CN105055769A

CN105055769A - Preparation method and application of Liuwei Dihuang pills

Info

Publication number: CN105055769A
Application number: CN201510434596.0A
Authority: CN
Inventors: 周勤文; 陈家进
Original assignee: TONGRENTANG PHARMACEUTICAL CO Ltd
Current assignee: TONGRENTANG PHARMACEUTICAL CO Ltd
Priority date: 2015-07-22
Filing date: 2015-07-22
Publication date: 2015-11-18
Anticipated expiration: 2035-07-22
Also published as: CN105055769B

Abstract

The invention belongs to the field of traditional Chinese medicine preparations and particularly relates to a preparation method of Liuwei Dihuang pills. The Liuwei Dihuang pills are made from 120g of prepared rehmannia root, 60g of common Macrocarpium fruit, 45g of root-bark of tree peony, 60g of common yam rhizome, 45g of Indian bread and 45g of rhizome of oriental water plantain. The Liuwei Dihuang pills are prepared with macroporous resin, cation exchange resin and anion exchange resin, so that contents are greatly increased and less usage is required. The invention further provides application of the Liuwei Dihuang pills in the preparation of medicaments for inhibiting proliferation of mouse embryonic fibroblast NIH3T3.

Description

A kind of preparation method of LIUWEI DIHUANG WAN and application

Technical field

The present invention relates to technical field of traditional Chinese medicine preparation, be specifically related to a kind of preparation method and application of LIUWEI DIHUANG WAN.

Background technology

LIUWEI DIHUANG WAN is recorded in Ministry of Public Health standard, is made up, decocts with water, make pill of Radix Rehmanniae Preparata 120g, Fructus Corni 60g, Cortex Moutan 45g, Rhizoma Dioscoreae 60g, Poria 45g, Rhizoma Alismatis 45g as crude drug.Enriching yin and nourishing kidney.For damage of kidney-YIN, dizziness and tinnitus, soreness of the waist and knees, osteopyrexia and fever, night sweat is passed out semen.In prior art, not yet have LIUWEI DIHUANG WAN extracting the report adopting resin technology in preparation, and adopt the method for soak by water, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, is inconvenient to take, and has had a strong impact on this product and has applied clinically.

In prior art, the heavy 1.44g of every 8 balls of former LIUWEI DIHUANG WAN, 8 balls, 3 times on the one, the heavy 1.2g of every 8 balls of the LIUWEI DIHUANG WAN adopting the present invention to be prepared into, but its active component contained increases greatly.

Summary of the invention

Goal of the invention: in order to solve the problem, the object of the present invention is to provide a kind of preparation method of LIUWEI DIHUANG WAN.

Another object of the present invention is to provide a kind of LIUWEI DIHUANG WAN to suppress the application in mouse embryo fibroblasts NIH3T3 hyperproliferation agent in preparation.

Technical scheme: the object of the invention is by following scheme realize:

A kind of preparation method of LIUWEI DIHUANG WAN, by Radix Rehmanniae Preparata 120g, Fructus Corni 60g, Cortex Moutan 45g, Rhizoma Dioscoreae 60g, Poria 45g, Rhizoma Alismatis 45g makes as crude drug, described method is made up of the following step: get Radix Rehmanniae Preparata 120g, Fructus Corni 60g, Cortex Moutan 45g, Rhizoma Dioscoreae 60g, Poria 45g, Rhizoma Alismatis 45g, add 8 times amount distilled water extraction twice, each 1.5 hours, merge extractive liquid, be concentrated into 0.4ml/g crude drug, adding ethanol makes alcohol content reach 80%, leave standstill 8 hours, sucking filtration, get supernatant, D1010 type macroporous adsorptive resins crossed by medicinal liquid, be washed to colourless, use 90% ethanol elution, collect eluent 10BV, namely evaporate to dryness obtains aminoacyl site, concentrated by water lotion after crossing macroporous resin, upper 732 type cation exchange resiies after medicinal liquid adjust ph to 2, are washed to colourless, and with 40% ethanol elution containing 3% ammonia, collect eluent, namely evaporate to dryness obtains B position, finally the water lotion crossing cationic resin is concentrated, upper 717 type anion exchange resin after adjust ph to 8, be washed to colourless, after concentrated hydrochloric acid desorption, use 40% ethanol elution, collect eluent, namely evaporate to dryness obtains C position, finally by three amount of powder mix homogeneously, pill, dry, polishing, the heavy 1.2g of every 8 balls.

Described LIUWEI DIHUANG WAN suppresses the application in mouse embryo fibroblasts NIH3T3 hyperproliferation agent in preparation, described method is made up of the following step: get Radix Rehmanniae Preparata 120g, Fructus Corni 60g, Cortex Moutan 45g, Rhizoma Dioscoreae 60g, Poria 45g, Rhizoma Alismatis 45g, add 8 times amount distilled water extraction twice, each 1.5 hours, merge extractive liquid, be concentrated into 0.4ml/g crude drug, adding ethanol makes alcohol content reach 80%, leave standstill 8 hours, sucking filtration, get supernatant, D1010 type macroporous adsorptive resins crossed by medicinal liquid, be washed to colourless, use 90% ethanol elution, collect eluent 10BV, namely evaporate to dryness obtains aminoacyl site, concentrated by water lotion after crossing macroporous resin, upper 732 type cation exchange resiies after medicinal liquid adjust ph to 2, are washed to colourless, and with 40% ethanol elution containing 3% ammonia, collect eluent, namely evaporate to dryness obtains B position, finally the water lotion crossing cationic resin is concentrated, upper 717 type anion exchange resin after adjust ph to 8, be washed to colourless, after concentrated hydrochloric acid desorption, use 40% ethanol elution, collect eluent, namely evaporate to dryness obtains C position, finally by three amount of powder mix homogeneously, pill, dry, polishing, the heavy 1.2g of every 8 balls.

Detailed description of the invention

Form by the following examples, foregoing of the present invention is described in further detail again, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example, all technology realized based on foregoing of the present invention all belong to scope of the present invention.

Embodiment 1: get Radix Rehmanniae Preparata 120g, Fructus Corni 60g, Cortex Moutan 45g, Rhizoma Dioscoreae 60g, Poria 45g, Rhizoma Alismatis 45g, adds 8 times amount distilled water extraction twice, each 1.5 hours, merge extractive liquid, is concentrated into 0.4ml/g crude drug, adds ethanol and makes alcohol content reach 80%, leave standstill 8 hours, sucking filtration, gets supernatant, and D1010 type macroporous adsorptive resins crossed by medicinal liquid, be washed to colourless, use 90% ethanol elution, collect eluent 10BV, namely evaporate to dryness obtains aminoacyl site; Concentrated by water lotion after crossing macroporous resin, upper 732 type cation exchange resiies after medicinal liquid adjust ph to 2, are washed to colourless, and with 40% ethanol elution containing 3% ammonia, collect eluent, namely evaporate to dryness obtains B position; Finally the water lotion crossing cationic resin is concentrated, upper 717 type anion exchange resin after adjust ph to 8, be washed to colourless, after concentrated hydrochloric acid desorption, use 40% ethanol elution, collect eluent, namely evaporate to dryness obtains C position, finally by three amount of powder mix homogeneously, pill, dry, polishing, the heavy 1.2g of every 8 balls.

Embodiment 2: LIUWEI DIHUANG WAN suppresses the experimentation data of mouse embryo fibroblasts NIH3T3 propagation

1 experiment material

1.1 experiment cell strain: mouse embryo fibroblasts NIH3T3, Nanjing Tongrentang Pharmaceutical Co., Ltd.'s laboratory cell storehouse, DMEM+10%FBS cellar culture.

1.2 Experimental agents: drugs: LIUWEI DIHUANG WAN of the present invention: prepare by embodiment 1 method.Medicinal liquid liquid storage: take 100mg LIUWEI DIHUANG WAN, is dissolved in 5ml dehydrated alcohol, 0.2 μm of frit, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, and simultaneously 0.2 μm of frit dehydrated alcohol is in order to the use of matched group.

1.3 experiment reagents: DMEM (GIBCO company Cat.No.12100-061Lot.No.758137); Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419); NaHCO3 (Shanghai hundred million chemical reagent company limited Cat.No.11810-033Lot.No.1088387 of a specified duration); Trypsin (AMRESCO company lot number: 2010/04); EDTA (AMRESCO company lot number: 2009/10); PenicillinGSodiumSalt (AMRESCO company lot number: 2010242); StreptomycinSulfate (AMRESCO company lot number: 2010382); Dehydrated alcohol (Nanjing Chemistry Reagent Co., Ltd.'s lot number: 080310182); MTT (Biosharp lot number: 0793); PBS (laboratory autogamy);

1.4 experiment equipments: Lycra inverted microscope (German Leica model: DM1L); Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX190); CO2 incubator (FORMA model: 3111); Super-clean bench (safe and sound Inc. of Su Jing group moulding number: SW-CJ-ZFD); Pure water instrument (Spring company of U.S. model: S/N020579); Accurate pipettor (French Gilson Inc model: P2); Electronic balance (German Sai Duolisi company limited model: BT323S); Full-automatic high-pressure autoclave (Japanese SANYO company model: MLS-3020); Table electrothermal air dry oven (the accurate experimental facilities company in Shanghai model: DHG9123A); Refrigerator (Siemens Company's model: KG18V21TI); Liquid nitrogen container (CBS model: 2001); Low speed centrifuge (Anting Scientific Instrument Factory, Shanghai's model: KA-1000); 0.2 μm of filter (MILLIPORE model: SLGP033RB); 10cm culture dish (NEST company), 96 well culture plates (NEST company); Cell counting count board; Centrifuge tube, pipet, Tips are some.

2 experimental techniques: 1) mouse embryo fibroblasts NIH3T3 DMEM+10%FBS in 37 DEG C, 5%CO2 carries out cellar culture (10cm culture dish), when Growth of Cells is to logarithmic (log) phase, collecting cell, discard culture fluid, PBS fine laundering 3 times, add 3ml0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, add 5ml complete medium neutralization reaction wherein, proceeded in centrifuge tube after piping and druming cell, the centrifugal 5min of 1000rpm, adjustment concentration of cell suspension 3 × 104/ml.2) enter in 96 well culture plates by cell kind, every hole adds cell suspension 180 μ l, and culture plate puts into cell culture incubator (37 DEG C, 5%CO2) cellar culture.3) according to cell growth status, generally grow to 50%-70%, add LIUWEI DIHUANG WAN solution, continue to cultivate 24h.4) add 20 μ lMTT solution (5mg/ml, i.e. 0.5%MTT) after 24h, continue to cultivate 4h.5) after 4h, buckle method removes supernatant, pats dry gently with absorbent paper, and every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, crystal is fully dissolved.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.6) background (do not add cell, only add culture fluid) is set, control wells (the medicine dissolution medium of cell, same concentrations, culture fluid, MTT, dimethyl sulfoxide) simultaneously, often organizes the multiple hole of setting 6.7) result represents with the suppression ratio of medicine to cell: cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD value)/control wells OD value × 100%.Experiment repetition 3 times.

3 experimental results: statistical result showed after mtt assay experiment, compare with matched group, when dosage reaches 5mg/ml, to mouse embryo fibroblasts NIH3T3 Proliferation Ability variant (P<0.05), dosage this difference when 10mg/ml has significance (P<0.01), has pole significant difference (P<0.001) when dosage reaches 15-20mg/ml.

Table 1 LIUWEI DIHUANG WAN is to mouse embryo fibroblasts NIH3T3 Proliferation Ability influence research (X ± SD)

Note: compare with matched group, * P<0.01; * P<0.001

4 experiment conclusion: LIUWEI DIHUANG WAN can suppress mouse embryo fibroblasts NIH3T3 to breed, reduce the Growth of Cells number of mouse embryo fibroblasts NIH3T3, this effect is dose dependent.

Claims

1. the preparation method of a LIUWEI DIHUANG WAN, by Radix Rehmanniae Preparata 120g, Fructus Corni 60g, Cortex Moutan 45g, Rhizoma Dioscoreae 60g, Poria 45g, Rhizoma Alismatis 45g makes as crude drug, it is characterized in that described method is made up of the following step: get Radix Rehmanniae Preparata 120g, Fructus Corni 60g, Cortex Moutan 45g, Rhizoma Dioscoreae 60g, Poria 45g, Rhizoma Alismatis 45g, add 8 times amount distilled water extraction twice, each 1.5 hours, merge extractive liquid, be concentrated into 0.4ml/g crude drug, adding ethanol makes alcohol content reach 80%, leave standstill 8 hours, sucking filtration, get supernatant, D1010 type macroporous adsorptive resins crossed by medicinal liquid, be washed to colourless, use 90% ethanol elution, collect eluent 10BV, namely evaporate to dryness obtains aminoacyl site, concentrated by water lotion after crossing macroporous resin, upper 732 type cation exchange resiies after medicinal liquid adjust ph to 2, are washed to colourless, and with 40% ethanol elution containing 3% ammonia, collect eluent, namely evaporate to dryness obtains B position, finally the water lotion crossing cationic resin is concentrated, upper 717 type anion exchange resin after adjust ph to 8, be washed to colourless, after concentrated hydrochloric acid desorption, use 40% ethanol elution, collect eluent, namely evaporate to dryness obtains C position, finally by three amount of powder mix homogeneously, pill, dry, polishing, the heavy 1.2g of every 8 balls.

2. LIUWEI DIHUANG WAN suppresses the application in mouse embryo fibroblasts NIH3T3 hyperproliferation agent in preparation according to claim 1, it is characterized in that described method is made up of the following step: get Radix Rehmanniae Preparata 120g, Fructus Corni 60g, Cortex Moutan 45g, Rhizoma Dioscoreae 60g, Poria 45g, Rhizoma Alismatis 45g, add 8 times amount distilled water extraction twice, each 1.5 hours, merge extractive liquid, be concentrated into 0.4ml/g crude drug, adding ethanol makes alcohol content reach 80%, leave standstill 8 hours, sucking filtration, get supernatant, D1010 type macroporous adsorptive resins crossed by medicinal liquid, be washed to colourless, use 90% ethanol elution, collect eluent 10BV, namely evaporate to dryness obtains aminoacyl site, concentrated by water lotion after crossing macroporous resin, upper 732 type cation exchange resiies after medicinal liquid adjust ph to 2, are washed to colourless, and with 40% ethanol elution containing 3% ammonia, collect eluent, namely evaporate to dryness obtains B position, finally the water lotion crossing cationic resin is concentrated, upper 717 type anion exchange resin after adjust ph to 8, be washed to colourless, after concentrated hydrochloric acid desorption, use 40% ethanol elution, collect eluent, namely evaporate to dryness obtains C position, finally by three amount of powder mix homogeneously, pill, dry, polishing, the heavy 1.2g of every 8 balls.