CN104983800B - A kind of gynopathic medicine of the treatment being made up of diuretic medicine and preparation method and detection method - Google Patents
A kind of gynopathic medicine of the treatment being made up of diuretic medicine and preparation method and detection method Download PDFInfo
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Abstract
The invention belongs to drug world, invention entitled a kind of gynopathic medicine of the treatment being made up of diuretic medicine and preparation method and detection method, this pharmaceutical composition is made up of the raw material of following weight portion: Semen Lepidii (Semen Descurainiae) 1~2 weight portion, Radix Stephaniae Tetrandrae 1~2 weight portion, Cortex Mori 1~2 weight portion.This pharmaceutical composition uses pharmaceutical methods conventional in Chinese materia medica to be prepared as tablet, pill, hard capsule, granule, oral liquid, externally used paste.The present invention also provides for this medicine assay method, and the method has high accuracy, stability and repeatability.This pharmaceutical composition can be used for preparation treatment hysteromyoma, the medicine of dysmenorrhea.
Description
Technical field
The invention belongs to drug world, relate to a kind of pharmaceutical composition treating endometriosis, be specifically related to one
Kind by being the pharmaceutical composition that raw material is made for Semen Lepidii (Semen Descurainiae), Radix Stephaniae Tetrandrae, Cortex Mori.
Background technology
Endometriosis belongs to the categories such as Chinese medicine " abdominal mass ", " dysmenorrhea ", " menoxenia ", is common gynecological disease.
Its pathogeny is mainly stagnation of blood stasis, and the main method for the treatment of is blood circulation promoting and blood stasis dispelling.But although gynecopathy is based on blood stasis,
But its pathogenesis is the most complicated, its concrete pattern of syndrome determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs during treatment, should be differentiated.
1. the Chinese medicine pathogeny of endometriosis
Endometriosis belongs to the categories such as Chinese medicine " abdominal mass ", " dysmenorrhea ", " menoxenia ", many traditional Chinese medical science scholars couple
Its etiology and pathogenesis is explored, and thinks that this disease is relevant with blood stasis more." blood stasis " is the key producing endometriosis.
In " Jing Yue's complete work married woman rule ", the associated description about endometriosis has: " be detained work, only married woman of blood stasis has it.Its
Card is then or by menstrual period, or by puerperal, all internal injuries raw food, or have a cold outward, or impairing the liver of being enraged, QI rising in reverse order and blood stays, or melancholy impairing the spleen,
The deficiency of vital energy and stasis, or it is long-pending weak to be overworked for a very long period, and gas is weak and not all right.When always being moved by blood, remaining blood is not clean, and one is the most inverse, then day of being detained is amassed
And gradually to become." say is that the symptom that uterus blood stasis is detained occurred in menstrual period or puerperal, internal injury is raw and cold or has a cold outward,
Or being that patient is angry and impairing the liver all can cause internal mechanism of qi to drive in the wrong direction, palace stasis is stayed;Worried, the thought pent-up meeting impairment of the spleen, Huan Zheqi
Void then blood operation has some setbacks and the stasis of blood is stagnant;Overworked then gas is weak, and gas is weak the most not all right.Menses rest on internal meeting and cause a series of
Pathological change, including blood savings, causes menoxenia, QI-blood circulation to be obstructed."Nei Jing" is described " stagnation of QI and blood may bring about pain ", and blood stasis hinders
Stagnant, so patient often suffers from abdominal pain or the lower abdomen pain of persistence, this is also the clinical common sympton of endometriosis.Blood
Operation be obstructed, meridians are impaired, can affect the function and then cause infertile of becoming pregnant of women.
2. the common pattern of syndrome of endometriosis
2.1 blood stasis type
Patient lives in menstrual period or puerperal and does not notes, experiences wind, cold, summer-heat, the exopathogen such as wet, dry, fiery all can cause chong channel
Sustaining damage with conception vessel, the dysfunction of blood stasis is discharged in uterus, although intermenstrual period menses also can be discharged, but not by positive normal practice
Road runs, and part menses can not be discharged, and causes " blood circulating out of vessels " to be detained in vivo, accumulates and become blood stasis, stagnation of blood stasis in pelvic cavity
Meridians then QI-blood circulation is not smooth, stagnation of QI and blood may bring about pain;In blood stasis is accumulated in, forms enclosed mass and then form the card of blood stasis.
2.2 qi stagnation and blood stasis type
Patient has the symptom of depression at ordinary times, or feels blue menstrual period, can cause depression of liver-QI, qi depression to blood stasis, and menses are transported
Unworkable freely, " stagnation of QI and blood may bring about pain ".Blood stasis in uterus gathers, depression and stagnation of QI, and menstrual period, palace blood filled, and added disorder of QI movement, stasis blocking
The most serious, clinical symptoms shows as qi stagnation and blood stasis type.
2.3 kidney deficiency blood stasis type
Kidney is the internal organs of store essential substances, plays an important role the growth promoter of human body with producing offspring, and collaterals of the uterus lies in kidney.Five true energy of the zang organ
Gas is kidney at all, and the kidney being the origin of congenital constitution, the residence of the root of the five internal organs, Source of life, vigour for positive what is called.So the five internal organs are if subjected to damage
Hurt and the most necessarily injure kidney, then decline of kidney-QI of suffering from a deficiency of the kidney, unable operation blood, cause blood stasis;Suffer from a deficiency of the kidney and cause punching to appoint the deficiency of vital energy,
Astringency inducing is neglected one's duty, it is impossible to consolidating semen becomes pregnant, thus causes infertile.The endometriosis course of disease is longer, easy damaged kidney qi, its symptom table
It it is now kidney deficiency blood stasis type.
2.4 cold blood stasis type
Patient's body constitution is yang deficiency, or prolonged illness injures yang-energy, causes in YIN-cold is born in, or catch cold when menstrual period, production, drench with rain,
Paddle, swim, or the thing of voracious raw food, or occupy residence humidity, cold-evil, damp invasion, the yang-energy of damage body, cold
Fleeing in vivo, stagnate vessels of the uterus, causes vessels of the uterus coagulation of QI-blood to block, stagnation of QI and blood may bring about pain, thus causes dysmenorrhea, forms cold blood stasis card
Type.
2.5 the mutual junction type of the hot stasis of blood
The body constitution of the hot stasis of blood mutual junction type patient mostly is the body of interior-heat, again because the time of blood stasis existence is relatively more long, accumulates heat of lighting a fire.
The pathogeny of endometriosis be blood stasis and the course of disease longer, the long heat-transformation of the easy stasis of blood, or the long-time stagnation of QI, five excessive emotions converting into internal fire, or
Pathogenic heat is invaded, and the bright blood vessels of heat, stagnant heat is contained in internal, and the hot stasis of blood is mutually entangled with, and pents up in the part of the body cavity below the umbilicus, housing the bladder, kidneys and bowels, is consequently formed.
3. the dialectical therapy of endometriosis and common drug
3.1 activating blood circulation and dissipating blood stasis methods
The basic cause of disease of endometriosis is blood stasis, uses activating blood circulation and dissipating blood stasis method can directly remove Basic disease cause, change
The situation of kind blood stasis.12 conventional taste Chinese medicines be Radix Paeoniae Rubra, Radix Salviae Miltiorrhizae, Rhizoma Curcumae, Radix Angelicae Sinensis, Hirudo, Carapax Trionycis, Rhizoma Corydalis, Semen Persicae, Rhizoma Sparganii,
Ramulus Cinnamomi, Pollen Typhae, Oletum Trogopterori, some of which Chinese medicine is medicine pair, has both strengthened drug effect, blood circulation promoting and blood stasis dispelling, has overcome again single drug not
Foot.Rhizoma Sparganii, Rhizoma Curcumae share, and can eliminate blood stasis, removing blood stasis circulation of qi promoting, removing food stagnancy pain relieving;Radix Angelicae Sinensis, Radix Salviae Miltiorrhizae share, can enrich blood, invigorate blood circulation,
Stimulate the menstrual flow;Pollen Typhae, Oletum Trogopterori share, can be with stasis-dispelling and pain-killing;Carapax Trionycis can be with hard masses softening and resolving, nourishing YIN for suppressing the hyperactive YANG, then is aided with Radix Paeoniae Rubra, is avoided that
In side, other the most pungent temperature of blood circulation promoting medicine drug effect causes body warm-dryness syndrome.
3.2 regulating qi to disperse stagnation methods
Gas dominates the operation of blood, gas row then blood, the stagnation of QI then blood stasis, and blood is the basic of gas, and the blood stasis of blood is stagnant necessarily to be led
Cause fate unworkable freely, so vital energy stagnation and blood stasis type endometriosis uses regulating qi to disperse stagnation method.12 the most frequently used taste Chinese medicines are
Radix Paeoniae Rubra, Radix Angelicae Sinensis, Radix Salviae Miltiorrhizae, Fructus Aurantii, Rhizoma Cyperi, Semen Persicae, Rhizoma Curcumae, Rhizoma Chuanxiong, Rhizoma Corydalis, Rhizoma Sparganii, Oletum Trogopterori, Flos Carthami.Wherein Rhizoma Cyperi, Rhizoma Corydalis
The effect of rope is depressed liver-energy dispersing and QI regulating, and Rhizoma Sparganii, Rhizoma Curcumae can eliminate blood stasis, removing blood stasis circulation of qi promoting, and the effect of Fructus Aurantii is promoting QI to circulate and dispersing the agglomeration of the pathogens, and other are several
The effect of taste Chinese medicine is promoting blood circulation and stopping pain.
3.3 Bushen Huayu methods
The kidney being the origin of congenital constitution, and for kidney deficiency blood stasis type patient, the kidney invigorating, blood stasis dispelling, to invigorate blood circulation be basic method for the treatment of.Modern many doctors
Research also confirms that, invigorating kidney, promoting blood circulation can improve the immunity of body.12 the most frequently used taste Chinese medicines be Semen Cuscutae, Rhizoma Curcumae, Rhizoma Sparganii, when
Return, Radix Paeoniae Rubra, Herba Epimedii, Rhizoma Cyperi, Radix Dipsaci, Cortex Moutan, Squama Manis, Radix Salviae Miltiorrhizae, Rhizoma Corydalis.Wherein tonic has Semen Cuscutae, Radix Angelicae Sinensis, celestial spirit
Spleen, Radix Dipsaci, Semen Cuscutae, Herba Epimedii, effect of Radix Dipsaci are invigorating the liver and kidney, and other each taste medicines have replenishing and activating blood effect.
3.4 warming the meridian blood stasis dispelling methods
Cold-evil stagnates and may result at passages through which vital energy circulates that the blood stasis of blood is stagnant, mechanism of qi blocks, stagnation of QI and blood may bring about pain.Described in "Nei Jing"
" Wen Ze disappears and goes it ", uses antalgic, activating blood circulation to dissipate blood stasis method to cold blood stasis type patient, can play and significantly treat effect
Really.12 conventional taste Chinese medicines are Radix Angelicae Sinensis, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Pollen Typhae, Oletum Trogopterori, Fructus Foeniculi, Rhizoma Zingiberis, Myrrha, Rhizoma Corydalis, Ramulus Cinnamomi, perfume (or spice)
Attached, Cortex Cinnamomi.Fructus Foeniculi, Rhizoma Zingiberis, the effect of Cortex Cinnamomi are warming spleen and stomach for dispelling cold, and in removing body, the cold stasis of blood is stagnant and pain relieving.Radix Angelicae Sinensis, Ramulus Cinnamomi are common
With, blood can be nourished with warm meridians, dispersing cold for relieving pain, makes venation unobstructed.Other each taste Chinese medicines all have blood stasis dispelling, circulation of qi promoting, pain relieving
Effect.
3.5 heat clearing and stasis removing therapy
The hot stasis of blood mutual junction type patient often has obvious heat syndrome, is embodied in that abdominal part is scorching hot, constipation with dry stool, often have low grade fever,
Or menstrual period body temperature rise high, therefore use heat clearing and stasis removing therapy.12 conventional taste Chinese medicines have Semen Persicae, Herba Patriniae, Radix Paeoniae Rubra, Rhizoma Cyperi, Radix Salviae Miltiorrhizae,
Pollen Typhae, Radix Glycyrrhizae, Concha Ostreae, Hirudo, worm, Semen Coicis, Rhizoma Curcumae.Herba Patriniae, Radix Paeoniae Rubra, Semen Coicis share, can play heat clearing away, removing toxic substances,
The effect of removing heat from blood, other each taste Chinese medicines have invigorate blood circulation, blood stasis dispelling, the curative effect of pain relieving, Radix Glycyrrhizae plays the effect of coordinating the actions of various ingredients in a prescription.
4. conclusion
Endometriosis is common gynecological disease, and pathogenesis is more complicated, uses tcm syndrome differentiation and treatment can obtain good therapeutic effect.
In addition to clinical syndrome differentiation classification therapy, old chin or cheek sums up Si Tuyi treatment endometriosis Chinese medical discrimination and experience, it is believed that menstrual period
Based on blood-activating analgetic, non-menstrual period are main blood circulation promoting and blood stasis dispelling of holding concurrently with qi-restoratives, with Bushen Huoxue Fang (Semen Cuscutae, Radix Dipsaci, Herba Taxilli, when
Return, Radix Salviae Miltiorrhizae etc.) be main treatment endometriosis dysmenorrhea, clinical obtain good curative effect.Yan Qingya sums up Xiayang and uses the cycle
Therapy for treating endometriosis card dysmenorrhea experience, based on activating blood and removing stasis Method, controls in conjunction with menstrual cycle different times opinion: non-warp
Phase delays Zhi Qiben with vital energy benefiting and the kidney invigorating, hard masses softening and resolving, use reinforcement and elimination in combination, the slow block side that disappears (Radix Stephaniae Tetrandrae, Radix Codonopsis, Semen Cuscutae, Rhizoma Polygonati,
Rhizoma Sparganii, Rhizoma Curcumae, Pseudobulbus cremastrae seu pleiones, Herba Scutellariae Barbatae, Ramulus Cinnamomi, Poria, Cortex Moutan, Radix Paeoniae Rubra, Semen Persicae, Spica Prunellae, Bulbus Fritillariae Thunbergii, Radix Salviae Miltiorrhizae, Squama Manis, prolong
Rhizoma Corydalis, Spina Gleditsiae), premenstrual 7d and menstrual period with dispelling cold by warming the meridian, regulating QI side of enriching blood (Radix Angelicae Sinensis, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Rhizoma Zingiberis Preparatum, Rhizoma Corydalis, five
Oletum Trogopterori, Fructus Foeniculi, Pollen Typhae, Ramulus Cinnamomi, Myrrha, Rhizoma Sparganii, Rhizoma Curcumae, Flos Carthami, Aspongopus, Eupolyphaga Seu Steleophaga, Sanguis Draxonis), carry out by menstrual cycle
Phase treatment, it is thus achieved that satisfactory effect.Research to endometriosis promotes the progress of clinical treatment gynecopathy, with the traditional Chinese medical science
Theoretical as instructing, open the new world for this disease of clinical treatment.
Medicine of the present invention is the most meticulously to develop through inventor, is mainly used in treating endometriosis.After deliberation
Showing, medicine of the present invention has preferable therapeutic effect to endometriosis.
Summary of the invention
It is an object of the invention to provide a kind of pharmaceutical composition treating endometriosis.
It is a further object of the present invention to provide the preparation method of this pharmaceutical composition.
The present invention also provides for the quality determining method of this pharmaceutical composition.
Present invention also offers the pharmaceutical applications of this pharmaceutical composition.
It is an object of the invention to be accomplished by:
A kind of pharmaceutical composition treating endometriosis, this pharmaceutical composition is by the raw material system of following weight portion
Become: Semen Lepidii (Semen Descurainiae) 1~2 weight portion, Radix Stephaniae Tetrandrae 1~2 weight portion, Cortex Mori 1~2 weight portion.
This pharmaceutical composition is preferably made up of the raw material of following weight portion: Semen Lepidii (Semen Descurainiae) 1 weight portion, Radix Stephaniae Tetrandrae 2 weight portion,
Cortex Mori 1 weight portion.
This pharmaceutical composition is preferably made up of the raw material of following weight portion: Semen Lepidii (Semen Descurainiae) 2 weight portion, Radix Stephaniae Tetrandrae 1 weight portion,
Cortex Mori 2 weight portion.
This pharmaceutical composition is preferably made up of the raw material of following weight portion: Semen Lepidii (Semen Descurainiae) 1 weight portion, Radix Stephaniae Tetrandrae 1 weight portion,
Cortex Mori 1 weight portion.
This pharmaceutical composition use pharmaceutical methods conventional in pharmacy be prepared as tablet, pill, hard capsule, granule,
Oral liquid, externally used paste.
This pharmaceutical composition hard capsule preferably employs following method to be prepared: take Semen Lepidii (Semen Descurainiae), Radix Stephaniae Tetrandrae, Cortex Mori, mixing, adds
The water soaking 0.5 of 3~15 times amount~2 hours, decoct 0.5~2 hour, decocts 2~4 times, and each 0.5~2 hour, extracting solution closed
And, filtering, filtrate concentrates, and dry, pulverize into fine powder, adds adjuvant, mixing, loads hard capsule, to obtain final product.
This pharmaceutical composition hard capsule is more highly preferred to adopt to be prepared with the following method: take Semen Lepidii (Semen Descurainiae), Radix Stephaniae Tetrandrae, Cortex Mori, mixing,
The water soaking of addition 11 times amount 1 hour, decocts 1 hour for the first time, and second time adds the water of 4 times amount, decocts 1 hour, merges decocting
Liquid, filters, and filtrate concentrates, and dry, pulverize into fine powder, adds adjuvant, mixing, loads hard capsule, to obtain final product.
This pharmaceutical composition emplastrum preferably employs following method to be prepared: take Semen Lepidii (Semen Descurainiae), Radix Stephaniae Tetrandrae, Cortex Mori, mixing, adds
The water soaking 0.5 of 3~15 times amount~2 hours, decoct 0.5~2 hour, decocts 2~4 times, and each 0.5~2 hour, extracting solution closed
And, filtering, filtrate concentrates, and adds normal hexane, vaseline, lanoline, continuously stirred in boiling water bath, is concentrated to give glue;To concentrate
After glue, spread in and scribble the special antiadhesion barrier surface of paraffin and dry, stick viscosity backing, be cut into suitable size, obtain plaster
Agent.
This pharmaceutical composition emplastrum is more highly preferred to adopt to be prepared with the following method: take Semen Lepidii (Semen Descurainiae), Radix Stephaniae Tetrandrae, Cortex Mori, mixing,
The water soaking of addition 11 times amount 1 hour, decocts 1 hour for the first time, and second time adds the water of 4 times amount, decocts 1 hour, merges decocting
Liquid, filters, and filtrate concentrates, and adds normal hexane, vaseline, lanoline, continuously stirred in boiling water bath, is concentrated to give glue;To concentrate
After glue, spread in and scribble the special antiadhesion barrier surface of paraffin and dry, stick viscosity backing, be cut into suitable size, obtain plaster
Agent.
The content assaying method of this pharmaceutical composition is: use high performance liquid chromatography to carry out tetrandrine containing measuring
Fixed:
(1) chromatographic condition: use HypersilDs chromatographic column;Flowing phase: ratio is the acetonitrile-water of 30:70;Detection ripple
Long: 200nm;Column temperature: 20 DEG C;Sensitivity: 0.1AUFS;Flow velocity: 1.0mL min-1;Sample size: 10 μ L;
(2) prepared by reference substance solution: precision weighs 80 DEG C and is dried to the tetrandrine reference substance of constant weight appropriate, adds methanol system
Become every 1mL solution containing 0.2mg;
(3) preparation of need testing solution: precision weighs medicine 10g of the present invention, adds methanol 40mL, is heated to reflux 4h, extracts
Liquid reflux solvent is also concentrated to dryness, residue add water 10mL dissolve, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, conjunction
And n-butanol extracting liquid, washing 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and it is molten that residue adds methanol
Solve and be transferred in 10mL volumetric flask, adding methanol to scale, shake up, filtering, take filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 5 μ L of reference substance solution respectively, inject high performance liquid chromatography
Instrument, detects.
This pharmaceutical composition may be used for the medicine of preparation treatment hysteromyoma.
This pharmaceutical composition can be also used for the medicine of preparation treatment dysmenorrhea.
Technique effect by the following experimentation checking present invention:
Experiment one: Chinese medicinal composition capsules Study on Preparation of the present invention
1 instrument and reagent
JA5003N type electronic balance (Shanghai Precision Scientific Apparatus Co., Ltd's production), RE-52A Rotary Evaporators (Shanghai
Ya Rong biochemical instrument factory produces), ZK-1 vacuum drying oven (productions of Tianjin medium ring testing furnace company limited), LC-10AT height
Effect liquid phase chromatogram instrument (Shimadzu Corporation of Japan produces);SPD-10A UV-detector (Shimadzu Corporation of Japan produces);The general Chinese in Shen, river
Word chromatographic work station.
Radix Notoginseng control medicinal material (Nat'l Pharmaceutical & Biological Products Control Institute), silica gel G (Qingdao Haiyang Chemical Industry Group Corp.);Semen Lepidii
Son, Radix Stephaniae Tetrandrae, Cortex Mori are commercially available prod, are provided with safe Drugstore chain by the Harbin Pharmaceutical Group people;Water is brand-new distilled water;
Adjuvant is pharmaceutic adjuvant;Acetonitrile is chromatographically pure, and other reagent are analytical pure.
2 test methods and result
2.1 extracting factor optimizations
2.1.1 orthogonal: selection amount of water, soak time, decocting time are as the factor of investigation, with dried cream yield
For evaluation index, use L9(34) orthogonal table arrangement test, selected factor level is shown in Table 1-1.
Table 1-1 extraction process orthogonal test factor level table
2.1.2 the mensuration of dried cream yield: precision measures extracting solution 50mL, puts and is dried to the evaporating dish of constant weight, water-bath
On be evaporated residue, in 105 DEG C of baking ovens be dried 3h, move in exsiccator cool down 30min, rapid precision weighing, calculate dry cream receive
Rate.
2.1.3 orthogonal experiments: weigh 9 parts of medical materials (every part of 50g) in prescription ratio precision, insert 1000mL flask
In, add ormal weight water, after being dipped to the stipulated time, decoct, reach to filter after the stipulated time medicinal residues, and add water and carry out
Decoct for 2 times, filter, merging filtrate, be concentrated into paste, calculate dried cream yield respectively.It is shown in Table 1-2.
Table 1-2 orthogonal experiments
The results of analysis of variance is shown in Table 1-3.From the result of table 1-3 range analysis, each factor cream dry to evaluation index is received
Rate affects primary and secondary order for B > C > A, the i.e. total amount of water of decocting time > soak time >.And A2 > A3 > A1, B2 > B1 >
B3, C2 > C3 > C1, and evaluation index all do not makes significant difference by each factor, therefore A2, B2, C2 should be selected, the most total amount of water:
11 times for the first time, for the second time 4 times, decocting time: be respectively 1h, soak time: 1h.
Table 1-3 the results of analysis of variance
F0.05(2,2)=19.00
Experiment two: Chinese medicinal composition capsules quality determining method of the present invention is studied
1 instrument and reagent
1.1 instrument
(G1314 is purple for Agilent110 high performance liquid chromatograph and work station, G1311Aquat pump for high performance liquid chromatograph
External detector).
1.2 reagent
Tetrandrine reference substance (China pharmaceutical biological product calibrating academy);Chinese medicinal composition capsules (reference of the present invention
Prepared by description of the invention embodiment 1);Chinese crude drug (the Harbin Pharmaceutical Group people provide with safe Drugstore chain);Methanol (chromatograph alcohol,
Biochemistry work auxiliary reagent factory, Shanghai);Other reagent are analytical pure.
2 methods and result
2.1 prescription
Semen Lepidii (Semen Descurainiae) 333g, Radix Stephaniae Tetrandrae 333g, Cortex Mori 333g.
2.2 preparation
Take dry Semen Lepidii (Semen Descurainiae) 333g, Radix Stephaniae Tetrandrae 333g, Cortex Mori 333g, add 11000mL water soaking 1 hour for the first time, decoct
Boiling 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, and filtrate concentrates, and is dried, to obtain final product.
The assay of 2.3 tetrandrine
2.3.1 HPLC chromatogram condition
Use HypersilDs(4.0mm × 125mm, 5 μm) chromatographic column;Flowing phase: ratio is the acetonitrile-water of 30:70;Inspection
Survey wavelength: 200nm;Column temperature: 20 DEG C;Sensitivity: 0.1AUFS;Flow velocity: 1.0mL min-1;Sample size: 10 μ L;At this chromatostrip
Under part, reference substance and sample chromatogram peak are good, noiseless to measuring without Radix Stephaniae Tetrandrae negative control.
2.3.2 the preparation of reference substance solution
Precision weighs 80 DEG C and is dried to the tetrandrine reference substance of constant weight appropriate, adds methanol and makes molten containing 0.2mg of every 1mL
Liquid.
2.3.3 need testing solution and the preparation of negative controls
Precision weighs medicine 10g of the present invention, adds methanol 40mL, is heated to reflux 4h, and extracting solution reflux solvent is also concentrated to dryness,
Residue add water 10mL dissolve, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, merging n-butanol extracting liquid, try with ammonia
Liquid washs 3 times, each 15mL, and n-butanol extracting liquid recycling design is to dry, and residue adds methanol and dissolves and be transferred to 10mL volumetric flask
In, add methanol to scale, shake up, filter, take filtrate and get final product;The granule negative control of Radix Stephaniae Tetrandrae is not separately contained in the preparation of prescription ratio
Product, are made in the same way of negative controls.
2.3.4 the drafting of standard curve
Precision weighs 80 DEG C and is dried to the tetrandrine reference substance of constant weight appropriate, makes 10.4 with methanol, and 20.8,41.6,
83.2,166.4 μ gmL-1The solution of series concentration, precision measures each 10 μ L of above-mentioned 5 kinds of strength solution respectively, injects efficient liquid phase
Chromatograph is measured.Carrying out linear regression with peak area ratio and concentration, obtaining regression equation is: A=21.2763C-0.1391,
r=0.9999.Show that tetrandrine is at 10.4~166.4 μ gmL-1In good linear relationship in concentration range.
2.3.5 stability test
Accurate absorption need testing solution 10 μ L, respectively at 0,1,2,4,8h sample introduction, and calculate tetrandrine content.Result 8h
Interior RSD is 0.45%(n=5).Show that sample solution is stable in 8h.
2.3.6 replica test
By 5 parts of need testing solution preparation method parallel processing sample, measure tetrandrine content in accordance with the law and calculate.Result is surveyed
Obtaining tetrandrine average content is 0.12mg-1, RSD is 1.3%.
2.3.7 Precision Experiment
Accurate tetrandrine reference substance solution of drawing, repetition sample introduction 5 times, measure peak area in accordance with the law.Result RSD is 0.23%
(n=5).Show that precision is preferable.
2.3.8 response rate experiment
Precision weighs 6 parts of the sample of the same lot number of known powder menispermine content, adds by high, medium and low concentration precision respectively
Enter appropriate tetrandrine reference substance solution, operate by under sample determination item, measure in accordance with the law, calculate the response rate.Result is averagely returned
Yield is 100.3%, and RSD is 0.45%(n=5).
2.3.9 sample size measures
Measuring reference substance solution respectively and need testing solution is appropriate, filter with microporous filter membrane, each sample introduction 10 μ L, by above-mentioned color
Spectral condition measures 3 batch samples, parallel assay 5 times.By external standard method with the content of calculated by peak area need testing solution tetrandrine.This
Product should be containing tetrandrine and indicate the 95%~105% of content, contain in terms of tetrandrine by every 1g sample, must not be less than 0.12mg.3 batches
Sample size is respectively 100.8%(RSD=1.2%), 101.7%(RSD=1.3%), 99.2%(RSD=1.1%).
Experiment three: the experimentation for the treatment of endometriosis
1 experiment material
1.1 animal
Health non-copulation female Wistar rat, body weight 200~250 g, dynamic purchased from Heilongjiang University of Chinese Medicine's experiment
Thing center.
1.2 medicine
(1) medicine of the present invention: take dry Semen Lepidii (Semen Descurainiae) 333g, Radix Stephaniae Tetrandrae 333g, Cortex Mori 333g, adds 11000mL for the first time
Water soaking 1 hour, decocts 1 hour, and second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, and filtrate concentrates, and is dried,
Add in distilled water, make the solution of 1.0g crude drug/mL concentration.
(2) drugs compared first (Semen Lepidii (Semen Descurainiae) and Radix Stephaniae Tetrandrae compositions): take dry Semen Lepidii (Semen Descurainiae) 500g, Radix Stephaniae Tetrandrae 500g, for the first time
Adding 11000mL water soaking 1 hour, decoct 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, filtrate
Concentrate, be dried, add in distilled water, make the solution of 1.0g crude drug/mL concentration.
(3) drugs compared second (Radix Stephaniae Tetrandrae and Cortex Mori compositions): take dry Radix Stephaniae Tetrandrae 500g, Cortex Mori 500g, for the first time
Adding 11000mL water soaking 1 hour, decoct 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, filtrate
Concentrate, be dried, add in distilled water, make the solution of 1.0g crude drug/mL concentration.
(4) drugs compared third (Semen Lepidii (Semen Descurainiae) and Cortex Mori compositions): take dry Semen Lepidii (Semen Descurainiae) 500g, Cortex Mori 500g, the
Once adding 11000mL water soaking 1 hour, decoct 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters,
Filtrate concentrates, and is dried, and adds in distilled water, makes the solution of 1.0g crude drug/mL concentration.
(5) positive control medicine danazol capsule: 200 mg/ grains, Jiangsu Lianhuan Pharmaceutical Co., Ltd. produces.
1.3 reagent and instrument
Estradiol (E2) put exempt from medicine box, progesterone (P) is put and is exempted from medicine box, Tianjin Jiuding Medical Biological Engineering Co., Ltd carry
Supply.ER(estrogen receptor), PR(progesterone receptor), VEGF(VEGF), NK cell (natural killer cell)
Immunohistochemical kit, is provided by Wuhan Boster Biological Technology Co., Ltd.;2008 G/r automatic counter for counting;Microscope:
OLYMPUS。
2 experimental techniques
2.1 modeling
Animal Model takes the adult female rats vaginal smear examination oestrous cycle.Take rat list of references in rutting period
Method (often warm up. the foundation of Endometriosis Model in Rats and pathological observation [J] thereof. clinical and experimental pathology magazine,
1998,14 (1): 67-69.) modeling, pentobarbital sodium 40 mg/kg intraperitoneal injection of anesthesia, the top that keeps left in hypogastric region center is done
One is about 1.5 cm otch, chooses the two ends of the uterus section that left uterine angle, ligation mesometrium blood vessel, then ligation need to be excised,
Cut the uterus of one section of 1.5 cm length.Wear separating uterus inner membrance in Crohn liquid aseptic, then cut 5 mm × 5 mm's
Internal film tissue's fragment, by epithelial layer facing to stomach wall, is sutured on stomach wall.After modeling 3 weeks, select rat in rutting period, open abdomen and see transplanting
Endometrium volume increases, and has liquid to gather and has angiopoietic transparent vesicles, with volume 20 mm in formation3, inner membrance
Transparent hydrops seen from vesicle is modeling Success criteria.
2.2 packets and administration
Take and model successfully 70 rats, be randomly divided into 7 groups by Ectopic Endometrium volume size, be i.e. model group, the danazol positive
Medicine group, medicine group of the present invention, drugs compared first group, drugs compared second group, drugs compared third group, separately take with batch normal 10 big
Mus as normal control, each group gastric infusion every day once, gavage volume 5 mL/kg, danazol group mg/kg every day 100 red that
Azoles gavage;Medicine group of the present invention gives medicine g/kg gavage every day 5 of the present invention;Drugs compared first group gives drugs compared first
Every day 5 g/kg gavage;Drugs compared second group gives drugs compared second g/kg gavage every day 5;Drugs compared third group gives
Drugs compared g/kg gavage the third every day 5;Normal group and model group such as give at the normal saline of capacity, all in modeling postoperative 35
D starts to be administered, every day 1 time, continuous 30 d.
2.3 index determining
1 h after last is administered, the anesthesia of each treated animal pentobarbital sodium, carotid artery takes blood, centrifugal (2000 r/min)
30 min, separate serum, to be checked.Followed by put to death animal, take normal group endometrium and respectively make the endometriotic tissues of film group,
Make tissue paraffin standard section (thick 3 μm).Section point 5 groups, is used for detecting ER, PR, VEGF, NK cytoactive and HE
Dyeing, measurement rat Ectopic Endometrium volume of cutting open the belly, calculate suppression ratio.
(1) serum measured by radioimmunoassay E2, P: operate by reagent kit determination step.
(2) Immunohistochemical Method measures ER, PR, VEGF, NK cell: operate according to SABC immunohistochemical staining test kit
Routine is carried out.Microscope inspection.Graphical analysis: every specimen after immunohistochemical staining, uses U.S. CMS800 color
Color image analyzes system, and the meansigma methods of the optical density (optical density/area) arbitrarily taking 3 visuals field on glandular epithelium surface represents
The content of ER, PR, VEGF, NK cell in this section glandular epithelium.
3 results
3.1 antithetical phrase endometriosis in rats serum E2, the impact (being shown in Table 3-1) of P
Table 3-1 antithetical phrase endometriosis in rats E2, the impact (mean ± standard deviation, n=10) of P
Note: compare with model control group, #P < 0.05, ##P < 0.01, ###P < 0.001;
Compare with Normal group, * P < 0.05, * * P < 0.01, * * * P < 0.001
3.2 impacts (being shown in Table 3-2) on experimental ectopic endometrium tissue ER, PR content
The table 3-2 impact (mean ± standard deviation, n=10) on experimental ectopic endometrium tissue ER, PR content
Note: compare with model control group, #P < 0.05, ##P < 0.01, ###P < 0.001;
Compare with Normal group, * P < 0.05, * * P < 0.01, * * * P < 0.001
3.3 impacts (being shown in Table 3-3) on experimental ectopic endometrium tissue VEGF, NK cytoactive
The table 3-3 impact (mean ± standard deviation, n=10) on experimental ectopic endometrium tissue VEGF, NK cell
Note: compare with model control group, #P < 0.05, ##P < 0.01, ###P < 0.001;
Compare with Normal group, * P < 0.05, * * P < 0.01, * * * P < 0.001
Under 3.4 optical microscopes, each group endometrial morphology is observed
(1) normal group: normal rats endometrium is simple columnar epithelium, it is seen that the normally knot such as body of gland, blood vessel, interstitial
Structure.
(2) model control group: have endometrial tissue, Glandular Dilatation by striped muscle, glandular epithelium be simple columnar, short cube
Shape or flat;Interstitial rich blood vessel, dilatation and congestion, it is seen that a small amount of hemosiderin and neutrophil infiltration.
(3) danazol group: compare with model group, lumen of gland reduces;The granulocyte infiltration having quantity not in interstitial and lumen of gland etc.
And hemosiderin.
(4) medicine group of the present invention: part lumen of gland reduces, and part glandular epithelium comes off, visible more neutral grain in part lumen of gland
Cell and foam cell, interstitial is relatively reduced, and wherein blood vessel number and caliber are relatively reduced, it is seen that more hemosiderin.
(5) drugs compared first group: part glandular epithelium comes off, visible neutrophilic granulocyte and histiocyte, interstitial blood in lumen of gland
Pipe quantity and caliber are relatively reduced, and in interstitial, inflammatory cell reduces, it is seen that hemosiderin.
(6) drugs compared second group: part lumen of gland relative decrease, is shown in neutrophilic granulocyte, interstitial vasodilation in part lumen of gland
Hyperemia, wherein visible hemosiderin and a small amount of lymphocyte, neutrophilic granulocyte.
(7) drugs compared third group: part lumen of gland relative decrease, is shown in neutrophilic granulocyte, interstitial vasodilation in part lumen of gland
Hyperemia, wherein visible hemosiderin and a small amount of lymphocyte, neutrophilic granulocyte.
3.5 impacts (being shown in Table 3-4) on Endometriosis Model in Rats
The table 3-4 impact (mean ± standard deviation, n=10) on Endometriosis Model in Rats
Note: compare with before medication, * P < 0.05, * * P < 0.01;Compare with model group, #P < 0.05, ##P < 0.01.
4 conclusions and discussion
4.1 endocrine regulation
4.1.1 reduce Estrogen and progestin level more and more testing proof endometriosis is estrogen-dependent
Disease.In this experiment, model control group serum Estrogen and progestin content is apparently higher than normal group, danazol and medicine group of the present invention
Can effectively reduce endometriosis rat blood serum E2, P level, illustrate medicine of the present invention can by suppression rat female,
The abnormal secretion of progestogen reaches to treat the purpose of endometriosis, and exercising result is similar to danazol.
4.1.2 reduce the growth of Estrogen and progesterone receptors content Ectopic Endometrium and maintain necessary gonadal hormone to need to pass through
Sex hormone receptor plays a role.Having ER, PR to express in endometriotic tissues, its content changes with menstrual cycle.This experiment
In, the expression of endometriosis rat rutting period ER, PR is slightly below the normal endometrium same period.Medicine of the present invention is permissible
By reducing the expression of ER, PR in Ectopic Endometrium, thus block Ectopic Endometrium to E2, the reaction of P, suppression Ectopic Endometrium is raw
Long.
4.2 improve immunologic function
4.2.1 improve NK cytoactive NK cell be class endometriosis generating process in uterus plays important
The immunocyte of immunosurveillance.NK cell is son to the defect of endometriotic tissues specific recognition and lethal effect
One of key link in Endometriosis pathogenesis.In an experiment, the activity of NK cell is had by medicine of the present invention
Notable ascending effect, effect is similar to danazol.The raising of NK Cell-mediated Immunity contributes to the identification to Ectopic Endometrium with clear
Removing, this is also one of the mechanism of Drug therapy endometriosis of the present invention.
4.2.2 suppressing the foundation of the blood vessel network that the angiogenesis of ectopic focus is new is that the endometrial implantation of adverse current becomes
Live and develop into the key of endometriosis.VEGF is the most key vascularization stimulating factor having now been found that, can
Direct and special stimulation vascular endothelial cell, induces its propagation, migrates, and increase vascular endothelial cell permeability, and induction is new
Angiogenic is formed.Research finds, endometriosis patients Ectopic Endometrium VEGF mRNA is high expressed, and Estrogen and progestin
The expression of VEGF can be promoted.To this end, VEGF is studied in zoopery.Result shows: experimental rat is different
In the internal film tissue of position, VEGF expresses and strengthens;Light Microscopic observation endometrial morphology: model control group interstitial medium vessels enriches, expands
Hyperemia, it was demonstrated that Ectopic Endometrium survive the support needing blood vessel network.And medicine group of the present invention and danazol group all can reduce
The expression of VEGF, under light microscopic, each treatment group blood vessel number reduces inconspicuous, but caliber reduces, the expansion of prompting Ectopic Endometrium blood vessel
Open hyperemia to be inhibited, consistent with the result that blood vessel surface after GnRH-a treats amasss minimizing.Consider that being probably medicine leads to
Cross the secretion of suppression Estrogen and progestin, reduce the generation of VEGF, so that the formation of Ectopic Endometrium blood vessel is inhibited.
Experiment four: the experimentation of medicine composite for curing hysteromyoma of the present invention
This research sets up Rat Experimental hysteromyoma model with Estrogen and progestin Load Method.Study drug regimen of the present invention
The thing intervention to it, observes Histomorphology and changes, and detect estrogen receptor (ER), progesterone receptor with Immunohistochemical Method
(PR), Bcl-2/Bax albumen expression in myometrial tissue, inquire into medicine composite for curing hysteromyoma of the present invention
Mechanism of action.
One, materials and methods
1. animals female unpregnancy SD rat, weight 210~250g, real by Heilongjiang University of Chinese Medicine's secondary animal
Test room and raise (SPF level), rearing conditions: room temperature (200 ± 20) DEG C, relative humidity (60 ± 5) %.
2. test medicine and reagent
(1) medicine of the present invention: take dry Semen Lepidii (Semen Descurainiae) 333g, Radix Stephaniae Tetrandrae 333g, Cortex Mori 333g, adds 11000mL for the first time
Water soaking 1 hour, decocts 1 hour, and second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, and filtrate concentrates, and is dried,
Add in distilled water, make the solution of 1.0g crude drug/mL concentration.
(2) drugs compared first (Semen Lepidii (Semen Descurainiae) and Radix Stephaniae Tetrandrae compositions): take dry Semen Lepidii (Semen Descurainiae) 500g, Radix Stephaniae Tetrandrae 500g, for the first time
Adding 11000mL water soaking 1 hour, decoct 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, filtrate
Concentrate, be dried, add in distilled water, make the solution of 1.0g crude drug/mL concentration.
(3) drugs compared second (Radix Stephaniae Tetrandrae and Cortex Mori compositions): take dry Radix Stephaniae Tetrandrae 500g, Cortex Mori 500g, for the first time
Adding 11000mL water soaking 1 hour, decoct 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, filtrate
Concentrate, be dried, add in distilled water, make the solution of 1.0g crude drug/mL concentration.
(4) drugs compared third (Semen Lepidii (Semen Descurainiae) and Cortex Mori compositions): take dry Semen Lepidii (Semen Descurainiae) 500g, Cortex Mori 500g, the
Once adding 11000mL water soaking 1 hour, decoct 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters,
Filtrate concentrates, and is dried, and adds in distilled water, makes the solution of 1.0g crude drug/mL concentration.
(5) positive control medicine GUIZHI FULING JIAONANG: Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov produces, specification: 0.31g.
Medicine in GUIZHI FULING JIAONANG is added the solution making 0.04g/mL concentration in distilled water.
2.6 rabbit anti-rat ER, PR, Bcl-2, Bax polyclonal antibody: purchased from Beijing Bo Aosen Bioisystech Co., Ltd.
3. modeling and packet
3.1 modeling
Set up Rat Experimental hysteromyoma model with Estrogen and progestin Load Method, select SD rat, be randomly divided into uterus muscle
Tumor modeling group and blank group two groups.Modeling method reference literature report (Yi Mingjuan. Gong Ning Oral Liquid treatment hysteromyoma
Experimentation. Pharmacology and Clinics of Chinese Materia Medica, 1996,12(4): 41-43), modeling group gives intramuscular injection estradiol benzoate
0.06mL/ only (content of dispersion 0.12mg), 3 times/week (Monday, three, five), totally 16 weeks, the 10th week added Progesterone 0.05mL/
(content of dispersion 1mg), 2 times/week (Tuesday, four), totally 7 weeks.Blank group is given and intramuscular injection high temperature sterilize Oleum Arachidis hypogaeae semen 0.05mL/
Only, 3 times/week, totally 16 weeks.Compared with blank group, the uterine smooth muscle hypertrophy of model group rats is obvious, and both have significance difference
Different (P < 0.001), illustrates program modeling success.
3.2 packet
After modeling terminates, it is randomly divided into model group 12, GUIZHI FULING JIAONANG group 12, pharmaceutical composition group 12 of the present invention
Only, Semen Lepidii (Semen Descurainiae) and Radix Stephaniae Tetrandrae compositions group 12, Radix Stephaniae Tetrandrae and Cortex Mori compositions group 12, Semen Lepidii (Semen Descurainiae) and Cortex Mori compositions group 12
Only, separately set blank group (sterilizing Oleum Arachidis hypogaeae semen modeling) 12.
4. it is administered
Modeling terminates rear gastric infusion.Pharmaceutical composition of the present invention gives pharmaceutical composition 10g crude drug/kg of the present invention, than
Medicine first group gives Semen Lepidii (Semen Descurainiae) and Radix Stephaniae Tetrandrae compositions 10g crude drug/kg, and drugs compared second group gives Radix Stephaniae Tetrandrae and Cortex Mori compositions
10g crude drug/kg, drugs compared third group gives Semen Lepidii (Semen Descurainiae) and Cortex Mori compositions 10g crude drug/kg, and GUIZHI FULING JIAONANG group is to osmanthus
Branch Indian buead capsule solution 0.6g/kg, model group, matched group give drinking water.Each group is administered volume and is 2mL/100g, 1 times/day
(10:00 in the morning), totally 4 weeks.Last is late 20 each group of Rat Fast 14h after being administered, and respectively organize rat 10% chloral hydrate morning next day
Intravenous anesthesia rear neck artery takes blood, uses cervical dislocation method put to death rat and take uterus, 10% phosphoric acid buffer Fu Er after taking blood
Malin fixes.
5. observation index
5.1 uterus histomorphologies are observed and are fixed uterus, specimens paraffin embedding slices with 10% formalin, and HE dyes, aobvious
Micro-Microscopic observation uterine histology metamorphosis.Smooth muscle cell proliferation degree standards of grading:
(-): myometrium has no and thickens, there are no inflammatory infiltration, and structure is normal.
(+): myometrium thickens inconspicuous, mild inflammatory infiltrates.
(++): myometrium slightly thickens, cell infiltration is more apparent.
(+++): myometrium substantially thickens, cell infiltration is heavier.
5.2 immunohistologies are observed
By ER, PR, Bcl-2/Bax expression feelings in Human Leiomyma in Immunohistochemical Method detection Leiomyoma Cell
Condition.ER: estrogen is the main stimulating factor of Uterine Leiomyoma, and finds that estrogen is to be played a role by its receptor
's.PR: progestogen play an important role in the morbidity of hysteromyoma, plays coordinative role with estrogen.Progesterone receptor get involved with
The growth of muscular tumor is relevant.Bcl-2 and Bax:Bcl-2 is a kind of proto-oncogene, can stop apoptosis and extend cell survival, Bax base
Because the activity of Bcl-2 can be resisted, there is the function directly facilitating apoptosis.
Grade scale: comprehensive positive cell proportion and staining power carry out sxemiquantitative judgement.Staining power is by following
Standard rating:
Feminine gender is 0 point;Weak though dyeing but be significantly stronger than negative control person is 1 point;The medium person of staining power is 2 points;Dyeing
Powerhouse is 3 points.
Positive cell institute accounting the evaluation criteria of dyeing is:
< 5% is negative to positive cell number: 0 point;6%~15% is the weak positive: 1 point;16%~30% is positive: 2 points;>31%
Person is strong positive: 3 points.
Comprehensive grading: two kinds of scorings are added, and result is divided into 4 grades:
0~1 is divided into (-);2 be divided into (+);3~4 are divided into (++);5~6 are divided into (+++).
Section index situation of change is evaluated by the grade of comprehensive grading.
6. statistical method
The statistical analysis application SPSS13.0 statistics software of data is added up, and measurement data represents with ± s, and examines with t
Test;Enumeration data rank test, P < 0.05 is that difference is statistically significant.
Two, result
The histopathology of rat model is affected by pharmaceutical composition the most of the present invention
Each group rat histopathology appraisal result is shown in Table 4-1.Blank group after modeling, hysteromyoma model group,
Both pathological proliferation in uterus have pole significant difference (P < 0.001), show modeling success.Compared with model group, cassia twig tuckahoe glue
The pathological proliferation of capsule group and pharmaceutical composition rat of the present invention significantly alleviates (P < 0.05 or P < 0.01), shows GUIZHI FULING JIAONANG
And the pathological proliferation of hysteromyoma rat model is had some improvement by pharmaceutical composition of the present invention, and medicine group of the present invention
Compound group is better than GUIZHI FULING JIAONANG group.Semen Lepidii (Semen Descurainiae) and Radix Stephaniae Tetrandrae compositions group, Radix Stephaniae Tetrandrae and Cortex Mori compositions group, Semen Lepidii (Semen Descurainiae) and Mulberry
It is not notable that Rhizoma Euonymus compositions group changes situation to the pathological proliferation of rat, prompting Semen Lepidii (Semen Descurainiae) and Radix Stephaniae Tetrandrae compositions, Radix Stephaniae Tetrandrae and Cortex Mori
Peel composition, Semen Lepidii (Semen Descurainiae) and Cortex Mori compositions are inconspicuous to the effect alleviating rat model pathology.Result display cassia twig tuckahoe
Capsule and pharmaceutical composition of the present invention are all significantly improved effect to the pathologic condition of hysteromyoma rat model, show that it is right
The Tumor like hyperplasia in rat model uterus has preferable therapeutical effect, and pharmaceutical composition of the present invention is better than GUIZHI FULING JIAONANG.
The table 4-1 impact (mean ± standard deviation) on rat model case scenario
Pharmaceutical composition the most of the present invention is on the impact of associated receptor in hysteromyoma rat model myometril cell
2.1 pharmaceutical compositions of the present invention, on the impact of ER content in Leiomyoma Cell, are shown in Table 4-2, with model group phase
Ratio, the difference that blank group ER is expressed is statistically significant (P < 0.01), and the mean rank order of model group is 15.38, high
In blank group (mean rank order is 6.85), illustrate that model group myometrium ER expresses level significantly raised.Medicine of the present invention
Compositions compared with model group, its difference statistically significant (P < 0.05).And the mean rank order of GUIZHI FULING JIAONANG group is
10.63, less than model group (mean rank order is 15.38), but its no significant difference.Result shows drug regimen of the present invention
Thing group rat uterus muscle layer ER express level relatively model group rats declined, and pharmaceutical composition of the present invention be better than Semen Lepidii (Semen Descurainiae) with
Radix Stephaniae Tetrandrae compositions, Radix Stephaniae Tetrandrae and Cortex Mori compositions, Semen Lepidii (Semen Descurainiae) and Cortex Mori compositions.GUIZHI FULING JIAONANG group rat uterus muscle layer
ER expresses level relatively model group rats without substantially changing.
Table 4-2 is on the impact (mean ± standard deviation) of ER content in Leiomyoma Cell
2.2 pharmaceutical compositions of the present invention, on the impact of PR content in myometril cell, are shown in Table 4-3, model group and blank right
Endochylema according to group myometril cell all there is positive particle to express, but model group muscular tumor cell positive and strong positive expression rate are up to
75%, and the corresponding expression rate of normal muscle tissue is only 30%, both compare difference statistically significant (P < 0.05).With model group
Comparing, pharmaceutical composition group of the present invention can substantially reduce PR content in hysteromyoma model Rat myocytes (P < 0.05).Osmanthus
In branch Indian buead capsule group model Rat myocytes, PR content reduces inconspicuous.Result shows that pharmaceutical composition of the present invention is to rat
Palace muscle layer PR is expressed level relatively model group rats and is decreased obviously, and pharmaceutical composition of the present invention is better than Semen Lepidii (Semen Descurainiae) and combines with Radix Stephaniae Tetrandrae
Thing, Radix Stephaniae Tetrandrae and Cortex Mori compositions, Semen Lepidii (Semen Descurainiae) and Cortex Mori compositions.GUIZHI FULING JIAONANG is without substantially changing.
Table 4-3 in myometril cell PR content impact (±)
2.3 pharmaceutical compositions of the present invention, on the impact of Bcl-2 content in myometril cell, are shown in Table 4-4, blank group
In the endochylema of rat uterus smooth muscle cell Bcl-2 protein positive cells many in the weak positive, be dispersed in distribution;Model group rats uterus
Bcl-2 protein positive cells quantity showed increased in the endochylema of smooth muscle cell, compares with blank group, the two Bcl-2 sun
Property express there were significant differences (P < 0.05).Compared with model group, pharmaceutical composition group rat uterus smooth muscle cell of the present invention
In endochylema, Bcl-2 protein positive cells quantity significantly reduces, and intensity substantially weakens, and there were significant differences (P < 0.05) with model group,
Result shows that pharmaceutical composition of the present invention can substantially reduce Bcl-2 protein content in hysteromyoma model Rat myocytes;Semen Lepidii
Son and Radix Stephaniae Tetrandrae compositions group, Radix Stephaniae Tetrandrae and Cortex Mori compositions group, Semen Lepidii (Semen Descurainiae) and Cortex Mori compositions group, compare with model group, nothing
Notable difference.In the endochylema of GUIZHI FULING JIAONANG group rat uterus smooth muscle cell, Bcl-2 protein content reduces inconspicuous.
Table 4-4 is on the impact (mean ± standard deviation) of Bcl-2 content in myometril cell
2.4 pharmaceutical compositions of the present invention, on the impact of Bax content in myometril cell, are shown in Table 4-5, model group rats Bax
Expression degree less than blank group, difference statistically significant (P < 0.05).Pharmaceutical composition group rat Bax's of the present invention
Expression degree is higher than model group, difference statistically significant (P < 0.05), illustrates that pharmaceutical composition of the present invention can increase uterus muscle
The expression of tumor rat model Bax.Semen Lepidii (Semen Descurainiae) and Radix Stephaniae Tetrandrae compositions group, Radix Stephaniae Tetrandrae and Cortex Mori compositions group, Semen Lepidii (Semen Descurainiae) and Cortex Mori
Compositions group, compares with model group, no significant difference.The expression degree of GUIZHI FULING JIAONANG group rat Bax is slightly above model group,
But no significant difference.
Table 4-5 is on the impact (mean ± standard deviation) of Bax content in myometril cell
Three, discussion and conclusion
Have various kinds of document to report, GUIZHI FULING JIAONANG for treatment hysteromyoma have definite curative effect, inventor as
Positive control medicine, compares with pharmaceutical composition of the present invention, shows medicine of the present invention from the pathological change situation of rat model
Compositions and GUIZHI FULING JIAONANG can the myometrial hypertrophy of antagonism (P < 0.05 or P < 0.01), and drug regimen of the present invention
Thing pharmaceutical composition of the present invention is better than GUIZHI FULING JIAONANG group.
Hysteromyoma is estrogen-sensitive tumor, is more common in the women of child-bearing age that gonadal hormone function is active, rare in menopause
Rear women.Clinic often find when exogenous give estrogen or gestation time, hysteromyoma increases rapidly;It is low that post menopausal occurs
Estradiol (E2) low progesterone (P) state can cause the reducing of muscular tumor, even disappear.Therefore for a long time, estrogen is recognized always
For being hysteromyoma generation and the accelerator of development, the most traditional " estrogen hypothesis ".But many studies have shown that at present, uterus
E2, P level in Hysteromyoma Patients Peripheral Circulation and normal person there is no difference, and E2, P content in meromyarian tumor tissue
But higher than the content in the normal smooth muscle tissue in same uterus.The most not can determine that blood serum E2, P level and hysteromyoma occur to send out
The direct relation of exhibition, and may is that the muscular tumor tissue local sensitivity to hormone.The main biological effect of hormone by with
Receptor just can be achieved after combining.Target tissue retain hormone number depend on containing of its intracellular corresponding receptor
Amount;Under same hormonal readiness, hormone produces the power of biological effect in target tissue and also depends on containing of receptor in target cell
Amount, illustrates that the growth of muscular tumor is closely related with the level of ER, PR.I.e. so-called receptor effect.This research is to Estrogen and progestin Load Method
The myometrial tissue of the experimental hysteromyoma rat of modeling has carried out the mensuration of ER, PR expression.Result shows: the present invention
Pharmaceutical composition group rat uterus muscle layer ER, PR express level relatively model group rats and have declined.GUIZHI FULING JIAONANG group rat
Myometrium ER, PR express level relatively model group rats without substantially changing.Show to reduce the expression of ER, PR in Human Leiomyma
Level is one of mechanism of medicine composite for curing hysteromyoma of the present invention.
Apoptosis is also referred to as procedural cell death, is the principal mode of cell ageing, death process.Apoptosis is body
One physiological regulatory mechanism of inner cell.Bcl-2 and Bax generally plays a role with dimeric forms in vivo.If wherein
Bcl-2 albumen forms then inhibited apoptosis with aggressiveness, and Bax albumen is formed with aggressiveness and then promotes apoptosis.The study find that:
In blank group rat uterus, Bcl-2 is low expression, and Bax is high expressed.This prompting is in normal rat myometrial tissue
Bax preponderates, and Bcl-2 expresses weak, may be allowed to more sensitive to apoptosis, is more easy to apoptosis.Bcl-2 in model group rats uterus
Expressing and increase, the expression of Bax reduces, and prompting Bcl-2 high expressed is probably the feature of muscular tumor, causes muscular tumor constantly to increase.This
Bright pharmaceutical composition can reduce the expression of hysteromyoma rat model Bcl-2, increases the expression of hysteromyoma rat model Bax,
Pharmaceutical composition of the present invention compares with model group that there were significant differences, and GUIZHI FULING JIAONANG group is expressed degree and slightly improved, but poor
Different not statistically significant.Pharmaceutical composition of the present invention may be difficult to escape wither by strengthening the muscular tumor cell sensitivity to apoptosis
Dying former stimulation, cause the muscular tumor cell survival phase to shorten, muscular tumor constantly reduces.
Experiment five: the experimentation of medicinal plaster of the present invention treatment endometriosis
1 materials and methods
1.1 object of study
Cleaning grade female sd inbred rats, body weight (180 ± 20) g, the non-copulation of sexual maturity, dynamic by Heilongjiang University of Chinese Medicine's experiment
Thing center provides, and raises in Heilongjiang University of Chinese Medicine's Experimental Animal Center.
1.2 modelling
Modeling is with reference to Vernon(Vernon MW, Wilson EM.Studies on the surgical inclusion
Of endom etriosis intherat [J] .FertilSteril, 1985,44:684-694) and method (Xiao of Xiao Chun etc.
Pure, Huang Guilin, Pengze China, etc. Rat Experimental model of endometriosis replicates [J]. Chinese Journal of Pathophysiology,
1995,11 (3): 332-333), operation transplantation method is used to set up model of endometriosis, after modeling 4 week, the 2nd time
The growing state of laparotomy inspection ectopic focus, to have following situations, person successfully indicates for model, and ectopic focus volume increases,
Transparent nodositas, cryptomere;Supernatant liquid is had to gather (hydrops height >=2mm);Sick inspection has intimal epithelium cell and interstitial raw
Long, there is secretory activity.The unsuccessful person of modeling rejects need not.Sham operated rats only uses tweezers tractive uterus, and remaining processes ibid.
Prepared by 1.3 medicines
Medicine of the present invention: take dry Semen Lepidii (Semen Descurainiae) 333g, Radix Stephaniae Tetrandrae 333g, Cortex Mori 333g, adds 11000mL water for the first time
Soaking 1 hour, decoct 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, and filtrate concentrates, and obtains extractum,
Add normal hexane, vaseline, lanoline, continuously stirred in boiling water bath, it is concentrated to give glue;Glue after concentrating, spreads in painting
The special antiadhesion barrier surface having paraffin is dried, and sticks viscosity backing, is cut into suitable size, obtains medicinal plaster of the present invention, and specification is
1cm × 1cm, mastic thickness about 0.75mm, every 100cm2Plaster 5.5g Han extractum.
Danazol capsule, Shanghai New Hualian Pharmaceutical Co., Ltd., traditional Chinese medicines quasi-word H31021681.
1.4 animal packet and processing methods
(1) normal group: conventional raising, does not make any experiment and processes;
(2) sham operated rats: conventional raising, does not make any treatment and processes;
(3) model group: do not make any treatment after modeling and process;
(4) medicinal plaster group of the present invention: 1cm × 1cm medicinal plaster of the present invention of applying ointment or plaster on Guanyuan acupoint and both sides SHENYU acupoint,
Every day 4 times, each 12h, continuously 4 weeks for the treatment of, conventional raising;
(5) danazol group: every rats gavaged danazol solution 0.3mL/d, molten with 0.9% normal saline containing crude drug 12mg(
Solve), successive administration 4 week.Each group rat is 10.
1.5 detection project and methods
(1) PGF2 alpha levels in detection blood plasma: use ELISA detection, operate (Jilin University's blood by test kit description
Research department provides).
(2) mensuration of ectopic focus volume and weight, rat abdominal cavity anesthesia is cut open the belly, and measures record ectopic focus with compasses
Size, and calculate its volume;Then take out ectopic focus, weigh.
(3) ectopic focus histopathologic examination, wins ectopic focus, and 10% formalin is fixed, and carries out conventional dehydration, stone
Wax embedding, HE dyeing, row light microscopy checking.
1.6 statistical method
In data, measurement data mean ± standard deviation represents, compares and checks with F, compare and examine with t before and after treatment between many groups
Test, with P < 0.05 for having statistical significance.
2 results
2.1 respectively organize PGF2 alpha levels measurement result
The blood plasma PGF2 alpha levels of model group apparently higher than normal group and sham operated rats, difference statistically significant (P <
0.05);Medicinal plaster group of the present invention and danazol group PGF2 alpha levels less than model group, difference statistically significant (P < 0.05),
And comparing difference not statistically significant, medicinal plaster group of the present invention, danazol between medicinal plaster group of the present invention and danazol group
Group compares not statistically significant (being shown in Table 5-1) respectively with normal group, sham operated rats.
Table 5-1 respectively organizes rat plasma PGF2 alpha levels and compares (mean ± standard deviation, pg/mL)
Note: compare with model group, * P < 0.05.
2.2 ectopic focus change in volume
Before treatment, (4 week after modeling) second time opens abdomen, measures three groups of rat ectopic focus volumes of modeling, and difference is without system
Meaning learned by meter, and after treatment, (8 week after modeling) third time opens abdomen, finds medicinal plaster group of the present invention and danazol group ectopic focus
Volume, compared with model group, has and significantly reduces (being shown in Table 5-2).
Ectopic focus volume ratio relatively (mean ± standard deviation, mm before and after three groups of rat treatments of table 5-2 modeling3)
Note: compare with model group, * P < 0.05;Compare with before treatment, #P < 0.05.
2.3 ectopic focus weight changes
After treatment, three groups of rat ectopic focuses of modeling are weighed, and find that medicinal plaster group of the present invention and danazol group dystopy are sick
Stove weight, compared with model group, has and significantly alleviates.It is shown in Table 5-3.
After three groups of rat treatments of table 5-3 modeling, ectopic focus weight ratio is relatively (mean ± standard deviation, mg)
Note: compare with model group, * P < 0.05.
2.4 histopathological examination
In 4 week after modeling, taking two modeling rat ectopic focuses at random and make histopathologic examination, result is shown in be had on inner membrance
Epidermal growth (Fig. 1).8 week after modeling, model group Ectopic Endometrium disease inspection see intimal epithelium cell well-grown, hypertrophy is prosperous
Contain;Body of gland number is many, and glandular epithelium is column or cube;Lumen of gland substantially expands, have in nodositas cryptomere (Fig. 2).The present invention
The inspection of medicinal plaster group Ectopic Endometrium disease is shown in that Ectopic Endometrium is that atrophic changes;Body of gland number reduces, and glandular epithelium is flat;Lumen of gland is little,
Intracavity has a small amount of pale red ambiguity material (Fig. 3).The inspection of danazol group Ectopic Endometrium disease is shown in that Ectopic Endometrium is that atrophic changes;Gland
Body number reduces, and glandular epithelium is flat;Lumen of gland is little, some without internal film tissue, only sees a small amount of foreign body reaction, body of gland fibroatrophy
(Fig. 4).
3 discuss and conclusion
Modern pharmacological research confirms, the principal agent Sanguis Draxonis of medicinal plaster of the present invention has and improves microcirculation, reduces whole blood and blood
The effect of slurry viscosity, it can make the blood being in hypercoagulability again circulate and play the effect of " blood circulation promoting and blood stasis dispelling ", can make again place
Solidify at angiorrhexis in hypocoagulability blood and play the effect of " astringing to arrest bleeding ", in terms for the treatment of blood disorder, there is two-way tune
Joint effect.Medicinal plaster of the present invention has its growth of suppression and promotes its effect absorbed of dissipating ectopic focus, and it treats effect
Fruit is similar to danazol.Medicinal plaster of the present invention has blood circulation promoting and blood stasis dispelling, the effect of inducing diuresis to remove edema, and pathological changes regional flow accelerates, blood
Flow increases, and improves pelvic cavity microcirculation, not only improves supplementing of nutrient substance, and histiocytic reparation, accelerates again
Local metabolic product, inflammatory factor, the removing of slough.This is conducive to implantatior cyst, the dissipation of tuberosity and blocks pain
Stimulating factor, improve uterus hypoxic-ischemic state, reduce the secretion of prostaglandin, reduce the abnormal PGF2 alpha levels raised, receive
To spasmolytic, the effect of pain relieving.
Accompanying drawing illustrates:
Fig. 1: 4W(HE × 100 after model group modeling)
Fig. 2: 8W(HE × 100 after model group modeling)
Fig. 3: medicine group (HE × 100) of the present invention
Fig. 4: danazol group (HE × 100)
Detailed description of the invention:
Embodiment 1:
Drug prescription: Semen Lepidii (Semen Descurainiae) 333g, Radix Stephaniae Tetrandrae 333g, Cortex Mori 333g
Preparation method: dry Semen Lepidii (Semen Descurainiae) 333g, Radix Stephaniae Tetrandrae 333g, Cortex Mori 333g, adds 11000mL water soaking 1 for the first time
Hour, decocting 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, and filtrate concentrates, and is dried, get Ben Fa
Bright medicine.
Use high performance liquid chromatography that tetrandrine carries out assay:
(1) chromatographic condition: use HypersilDs chromatographic column;Flowing phase: ratio is the acetonitrile-water of 30:70;Detection ripple
Long: 200nm;Column temperature: 20 DEG C;Sensitivity: 0.1AUFS;Flow velocity: 1.0mL min-1;Sample size: 10 μ L;
(2) prepared by reference substance solution: precision weighs 80 DEG C and is dried to the tetrandrine reference substance of constant weight appropriate, adds methanol system
Become every 1mL solution containing 0.2mg;
(3) preparation of need testing solution: precision weighs medicine 10g of the present invention, adds methanol 40mL, is heated to reflux 4h, extracts
Liquid reflux solvent is also concentrated to dryness, residue add water 10mL dissolve, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, conjunction
And n-butanol extracting liquid, washing 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and it is molten that residue adds methanol
Solve and be transferred in 10mL volumetric flask, adding methanol to scale, shake up, filtering, take filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 5 μ L of reference substance solution respectively, inject high performance liquid chromatography
Instrument, detects, testing result be the content of tetrandrine be 0.1823mg/g.
Function cures mainly: for endometriosis, hysteromyoma, dysmenorrhea.
Embodiment 2:
Drug prescription: Semen Lepidii (Semen Descurainiae) 333g, Radix Stephaniae Tetrandrae 333g, Cortex Mori 333g
Preparation method: dry Semen Lepidii (Semen Descurainiae) 333g, Radix Stephaniae Tetrandrae 333g, Cortex Mori 333g, adds 11000mL water soaking 1 for the first time
Hour, decocting 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, and filtrate concentrates, and dry, pulverize into
Fine powder, adds adjuvant, mixing, loads hard capsule.
Use high performance liquid chromatography that tetrandrine carries out assay:
(1) chromatographic condition: use HypersilDs chromatographic column;Flowing phase: ratio is the acetonitrile-water of 30:70;Detection ripple
Long: 200nm;Column temperature: 20 DEG C;Sensitivity: 0.1AUFS;Flow velocity: 1.0mL min-1;Sample size: 10 μ L;
(2) prepared by reference substance solution: precision weighs 80 DEG C and is dried to the tetrandrine reference substance of constant weight appropriate, adds methanol system
Become every 1mL solution containing 0.2mg;
(3) preparation of need testing solution: precision weighs medicine 10g of the present invention, adds methanol 40mL, is heated to reflux 4h, extracts
Liquid reflux solvent is also concentrated to dryness, residue add water 10mL dissolve, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, conjunction
And n-butanol extracting liquid, washing 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and it is molten that residue adds methanol
Solve and be transferred in 10mL volumetric flask, adding methanol to scale, shake up, filtering, take filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 5 μ L of reference substance solution respectively, inject high performance liquid chromatography
Instrument, detects, testing result be the content of tetrandrine be 0.1843mg/g.
Function cures mainly: for endometriosis, hysteromyoma, dysmenorrhea.
Embodiment 3:
Drug prescription: Semen Lepidii (Semen Descurainiae) 250g, Radix Stephaniae Tetrandrae 500g, Cortex Mori 250g
Preparation method: dry Semen Lepidii (Semen Descurainiae) 250g, Radix Stephaniae Tetrandrae 500g, Cortex Mori 250g, adds 11000mL water soaking 1 for the first time
Hour, decocting 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, and filtrate concentrates, and dry, pulverize into
Fine powder, adds adjuvant, mixing, loads hard capsule.
Use high performance liquid chromatography that tetrandrine carries out assay:
(1) chromatographic condition: use HypersilDs chromatographic column;Flowing phase: ratio is the acetonitrile-water of 30:70;Detection ripple
Long: 200nm;Column temperature: 20 DEG C;Sensitivity: 0.1AUFS;Flow velocity: 1.0mL min-1;Sample size: 10 μ L;
(2) prepared by reference substance solution: precision weighs 80 DEG C and is dried to the tetrandrine reference substance of constant weight appropriate, adds methanol system
Become every 1mL solution containing 0.2mg;
(3) preparation of need testing solution: precision weighs medicine 10g of the present invention, adds methanol 40mL, is heated to reflux 4h, extracts
Liquid reflux solvent is also concentrated to dryness, residue add water 10mL dissolve, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, conjunction
And n-butanol extracting liquid, washing 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and it is molten that residue adds methanol
Solve and be transferred in 10mL volumetric flask, adding methanol to scale, shake up, filtering, take filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 5 μ L of reference substance solution respectively, inject high performance liquid chromatography
Instrument, detects, testing result be the content of tetrandrine be 0.1891mg/g.
Function cures mainly: for endometriosis, hysteromyoma, dysmenorrhea.
Embodiment 4:
Drug prescription: Semen Lepidii (Semen Descurainiae) 400g, Radix Stephaniae Tetrandrae 200g, Cortex Mori 400g
Preparation method: dry Semen Lepidii (Semen Descurainiae) 400g, Radix Stephaniae Tetrandrae 200g, Cortex Mori 400g, adds 11000mL water soaking 1 for the first time
Hour, decocting 1 hour, second time adds 4000mL, decocts 1 hour, merges decocting liquid, filters, and filtrate concentrates, and dry, pulverize into
Fine powder, adds adjuvant, mixing, makes granule.
Use high performance liquid chromatography that tetrandrine carries out assay:
(1) chromatographic condition: use HypersilDs chromatographic column;Flowing phase: ratio is the acetonitrile-water of 30:70;Detection ripple
Long: 200nm;Column temperature: 20 DEG C;Sensitivity: 0.1AUFS;Flow velocity: 1.0mL min-1;Sample size: 10 μ L;
(2) prepared by reference substance solution: precision weighs 80 DEG C and is dried to the tetrandrine reference substance of constant weight appropriate, adds methanol system
Become every 1mL solution containing 0.2mg;
(3) preparation of need testing solution: precision weighs medicine 10g of the present invention, adds methanol 40mL, is heated to reflux 4h, extracts
Liquid reflux solvent is also concentrated to dryness, residue add water 10mL dissolve, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, conjunction
And n-butanol extracting liquid, washing 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and it is molten that residue adds methanol
Solve and be transferred in 10mL volumetric flask, adding methanol to scale, shake up, filtering, take filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 5 μ L of reference substance solution respectively, inject high performance liquid chromatography
Instrument, detects, testing result be the content of tetrandrine be 0.1858mg/g.
Embodiment 5:
Drug prescription: Semen Lepidii (Semen Descurainiae) 333g, Radix Stephaniae Tetrandrae 333g, Cortex Mori 333g
Preparation method: dry Semen Lepidii (Semen Descurainiae) 333g, Radix Stephaniae Tetrandrae 333g, Cortex Mori 333g, adds 15000mL water soaking 1 for the first time
Hour, decocting 1 hour, second time adds 5000mL, decocts 1 hour, merges decocting liquid, filters, and filtrate concentrates, addition normal hexane,
Vaseline, lanoline, continuously stirred in boiling water bath, it is concentrated to give glue;Glue after concentrating, spreads in and scribbles the special of paraffin
Antiadhesion barrier surface is dried, and sticks viscosity backing, is cut into suitable size, obtains emplastrum.
Use high performance liquid chromatography that tetrandrine carries out assay:
(1) chromatographic condition: use HypersilDs chromatographic column;Flowing phase: ratio is the acetonitrile-water of 30:70;Detection ripple
Long: 200nm;Column temperature: 20 DEG C;Sensitivity: 0.1AUFS;Flow velocity: 1.0mL min-1;Sample size: 10 μ L;
(2) prepared by reference substance solution: precision weighs 80 DEG C and is dried to the tetrandrine reference substance of constant weight appropriate, adds methanol system
Become every 1mL solution containing 0.2mg;
(3) preparation of need testing solution: precision weighs medicine 10g of the present invention, adds methanol 40mL, is heated to reflux 4h, extracts
Liquid reflux solvent is also concentrated to dryness, residue add water 10mL dissolve, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, conjunction
And n-butanol extracting liquid, washing 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and it is molten that residue adds methanol
Solve and be transferred in 10mL volumetric flask, adding methanol to scale, shake up, filtering, take filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 5 μ L of reference substance solution respectively, inject high performance liquid chromatography
Instrument, detects, testing result be the content of tetrandrine be 0.1875mg/g.
Function cures mainly: for endometriosis, hysteromyoma, dysmenorrhea.
Embodiment 6:
Drug prescription: Semen Lepidii (Semen Descurainiae) 250g, Radix Stephaniae Tetrandrae 500g, Cortex Mori 250g
Preparation method: dry Semen Lepidii (Semen Descurainiae) 250g, Radix Stephaniae Tetrandrae 500g, Cortex Mori 250g, adds 7000mL water soaking 0.5 for the first time
Hour, decocting 1.5 hours, add 6000mL for the second time, decoct 0.5 hour, merge decocting liquid, filter, filtrate concentrates, and adds just own
Alkane, vaseline, lanoline, continuously stirred in boiling water bath, it is concentrated to give glue;Glue after concentrating, spreads in and scribbles paraffin
Special antiadhesion barrier surface is dried, and sticks viscosity backing, is cut into suitable size, obtains emplastrum.
Use high performance liquid chromatography that tetrandrine carries out assay:
(1) chromatographic condition: use HypersilDs chromatographic column;Flowing phase: ratio is the acetonitrile-water of 30:70;Detection ripple
Long: 200nm;Column temperature: 20 DEG C;Sensitivity: 0.1AUFS;Flow velocity: 1.0mL min-1;Sample size: 10 μ L;
(2) prepared by reference substance solution: precision weighs 80 DEG C and is dried to the tetrandrine reference substance of constant weight appropriate, adds methanol system
Become every 1mL solution containing 0.2mg;
(3) preparation of need testing solution: precision weighs medicine 10g of the present invention, adds methanol 40mL, is heated to reflux 4h, extracts
Liquid reflux solvent is also concentrated to dryness, residue add water 10mL dissolve, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, conjunction
And n-butanol extracting liquid, washing 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and it is molten that residue adds methanol
Solve and be transferred in 10mL volumetric flask, adding methanol to scale, shake up, filtering, take filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 5 μ L of reference substance solution respectively, inject high performance liquid chromatography
Instrument, detects, testing result be the content of tetrandrine be 0.1991mg/g.
Function cures mainly: for endometriosis, hysteromyoma, dysmenorrhea.
In medicinal plaster of the present invention, the Ji Yuan of crude drug is as follows:
Semen Lepidii (Semen Descurainiae) is Cruciferae separate row Vegetable spp plant Semen LepidiiLepidum apetalum Willd., qin leaf Semen LepidiiL. Virginicum L.With Descurainia plant descurainia sophia (l.) webb ex prantlDescurainia sophia(L.) Webb ex Prantl [Sisymbrium sophia L.]Seed.
Radix Stephaniae Tetrandrae is Menispermaceae stephania plant Radix stephaniae tetrandraeStephania tetrandra S. MooreTuber.
Cortex Mori is Moraceae Mulberry plant MulberryMorus alba L.Dry root bark.
Although moreover, it will be appreciated that this specification is been described by according to embodiment, but the most each embodiment only wraps
Containing an independent technical scheme, this narrating mode of description is only that for clarity sake those skilled in the art should
Description can also be formed those skilled in the art through appropriately combined as an entirety, the technical scheme in each embodiment
May be appreciated other embodiments.
Claims (5)
1. the pharmaceutical composition treating endometriosis, it is characterised in that this pharmaceutical composition is by following weight
The raw material of part is made: Semen Lepidii (Semen Descurainiae) 1 weight portion, Radix Stephaniae Tetrandrae 1 weight portion, Cortex Mori 1 weight portion.
2. pharmaceutical composition as claimed in claim 1, it is characterised in that this pharmaceutical composition uses pharmacy conventional in pharmacy
Method is prepared as tablet, pill, hard capsule, granule, oral liquid, externally used paste.
3. pharmaceutical composition as claimed in claim 2, it is characterised in that this pharmaceutical composition is adopted and prepared with the following method: take
Semen Lepidii (Semen Descurainiae), Radix Stephaniae Tetrandrae, Cortex Mori, mixing, the water soaking 0.5 of addition 3~15 times amount~2 hours, decoct 0.5~2 hour, decoct 2
~4 times, each 0.5~2 hour, extracting solution merged, and filtered, and filtrate concentrates, and dry, pulverize into fine powder, added adjuvant, mixed,
Load hard capsule, to obtain final product.
4. pharmaceutical composition as claimed in claim 2, it is characterised in that this pharmaceutical composition is adopted and prepared with the following method: take
Semen Lepidii (Semen Descurainiae), Radix Stephaniae Tetrandrae, Cortex Mori, mixing, the water soaking 0.5 of addition 3~15 times amount~2 hours, decoct 0.5~2 hour, decoct 2
~4 times, each 0.5~2 hour, extracting solution merged, and filtered, and filtrate concentrates, and added normal hexane, vaseline, lanoline, boiling water bath
In continuously stirred, be concentrated to give glue;Glue after concentrating, spreads in and scribbles the special antiadhesion barrier surface of paraffin and dry, stick
Viscosity backing, is cut into suitable size, obtains emplastrum.
5. pharmaceutical composition as claimed in claim 1, it is characterised in that the content assaying method of this pharmaceutical composition is: adopt
The assay of tetrandrine is carried out by high performance liquid chromatography:
(1) chromatographic condition: use Hypersil Ds chromatographic column;Flowing phase: ratio is the acetonitrile-water of 30:70;Detection wavelength:
200nm;Column temperature: 20 DEG C;Sensitivity: 0.1AUFS;Flow velocity: 1.0mL min-1;Sample size: 10 μ L;
(2) prepared by reference substance solution: precision weighs 80 DEG C and is dried to the tetrandrine reference substance of constant weight appropriate, adds methanol and makes often
The 1mL solution containing 0.2mg;
(3) preparation of need testing solution: precision weighs the pharmaceutical composition 10g described in claim 1, adds methanol 40mL, heating
Backflow 4h, extracting solution reflux solvent is also concentrated to dryness, residue add water 10mL dissolve, with water saturated n-butyl alcohol shaking extraction 5 times,
20mL every time, merges n-butanol extracting liquid, washs 3 times with ammonia solution, each 15mL, and n-butanol extracting liquid recycling design is the most dry,
Residue adds methanol and dissolves and be transferred in 10mL volumetric flask, adds methanol to scale, shakes up, and filters, takes filtrate, obtain test sample molten
Liquid;
(4) measure: precision measures above-mentioned need testing solution, each 5 μ L of reference substance solution respectively, inject high performance liquid chromatograph, enter
Row detection.
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