CN102366621B - Plant estrogenic effect of acanthopanax biochemical traditional Chinese medicine compound extract and its application - Google Patents
Plant estrogenic effect of acanthopanax biochemical traditional Chinese medicine compound extract and its application Download PDFInfo
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Abstract
The invention relates to a traditional Chinese medicine compound extract with plant estrogenic effect. The traditional Chinese medicine compound extract takes six traditional Chinese medicines of acanthopanax, Chinese angelica, hemlock parsley, peach seed, dry ginger and licorice as raw materials, a carbon dioxide supercritical extraction technology and a macroporous adsorption resin enrichment technology are employed, the extract is prepared under certain technological parameter. The extract has obvious plant estrogenic effect, enables bidirectional adjustment of organism estrogenic level, prolongs the arrival of climacterium, and is suitable for improving the perimenopausal syndrome of female, such as menstrual disorder, vasomotorial symptom (flush, hot flash), mental and nervous disorder (depression, anxiety and easy excitement and insomnia), bone and joint symptom (osteoporosis, arthralgia and the like), urogenital tract symptom and the like. The extract can be directly and safely used for preparing the medicinal preparation and health food for treating perimenopausal syndrome.
Description
Technical field
The present invention relates to a kind of Chinese medicinal compound extract, specifically relate to a kind of employing carbon dioxide supercritical fluid extraction technology and macroporous adsorbing resin for purification technology, the extract that makes as raw material take Radix Angelicae Sinensis, Rhizoma Chuanxiong, Semen Persicae, Rhizoma Zingiberis (big gun), Radix Glycyrrhizae and Radix Et Caulis Acanthopanacis Senticosi, this extract has the effect of obvious phytoestrogen sample, is applicable to improve women's perimenopausal syndrome.The invention still further relates to the application of this extract in preparation treatment perimenopausal syndrome pharmaceutical preparation and health food.
Background technology
Along with the day by day raising of social population's aging degree, old people's quality of life has been subject to the extensive attention of society, and women's average life rises year by year, means that they will have time of 1/3 to tide over after climacteric and menopause.Climacteric refer to from endocrine, biology and the Clinical symptoms relevant with menopause occur near menopause (menopause) to the menopause 1 year during.Perimenopausal syndrome refers to the syndrome that estrogen decrease is the sings and symptoms of each organ due to main neuroendocrine, psychology and the metabolic alterations occur at climacteric, cardinal symptom: menoxenia, vasomotor symptoms (flushing, hectic fever), spiritual neurological disorders (depressed, worried, emotional and have a sleepless night), cardiovascular and cerebrovascular symptoms (hypertension, hyperlipidemia, cardiopalmus, atherosclerosis, coronary heart disease, senile dementia etc.), bone and joint symptom (osteoporosis, arthralgia etc.), urogenital tract symptom etc.These diseases have had a strong impact on the quality of life of middle and aged women.Complementing estrogen can obviously be improved above-mentioned symptom.
Chemosynthesis estrogen is brought into play very important effect aspect the anti-many ill symptomses of pre-postmenopausal women.Yet, because estrogenic long-term single use increases prevalence and other untoward reaction of ovarian cancer, breast carcinoma, carcinoma of endometrium, thereby limited its application.Therefore, controversies in hormone replacement in the elderly (selective estrogen receptor modulators) comes into one's own for these diseases of control.
Phytoestrogen is the selective estrogen receptor modulators that is present in the plant, can in conjunction with and activate mammal and the mankind estrogen receptor, thereby performance estrogen-like or the effect of estrogen antagonist sample, be that the body estrogen level shows as estrogen-like effects when low, otherwise show as the effect of estrogen antagonist sample, therefore have certain safety.In view of the potential danger of chemosynthesis hormonotherapy, seek safety, effectively, can be particularly important for the high natural plants estrogenic of usefulness of life-time service, also be one of pharmacy worker current research focus.
" SHENGHUATANG " comes from "Fu Qingzhu works on obstetric", is comprised of Radix Angelicae Sinensis, CHUANSHAO, Semen Persicae, Radix Glycyrrhizae Preparata and five kinds of Chinese medicines of Rhizoma Zingiberis Preparatum.The prescription of extract of the present invention is to have increased on the basis of SHENGHUATANG to consolidate the antasthenic Radix Et Caulis Acanthopanacis Senticosi, so be called slender acanthopanax biochemistry side, extract is called the slender acanthopanax biochemical extract.The slender acanthopanax Biochemical Drugs of mentioning among the CN 1415363A is similar to prescription of the present invention, but preparation technology is different, and, do not have the effect of phytoestrogen sample in its function of mentioning and in curing mainly, do not treat the effect of women's perimenopausal syndrome yet.The present invention finds that first slender acanthopanax biochemistry side has the effect of phytoestrogen sample.
Summary of the invention
An object of the present invention is to provide a kind of Chinese medicinal compound extract with the effect of phytoestrogen sample.This Chinese medicinal compound extract is a kind of selective estrogen receptor modulators.
Another object of the present invention is to propose the pharmaceutical preparation of the perimenopausal syndrome that this Chinese medicinal compound extract causes at preparation treatment estrogen deficiency and the application in the health food.
In order to achieve the above object, the present invention has taked following technical scheme:
Prescription medical material and ratio are: 5 parts of Radix Angelicae Sinensis, 3 parts of Rhizoma Chuanxiongs, 2 parts in Semen Persicae, 1 part of Rhizoma Zingiberis (big gun), 1 part in Radix Glycyrrhizae, 100 parts of Radix Et Caulis Acanthopanacis Senticosis.
According to 5 parts of Radix Angelicae Sinensis, 3 parts of Rhizoma Chuanxiongs, 2 parts in Semen Persicae, 1 part of Rhizoma Zingiberis (big gun), 1 part in Radix Glycyrrhizae, it is an amount of to take by weighing each medical material, co-grinding, cross No. 2 sieves of pharmacopeia, medicated powder is packed in the carbon dioxide supercritical fluid extraction device, adds the entrainer dehydrated alcohol, and consumption is 0.5ml/g medicated powder, setting extraction temperature is 45 ℃, extracting pressure is 40MPa, and resolving I pressure is that 10MPa, temperature are 40 ℃, and resolving II pressure is that 4MPa, temperature are 25 ℃, extraction time is 2h, gets brown color thickness grease.Other gets 100 parts of Radix Et Caulis Acanthopanacis Senticosi coarse powder, mix with the medicinal residues behind the previous step carbon dioxide supercritical fluid extraction, added 10 times of amount 70% soak with ethanol 12 hours, heating and refluxing extraction 2 hours, filter, in medicinal residues, add again 5 times of amount 70% alcohol heating reflux extraction 2 hours, filter, merging filtrate, drying under reduced pressure gets extractum.Extractum added after the suitable quantity of water dilution to mix with isopyknic D-101 type macroporous adsorbent resin mix thoroughly, be splined in the D-101 type macroporous adsorptive resins (applied sample amount is calculated as 1: 2 with the ratio of crude drug amount and amount of resin), static adsorption 30 minutes, then carry out eluting with 20% ethanol of the water of 10 column volumes, 10 column volumes and 50% ethanol of 10 column volumes successively, collect 50% ethanol elution, pulverize behind the drying under reduced pressure, make brown ceramic powder.The brown color thickness grease that this powder and carbon dioxide supercritical fluid extraction are made mixes, and namely gets Chinese medicinal compound extract of the present invention.
As to improvement of the technical scheme with replenish:
1, the relative quantity of prescription Chinese crude drug can according to: Radix Angelicae Sinensis 4-6 part, Rhizoma Chuanxiong 2-4 part, Semen Persicae 1-3 part, Rhizoma Zingiberis (big gun) 1-2 part, Radix Glycyrrhizae 1-2 part, the ratio of Radix Et Caulis Acanthopanacis Senticosi 50-150 part is adjusted.
2, when carrying out carbon dioxide supercritical fluid extraction, extraction temperature can be set as 30-60 ℃.
3, when carrying out carbon dioxide supercritical fluid extraction, extracting pressure can be set as 20-60MPa.
4, when carrying out carbon dioxide supercritical fluid extraction, extraction time can be set as 1h or more than.
5, when carrying out carbon dioxide supercritical fluid extraction, entrainer can be ethanol more than 95% for ethanol or concentration, and consumption can be set as 0.1-1ml/g medicated powder.
6, when with Rotary Evaporators carbon dioxide supercritical fluid extraction liquid being carried out the concentrating under reduced pressure Recycled ethanol, temperature can be set as 45 ℃ or following.
7, when the medicinal residues behind Radix Et Caulis Acanthopanacis Senticosi coarse powder and the carbon dioxide supercritical fluid extraction are extracted, extracting solvent can be 30%-100% ethanol.
8, when the medicinal residues behind Radix Et Caulis Acanthopanacis Senticosi coarse powder and the carbon dioxide supercritical fluid extraction are extracted, extracting mode can be that heating and refluxing extraction, supersound extraction or percolation extract.
9, when ethanol extraction extractum is carried out macroporous adsorbing resin for purification, the post fat of use can be D-101 type or AB-8 type, and other are nonpolar, low pole or middle polarity macroporous adsorbent resin.
10, when ethanol extraction extractum is carried out macroporous adsorbing resin for purification, eluting solvent can be the ethanol of water or variable concentrations, according to concentration of alcohol eluting successively from low to high.
By the extract that technique scheme prepares, have the effect of obvious phytoestrogen sample, can be for the preparation of pharmaceutical preparation and the health food of the perimenopausal syndrome that causes for the treatment of estrogen deficiency.
When the said extracted thing being mixed into pharmaceutical preparation with the perimenopausal syndrome effect that causes for the treatment of estrogen deficiency and health food, except these extracts, in the scope of not overslaugh effect of the present invention, can suitably be mixed for as required in other compositions of common pharmaceutical preparation and health food, such as: add adjuvant in crospolyvinylpyrrolidone, carboxymethylstach sodium, microcrystalline Cellulose, lactose, aspartame, the magnesium stearate etc., add adjuvant and coating adjuvant.Its type agent can be any pharmaceutically said dosage form, preferred pill, tablet, capsule or oral liquid.
Effect experiment subsequently will prove, the perimenopausal syndrome that the alternative estrin treatment estrogen deficiency of Chinese medicinal compound extract of the present invention causes.
Chinese medicinal compound extract of the present invention is the plant extract of the pure natural of dietotherapeutic, selective estrogen receptor modulators effect with phytoestrogen, estrogen is had dual regulation, be different from the estrogenic of chemosynthesis, do not increase the side effect of breast carcinoma, uterus carcinoma.
Description of drawings
Fig. 1 is the aspect graph that mice diestrus vaginal smear microscopically is observed
Fig. 2 is the aspect graph that mice proestrus vaginal smear microscopically is observed
Fig. 3 is the aspect graph that mice vaginal smear in rutting period microscopically is observed
Fig. 4 is the aspect graph that mice metoestrus vaginal smear microscopically is observed
Fig. 5 is the morphosis figure that microscopically is observed after removal ovary (OVX) the group Mouse Uterus pathological section HE dyeing
Fig. 6 gives the TQW group Mouse Uterus pathological section HE dyeing morphosis figure that rear microscopically is observed behind the removal ovary (OVX)
Fig. 7 is the morphosis figure that microscopically is observed after sham-operation (Sham) the group Mouse Uterus pathological section HE dyeing
Fig. 8 gives the nilestriol group Mouse Uterus pathological section HE dyeing morphosis figure that rear microscopically is observed behind the removal ovary (OVX)
Fig. 9 is the morphosis figure that microscopically is observed behind removal ovary (OVX) the group Mouse Uterus pathological section ER immunohistochemical staining
Figure 10 gives the morphosis figure that microscopically is observed behind the TQW group Mouse Uterus pathological section ER immunohistochemical staining behind the removal ovary (OVX)
Figure 11 is the morphosis figure that microscopically is observed behind sham-operation (Sham) the group Mouse Uterus pathological section ER immunohistochemical staining
Figure 12 gives the morphosis figure that microscopically is observed behind the nilestriol group Mouse Uterus pathological section ER immunohistochemical staining behind the removal ovary (OVX)
Figure 13 is the morphosis figure that microscopically is observed behind the staining pathologic section of blank aging rats group uterus
Figure 14 gives the TQW aging rats to give the morphosis figure that microscopically is observed behind the staining pathologic section of group uterus
Figure 15 gives the morphosis figure that microscopically is observed behind the staining pathologic section of estradiol aging rats group uterus
Figure 16 is the morphosis figure that microscopically is observed behind the blank aging rats group vagina staining pathologic section
Figure 17 gives the TQW aging rats to give the morphosis figure that microscopically is observed behind the group vagina staining pathologic section
Figure 18 gives the morphosis figure that microscopically is observed behind the estradiol aging rats group vagina staining pathologic section
Figure 19 is the morphosis figure that microscopically is observed after the section statining of blank aging rats group mammary gland pathological
Figure 20 gives the TQW aging rats to give the morphosis figure that microscopically is observed after the section statining of group mammary gland pathological
Figure 21 gives the morphosis figure that microscopically is observed after the section statining of estradiol aging rats group mammary gland pathological
Figure 22 is the Western blot experimental result picture that blank group, TQW group and estradiol group aging rats mammary gland proliferating cell nuclear antigen (PCNA) are expressed
The specific embodiment
Below in conjunction with specific embodiments and the drawings, further set forth the present invention.These embodiment are interpreted as only being used for explanation the present invention and are not used in restriction protection scope of the present invention.After the content of having read the present invention's record, those skilled in the art can make various changes or modifications the present invention, and these equivalences change and modification falls into claims limited range of the present invention equally.For convenience of explanation, below with extract of the present invention referred to as TQW.
Embodiment 1 (the body inner estrogen sample effect research of TQW)
Experimentation adopts the internationally recognized three kinds of classical animal models of research estrogen action in the TQW estrogen-like effects body: immaturity mice, ovariectomized mouse, aging rats model are simulated respectively three low physiological stages of women's estrogen in all one's life.
One, TQW is on the impact of immaturity mice target organ
It 21 days was the mice of just having weaned that the immaturity mice is selected birth, and this moment, mice did not also reach sexual maturity and body maturation, and the medicine with estrogen-like effects can promote its Development of Reproductive System ripe.
1 materials and methods
1.1 material
KM kind mice, ♀, 21 ages in days are available from Heilongjiang University of Chinese Medicine GLP center.
The TQW suspension: in TQW suspendible distilled water, ig dosage is 15mg/kg.
Nilestriol suspension (Nilestrdiol): nilestriol (available from Pharmaceutical Group Hua Lian, Shanghai pharmaceutical factory, the accurate word of traditional Chinese medicines: H3102164), and in the suspendible distilled water, ig 0.5mg/kg.
1.2 method
1.2.1 TQW is to the estrogen-like effects of immaturity mice target organ
1.2.1.1 the impact on immaturity Mouse Oestrous Cycle (vaginal epithelial cell)
Get mice and do first vaginal smear, choose continuous 5 days and diestrous mice occurs every day 1 time, be divided at random 3 groups, 10 every group, be respectively the blank group: the gavage distilled water; TQW suspension group: gavage TQW suspension (15mg/kg); Positive controls: gavage nilestriol (0.5mg/kg).Every day 1 time, successive administration 5 days, 24h respectively does vaginal smear one time after the 2nd, 3,4,5 day evening and the last administration after administration.Emotionally number of elements and the percentage of mice occur after calculating medication, compare with the blank group.
1.2.1.2 on immaturity mouse uterine weight and the impact of taking the photograph the water rate
Get and above-mentionedly do the vaginal cornification test mice after finishing vaginal smear for the last time, take off cervical vertebra and put to death, cut the abdominal cavity open, win uterus and vagina with shears, be put on the filter paper, remove fatty tissue, ovary and vagina, claim rapidly its weight in wet base, calculate the uterus weight percentage rate.Then the uterus is put into 100 ℃ of baking ovens and dried 24h, again weigh.Relatively each experimental group and the percentile difference of blank group uterus weight and water intake increase percentile difference
Organ weights percentage rate=internal organs weight in wet base/every 100g body weight * 100%
Water intake increases percentage rate=(uterus weight in wet base-uterus dry weight)/uterus weight in wet base * 100%
2 results
2.1 the impact on the immaturity Mouse Oestrous Cycle
Experimental result sees Table 1.This experimental selection 21 ages in days (namely just wean) mice that is born does not reach sexual maturity and body maturation, this in stage mice be in dioestrus, the material with estrogen-like effects can promote it to enter rutting period in advance.Mouse Oestrous Cycle vaginal smear cytology variation characteristic: (seeing Figure of description 1-4) diestrus, see a large amount of multinuclear leucocytes under the mirror, a small amount of epithelial cell.Proestrus, a large amount of epithelial cells, a small amount of superficial cell (seedless) is without leukocyte.Rutting period, a large amount of superficial cells, shape is large and irregular, and a small amount of epithelial cell is still arranged.Metoestrus, a large amount of leukocyte still have the superficial cell of fusion.The immaturity mice gives behind the TQW 15mg/kg 100% and enters rutting period in advance, and is similar to the positive control drug of classics, yet the blank group only has one to enter rutting period, therefore infers that TQW has estrogenic activity.
Table 1 TQW is on the impact of underage mouse oestrous cycle
Annotate: compare * * * P＜0.001 with the blank group; ###P＜0.001
2.2 to immaturity mouse uterine weight and water intake experiment
The uterus is the tissue the most responsive to estrogen response as estrogenic target organ, and estrogen can promote the target organ protein synthesis, and its weight is increased, and can promote the uterine vascular expansion simultaneously, and the permeability changes water intake increases.TQW group as shown in table 2 compares with blank group, and uterus weight and water intake rate significantly increase, and are higher than respectively the nilestriol group.Prompting TQW has estrogen-like effects.
Table 2 TQW is on the impact of underage mouse uterus weight
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; With nilestriol group comparison ▲ P＜0.05, ▲ ▲ P＜0.01
Two, TQW is on the impact of ripe ovariectomized mouse
Adopt ovariectomized mouse experimental animal model research TQW quasi-estrin sample biological activity, removal ovary can make body inner estrogen level descend rapidly, can simulate the postclimacteric physiological and pathological feature of women.
1 materials and methods
1.1 material
KM kind mice, ♀, in 12 ages in week, unpregnancy is available from Heilongjiang University of Chinese Medicine GLP center.
The TQW suspension: in TQW suspendible distilled water, ig dosage is 15mg/kg.
Nilestriol suspension (Nilestrdiol): nilestriol (available from Pharmaceutical Group Hua Lian, Shanghai pharmaceutical factory, the accurate word of traditional Chinese medicines: H3102164), and in the suspendible distilled water, ig 0.5mg/kg.
1.2 method
1.2.1 the preparation of ovariectomized mouse model
Get mice, the collaborative anesthesia of 30mg/kg pentobarbital, 500mg/kg urethane, two skidding bilateral ovaries are extractd in the spinal column axis, near the fat the sham operated rats spay, spay not, postoperative is sewed up, the iodine disinfection wound.(draw normal saline 0.1～0.2ml with dropper by only carrying out vaginal smear, insert gently mouse vagina 2～3mm, aspirate smear 2～3 times, 95% ethanol is 10min fixedly, methylene blue solution-dyed 10min, water washes gently, drenches to do, in order to reading sheet), every day 1 time, observe vaginal epithelial cell and be in dioestrus in continuous 5 days, with the success of proof castration.
1.2.2 animal grouping and administration
Be divided into sham operated rats (Sham group), simple ovarian resection group (OVX group), OVX+TQW suspension (15mg/kg), OVX+ nilestriol (0.5mg/kg).1 time/d of gavage, 4 weeks of successive administration.
1.2.3 the detection of oestrous cycle and organ index
Microscopically was observed the mouse vagina cellular change and was determined whether to enter rutting period by only row vaginal smear procuratorial work every day.Broken end is got blood, and separation of serum is peeled off uterus, adrenal gland and claimed its weight in wet base, calculates organ index, respectively organizes organ index difference.
1.2.4 ELISA detects E in the blood
2, FSH
After the mice broken end is got blood, collect serum, adopt mice E
2FSH scientific research test kit detect, the 450nm wavelength is surveyed its absorbance.
1.2.5 Histomorphology inspection
With every right side of mice cornua uteri, do pathological section, HE dyeing, microscopically is observed.
1.2.6 uterus ER immunohistochemical staining and quantitative analysis
Take the S.ABC method to carry out immunohistochemical staining, microscopically is observed.Analyze the expression of corresponding antibodies with the OS-9 data analysis software.Every group of section of getting 10 animals, 5 visuals field are selected in every section, and its meansigma methods is the gray value of this section.Each organizes the data
Expression.
2 results
2.1 the impact on ripe ovariectomized mouse vagina epithelium keratinization (rutting period)
Experimental result sees Table 3.Behind the mice removal ovary, its normal continuous 5 days oestrous cycle disappears, and enters dioestrus, and OVX organizes being leukocyte from removal ovary to putting to death the provagina cell, is in dioestrus always.And TQW group and nilestriol group mice all can recover rutting period in administration in 7 days, and are in rutting period before putting to death always, and the Sham group is in the normal continuous 5 day oestrous cycle, and prompting TQW has estrogen-like effects.
Table 3 TQW is on the impact of ripe ovariectomized mouse vagina epithelium keratinization (rutting period)
Annotate: compare * * * p＜0.001 with the OVX group
2.2 the impact on ripe ovariectomized mouse uterus weight
Experimental result sees Table 4.Mouse Uterus atrophy behind the removal ovary, TQW group and nilestriol group be energy antagonism metratrophia all, significantly increases uterus weight (p＜0.001), and prompting TQW has estrogen-like effects.
Table 4 TQW is on the impact of ripe ovariectomized mouse uterus weight
Annotate: compare * * * with the OVX group
2.3 the impact on ripe ovariectomized mouse Histomorphology
Experimental result is seen Figure of description 5-9.The atrophy that attenuates of Mouse Uterus atrophy smaller volume behind the removal ovary, uterine cavity, and endometrium attenuation, the body of gland atrophy reduces.And TQW and Sham group endometrium thicken than the OVX group, and uterine glands increases, increases, and lumen of gland enlarges, and wherein part has been full of red secretions (acidophilia's material).Nilestriol group lamina propria short texture, intercellular substance increases, and lumen of gland enlarges, and wherein has been full of red secretions and has been in the secretory phase.TQW has the metratrophia that the antagonism OVX similar with nilestriol causes, prompting TQW has estrogen-like effects.
2.4 to ripe ovariectomized mouse serum E
2Impact with LH
Experimental result sees Table 5.E in the blood behind the mice removal ovary
2Concentration significantly reduces (p＜0.05), and TQW can increase serum E
2Content, and with OVX group significant difference (p＜0.05) is arranged relatively.Behind the mice removal ovary in the blood LH concentration significantly raise (p＜0.05), TQW group can leniently reduce the content of LH in the blood, and with the OVX group significant difference (p＜0.05) is arranged relatively.The TQW effect is similar to nilestriol, and prompting TQW directly shows estrogen-like effects.
Table 5 TQW is on the impact of E2 and LH in the ripe ovariectomized mouse blood
Annotate: compare * p＜0.05 with the OVX group, compare with the Sham group
▲P＜0.05
2.5 immunohistochemical staining and quantitative analysis to ripe ovariectomized mouse uterus ER
Experimental result is seen Figure of description 9-12 and table 6.The immunohistochemical method evaluation of impact that TQW expresses the ovariectomized mouse uterine estrogen receptor, use the simultaneously antibody of anti-ER α and ER β, ERs is distributed in endometrial epithelial cell, smooth muscle cell, Interstitial cell, and brown color represents estrogen receptor positive and expresses.Ovariectomized mouse uterus ERs expresses lower, and TQW and nilestriol can obviously improve the expression of ERs.Inferring that TQW can increase estrogen receptor content, is one of its performance estrogen-like effects mechanism.
Table 6 TQW is to the quantitative analysis of the immunohistochemical staining of ripe ovariectomized mouse uterus ER
Annotate: compare * p＜0.05 with the OVX group, compare with the Sham group
▲P＜0.05
Three, TQW is on the impact of aging rats
12 monthly age of this experimental selection, the naturally-aged rat was as model, administration 3 months, to 15 monthly ages of rat,, this in stage rat can well simulate 45～50 years old physiological stage of women, meet climacteric women's physiological feature.
1 materials and methods
1.1 material
The SD rat, ♀, 12 monthly ages are available from Heilongjiang University of Chinese Medicine GLP center.
The TQW suspension: in TQW suspendible distilled water, ig dosage is 15mg/kg.
Nilestriol suspension (Nilestrdiol): nilestriol (available from Pharmaceutical Group Hua Lian, Shanghai pharmaceutical factory, the accurate word of traditional Chinese medicines: H3102164), and in the suspendible distilled water, ig0.5mg/kg.
1.2 method
1.2.1 animal grouping and administration
Get rat, be divided at random 4 groups: blank group: gavage distilled water; TQW group: gavage TQW suspension 15mg/kg; Estradiol group: gavage estradiol 0.7mg/kg, 1 times/day, 12 weeks of successive administration.
1.2.2 on the adrenal impact in aging rats uterus
After the last administration, the abdomen cardinal vein is got blood, peels off uterus, vagina, adrenal gland, ovary, mammary gland tissue, weighing uterus, adrenal weight.
1.2.3 to E in the aging rats serum
2Impact
According to E in the ELISA rat scientific research test kit operation detection serum
2Content.
1.2.4 to aging rats uterus, vagina, mammary gland tissue Morphology Effects
Get right side, uterus cornua uteri, be connected in that 0.4cm place vagina, mammary gland are fixed in 4% paraformaldehyde on the anus, dehydration, transparent, waxdip and embedding, section, paster and roasting sheet, cut into slices dewaxing and aquation, dyeing, section are dewatered, transparent and mounting.Microscopically is observed.
1.2.5 the impact on aging rats mammary gland PCNA expression of receptor
Western blot detects uterus, the ER of vagina and the expression of mammary gland PCNA receptor.
2 results
2.1 the impact on the aging rats uterus weight
Experimental result sees Table 7.Aging rats give TQW12 after week uterus weight significantly increase, this acts on a little less than estradiol.But the atrophy of prompting TQW delaying senility rat uterus has estrogen-like effects.
Table 7 TQW is on the impact of aging rats uterus weight
Annotate: compare * p＜0.05 with the blank group
2.2 the impact on the aging rats Histomorphology
Experimental result is seen Figure of description 13-15.The attenuation of aging rats endometrium, the body of gland atrophy reduces.And TQW administration group and E
2The blank group of group endometrium thickens, and uterine glands increases, increases, and lumen of gland enlarges, the lamina propria short texture, and intercellular substance increases, and is in the secretory phase.But the metratrophia that the aging of prompting TQW antagonism causes, prompting TQW has estrogen-like effects.
2.3 on the morphologic impact of aging rats vagina
Experimental result is seen Figure of description 16-18.The atrophy of aging rats vaginal epithelial cell is comprised of pinacocyte; E
2The group vaginal epithelial cell is typical squamous multilamellar epithelial cell, and TQW group vaginal epithelial cell is loose, hypertrophy, the epithelial layer keratinization, and a large amount of Cytoplasm cavitys appear on the upper strata.But the aging of prompting TQW antagonism causes vaginal atrophy, and it has estrogen-like effects prompting TQW.
2.4 on the morphologic impact of aging rats mammary gland
Experimental result is seen Figure of description 19-21.Aging rats mammary gland deep fat is lined with dense epithelial cell and net cavity configuration not, and TQW group epithelial cell is with a small amount of net cavity configuration but there is not secretions.E
2Group is with a large amount of net cavity configurations and secretions is arranged.But the mammary gland atrophy that the aging of prompting TQW antagonism causes points out it to have estrogen-like effects.
2.5 the impact on aging rats mammary gland PCNA expression
Experimental result is seen Figure of description 22.Aging rats after 12 weeks of administration, E
2The visible obviously hypertrophy of group aging rats mammary gland naked eyes, and in mammary gland morphology research, find E
2Group mammary gland deep fat pad epithelial cell is with a large amount of net cavity configurations and secretions is arranged, and shows to be in the secretory phase.Proliferating cell nuclear antigen (PCNA) is a sensitive indicator of breast cancer research, and mammary glandular cell transition propagation is the risk factor that breast carcinoma occurs.Therefore I observe the PCNA protein expression, and to investigate prolonged application TQW to the safety of mammary gland, Western blot result shows E
2Increase the expression of aging rats mammary gland PCNA, and TQW has no significant effect to the expression of PCNA.Prompting TQW long-term taking is safe and effective.
2.6 the impact on aging rats adrenal gland weight
Experimental result sees Table 8.TQW has gentle increasing action to aging rats adrenal gland weight, TQW group and E
2Group relatively has significant difference (p＜0.05) with the blank group.Ovary and adrenal gland are the estrogenic major organs of secretion.Prompting TQW may play a role by hypothalmus-pituitary-adrenal axis.
Table 8 TQW is on the impact of aging rats adrenal gland weight
Annotate: compare p＜0.05 with the blank group
2.7 to aging rats serum E
2Impact
Experimental result sees Table 9.TQW and E
2All can increase E in the aging rats blood
2Content, and with old and feeble matched group significant difference (p＜0.05) is arranged relatively.Prompting TQW directly translates into estrogen-like effects.
Table 9 TQW is on the impact of aging rats serum E2
Annotate: compare p＜0.05 with the blank group
Embodiment 2 (the external estrogen-like effects of TQW)
The MCF-7 cell is the human breast cancer cell strain of the ER positive, can be subjected to specifically estrogen or estrogenic activity material to regulate and breed.MCF-7 cell estrogen-like proliferation test is very sensitive, simple and easy to do, is widely used in rapid screening and estimates phytoestrogen.
1 method
1.1 TQW is to the MCF-7 proliferation function
1.1.1 cell culture
The MCF-7 cell culture is in the high sugar juice of the DMEM that contains 10% hyclone, and condition of culture is 37 ℃, 5%CO
2, relative saturation humidity.
1.1.2 MTT cell proliferation test
After 2d washs 2 times with cell with PBS before the experiment beginning, change in without phenol red DMEM (containing 5%CD-FBS) and cultivating, to exhaust the estrogen that stores in the cell.Choose the exponential phase cell, use 0.25% trypsin digestion cell, with 2 * 10
3Individual/hole inoculating cell on 96 well culture plates, every pore volume 180 μ l, the 24h cell attachment is established respectively blank group (culture fluid that contains 1 ‰ DMSO), 17 beta estradiol positive controls (1 * 10
-9M), TQW organizes (0.1/mlTQW), every row are established 8 multiple holes, continue after the dosing to observe and cultivate 48h.
Colour developing and colorimetric: after 48h is cultivated in dosing, take out culture plate, every hole adds MTT solution (5mg/ml) 20 μ l, 37 ℃, 95%CO
2Continuing to cultivate 4h ends to cultivate.The careful suction abandoned culture supernatant in the hole, and every hole adds 150 μ lDMSO vibration 10min, and crystal is fully dissolved, and selects 490nm to measure each hole absorbance value (OD), three independent repeated experiments in automatic microplate reader.
PR%=experimental group OD value/group of solvents OD value * 100
2. result's (on impact of the propagation of MCF-7 cell)
Experimental result sees Table 10.TQW can significantly promote the MCF-7 cell proliferation, and and 1 * 10
-917 β of M-estrodiol effect is similar.Infer that TQW has estrogen-like effects.
Table 10 TQW is on the impact of the propagation of MCF-7 cell
Annotate: compare * p＜0.05 with the blank group
Embodiment 3 (research that improves perimenopausal syndrome of TQW)
Observe TQW to the impact of the related symptoms of perimenopausal syndrome such as hectic fever, insomnia, hyperlipidemia.
One, TQW is on the impact of ovariectomized mouse adrenal gland weight and anus temperature
The test of employing ovariectomized mouse, removal ovary can make body inner estrogen level descend rapidly, can simulate the postclimacteric physiological and pathological feature of women.
1 materials and methods
1.1 material
KM kind mice, ♀, in 12 ages in week, unpregnancy is available from Heilongjiang University of Chinese Medicine GLP center.
The TQW suspension: in TQW suspendible distilled water, ig dosage is 15mg/kg.
Nilestriol suspension (Nilestrdiol): nilestriol (available from Pharmaceutical Group Hua Lian, Shanghai pharmaceutical factory, the accurate word of traditional Chinese medicines: H3102164), and in the suspendible distilled water, ig 0.5mg/kg.
1.2 method
1.2.1 the preparation of ovariectomized mouse model
Get mice, the collaborative anesthesia of 30mg/kg pentobarbital, 500mg/kg urethane, two skidding bilateral ovaries are extractd in the spinal column axis, near the fat the sham operated rats spay, spay not, postoperative is sewed up, the iodine disinfection wound.(draw normal saline 0.1～0.2ml with dropper by only carrying out vaginal smear, insert gently mouse vagina 2～3mm, aspirate smear 2～3 times, 95% ethanol is 10min fixedly, methylene blue solution-dyed 10min, water washes gently, drenches to do, in order to reading sheet), every day 1 time, observe vaginal epithelial cell and be in dioestrus in continuous 5 days, with the success of proof castration.
1.2.2 animal grouping and administration
Be divided into sham operated rats (Sham group), simple ovarian resection group (OVX group), OVX+TQW suspension (15mg/kg), OVX+ nilestriol (0.5mg/kg).1 time/d of gavage, 4 weeks of successive administration.
1.2.3 the detection of oestrous cycle and anus temperature
Every day is by only row vaginal smear procuratorial work, microscopically is observed the mouse vagina cellular change and is determined whether to enter rutting period, and 1h-2h surveys its anus temperature after the last administration: clinical thermometer is coated vaseline, insert the mice anus after making it lubricated, about 1cm, stable rear newspaper song reading its anus temperature of electric body-temperature instrumentation that occurs.
2 results (on the impact of ripe ovariectomized mouse adrenal gland weight and anus temperature)
Experimental result sees Table 11.TQW has gentle increasing action to ovariectomized mouse adrenal gland weight.Ovary and adrenal gland are the estrogenic major organs of secretion, and the adrenal gland becomes the estrogenic major organs of secretion behind the removal ovary.Infer that TQW starts the positive feedback of body hypothalamic-pituitary-adrenal element axle (HPA), adrenal cortex reinforcing is secreted estrogenic function.The TQW group can obviously reduce OVX mice anus temperature, relatively has statistical significance (p＜0.001) with the OVX mice.The effect of this reduction anus temperature is similar to Nilestriol, maintains 2h after the administration.This results suggest TQW has the effect of alleviating climacteric women hectic fever symptom.
Table 11 TQW is on the impact of ripe ovariectomized mouse adrenal gland weight and anus temperature
Annotate: compare * p＜0.05, * * p＜0.01, * * * p＜0.001 with the OVX group
Two, TQW is on the impact of mice sleep
1 materials and methods
1.1 material
KM kind mice, ♀, 20 ± 2g is available from Heilongjiang University of Chinese Medicine GLP center.
The TQW suspension: in TQW suspendible distilled water, ig dosage is 15mg/kg.
Stable suspension: diazepam is pulverized and is suspended in the distilled water, and ig dosage is 0.1g/kg.
1.2 method
1.2.1 the impact on sub-threshold dose pentobarbital sodium hypnosis rate
Get mice and be divided at random 3 groups, every group 10, be respectively blank group: the ig distilled water, the TQW group: igTQW suspension 15mg/kg, the stable 0.1g/kg of positive controls: ig is once a day, successive administration 7 days, 1 time/d, behind the last administration 60min, every mouse peritoneal injection gives the pentobarbital sodium of 30mg/kg, reach more than the 1min as sleeping criterion with the mice righting reflex loss, observe the number that sleep relatively occurs to administration treated animal in the pentobarbital sodium 30min and blank group.
1.2.2 on the impact of hypnosis time of threshold dose pentobarbital sodium
Get mice and be divided at random 3 groups, every group 10, be respectively blank group: the ig distilled water, the TQW group: igTQW suspension 15mg/kg, positive controls: ig stabilizes 0.1g/kg, once a day, successive administration 7 days, behind the last administration 60min, every mouse peritoneal injection gives the pentobarbital sodium of 50mg/kg, reach more than the 1min as sleeping criterion with the mice righting reflex loss, observe the time that sleep relatively occurs to administration treated animal in the pentobarbital sodium 30min and blank group.
2 results
2.1 the impact on mice sub-threshold dose pentobarbital sodium sleep effect
Experimental result sees Table 12.The TQW group compares with blank group, can increase sub-threshold dose pentobarbital sodium mice sleep number, significantly shortens the mice time for falling asleep, can improve 40% sleep rate.The climacteric women difficulty falling asleep appears and easily wake up and wake up after the E in sleep disorder and this physiological stage women serum such as have difficulty in going to sleep
2Level reduces relevant, and estrogen shortens Sleep latency, and sleeping rear awakening number of times reduces, and increases the tendency of total sleep time.Former studies shows that estrogen replacement therapy can make objective sleep quality improve, and can effectively improve subjective sleep quality.This results suggest TQW may improve sleep by the performance estrogen-like effects.
Table 12 TQW is on the impact of sub-threshold dose pentobarbital sodium hypnosis
Annotate: compare * p＜0.05, * * * p＜0.001 with the blank group
2.2 the impact on mice threshold dose pentobarbital sodium sleep effect
Experimental result sees Table 13.TQW organizes with blank group and compares, and TQW energy significant prolongation threshold dose is without the length of one's sleep of barbital sodium mice, and prompting TQW can sleep by the improvement of performance estrogen-like effects.
Table 13 TQW is on the impact of threshold dose pentobarbital sodium hypnosis
Annotate: compare * p＜0.05, * * p＜0.01 with the blank group
Three, TQW is on the impact of ovariectomized mouse blood fat
1 materials and methods
1.1 material
KM kind mice, ♀, 12 ages in week are available from Heilongjiang University of Chinese Medicine GLP center.
The TQW suspension: in TQW suspendible distilled water, ig dosage is 15mg/kg.
Nilestriol suspension (Nilestrdiol): nilestriol (available from Pharmaceutical Group Hua Lian, Shanghai pharmaceutical factory, the accurate word of traditional Chinese medicines: H3102164), and in the suspendible distilled water, ig 0.5mg/kg.
1.2 method
Get healthy mature 4 month female mices, the collaborative urethane anesthesia of ether, the row bilateral ovaries is extractd, and postoperative is sewed up, the iodine disinfection wound.By only carrying out vaginal smear, observe vaginal epithelial cell and be in dioestrus in continuous 5 days every day 1 time, with the success of proof castration.Be divided into sham operated rats (Sham) group, near the fat the spay, not spay; Simple ovarian resection group (OVX group), OVX+TQW organize (ig TQW suspension 15mg/kg), OVX+ nilestriol (0.5mg/kg).1 time/d of gavage, 4 weeks of successive administration.
After the last administration, extract eyeball and get blood, centrifugal.Survey the content of its serum total cholesterol (TC), triglyceride (TG) by the test kit method.
2 results (on the impact of ovariectomized mouse serum TC, TG)
Experimental result sees Table 14.Serum TC and TG content are organized apparently higher than sham behind the mice removal ovary, prove that Estrin drop causes the disorder of lipid metabolism in the body.And TQW can significantly reduce the content of serum TC, TG, acts on suitable with nilestriol.Women's blood lipid metabolism gets muddled after menopause, and the levels such as serum TC, TG that show as increase, and Incidence of CHD obviously increases.These occur changes main relevant with postmenopausal women's decrease in estrogen.This results suggest TQW can bring into play estrogen-like effects, improves metabolism disorder of blood lipid.
Table 14 TQW is on the impact of ovariectomized mouse serum TC, TG
Annotate: compare * p＜0.05, * * p＜0.01 with the OVX group
Embodiment 5 (the body inner estrogen sample effect antagonistic experiment of TQW)
1 materials and methods
1.1 material
KM kind mice, ♀, 21 ages in days are available from Heilongjiang University of Chinese Medicine GLP center.
The TQW suspension: in TQW suspendible distilled water, ig dosage is 15mg/kg.
Nilestriol suspension (Nilestrdiol): nilestriol (available from Pharmaceutical Group Hua Lian, Shanghai pharmaceutical factory, the accurate word of traditional Chinese medicines: H3102164), and in the suspendible distilled water, ig 0.5mg/kg.
1.2 method
Choose 11-13g Healthy female 21 age in days white mice, be divided at random blank group, nilestriol suspension group (0.5mg/kg), nilestriol+TQW suspension group (Chinese medicine antagonism group, Nilestriol0.5mg/kg+TQW15mg/kg), respectively organize every day in the morning 9 gavage distilled water and nilestriol, Chinese medicine antagonism group is in 5 gavage TQW suspension in afternoon, continuous 7 days, 24h weighs after the last administration, takes off cervical vertebra and puts to death, and cuts the abdominal cavity open, win uterus and vagina with shears, be put on the filter paper, remove fatty tissue, ovary and vagina claim rapidly its weight in wet base, calculate the uterus weight percentage rate, relatively the difference of each group.
2 results
In view of present chemosynthesis estrogen can cause the potential risks such as breast carcinoma, carcinoma of endometrium, phytoestrogen is because it has the focus that dual regulation becomes research, and the simultaneously safety of going deep into phytoestrogen along with research also remains to be confirmed.This experiment is intended to investigate the effect that TQW brings into play under the high estrogen environment in vivo, as shown in Table 15, the nilestriol group relatively makes the immaturity mouse uterine weight increase by 200% with blank group, and this increasing action is by antagonism 50% when giving TQW (Chinese medicine antagonism group) simultaneously.The prompting TQW effectively the antagonism nilestriol to the uterus weight effect of immaturity mice.When body inner estrogen level is low, show as estrogen-like effects, when body inner estrogen level is high, occupy the binding site of estrogen receptor (ERs), show as the antagonism estrogen-like effects.In conjunction with the experimental result of front, prompting TQW has estrogen-like effects and the effect of estrogen antagonist sample simultaneously, meets the dual regulation characteristics of phytoestrogen, proves the safety of its use.
Table 15 TQW antagonism nilestriol is to immaturity mice estrogen-like effects
Annotate: compare * * * p＜0.001 with the blank group; Compare with the nilestriol group
▲P＜0.05,
▲ ▲ ▲P＜0.001
Embodiment 6 (preparation of tablet)
By weight, get TQW5～9 part, add 1～2 part of Pulvis Talci, 1～2 part of starch, 1～2 part of carboxymethyl starch sodium sprays into 95% ethanol moistening, wet granulation, drying adds 0.5～1% magnesium stearate, mix homogeneously, tabletting; Add 15% Opadry coating solution coating, 7% coating that increases weight gets Film coated tablets.
Embodiment 7 (preparation of capsule)
By weight, get TQW8～9 part, add 1～2 part of Pulvis Talci, spray into 95% ethanol moistening, wet granulation, drying adds 0.5～1% magnesium stearate, mix homogeneously, packing gets capsule.
Claims (7)
1. Chinese medicinal compound extract with the effect of phytoestrogen sample, its extracting method is as follows:
In weight portion, according to Radix Angelicae Sinensis 4-6 part, Rhizoma Chuanxiong 2-4 part, Semen Persicae 1-3 part, gun ginger 1-2 part, Radix Glycyrrhizae 1-2 part, it is an amount of to take by weighing each medical material, co-grinding is crossed No. 2 sieves of pharmacopeia, and medicated powder carries out carbon dioxide supercritical fluid extraction, take ethanol as entrainer, under certain technological parameter, extract to get brown color liquid, concentrating under reduced pressure Recycled ethanol, the final brown color thickness grease that gets; Take by weighing Radix Et Caulis Acanthopanacis Senticosi 50-150 part, coarse powder mixes with medicinal residues behind the previous step carbon dioxide supercritical fluid extraction again, with extracting after the debita spissitudo soak with ethanol, the dry extractum that gets of extracting solution, extractum utilizes macroporous adsorptive resins to carry out enrichment, makes brown ceramic powder under certain technological parameter; The brown color thickness grease that this powder and carbon dioxide supercritical fluid extraction are made mixes, and namely gets Chinese medicinal compound extract; Wherein, when Radix Angelicae Sinensis, Rhizoma Chuanxiong, Semen Persicae, gun ginger and Radix Glycyrrhizae medicated powder are carried out carbon dioxide supercritical fluid extraction, extraction temperature is 30-60 ℃, extracting pressure is 20-60MPa, extraction time is more than the 1h, and entrainer is that dehydrated alcohol or concentration are the alcoholic solution more than 95%, and its consumption is 0.1-1ml/g medicated powder, resolving pressure is below the 20MPa, and resolution temperature is below 40 ℃; Wherein be when the medicinal residues behind Radix Et Caulis Acanthopanacis Senticosi coarse powder and the carbon dioxide supercritical fluid extraction are extracted, extracting solvent is 30%-100% ethanol, consumption is 10 times of amounts for the first time, soaks 12 hours, and heating and refluxing extraction or supersound extraction or percolation extracted 2 hours, filter, in medicinal residues, add again 5 times of amount 30%-100% ethanol, extracted again 2 hours, filter, merging filtrate, drying under reduced pressure gets extractum; When wherein extractum utilizes macroporous adsorbing resin for purification, extractum added after the suitable quantity of water dilution to mix with isopyknic D-101 type macroporous adsorbent resin mix thoroughly, be splined on D-101 type or AB-8 type, and other are nonpolar, in low pole or the middle polarity macroporous adsorptive resins, applied sample amount is calculated as 1: 2 with the ratio of crude drug amount and amount of resin, static adsorption 30 minutes, then carry out eluting with the ethanol of the variable concentrations of 10 column volumes successively, collect the 20%-50% ethanol elution, pulverize behind the drying under reduced pressure, make brown ceramic powder.
2. Chinese medicinal compound extract according to claim 1 is characterized in that in weight portion, wherein 5 parts of Radix Angelicae Sinensis, 3 parts of Rhizoma Chuanxiongs, 2 parts in Semen Persicae, 1 part of gun ginger, 1 part in Radix Glycyrrhizae, 100 parts of Radix Et Caulis Acanthopanacis Senticosis.
3. Chinese medicinal compound extract according to claim 1 is characterized in that must carrying out in Rotary Evaporators to carbon dioxide supercritical fluid extraction liquid concentrating under reduced pressure Recycled ethanol the time, and temperature is below 45 ℃.
4. Chinese medicinal compound extract according to claim 1, it is characterized in that when carrying out carbon dioxide supercritical fluid extraction, extraction temperature is 45 ℃, extracting pressure is 40MPa, extraction time is 2h, the consumption of entrainer dehydrated alcohol is 0.5ml/g medicated powder, and resolving I pressure is that 10MPa, temperature are 40 ℃, and resolving II pressure is that 4MPa, temperature are 25 ℃.
5. Chinese medicinal compound extract according to claim 1, when it is characterized in that the medicinal residues behind Radix Et Caulis Acanthopanacis Senticosi coarse powder and the carbon dioxide supercritical fluid extraction are extracted, added 10 times of amount 70% soak with ethanol 12 hours, heating and refluxing extraction 2 hours, filter, in medicinal residues, add again 5 times of amount 70% alcohol heating reflux extraction 2 hours, filter, merging filtrate, drying under reduced pressure gets extractum.
6. Chinese medicinal compound extract according to claim 1, when it is characterized in that ethanol extraction extractum carried out macroporous adsorbing resin for purification, the ethanol elution solvent of variable concentrations is according to concentration of alcohol eluting successively from low to high.
7. any one described Chinese medicinal compound extract is used in the pharmaceutical preparation for preparing the treatment perimenopausal syndrome and health food among the claim 1-6.
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| CN1415363A (en) * | 2002-11-14 | 2003-05-07 | 黑龙江省佳木斯晨星药业有限责任公司 | Biochemical medicament of araliaceae plant and its preparation method |
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