
| ??0.14 | ????667±150## | |
| ??0.28 | ????689±146## |
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.LWD-b 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 LWD-b after implanted tumor cells, and continuous 10 days, experimentize, the mouse boosting cell breeder reaction is undertaken by preceding method.The result shows, with matched group relatively, tumor-bearing mice is obviously low by the inductive splenocyte breeder reaction of ConA, oral LWD-b (0.07,0.14g/kg) has improvement effect significantly (the results are shown in Figure 11) to the low splenic lymphocytes of tumor-bearing mice.
4. oral LWD-b is to the influence of campylobacter jejuni sensitized mice splenocyte breeder reaction
CJ sensitization autoimmune disease mouse model preparation is carried out as follows: twice (4000rpm * 30min), transferring 540nm OD value is 1.25, is equivalent to campylobacter jejuni 3 * 10 with the campylobacter jejuni (CJ) of normal saline washing formolation
9CFU/ml, with the mixing emulsifying in 1: 1 of Fu Shi Freund's complete adjuvant, mouse insole injection 0.05ml.Back 15 days of immunity, the above-mentioned bacteria suspension 0.2ml of tail vein injection, the while oral administration, and set up blank and adjuvant matched group.After 30 days (administration 15 days) carry out immunologic function and detect.The mouse boosting cell breeder reaction is undertaken by preceding method.The result shows, compares with matched group, and the breeder reaction of CJ sensitized mice splenocyte obviously strengthens.Spontaneous breeder reaction of splenocyte and the inductive splenocyte breeder reaction of Con A that oral LWD-b (0.07,0.14g/kg) increases unusually to the CJ sensitized mice all have tangible restitution (the results are shown in Figure 12).
5. oral LWD-b is to the influence of CJ sensitized mice serum antinuclear antibody level
Preparation of CJ sensitization autoimmune disease mouse model and medication are the same, and administration was put to death mice after 15 days, and separation of serum is measured serum antinuclear antibody level.The serum antinuclear antibody adopts following method to measure: with the rat hepatocytes nucleoprotein coating buffer bag of 100 μ g/ml by 96 hole elisa plates (37 ℃ 1 hour, 4 ℃ are spent the night).1%FBS sealase yoke plate is after 1 hour, and PBS-T liquid is washed plate 3 times, dries.Every hole adds the test serum sample of 50 μ l dilution in 1: 100, puts 37 ℃ of incubations 1 hour.PBS-T liquid is washed plate 3 times, dries.Every hole adds the horseradish peroxidase-labeled goat anti-mouse igg antibody of 100ul dilution in 1: 1000, and the zeroing hole adds 100 μ l antibody diluents, and 37 ℃ act on 30 minutes.PBS-T liquid is washed plate 3 times, dries.Every hole adds the TMB solution of 100ul0.1mg/ml, and 37 ℃ were developed the color 10-20 minute.Every hole adds 50ul H
2SO
4(2mol/L) color development stopping is measured optical density value (450nm) on enzyme connection detector.The result shows that the antinuclear antibody level obviously raises than matched group in the CJ sensitized mice serum, oral (0.07,0.14g/kg, continuous 15 days) has tangible reduction effect to the antinuclear antibody level that increases in the CJ sensitized mice serum.
Above-mentioned experimental result shows that orally give LIUWEI DIHUANG TANG extract LWD-b all has obvious facilitation to spontaneous breeder reaction of normal mouse boosting cell and the inductive breeder reaction of ConA, and prompting LWD-b has potentiation to normal body's immunity.Simultaneously, orally give LWD-b to the chemotherapeutic cyclophosphamide handle mice, the equal tool of immunologic hypofunction that tumor-bearing mice showed improves significantly, and shows that LWD-b has obvious improvement effect to chemotherapeutic and the caused immunologic hypofunction of lotus tumor.In addition, orally give LWD-b has obvious inhibitory action to the splenocyte breeder reaction that CJ sensitization autoimmune disease model mice increases unusually, and can obviously reduce the level of antinuclear antibody in the serum, show that LWD-b has obvious role of correcting to the inductive immunologic function disorder of CJ.Above-mentioned result of study prompting LWD-b is clinical to can be used for multiple treatment of diseases or auxiliary treatment with immunologic hypofunction or the disorderly performance of balance, as in tumor, tumor chemistry or the radiation therapy process, leukopenia, aging immunologic hypofunction, infection etc.; Autoimmune disease such as systemic lupus erythematosus (sle), nephrotic syndrome, nephritis, bronchitis and bronchial asthma, prostatitis, aplastic anemia, thrombocytopenic purpura, myasthenia gravis, and the immunologic function disorder that the patient showed such as Senile disease etc.
Two, LWD-b is to the effect of reproductive endocrine system
1.LWD-b effect to female " deficiency of the kidney yin " mouse hypothalamus-hypophysis-hypothalamic pituitary ovarium axis dysfunction
(1) LWD-b is to the influence of " deficiency of the kidney yin " mouse ovarian weight
The preparation and the medication of " deficiency of the kidney yin " mouse model are the same.The sacrificed by decapitation mice is got ovary and weighs.The result shows, handles with corticosterone that mouse ovarian weight obviously reduces than matched group after 5 days.Reduction has obvious improvement effect to orally give LW to corticosterone induced mice ovary weight, and orally give LWD-b also can obviously improve the ovary weight (table 3) of model mice.
Table 3.LW-AFC handles the influence of (deficiency of the kidney yin) mouse ovarian weight to corticosterone
| Group | Dosage (g/kg) | Ovary weight (mg) |
| Matched group | - | ????6.10±0.88 |
| Model group | - | ????4.32±0.67 ** |
| Model+ | 10 | ????5.44±1.20# |
| Model+LWD-b group | 0.07 | ????5.63±0.97 ## |
| 0.14 | ????5.12±0.89 # | |
| 0.28 | ????4.48±1.10 |
Annotate:
*Compare with the normal control group P＜0.01; #p＜0.05, compare with model group ##p＜0.01; Mean ± SD, n=12-17
(2) LWD-b is to the influence of " deficiency of the kidney yin " mice serum E2 level
Application of radiation immunoassay detection kit (Tianjin consonance biotechnology research institute product) detects mice serum E2 level.Specific operation process is undertaken by the test kit explanation, and (μ l) is as shown in table 4 for operation sequence and application of sample amount, and standard substance, NSB, T are two-tube.
Table 4 mice serum E2 detecting operation program
| ???T | ???T | ???N | ???N | ???0 | ???0 | ??S1-S6 | Quality-control product and testing sample | |
| Remove the hormone standard serum | ??- | ??- | ?200 | ?200 | ??200 | ??200 | - | ??- |
| Standard serum | ??- | ??- | ?- | ?- | ??- | ??- | 200 | ??- |
| Quality-control product and testing sample | ??- | ??- | ?- | ?- | ??- | ??- | - | ??200 |
| Anti-E 2Serum | ??- | ??- | ?- | ?- | ??100 | ??100 | 100 | ??100 |
| Mixing, 37 ℃ of water-bath incubation 2h | ||||||||
| 125I-E 2 | ??10 | ??10 | ?100 | ?100 | ??100 | ??100 | 100 | ??100 |
| ??0 | ??0 | |||||||
| Mixing, 37 ℃ of water-bath incubation 1h | ||||||||
| Separating medium (cold) | ??- | ??- | ?500 | ?500 | ??500 | ??500 | 500 | ??500 |
| Fully shake up, centrifugal 3000g * 20min, 4 ℃ of supernatant precipitate counting cpm values of inclining, according to standard curve, with COMPUTER CALCULATION E2 concentration | ||||||||
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 (table 5) to orally give LWD-b to model mice serum E2 level.
Table 5.LWD-b is to the influence of " deficiency of the kidney yin " mice serum E2 level
| Group | Dosage (g/kg) | Serum estradiol (pg/ml) |
| Matched group | ????- | ????27.19±8.13 |
| Model group | ????- | ????18.36±4.36 ** |
| Model+LW group | ????10 | ????25.51±3.84 # |
| Model+LWD-b group | ????0.07 | ????22.13±9.04 |
| ????0.14 | ????25.75±9.63 | |
| ????0.28 | ????26.47±6.23 # |
Annotate:
*Compare with the normal control group P＜0.01; #p＜0.05, compare with model group ##p＜0.01; Mean ± SD, n=12-17
(3) LWD-b is to the influence of " deficiency of the kidney yin " mice 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.The hypophysis sample was by 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 that the normal matched group of the content of model mice hypophysis LH obviously descends, and oral LIUWEI DIHUANG TANG and LWD-b all can obviously improve the content (table 6) of model mice hypophysis LH.
Table 6.LWD-b is to " deficiency of the kidney yin " mice serum E2, hypophysis LH, hypothalamus GnRH water
Flat influence
| Group | Dosage (g/kg) | Hypophysis LH concentration (U/g tissue) |
| Matched group | ??- | ????2086.8±802.8 |
| Model group | ??- | ????1158.0±491.8 * |
| Model+LW group | ??10 | ????1889.3±516.5 # |
| Model+LWD-b group | ??0.07 | ????1359.0±499.5 |
| ??0.14 | ????1730.0±693.2# | |
| ??0.28 | ????2225.1±696.6 ## |
Annotate:
*Compare with the normal control group P＜0.01; #p＜0.05, compare with model group ##p＜0.01; Mean ± SD, n=12-17
(4) LWD-b is to the influence of " deficiency of the kidney yin " mouse 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 mice sacrificed by decapitation is separated and is taken 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, add 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, T, NSB, standard substance are two-tube, and (μ l) is as shown in table 7 for operation sequence and application of sample amount.
Table 7 mouse hypothalamus GnRH assay operation sequence
| ????T | ??T | ??N | ??N | ??0 | ??0 | ???S1-S | Testing sample | |
| Standard substance | ??8 | |||||||
| ????- | ?- | ??- | ?- | ??- | ??- | ??100 | ?- | |
| Testing sample | ????- | ?- | ??- | ?- | ??- | ??- | ??- | ?200 |
| Anti-GnRH serum | ????- | ?- | ??- | ?- | ??10 | ??10 | ??100 | ?100 |
| ????- | ?- | ??- | ?- | ??0 | ??0 | |||
| Buffer | ????- | ?- | ??30 | ?30 | ??20 | ??20 | ??100 | ?- |
| ????- | ?- | ??0 | ?0 | ??0 | ??0 | |||
| Mixing is placed 24h for 4 ℃ | ||||||||
| 125I-GnRH | ????10 | ?10 | ??10 | ?10 | ??10 | ??10 | ??100 | ?100 |
| ????0 | ?0 | ??0 | ?0 | ??0 | ??0 | |||
| Mixing, 37 ℃ of water-bath incubation 1h | ||||||||
| Separating medium (cold) | ????- | ?- | ??50 | ?50 | ??50 | ??50 | ??500 | ?500 |
| ??0 | ?0 | ??0 | ??0 | |||||
| Fully shake up, room temperature is placed 15min, 4 ℃ of centrifugal 3000g * 15min supernatant precipitate counting cpm value of inclining, according to standard curve, with COMPUTER CALCULATION GnRH concentration | ||||||||
The result shows that the normal matched group of model mice hypothalamus GnRH content obviously descends, and oral LIUWEI DIHUANG TANG and LWD-b can obviously improve model mice hypothalamus GnRH content (table 8).
Table 8.LWD-b is to " deficiency of the kidney yin " mice serum E2, hypophysis LH, hypothalamus GnRH water
Flat influence
| Group | Dosage (g/kg) | Hypothalamus GnRH (ng/g tissue) |
| Matched group | ??- | ??118.4±33.1 |
| Model group | ??- | ??86.8±20.2 * |
| Model+LW group | ??10 | ??124.1±45.1 # |
| Model+LWD-b group | ??0.07 | ??81.4±11.19 |
| ??0.14 | ??110.9±32.9 | |
| ??0.28 | ??123.0±21.6 # |
Annotate:
*Compare with the normal control group P＜0.01; #p＜0.05, compare with model group ##p＜0.01; Mean ± SD, n=12-17
3.LWD-b influence to male " 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 is got testis and is weighed, and calculates 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, measure automatically, and calculate serum testosterone content by instrument.The result shows, the model mice level of serum testosterone obviously reduces than matched group, 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 LWD-b has obvious improvement effect (table 9) to the decline of model mice level of serum testosterone.
Table 9.LW-AFC is to the influence of " deficiency of the kidney yin " mouse testis weight and serum corticosterone level
| Grouping | Dosage (g/kg) | Testis | ||
| Weight (mg) | Coefficient | Testosterone (ng/ml) | ||
| Matched group | ??/ | ??162.0±1?0.7 | ??7.7±0.7 | ??9.13±4.54 |
| Model group | ??/ | ??150.4±16.0 | ??8.2±0.8 | ??5.37±3.93 ** |
| Model+LW | ??10.0 | ??150.1±13.4 | ??8.2±0.7 | ??6.96±3.63 |
| Model+LWD-b | ??0.8 | ??120.0±50.3 | ??6.5±2.7 | ??8.97±2.63# |
| ??1.6 | ??154.6±10.2 | ??8.2±0.5 | ??7.19±3.51# | |
| ??3.2 | ??151.5±12.2 | ??8.2±0.8 | ??6.92±3.02 | |
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.
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.
This experimental result shows, orally give LWD-b to female " deficiency of the kidney yin " model mice ovary weight due to the adrenocortical hormone reduce, the reduction of serum E2 and hypophysis LH level also has 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 LWD-b.The The above results prompting, LWD-b to HPG s function due to 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 (10)
1. LIUWEI DIHUANG TANG (glucosides class active site LWD-b) extract, it comprises, in extract weight, 12.5-15.3 the total glycosides of weight % (morroniside, meliatin, peoniflorin), wherein LIUWEI DIHUANG TANG by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria Six-element Chinese medicine with 8: 4: 4: 3: 3: 3 part by weight compatibility is formed.
2. pharmaceutical composition, it comprises total glycosides (morroniside, meliatin, peoniflorin) LIUWEI DIHUANG TANG extract and the pharmaceutical carrier that contains 12.5-15.3% weight %.
3. the preparation method of LIUWEI DIHUANG TANG extract, it comprises:
I) will be by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria by 8: 4: 4: the mixture of forming at 3: 3: 3 be concentrated into extractum then with boiling water steaming and decocting twice;
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 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.
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 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, senile osteoporosis, the extract of autonomic nervous dysfunction.
6. the pharmaceutical composition of claim 2, wherein said pharmaceutical composition 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, senile osteoporosis, the pharmaceutical composition of autonomic nervous dysfunction.
7. the extract of claim 1 is used for the treatment of 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, thrombocytopenic purpura, 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.
8. the extract of claim 1, in extract weight, it contains 6.2-7.6 weight % morroniside.
9. the extract of claim 1, in extract weight, it contains 3.3-4.1 weight % meliatin.
10. the extract of claim 1, in extract weight, it contains 3.0-3.6 weight % peoniflorin.
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| CN107411087A (en) * | 2017-05-22 | 2017-12-01 | 杭州济佰氏生物科技有限公司 | A kind of food for being used to nurse one's health women ovarian function |
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| CN107411087A (en) * | 2017-05-22 | 2017-12-01 | 杭州济佰氏生物科技有限公司 | A kind of food for being used to nurse one's health women ovarian function |
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