CN105055769B - A kind of preparation method and application of Liuwei Dihuang Wan - Google Patents

Abstract

The invention belongs to field of traditional Chinese, a kind of preparation method of Liuwei Dihuang Wan is specifically provided, it is made up of prepared rhizome of rehmannia 120g, Fructus Corni 60g, root bark of tree peony 45g, Chinese yam 60g, Poria cocos 45g, rhizoma alismatis 45g as bulk drug, it is prepared using macroreticular resin, cationic ion-exchange resin and anion exchange resin, so that content improves a lot, dosage is reduced, and present invention also offers Liuwei Dihuang Wan to prepare the application in suppressing MEC NIH3T3 hyperproliferation agents.

Description

A kind of preparation method and application of Liuwei Dihuang Wan

Technical field

The present invention relates to technical field of traditional Chinese medicine preparation, and in particular to a kind of preparation method and application of Liuwei Dihuang Wan.

Background technology

Liuwei Dihuang Wan is recorded in hygienic ministerial standard, by prepared rhizome of rehmannia 120g, Fructus Corni 60g, root bark of tree peony 45g, Chinese yam 60g, Fu Siberian cocklebur 45g, rhizoma alismatis 45g are made as bulk drug, add water to cook, pill is made.Enriching yin and nourishing kidney.For damage of kidney-YIN, dizziness and tinnitus, Soreness and weakness of waist and knees, osteopyrexia and fever, night sweat seminal emission.In the prior art, there has been no Liuwei Dihuang Wan to use resin in terms of preparation is extracted The report of technology, and the method boiled using decocting, technique is coarse, falls behind, and impurity is more, causes patient's dosage excessive, it has not been convenient to takes With having had a strong impact on that this product is clinically applied.

The content of the invention

Goal of the invention：In order to solve the above problems, it is an object of the invention to provide a kind of preparation side of Liuwei Dihuang Wan Method.

It is another object of the present invention to provide a kind of Liuwei Dihuang Wan to prepare suppression MEC Application in NIH3T3 hyperproliferation agents.

Technical scheme：The purpose of the present invention is realized by following scheme：

A kind of preparation method of Liuwei Dihuang Wan, by prepared rhizome of rehmannia 120g, Fructus Corni 60g, root bark of tree peony 45g, Chinese yam 60g, Poria cocos 45g, rhizoma alismatis 45g are made as bulk drug, and described method is made up of the following steps：Take prepared rhizome of rehmannia 120g, Fructus Corni 60g, pellet Skin 45g, Chinese yam 60g, Poria cocos 45g, rhizoma alismatis 45g, add 8 times of amount distilled water extractions twice, 1.5 hours every time, merge extract solution, it is dense 0.4ml/g crude drugs are reduced to, add ethanol alcohol content is stood 8 hours up to 80%, is filtered, take supernatant, it is big that decoction crosses D1010 types Macroporous adsorbent resin post, is washed to colourless, with 90% ethanol elution, collects eluent 10BV, is evaporated and produces A positions；By macropore Water lotion concentration after resin, decoction adjust pH value to upper 732 type cationic ion-exchange resin after 2, be washed to it is colourless, with containing 3% 40% ethanol elution of ammoniacal liquor, eluent is collected, is evaporated and produces B positions；Finally the water lotion for crossing resin cation is concentrated, adjusted PH value is saved to upper 717 type anion exchange resin after 8, be washed to it is colourless, with 40% ethanol elution, collection after concentrated hydrochloric acid desorption Eluent, it is evaporated and produces C positions, be finally well mixed three parts powder, pill, dries, polishing, every 8 ball weight 1.2g.

The Liuwei Dihuang Wan is preparing the application in suppressing MEC NIH3T3 hyperproliferation agents, described Method be made up of the following steps：Prepared rhizome of rehmannia 120g, Fructus Corni 60g, root bark of tree peony 45g, Chinese yam 60g, Poria cocos 45g, rhizoma alismatis 45g are taken, Add 8 times of amount distilled water extractions twice, 1.5 hours every time, merge extract solution, be concentrated into 0.4ml/g crude drugs, add ethanol to make alcohol content Up to 80%, stand 8 hours, filter, take supernatant, decoction crosses D1010 type large pore resin absorption columns, be washed to it is colourless, with 90% Ethanol elution, eluent 10BV is collected, is evaporated and produces A positions；Water lotion after macroreticular resin excessively is concentrated, decoction regulation pH value Upper 732 type cationic ion-exchange resin after to 2, is washed to colourless, with 40% ethanol elution containing 3% ammoniacal liquor, collects eluent, steams It is dry to produce B positions；Finally the water lotion for crossing resin cation is concentrated, regulation pH value to upper 717 type anion exchange tree after 8 Fat, is washed to colourless, with 40% ethanol elution after concentrated hydrochloric acid desorption, collects eluent, is evaporated and produces C positions, finally by three Amount of powder is well mixed, pill, is dried, polishing, every 8 ball weight 1.2g.

In the prior art, the every 8 ball weight 1.44g of former Liuwei Dihuang Wan, 8 balls, 3 times a day, is prepared into using the present invention Liuwei Dihuang Wan every 8 ball weight 1.2g, but its active component contained greatly increases.

Embodiment

Form by the following examples, the above of the present invention is described in further detail again, but should not be by this The scope for being interpreted as the above-mentioned theme of the present invention is only limitted to following example, and all technologies for being realized based on the above of the present invention are equal Belong to the scope of the present invention.

Embodiment 1：Prepared rhizome of rehmannia 120g, Fructus Corni 60g, root bark of tree peony 45g, Chinese yam 60g, Poria cocos 45g, rhizoma alismatis 45g are taken, adds 8 times Measure distilled water extraction twice, 1.5 hours every time, merge extract solution, be concentrated into 0.4ml/g crude drugs, add ethanol to reach alcohol content 80%, stand 8 hours, filter, take supernatant, decoction crosses D1010 type large pore resin absorption columns, be washed to it is colourless, with 90% second Alcohol elutes, and collects eluent 10BV, is evaporated and produces A positions；By cross macroreticular resin after water lotion concentrate, decoction adjust pH value to Upper 732 type cationic ion-exchange resin after 2, is washed to colourless, with 40% ethanol elution containing 3% ammoniacal liquor, collects eluent, is evaporated Produce B positions；Finally the water lotion for crossing resin cation is concentrated, regulation pH value to upper 717 type anion exchange resin after 8, It is washed to colourless, with 40% ethanol elution after concentrated hydrochloric acid desorption, collects eluent, be evaporated and produce C positions, finally by three parts Powder is well mixed, pill, is dried, polishing, every 8 ball weight 1.2g.

Embodiment 2：Liuwei Dihuang Wan suppresses the experimental study data of MEC NIH3T3 propagation

1 experiment material

1.1 experiment cell lines：MEC NIH3T3, Nanjing Tongrentang Pharmaceutical Co., Ltd. are real Test ventricular cell storehouse, DMEM+10%FBS cellar cultures.

1.2 Experimental agents：Study medicine：Liuwei Dihuang Wan of the present invention：Prepared by the method for embodiment 1.Decoction liquid storage：Weigh 100mg Liuwei Dihuang Wans, it is dissolved in 5ml absolute ethyl alcohols, 0.2 μm of filter filtering, the packing of 500 μ l doff pipes, -20 DEG C of storages, together When 0.2 μm of filter filter absolute ethyl alcohol in case control group is used.

1.3 experiment reagent：DMEM (GIBCO company Cat.No.12100-061Lot.No.758137)；Hyclone (Zhejiang Sky over the river Hangzhoupro bio tech ltd Lot.No.100419)；NaHCO3 (the long hundred million chemical reagent Co., Ltds in Shanghai Cat.No.11810-033Lot.No.1088387)；Trypsin (AMRESCO companies lot numbers：2010/04)；EDTA(AMRESCO Company's lot number：2009/10)；Penicillin G Sodium Salt (AMRESCO companies lot numbers：2010242)； Streptomycin Sulfate (AMRESCO companies lot numbers：2010382)；Absolute ethyl alcohol (Nanjing Chemistry Reagent Co., Ltd. batch Number：080310182)；MTT (Biosharp lot numbers：0793)；PBS (laboratory autogamy)；

1.4 experiment equipment：Lycra inverted microscope (German Leica models：DM1L)；It can be seen that-ultraviolet light microwell plate detects Instrument (MD companies of U.S. model：SPECTRA MAX 190)；CO2 incubators (FORMA models：3111)；Super-clean bench (Su Jing groups Safe and sound company system moulding number：SW-CJ-ZFD)；Pure water meter (Spring companies of U.S. model：S/N 020579)；Accurate pipettor (French Gilson Inc's model：P2)；Electronic balance (German Sai Duolisi Co., Ltds model：BT323S)；Full-automatic high-pressure Autoclave (Japanese SANYO companies model：MLS-3020)；Table electrothermal air dry oven (Shanghai precision experimental facilities company type Number：DHG9123A)；Refrigerator (Siemens Company's model：KG18V21TI)；Liquid nitrogen container (CBS models：2001)；Low speed centrifuge (Anting Scientific Instrument Factory, Shanghai's model：KA-1000)；0.2 μm of filter (MILLIPORE model：SLGP033RB)；10cm is cultivated Ware (NEST companies), 96 well culture plates (NEST companies)；Cell counting count board；Centrifuge tube, pipette, Tips are some.

2 experimental methods：1) MEC NIH3T3 is carried out often with DMEM+10%FBS in 37 DEG C, 5%CO2 Rule culture (10cm culture dishes), when cell growth to logarithmic phase, cell is collected, discard nutrient solution, PBS is rinsed 3 times, is added After 3ml 0.25% trypsase -0.04%EDTA, 37 DEG C of digestion 2min, 5ml complete medium neutralization reactions are added thereto, It is transferred to after piping and druming cell in centrifuge tube, 1000rpm centrifugation 5min, 3 × 104/ml of adjustment concentration of cell suspension.2) will be thin Born of the same parents are planted in 96 well culture plates, and the μ l of cell suspension 180 are added per hole, and culture plate is put into cell culture incubator (37 DEG C, 5%CO2) Cellar culture.3) it is general long to 50%-70% according to cell growth status, Liuwei Dihuang Wan solution is added, continues to cultivate 24h. 4) 20 μ l MTT solution (5mg/ml, i.e. 0.5%MTT) are added after 24h, continue to cultivate 4h.5) buckle method removes supernatant after 4h, uses Blotting paper is gently patted dry, and 200 μ l dimethyl sulfoxide (DMSO)s are added per hole, low-speed oscillation 10min on shaking table is put, makes crystal fully molten Solution.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm.6) while background is set (to be not added with cell, only plus cultivate Liquid), control wells (cell, the medicine dissolving medium of same concentrations, nutrient solution, MTT, dimethyl sulfoxide (DMSO)), every group of setting 6 is again Hole.7) result is represented the inhibiting rate of cell with medicine：Cell proliferation inhibiting rate (%)=(control wells OD values-dosing holes OD Value)/control wells OD value × 100%.Experiment is repeated 3 times.

3 experimental results：Statistical result showed after mtt assay experiment is right when dosage reaches 5mg/ml compared with control group Variant (the P of MEC NIH3T3 Proliferation Abilities<0.05), dosage difference in 10mg/ml has notable Property (P<0.01), there is pole significant difference (P when dosage reaches 15-20mg/ml<0.001).

The Liuwei Dihuang Wan of table 1 influences research (X ± SD) to MEC NIH3T3 Proliferation Abilities

Note：Compared with control group, * P<0.01；**P<0.001

4 experiment conclusions：Liuwei Dihuang Wan can suppress MEC NIH3T3 propagation, reduce mice embryonic Fibroblast NIH3T3 cell growth number, the effect are in dose dependent.

Claims (1)

1. Liuwei Dihuang Wan is preparing the application in suppressing MEC NIH3T3 hyperproliferation agents, it is characterised in that The preparation method of the Liuwei Dihuang Wan is made up of the following steps：Take prepared rhizome of rehmannia 120g, Fructus Corni 60g, root bark of tree peony 45g, Chinese yam 60g, Poria cocos 45g, rhizoma alismatis 45g, add 8 times of amount distilled water extractions twice, 1.5 hours every time, merge extract solution, be concentrated into 0.4ml/ G crude drugs, ethanol is added alcohol content is stood 8 hours up to 80%, is filtered, take supernatant, decoction crosses D1010 type macroporous absorbent resins Post, is washed to colourless, with 90% ethanol elution, collects eluent 10BV, is evaporated and produces A positions；By the water after macroreticular resin excessively Washing lotion concentrates, and decoction adjusts pH value to upper 732 type cationic ion-exchange resin after 2, be washed to it is colourless, with 40% containing 3% ammoniacal liquor Ethanol elution, eluent is collected, is evaporated and produces B positions；Finally the water lotion for crossing resin cation is concentrated, regulation pH value to 8 Upper 717 type anion exchange resin afterwards, is washed to colourless, with 40% ethanol elution after concentrated hydrochloric acid desorption, collects eluent, steams It is dry to produce C positions, finally three parts powder is well mixed, pill, dried, polishing, every 8 ball weight 1.2g.