CN101897776A - New application of Chinese flowing crabapple extractive and phlorizin - Google Patents
New application of Chinese flowing crabapple extractive and phlorizin Download PDFInfo
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Abstract
The invention provides a new application of Chinese flowing crabapple extractive and phlorizin, in particular relating to an application in preparing a drug or a health care food for adjusting estrogen. The Chinese flowing crabapple extractive and phlorizin in the invention has the function of adjusting the estrogen in a two-way manner, definite potency and strong controllability, and provides a new choice for clinic drugs or health care foods.
Description
Technical field
The present invention relates to the new purposes of crab apple extract and phlorhizin, particularly, is the purposes in preparation estrogenic medicine of adjusting or health food, belongs to drug world.
Background technology
Hubei Chinese flowering crabapple [Malus hupehensis (Pamp) Rehd] originates in Hubei, Shaanxi, Hunan, Shanxi, various places, Chongqing.Its tender leaf can be commonly called as " premium tea " for tea, is beverage commonly used among the people, existing more than 400 year drink history, be rare expelling summer-heat herbal tea in midsummer, its root can be controlled muscles and bones spraining, fruit can be used as medicine and be controlled gastropathy and can make wine.Hubei Chinese flowering crabapple has multiple pharmacological effect (Wang Lam such as blood sugar lowering, blood fat reducing, anti-inflammation, anoxia enduring, resisting fatigue and plants, Yang Yuanbing. the quality research of Hubei Chinese flowering crabapple beverage, food research and development, 200223 (2): 5961), wherein, the main chemical compositions phlorhizin have certain non-oxidizability and antioxidation characteristics (Dong Huaqiang, Ning Zhengxiang. phlorhizin and diabetes control, food science and technology, 2006 (12): 192-194).Bibliographical information the antioxidant activity of Hubei Chinese flowering crabapple different parts and phlorhizin.(Zhang Hongqi Wang Lam is planted, the Study on Its Antioxidant Activity in Vitro of Hubei Chinese flowering crabapple extract, " food science and technology ", 2008 33 11 phases of volume); In addition, Lv Jiangming, hypericum ascyron extract anti-inflammatory and antalgic Study on Effect, " the practical combination of Chinese and Western medicine is clinical ", 2008 84 phases of volume, the document has reported that the hypericum ascyron extract has stronger anti-inflammatory and antalgic effect.
Still there is not at present the pertinent literature report that crab apple extract and phlorhizin are used to regulate estrogenic medicine or health food.
Summary of the invention
Technical scheme of the present invention has provided the new purposes of crab apple extract and phlorhizin, particularly, is the purposes in preparation estrogenic medicine of adjusting or health food.
The invention provides the purposes of crab apple extract in preparation estrogenic medicine of adjusting or health food.
Wherein, described medicine or health food are estrogenic medicine of two-ways regulation or health food.
Wherein, described crab apple extract derives from Hubei Chinese flowering crabapple Malus hupehensis Rehd., Flos Mali Hallianae Malus Halliana Koehne, Malus toringoides (Rehd.) Hughes Malus toringoides (Rehd.) Hughes.
Described crab apple extract is to be prepared from by following method: get Caulis et folium euphorbiae milii, add 10-30 water 50-100 ℃ heating extraction 2-3 time or 10-30 times 50-95% (v/v) alcohol reflux 2-3 time doubly, promptly get crab apple extract.
The present invention also provides the purposes of phlorhizin in preparation estrogenic medicine of adjusting or health food.
Wherein, described medicine or health food are estrogenic medicine of two-ways regulation or health food.
Wherein, described phlorhizin extraction separation gained or come from other plant that contains phlorhizin from Malus such as Hubei Chinese flowering crabapple Malus hupehensis Rehd., Flos Mali Hallianae MalusHalliana Koehne, Malus toringoides (Rehd.) Hughes Malus toringoides (Rehd.) Hughes, or the synthetic chemical compound of chemical method usually.
Described medicine is treatment estrogen deficiency syndrome in caused menopause, dysplasia, and menoxenia, sclerotin sulfur pine, cardiovascular system diseases, tumor, Alzheimer, cataract, macula retinae become.Specific as follows:
One. menopause syndrome:
Menopausal women is because ovarian function decline, and the body inner estrogen lowly causes " menopausal syndrome ", shows as: hectic fever, night sweat, agitation, insomnia, cardiopalmus, mastatrophy, skin exsiccation crumple, lack flexibility and gloss, various pigmentations etc.
Two. dysplasia, menoxenia: can promote breast development, the low dysplasia that causes of treatment body inner estrogen, menoxenia etc.
Three. sclerotin sulfur pine: menopausal women is because of decrease in estrogen, and bone absorption is accelerated, and causes osteoporosis and decalcification of bone, loss of tooth.
Four. cardiovascular system diseases
The low sclerosis of blood vessels that causes of body inner estrogen, blood fat rising, coronary heart disease etc.
Five. tumor
The low colon cancer that causes of body inner estrogen.
Six, others: as the low Alzheimer that causes of body inner estrogen, cataract, macula retinae change etc.
Described medicine is to be used for the treatment of the too much or caused cancer of controversies in hormone replacement in the elderly of endogenous estrogen, gallbladder disease, carbohydrate tolerance to change.
Specific as follows:
One, cancer
Carcinoma of endometrium, breast carcinoma, ovarian cancer, hepatocarcinoma and vaginal hemorrhage etc. that prolonged application estrogen or endogenous estrogen too much cause.
Two, gallbladder disease
Gallbladder disease due to the prolonged application estrogen such as cholecystitis, cholangitis are sent out, obstructive jaundice etc.
Three, carbohydrate tolerance changes
Estrogen can cause that carbohydrate tolerance increases.
The present invention also provides estrogenic medicine of a kind of adjusting or health food, and it is to be active component by the crab apple extract of effective dose or phlorhizin, adds the medicament that acceptable accessories or complementary composition are prepared from.
Wherein, described medicament is oral formulations or ejection preparation.
Hubei Chinese flowering crabapple is the conventional tea beverage, and main component phlorhizin approved abroad is a food additive.Crab apple extract and phlorhizin action temperature and, safety, can take for a long time, thereby take crab apple extract and phlorhizin, particularly drinking Hubei Chinese flowering crabapple tea is one of best complementing estrogen mode.Medicine crab apple extract of the present invention and phlorhizin have the estrogenic effect of two-ways regulation, and drug effect is clear and definite, and controllability is strong, for clinical a kind of new medicine or the health food of providing selected.
Description of drawings
Fig. 1 phlorhizin chromatogram (8.57min)
Fig. 2 phloretin chromatogram (13.7min)
Fig. 3 serum alcohol soluble substance chromatogram
Fig. 4 uterus ethanol extract chromatogram
Fig. 5 phlorhizin, phloretin, uterus ethanol extract mixing sample introduction chromatogram
Fig. 6 tartaric-resistant figure
The specific embodiment
The preparation of embodiment 1 crab apple extract of the present invention, phlorhizin
Get Caulis et folium euphorbiae milii, add 10-30 water 50-100 ℃ heating extraction doubly 2-3 time.It is standby that filtrate normal pressure or concentrating under reduced pressure get extractum.
Phlorhizin: separate from Hubei Chinese flowering crabapple, structure is identified as follows:
Phlorhizin, the Lycoperdon polymorphum Vitt acicular crystal.Its ESI-MS mass spectrum shows its quasi-molecular ion peak 449[M+Na]
+, in conjunction with the NMR data, drawing its molecular formula is C
21H
24O
10Its one dimension carbon spectrum only demonstrates 19 carbon signals, obviously has two carbon overlapping, and this signal that provides from its unidimensional hydrogen spectrum as can be seen, chemical shift be 7.04 (d, 8.5,2H), 6.64 (d, 8.5 two hydrogen signals 2H) obviously are ortho-hydrogens signals on the phenyl ring, its coupling constant is 8.5Hz, and its integral area is 2, show in the phlorhizin to have one 1, the phenyl ring of 4-para-orientation, in addition, chemical shift is 6.12 (d, 2.5,1H), 5.92 (d, 2, the position replaced between two hydrogen signals 1H) were judged on the phenyl ring according to its coupling constant, and its chemical shift only is 6.12, and this illustrates that the ortho position of this two hydrogen signal all has hydroxyl and replaces 5.92ppm, also there is another one 1 in the The above results proof phlorhizin, 2,3, the quaternary phenyl ring of 5-exists.Find out from its one dimension carbon spectrum and hydrogen spectrum and also to have a glucose existence the phlorhizin, have 100.8 in its carbon spectrum, 77.2,76.6,73.1,69.4,60.5ppm signal, and have a series of hydrogen signal, 4.92 (d in the hydrogen spectrum from 3.20-4.92ppm, 7) hydrogen signal of ppm judges according to its coupling constant that for being the anomer hydrogen of glucose the anomer hydrogen of glucose is a beta comfiguration.Also there are the carbon and the hydrogen of two High-Fields in its one dimension carbon spectrum and hydrogen spectrum in addition, and its chemical shift is respectively 44.5,28.9 and 3.40,3.30; 2.78ppm.The signal 204.6ppm that also has a ketone carbonyl in the one dimension carbon spectrum by contrasting with document, finds that the spectral data of this chemical compound and phlorhizin is about the same, thereby proves that this chemical compound is a phlorhizin.
Table1：The?NMR?data?of?ph(DMSO-d
6)
The structure of phlorhizin
The preparation of embodiment 2 crab apple extracts of the present invention
Get Caulis et folium euphorbiae milii, add 10-30 50-95% (v/v) alcohol reflux doubly 2-3 time.It is standby that filtrate normal pressure or concentrating under reduced pressure get extractum.
The preparation of embodiment 3 crab apple extracts of the present invention
Get Caulis et folium euphorbiae milii, add 50 ℃ of heating extraction of water 2 times of 10 times.It is standby that filtrate normal pressure or concentrating under reduced pressure get extractum.
The preparation of embodiment 4 crab apple extracts of the present invention
Get Caulis et folium euphorbiae milii, add 100 ℃ of heating extraction of water 3 times of 30 times.It is standby that filtrate normal pressure or concentrating under reduced pressure get extractum.
The preparation of embodiment 5 crab apple extracts of the present invention
Get Caulis et folium euphorbiae milii, add 50% (v/v) alcohol reflux 3 times of 10 times.It is standby that filtrate normal pressure or concentrating under reduced pressure get extractum.
The preparation of embodiment 6 crab apple extracts of the present invention
Get Caulis et folium euphorbiae milii, add 95% (v/v) alcohol reflux 2 times of 30 times.It is standby that filtrate normal pressure or concentrating under reduced pressure get extractum.
Embodiment 7 medicines of the present invention or health food Caulis et folium euphorbiae milii teabag processing method
Get the Caulis et folium euphorbiae milii leaf and give birth to product, pulverize the back and cross the 20-60 mesh sieve, get powder with filling machine or manually fill become bag, add outer package, make finished product.Every bag contains tea powder end 1-3g.
Embodiment 9 medicines of the present invention or the instant tea process of health food Caulis et folium euphorbiae milii
Get Chinese flowering crabapple tea, add 10-30 water 50-100 ℃ heating extraction 2-3 time or 10-30 times 50-95% (v/v) alcohol reflux 2-3 time doubly.Filtrate is handled through one of following method.
1, normal pressure or concentrating under reduced pressure get dry extract, and extractum is through being ground into powder, and it is standby to cross the 80-200 mesh sieve.
2, normal pressure or concentrating under reduced pressure get thick extractum, and thick extractum lyophilization gets powder, and it is standby to cross the 80-200 mesh sieve.
3, normal pressure or be evaporated to solid content 20-40%, spray drying gets powder, and it is standby to cross the 80-200 mesh sieve.Spray drying condition: solid content 15-30%; The baking temperature inlet temperature is selected for use about 160-200 ℃, about outlet temperature 70-110 ℃.Flow is regulated according to equipment performance, guarantees out temperature.
Above-mentioned powder directly or add adjuvant (one or more in the adjuvant that used adjuvant can soluble starch, lactose etc. is commonly used).Aseptic subpackaged, every bag of 0.5-3g.Make every bag to contain phlorhizin 60-300mg
The preparation of embodiment 10 medicinal tablets of the present invention
Get the crab apple extract of embodiment 1 preparation, powder is direct or add adjuvant, is pressed into ordinary tablet or effervescent tablet, makes every to contain phlorhizin 60-300mg.(used adjuvant can be one or more in tablets such as microcrystalline Cellulose, modified starch, ethyl cellulose, methylcellulose, hydroxyethyl-cellulose, hydroxypropyl emthylcellulose, the sodium carboxymethyl cellulose adjuvant commonly used).
The preparation of embodiment 11 medicine capsules of the present invention
Get the direct or adding adjuvant of crab apple extract powder of embodiment 2 preparations, encapsulated, contain phlorhizin 60-30mg in every capsules.(used adjuvant can be one or more in capsules such as microcrystalline Cellulose, modified starch, ethyl cellulose, methylcellulose, hydroxyethyl-cellulose, hydroxypropyl emthylcellulose, the sodium carboxymethyl cellulose adjuvant commonly used).
Below prove beneficial effect of the present invention by concrete pharmacodynamics test.
The estrogen action of test example 1 crab apple extract, phlorhizin
1, experiment material
Crab apple extract: by embodiment 3 preparations
Phlorhizin: separate from Hubei Chinese flowering crabapple, structure is identified and is seen Appendix.
Experiment reagent: DMEM culture fluid; New-born calf serum; 17 beta estradiols.
Experimental apparatus: microplate reader; CO
2Incubator etc.
2, experimental technique and result
The take the logarithm MCF-7 cell (human breast cancer cell is to the estrogen sensitivity) of trophophase is adjusted cell concentration to 1 * 10
4Individual/ml, be inoculated in 96 orifice plates, made it adherent in 24 hours with the DMEM culture fluid cultivation that contains 10% calf serum.Change culture medium into no phenol red culture medium, add the variable concentrations medicine then, the medicinal E2 of positive control (17 beta estradiol), each medicine is established 4 concentration.37 ℃, 5%CO in incubator
2Cultivated 4 days, and measured cell proliferation with mtt assay: the sucking-off culture medium, every hole adds 0.2%MTT 30 μ l, continues to cultivate 4h, sucking-off MTT, every hole adds DMSO100 μ l, measures OD in 540nm.The results are shown in Table 1, all data are all represented with mean+SD, carry out statistical procedures with variance analysis.
Table 1. medicine to the value-added influence of MCF-7 cell (
N=6)
With blank group ratio
*P＜0.05,
*P＜0.01
The take the logarithm MDA-MB-231 cell (human breast cancer cell, lack estrogen receptor) of trophophase is adjusted cell concentration to 1 * 10
4Individual/ml, be inoculated in 96 orifice plates, made it adherent in 24 hours with the L-15 culture fluid cultivation that contains 10% calf serum.Change culture medium into no phenol red culture medium, add the variable concentrations medicine then, the medicinal E2 of positive control (17 beta estradiol), each medicine is established 4 concentration.37 ℃, 5%CO in incubator
2Cultivated 4 days, and measured cell proliferation with mtt assay: the sucking-off culture medium, every hole adds 0.2%MTT 30 μ l, continues to cultivate 4h, sucking-off MTT, every hole adds DMSO100 μ l, measures OD in 540nm.The results are shown in Table 2, all data are all represented with mean+SD, carry out statistical procedures with variance analysis.
Table 2. medicine to the value-added influence of 231 cells (
N=6)
Crab apple extract and main component phlorhizin have the estrogen action of going out, and when estrogen deficiency, estrogen sensitive cells strain (MCF-7) are had with the same promotion cel l proliferation of estrogen.The breast cancer cell (MDA-MB-231) that lacks estrogen receptor is not promoted cell growth effect, show that its estrogen action mainly is to play a role by estrogen receptor.
The estrogenic antagonist of test example 2 medicine crab apple extracts of the present invention, phlorhizin
1, experiment material
Experimental raw: Hubei Chinese flowering crabapple (Malus hupehensis Rehd.), Flos Mali Hallianae MalusHalliana Koehne, Malus toringoides (Rehd.) Hughes Malus toringoides (Rehd.) Hughes.Crab apple extract is by embodiment 5 preparations, phloretin (Phloretin), phlorhizin (Phloridzin).
Experiment reagent: DMEM culture fluid; New-born calf serum; 17 beta estradiols; Zitazonium.
Experimental apparatus: microplate reader; CO
2Incubator etc.
Two, experimental technique and result
The take the logarithm MCF-7 cell (human breast cancer cell is to the estrogen sensitivity) of trophophase is adjusted cell concentration to 1 * 10
4Individual/ml, be inoculated in 96 orifice plates, cultivate and made it adherent in 24 hours.Change culture medium into no phenol red culture medium, add the variable concentrations medicine then and add 10 simultaneously
-1017 beta estradiols of M, each medicine are established 4 concentration.Contrast with the zitazonium effect.37 ℃, 5%CO in incubator
2Cultivated 4 days, and measured cell proliferation with mtt assay: the sucking-off culture medium, every hole adds 0.2%MTT 30 μ l, continues to cultivate 4h, sucking-off MTT, every hole adds DMSO100 μ l, measures OD in 540nm.The results are shown in Table 3, all data are all represented with mean+SD, carry out statistical procedures with variance analysis.
Table 3. medicine to the value-added influence of MCF-7 cell (
N=3)
With estradiol group ratio
*P＜0.01
Table 4. zitazonium to the value-added influence of MCF-7 cell (
N=3)
With blank group ratio
*P＜0.05,
*P＜0.01
Crab apple extract and main component phlorhizin have estrogenic antagonist, and when hyperestrogenism, strain has same estrogenic antagonist to the estrogen sensitive cells, suppress cell proliferation.
Test example 1 and test example 2 proofs, crab apple extract of the present invention and phlorhizin have the two-ways regulation function, when endogenous estrogen lacks, show estrogen activity; When hyperestrogenism, has estrogenic antagonist again.
The effect test of test example 3 medicines of the present invention and estrogen receptor
Measure medicine and estrogen receptor binding ability with yeast two-hybrid system, change selective medium (lacking tryptophan and leucine) that the estrogen receptor yeast cells adds 10ml in (the 200r/min cultivation 16～24h) of spending the night of 30 ℃ of shaking tables.Get per 50 μ l culture fluid and add the new culture medium adding of 200 μ l medicine 2.5 μ 1 continuation cultivation 4h.Get 150 μ l and put into 96 orifice plates, survey the OD value as yeast cells density in 600nm.Remaining part is used to measure the activity of beta galactosidase.Crab apple extract is by embodiment 6 preparations; The determination of activity of beta galactosidase:
1, centrifugal: 12000rpm, centrifugal 5min; The careful suction removed supernatant, collects yeast cells.
2, breaking cellular wall: yeast cells adds in the 200 μ l Z buffer (0.1M sodium phosphate [pH7.0], 10mMKCl, 1mM MgSO4), causes final concentration 1mg/ml with preceding adding zymolyaseg, and it is even to shake, 37 ℃ of incubation 15min.
3, enzyme reaction: the 2-Nitrobenzol β-D-galactoside that adds 40 μ l 4mg/ml concentration begins enzymatic reaction.30min。
4, enzyme assay: add 100 μ l 1MNa
2CO
3Stopped reaction.Get 150 μ l and add mensuration OD value in 96 orifice plates, calculate enzymatic activity by following formula.
U＝1000×(OD
420-1.75×OD
550)/(t×v×OD
600)
Annotate: T response time (min), the culture fluid volume (ml) that v is used to measure, OD
600Density during raji cell assay Raji, OD
420Absorbance after the 2-Nitrobenzol has reacted, OD
550Absorbance after having reacted.The results are shown in Table 5, table 6
The effect of table 5. medicine pair and estrogen receptor alpha (
N=3)
With blank group ratio
*P＜0.01
The effect of table 6. medicine pair and erss (
N=3)
With blank group ratio
*P＜0.01
Crab apple extract and main component phlorhizin and estrogen receptor alpha binding ability are low, have significant binding ability with erss, mainly produce estrogen and estrogen antagonist dual regulation by combining with erss.
Experiment in the test example 4 medicine estrogen-like effects bodies of the present invention
Laboratory animal: Kunming kind female mice, body weight 9-12g, the cleaning level is available from Wuhan Tongji Medical Institute Experimental Animal Center.Adopt the special feedstuff of no Semen sojae atricolor to raise.
The experiment medicine: the Hubei Chinese flowering crabapple extract, by embodiment 4 preparations, phlorhizin, diethylstilbestrol
Grouping and medication: animal random packet, gastric infusion.The medicine group gives variable concentrations equal-volume medicinal liquid, and matched group gives the equal-volume distilled water.Administration 6 days was plucked eyeball and is got blood, separation of serum on the 7th day.Put to death animal, open the abdominal cavity, remove periuterine fatty tissue, weighing is cut off in uterus and vagina intersection in the back.Calculate uterus coefficient [uterus wet weight (mg)/body weight (g)], adopt enzyme linked immunological ELISA test kit to detect mice serum E2.
The results are shown in Table 7.
Table 7. medicine to the influence of Mouse Uterus index and serum E2 content (
N=10)
With the matched group ratio
*P＜0.01
Experiment distributes in the body of test example 5 medicine phlorhizin of the present invention
Laboratory animal: Kunming kind female mice, body weight 18-22g, the cleaning level is available from Tongji University of Central China University of Science and Technology medical board Experimental Animal Center.
Experiment medicine: phlorhizin, phloretin
Medication: get three of kunming mices, irritate stomach with 80mg/kg dosage and give phlorhizin 2 times, two minor ticks 4 hours every day, administration 3 days is plucked eyeball behind the last administration 30min and is got blood, separation of serum, rotary evaporation adds 95% dissolve with ethanol, with 0.45 μ m membrane filtration to doing.Put to death animal, open the abdominal cavity, remove periuterine fatty tissue, cut off in uterus and vagina intersection the back.The uterus is with 95% ethanol extraction.Concentrate with 0.45 μ m membrane filtration, measure through high performance liquid chromatogram.The chromatographic column octadecylsilane chemically bonded silica be filler (250 * 4.6mm), particle diameter 5 μ m, mobile phase is that acetonitrile-water is (from 0 to 20min, the acetonitrile volume ratio becomes 80% from 10), sample size 10.0 μ L, flow velocity 1.0mL/min, the detection wavelength is 284nm, 25 ℃ of column temperatures.The results are shown in Figure 1-Fig. 5.
Experiment shows that the phlorhizin oral administration mainly absorbs with prototype and enters blood, and small part metabolism in liver is a phloretin.In target organ, mainly exist with the phlorhizin prototype.The pharmacological action that shows phlorhizin mainly is the direct effect of phlorhizin, rather than is metabolized to phloretin, is played a role by phloretin.
Experiment in vivo and vitro proves: crab apple extract and main component phlorhizin have estrogen and estrogen antagonist dual regulation, when hyperestrogenism, when endogenous estrogen lacks, show estrogen activity, can promote the endogenous estrogen secretion simultaneously; When hyperestrogenism, have estrogenic antagonist again, reduce the endogenous estrogen secretion simultaneously.Crab apple extract and main component phlorhizin and estrogen receptor alpha binding ability are low, have significant binding ability with erss, mainly produce estrogen and estrogen antagonist dual regulation by combining with erss.
Above-mentioned evidence, medicine crab apple extract of the present invention and phlorhizin have the estrogenic effect of two-ways regulation, and drug effect is clear and definite.
Test example 6 medicines of the present invention are to the influence of rat osteoblast propagation and differentiation
1, experiment material
Laboratory animal: SD rat in the birth 24h, the sex body weight is not limit, and the cleaning level is available from Tongji University of Central China University of Science and Technology medical board Experimental Animal Center.
Experimental raw: Hubei Chinese flowering crabapple extract (by embodiment 3 preparations), phlorhizin.
Experiment reagent: a-MEM culture fluid; New-born calf serum; Trypsin GiBCO), collagenase II (Sigma), tetrazolium bromide (MTT, sigma), alkaline phosphatase enzyme reagent kit (Nanjing is built up).
Experimental apparatus: superclean bench; Microplate reader; CO
2Incubator etc.
Two, experimental technique and result
Under the aseptic condition, take out the rat cranium of giving birth in the 24h, with the balanced salt solution flushing for several times, put into 0.25% trypsin solution, reject bone surface tunicle and soft tissue, osteocomma is cut into 1-3mm
3The skeletal grain of size discards trypsin solution.Room temperature digestion 45min discards Digestive system in 0.1% collagenase II.Skeletal grain is scattered in the 100mL culture bottle, add contain the a-MEM culture medium culturing 7d of 20% newborn calf serum after, skeletal grain is discarded the cell cultivation of going down to posterity.Cell reached for the 5th generation, with the cell trypsinization, behind the cell counting, was diluted to 4 * 10 with the a-MEM culture medium that contains 10% calf serum
4The cell suspension of/mL.Add 96 well culture plates with every hole 0.2mL, every hole 0.5mL adds 24 well culture plates, and 37 ℃, 5%CO
2Cultivate in the incubator, mirror is observed down when treating that cell 70% merges, and will contain the blood serum medium suction and abandon, and balanced salt solution flushing twice adds the serum-free medium that contains the variable concentrations Hubei Chinese flowering crabapple, continues to cultivate the propagation and the differentiation of different time detection cell.
Identify that by morphological observation and alkali phosphatase cytochemical staining used cell is an osteoblast.Mtt assay detects cell proliferation.By alkali phosphatase (ALP) (p-nitrophenyl disodium hydrogen phosphate substrate KINETIC METHOD) is measured cell differentiation.
The results are shown in Table 8, all data are all represented with mean+SD, carry out statistical procedures with variance analysis.
The influence that table 8. medicine is bred rat osteoblast (n=10,
)
With blank group ratio
*P＜0.05
Table 9 medicine to the influence of rat osteoblast alkaline phosphatase activities (n=8,
)
With blank group ratio
*P＜0.05
MTT shows that crab apple extract and phlorhizin can promote osteoblastic proliferation, and has dose dependent; Hubei Chinese flowering crabapple extract low dosage significantly improves phosphatase activity at 48h and 72h.Illustrate that crab apple extract has the osteoplastic effect of promotion.
Test example 7 medicines of the present invention are to the influence of differentiation of rat osteoclast and bone resorption activity
1, experiment material
Laboratory animal: SD rat in the birth 24h, the sex body weight is not limit, available from Tongji University of Central China University of Science and Technology medical board Experimental Animal Center.
Experimental raw: Hubei Chinese flowering crabapple (Malus hupehensis Rehd.), phlorhizin (Phloridzin).
Crab apple extract is by embodiment 5 preparations.
Experiment reagent: RPMI-1640; New-born calf serum; Anti-tartaic acid phosphatase dye liquor (Nanjing is built up).
Experimental apparatus: superclean bench CO
2Incubator etc.
Two, experimental technique and result
With put to death 75% soak with ethanol 5min behind the distilled water flushing Mus body with dislocation of cervical vertebra.Aseptic condition separates femur and humerus down, cuts off both sides epiphysis end, with injection needle a-MEM is washed medullary cavity gently, collects the medullary cell mixed liquor and is seeded to and cultivate 45min in the culture bottle.Abandon supernatant, with DMEM culture fluid re-suspended cell, counting is also adjusted cell concentration＞1 * 10
6/ ml.The cell mixture immigration is had in 24 well culture plates of slide, and insert in the incubator and cultivate.Change liquid 1 time every 2d, change liquid 50% at every turn.In 7d slide is taken out observation.
Influence to the anti-tartaic acid phosphatase activity of osteoclast: cell climbing sheet is taken out, with twice of phosphate buffer flushing, 10min is with dissolved cell in 0.1%TritonX-100 (10 μ L) effect, (in the 100mL deionized water, add the 0.4g disodium phenyl phosphate at dropping 100 μ L matrix solutions on the slide, 2.0g antimony potassium tartrate adds deionized water then to 150mL, adjusting pH value with 1mol/L HCl is 3.5, add deionized water again to 200mL), in 37 ℃ of incubators, hatch 30min.With 1mol/L NaOH (100 μ L) cessation reaction, with its absorptance of 405nm wavelength measurement, micromole's amount of counting nitrophenol.
The morphologic observation of osteoclast and evaluation: tartaric-resistant, om observation.
Detection method: the 1. influence of the anti-tartaic acid phosphatase activity of pair osteoclast
2. to the influence of osteoclastic bone absorption lacuna area
Result: tartaric-resistant such as Fig. 6
Table 10 medicine is to the influence of anti-tartaic acid phosphatase activity of osteoclast and bone resorption lacuna area
*The expression with the matched group comparing difference have significance p＜0.05 (
*), p＜0.01 (
*)
Crab apple extract and phlorhizin can suppress the bone active of having a liking for of osteoclast: bone resorption lacuna area is compared with the blank group, and the decline of variable concentrations is all arranged; Suppress the formation and the differentiation of osteoclast: along with the increase of Caulis et folium euphorbiae milii concentration, the TRAP activity reduces gradually.
Test example 6,7 proof crab apple extracts and phlorhizin can promote bone formation, suppress bone resorption, thus the treatment osteoporosis.
Above-mentioned pharmacodynamics test proves that medicine crab apple extract of the present invention and phlorhizin have the estrogenic effect of two-ways regulation, can be used for treating osteoporosis, and drug effect is clear and definite, and controllability is strong, for clinical a kind of new medicine or the health food of providing selected.
Claims (11)
1. crab apple extract is preparing the purposes of regulating in estrogenic medicine or the health food.
2. purposes according to claim 1 is characterized in that: described medicine or health food are estrogenic medicine of two-ways regulation or health food.
3. purposes according to claim 1 and 2 is characterized in that: described crab apple extract derives from Hubei Chinese flowering crabapple Malus hupehensis Rehd., Flos Mali Hallianae Malus Halliana Koehne, Malus toringoides (Rehd.) Hughes Malus toringoides (Rehd.) Hughes.
4. according to any described purposes of claim 1-3, it is characterized in that: described crab apple extract is to be prepared from by following method: get Caulis et folium euphorbiae milii, add 10-30 water 50-100 ℃ heating extraction 2-3 time or 10-30 times 50-95% (v/v) alcohol reflux 2-3 time doubly, promptly get crab apple extract.
5. phlorhizin is preparing the purposes of regulating in estrogenic medicine or the health food.
6. purposes according to claim 5 is characterized in that: described medicine or health food are estrogenic medicine of two-ways regulation or health food.
7. according to claim 5 or 6 described purposes; it is characterized in that: described phlorhizin extraction separation gained or come from other plant that contains phlorhizin from Hubei Chinese flowering crabapple Malus hupehensis Rehd., Flos Mali Hallianae Malus Halliana Koehne, Malus toringoides (Rehd.) Hughes Malus toringoides (Rehd.) Hughes Malus, or the synthetic chemical compound of chemical method usually.
8. according to any described purposes of claim 1-7, it is characterized in that: described medicine is treatment estrogen deficiency syndrome in caused menopause, dysplasia, and menoxenia, sclerotin sulfur pine, cardiovascular system diseases, tumor, Alzheimer, cataract, macula retinae become.
9. according to any described purposes of claim 1-7, it is characterized in that: described medicine is to be used for the treatment of the too much or caused cancer of controversies in hormone replacement in the elderly of endogenous estrogen, gallbladder disease, carbohydrate tolerance to change.
10. regulate estrogenic medicine or health food for one kind, it is to be active component by the crab apple extract of effective dose or phlorhizin, adds the medicament that acceptable accessories or complementary composition are prepared from.
11. medicine according to claim 10 or health food is characterized in that: described medicament is oral formulations or ejection preparation.
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| CN2010101929290A CN101897776A (en) | 2009-06-01 | 2010-05-27 | New application of Chinese flowing crabapple extractive and phlorizin |
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| Application Number | Priority Date | Filing Date | Title |
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| CN200910059475.7 | 2009-06-01 | ||
| CN200910059475 | 2009-06-01 | ||
| CN2010101929290A CN101897776A (en) | 2009-06-01 | 2010-05-27 | New application of Chinese flowing crabapple extractive and phlorizin |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103118688A (en) * | 2010-10-14 | 2013-05-22 | 天津中医药大学 | New use of chemical ingredients in cynomorium as phytoestrogen |
| CN103444952A (en) * | 2013-08-29 | 2013-12-18 | 河南大学 | Filamental flowering crab tea and aqueous extract thereof as well as application of aqueous extract |
| CN104447630A (en) * | 2014-10-24 | 2015-03-25 | 成都普思生物科技有限公司 | Compound extracted and separated from Tibetan medicine malus toringoides as well as method and application of compound |
| CN104825810A (en) * | 2015-04-24 | 2015-08-12 | 王爱玲 | Pharmaceutical composition for treating osteoporosis |
| CN104839382A (en) * | 2014-06-20 | 2015-08-19 | 三峡大学 | Lucid ganoderma and Chinese flowering crabapple tea as well as preparation process and application thereof |
| CN114432370A (en) * | 2020-11-02 | 2022-05-06 | 四川智佳成生物科技有限公司 | Russian fruit extract and pharmaceutical application thereof |
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| CN1880325A (en) * | 2006-04-21 | 2006-12-20 | 西斯汀财产有限公司 | Preparation and application of phlorizin composition |
| JP2007106712A (en) * | 2005-10-14 | 2007-04-26 | Maruzen Pharmaceut Co Ltd | Skin cosmetic and hair cosmetic |
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| JP2007106712A (en) * | 2005-10-14 | 2007-04-26 | Maruzen Pharmaceut Co Ltd | Skin cosmetic and hair cosmetic |
| CN1880325A (en) * | 2006-04-21 | 2006-12-20 | 西斯汀财产有限公司 | Preparation and application of phlorizin composition |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103118688A (en) * | 2010-10-14 | 2013-05-22 | 天津中医药大学 | New use of chemical ingredients in cynomorium as phytoestrogen |
| CN103444952A (en) * | 2013-08-29 | 2013-12-18 | 河南大学 | Filamental flowering crab tea and aqueous extract thereof as well as application of aqueous extract |
| CN104839382A (en) * | 2014-06-20 | 2015-08-19 | 三峡大学 | Lucid ganoderma and Chinese flowering crabapple tea as well as preparation process and application thereof |
| CN104839382B (en) * | 2014-06-20 | 2018-01-16 | 三峡大学 | A kind of ganoderma lucidum Chinese flowering crabapple tea and preparation technology and its application |
| CN104447630A (en) * | 2014-10-24 | 2015-03-25 | 成都普思生物科技有限公司 | Compound extracted and separated from Tibetan medicine malus toringoides as well as method and application of compound |
| CN104825810A (en) * | 2015-04-24 | 2015-08-12 | 王爱玲 | Pharmaceutical composition for treating osteoporosis |
| CN114432370A (en) * | 2020-11-02 | 2022-05-06 | 四川智佳成生物科技有限公司 | Russian fruit extract and pharmaceutical application thereof |
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