CN1837226B - Separation of medical derivatives from phoenix-tail fern and use thereof - Google Patents
Separation of medical derivatives from phoenix-tail fern and use thereof Download PDFInfo
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- CN1837226B CN1837226B CN2005100245546A CN200510024554A CN1837226B CN 1837226 B CN1837226 B CN 1837226B CN 2005100245546 A CN2005100245546 A CN 2005100245546A CN 200510024554 A CN200510024554 A CN 200510024554A CN 1837226 B CN1837226 B CN 1837226B
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Abstract
The invention relates to relates to Yangqinzisu-4'-alphs-L- rhamnoside, a novel glucoside of Yangqinzisu extracted from phoenix-tail fern. Tests show that, the Yangqinzisu-4'-alpha-L-rhamnose-7-beta-D-heteroside and docosanoic acid has very strong inhibitory action to the proliferation of cultivated prostate gland cells, thus can be applied into the development of medicaments for treating prostatehyperplasia and prostatitis.
Description
Technical field
The present invention relates to from herbal medicine, separate effective ingredient, more specifically relate to from Herba Pteridis Multifidae separating and obtain plain derivative of foreign celery and their purposes.
Background technology
(benign prostatic hyperhlasia is the male sex's a old common disease BPH) to benign prostate hyperplasia, and along with social senilization, its sickness rate rises year by year.According to statistics, in 60～80 years old the male sex, have 50% can be proved by histopathologic examination and suffer from BPH approximately, its cause of disease is unclear fully as yet.The relevant expert is taken place BPH and the matrix of development has proposed three kinds of hypothesis in recent years, and promptly sexual hormoue theory, embryo wake theory and the prostate epithelial cell group theory that increases again up.Internal secretion related substanceses such as sexual hormoue are the external causes of regulation and control prostate gland growth, it realizes by various peptide class somatomedins such as epithelial cell growth factor (EGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF) and transforming growth factor (FGF) prostatic biological action, and prostatic cell proliferation and apoptosis unbalance be its basic reason.In the generating process of prostate gland normal development and hypertrophy and tumour, the interaction between inoblast and the epithelial cell is very important.External cause and internal cause run through the pathogenic process of BPH.And proved that at present bacterium exists and benign prostatic hyperplasia is related.Bacterium itself can be used as a kind of antigen and discharges some material, directly or indirectly induce the form of a prostatic epithelium and a matter and the change of growth pattern, the regulatory mechanism complexity of prostate gland growth, under normal circumstances keep relative equilibrium, in case unbalancely just can produce hyperplasia, prostatitis might be to break the important channel that this balance causes hyperplasia of prostate.
The medicine of BPH mainly is divided into α
1-AR retarding agent, 5 inhibitor, LHRH promotor and antagonist also have estrogen antagonist medicine and plant medicine in addition.
It is one of effective means of the BPH of screening treatment at present medicine that prostatic cell is cultivated.Underage rat prostatic cell serum-free culture is adopted in this research, utilizes epithelial cell and fibroblastic interaction, and adds cell growth factors such as Regular Insulin, EGF, Transferrins,iron complexes, prolactin, the process of simulation prostatic cell proliferation.The propagation that adopts compound that this model discrimination goes out or monomer can effectively suppress prostatic cell has important meaning for further research and development treatment hyperplasia of prostate and prostatitic new drug.
Summary of the invention
The purpose of this invention is to provide from Herba Pteridis Multifidae and separate plain-the 4 '-alpha-L-rhamnoside of celery of going abroad, plain-the 4 '-α of foreign celery-L-rhamnosyl-7-β-D-grape glycosides and mountain Yu acid, palmitinic acid, luteolin.
Another object of the present invention is the preparation method who discloses them.
A further object of the present invention provides the purposes of this compounds.
Herba Pteridis Multifidae has another name called spire Herba Pteridis Multifidae, leaflet Herba Pteridis Multifidae, is Pteridaceae plant Pteris multifida Poir., and another name has halymenia dentata, golden pheasant grass, Herba Pteridis multifidae, well limit phoenix tail, curb grass, Herba Pteridis creticae, cinquefoil etc.Belong to per nnial herb, be born in the half dark and damp rock and cornerstone crack more, be distributed in ground such as Yunnan, Sichuan, Guangdong, Guangxi, Hunan, Jiangxi, Zhejiang, Anhui, Jiangsu, Fujian, Taiwan.As the then lightly seasoned little hardship of Chinese medicine, cold in nature.But the function clearing heat and promoting diuresis, cooling blood for hemostasis, subduing swelling and detoxicating.
We assign to the plain new glucosides of new compound ocean celery-plain-the 4 '-alpha-L-rhamnoside of foreign celery and plain-the 4 '-α of the foreign celery-L-rhamnosyl-7-β-D-heteroside that can obviously suppress prostatic cell proliferation from Herba Pteridis Multifidae, and mountain Yu acid, palmitinic acid, luteolin.
Embodiment
The present invention implements through the following steps.
Dried Herba Pteridis Multifidae meal is with 20% extraction using alcohol three times, united extraction liquid is evaporated to the rare medicinal extract of no alcoholic acid, by the rare HPD100 macroporous resin column of benzene second, wash with water earlier and remove the desaccharification class, constituents such as protein and inorganic salt, use 50% water-ethanol and ethanol elution again, after concentrating, collection passes through the LH-20 gel filtration chromatography more respectively, and use methyl alcohol and methyl alcohol respectively: chloroform=7: 3 stepwise elutions, thin layer detects, collect and merge the stream part that contains same blob on the silica gel thin-layer plate, recycle silicon glue behind the concentrating under reduced pressure (Haiyang Chemical Plant, Qingdao produces, the 200-300 order) column chromatography is with CHCI
3: MeOH: H
2O=40: 10: 1 and CHCI
3: MeOH: H
2O=70: 25: 5 wash-outs, further separation and purification obtains thick PM-7 and PM-8, with the pure PM-7 and the PM-8 that get yellow or sundown powder behind the refining methanol, the Rf value of their silica gel thin-layer chromatography (thin layer plate also is that chromatography Haiyang Chemical Plant, Qingdao produces) is about 0.7 and 0.3, and (the used developping agent of thin-layer chromatography is CHCI again
3: MeOH: H
2O=70: 30: 5).
Identify through methods such as chemistry, spectrum, make structure and be:
Compound P M 7
Compound P M 8
PM 8 is a yellow powder shape material, mp.223～224 ℃, [α]
D 20-116.8 ° (c=0.23, pyrrole heavy stone used as an anchor).The IR spectrum shows in its molecule have-OH (3384.5cm
-1), carbonyl (1662.4cm
-1), and the eigen vibration (1606.4cm of the two keys of phenyl ring is arranged
-1, 1496.5cm
-1).FAB-MS provides quasi-molecular ion peak [M+H]
+M/z:579 (22%); HR ESIMS (m/z579.1712[M+H]
+) determine that its molecular formula is C
27H
30O
14(calculated value 579.1708[M+H]
+).
13C NMR provides 25 carbon spectral lines, wherein δ altogether
C128.8 not only show very high abundance with the CH of 117.4ppm, correspondingly
1Therefore a pair of symmetric ddd peak has also appearred in H NMR, respectively shows as the integration of two H atoms, thinks to have 27 C in this compound, and the molecular formula that this and ESI high resolution mass spectrum provide matches.From
13Also in the molecule two glycosyls should be arranged as can be seen in the C NMR spectrum, one of them glycosyl is β-D-glucose (δ
C: 101.9d, 71.3d, 78.6d, 75.0d, 79.4d, 62.5t), another glycosyl should be α-L-rhamnosyl (δ
C: 100.0d, 71.9d, 72.6d, 73.7d, 71.3d, 18.7q), except that glycosyl part, the chemical shift of glucoside unit has the feature of chromocor compound, infers that therefore Compound P M 8 is the flavonoid glycoside with two glycosyls.From
1The integration of H NMR, except that two glycosyls, glucoside unit part has the integration of 7 hydrogen.Because C-5 position hydroxyl provides in low place and has the wide unimodal of 1/2 hydrogen, remove in addition
1H NMR provides unimodal (δ 7.04) that a group inte gration is 1H, and two groups respectively is d peak (the long-range coupling constant of 1H
4J is 2.1Hz) and a pair ofly respectively be the ddd peak of 2H integration, infer from the basic framework of flavones, two d peaks are respectively C-6 and C-8 position H, one group of unimodal C-3 position H that should be, a pair of result who respectively then should be 4 ' replacement back mutual coupling of 3 ', 5 ' and 2 ', 6 ' the symmetrical H of C ring and long-range coupling for the ddd peak of 2H integration.Glycosylation should be in two positions of C-7 and C-4 ' position, and which glycosyl which position connects actually, then can obtain proof by 2D NMR, and this HNBC spectrum has been provided very tangible proof.
Can find all glycosides millions from the HNBC spectrum, the reference point that β-D-glucosyl group and α-the L-rhamanopyranosyl should occur, and two glycosyls are connected in C-7 position and C-4 ' position respectively as can be seen, because (δ 5.94 (the 1H of the hydrogen on the end of the bridge carbon of β-D-glucosyl group, d, J=7.5Hz)) with C-7 position quaternary carbon (δ 164.3) appearance reference point clearly, hydrogen (δ 6.24 (1H on α-L-rhamanopyranosyl end of the bridge carbon, d, J=1.3Hz)) with C-4 ' position carbon (δ 160.4) tangible reference point appears too, Compound P M 8 is accredited as 4H-1-Benzopyran-4-one thus, [(2-β-D-glucopyranosyl) oxy] 5-hydroxy-2-(oxy of 4-α-L-rhamnosyl)] phenyl-, that is: apiolin-7-β-D-glucose-4 '-α-L-rhamnoside, its C, the H ownership sees Table 1.
The correlationship that the HMBC spectrum of Compound P M 8 discloses
(only listed the crucial reference point of definite sugar-base binding site, other reference point omits, and the arrow direction is from H to C)
PM 7 is the tawny powdery substance, mp.281 ℃ (decomposition point), [α]
D 20-61.3 ° (c=0.13, DMSO).The IR spectrum shows in its molecule have-OH (3232.2cm
-1), carbonyl (1656.6cm
-1), and the eigen vibration (1608.4cm of the two keys of phenyl ring is arranged
-1, 1579.4cm
-1, 1502.3cm
-1).ESIMS provides quasi-molecular ion peak [M+H]
+M/z:417.1 (100%), [M-H]
+M/z:415.2 (100%); HR ESIMS (m/z 417.1182[M+H]
+) determine that its molecular formula is C
21H
20O
9(calculated value 417.1180[M+H]
+).Obviously can see in its carbon spectrum and contain a α-L-rhamanopyranosyl in the molecule, and three-OH in this glycosyl exists
1The d peak that to provide three groups of each integrations among the H NMR be 1/2H, coupling constant is approximately about 5Hz, the information that combined carbon spectrum and hydrogen spectrum provide, this compound also is apiolin-α-L-rhamnoside compounds, because its hydrogen spectrum and carbon are composed and are all shown and should contain the fragrant hydrogen symmetrical structure the same with PM 8 in the molecule, promptly at δ
C128.3 and each overlapping two CH of 116.7ppm, δ
H8.01 (d, J=9.0Hz) and 7.19 (d, J=9.0Hz) two of ppm place groups of peaks respectively are the 2H integration, the wide unimodal of C-5 position hydroxyl 1/2 hydrogen integration also occurred in low place, the position of substitution of α-L-rhamnosyl is also definite by HMBC.
The correlationship of definite sugar-base binding site that the HMBC spectrum of Compound P M 7 discloses
Compound P M 7 finally is accredited as: 4H-1-Benzopyran-4-one, the oxy of 2-[(4-α-L-rhamnosyl)] phenyl-, i.e. apiolin-4 '-α-L-rhamnoside, its C, H ownership sees Table 1.
The NMR (Nuclear Magnetic Resonance) spectrum of C, H ownership (400MHz, TMS, δ ppm) in table 1 Compound P M 7 and PM 8 structures:
*All ownership are all confirmed by the two dimensional NMR spectrum
Make solvent with deuterium for the inferior phenol of diformazan
Other gets acetone extraction three times of dried Herba Pteridis Multifidae meal, united extraction liquid is evaporated to medicinal extract, pass through silica gel column chromatography earlier, Fractional Collections stream part, again respectively by the LH-20 gel filtration chromatography, with sherwood oil: acetone=8: 2 and chloroform: methyl alcohol=7: 3 wash-outs, with the glue thin layer analyse be according to and same stream part, the recycle silicon plastic column chromatography is with CHCI behind the concentrating under reduced pressure
3: MeOMe=7: 3 wash-outs, further separation and purification obtains PM-15 respectively, PM-20, PM-22 compound.
Compound P M's 15
1H NMR is low, and the place has 6 hydrogen,
13Provide 15 carbon spectral lines altogether in the C NMR spectrum, infer that from two carbon spectral lines of δ 147.51 and 151.41ppm this compound should belong to the Luteolin flavonoids, through contrasting with document, its spectral data is consistent with Luteolin, therefore identifies that this compound is a luteolin.
Compound P M's 20
1H NMR only provides four groups of proton signals, δ 2.34 (2H, t, J=7.7Hz), 1.63 (2H, p, J=14.7,7.3Hz), 1.25 (24H, brs.), 0.88 (3H, t, J=6.6Hz).Wide unimodal from 1.25ppm should have a fatty long-chain in the molecule, this is from it
13The strong unimodal conclusive evidence that obtains at δ 29.36 places in the C NMR spectrum, except that δ 14.10 places were the carbonyl of monomethyl and δ 180.34, remaining several carbon spectral lines were CH in the carbon spectrum
2, the molecular weight that EIMS provides is 256, identifies that by these information this compound is Palmitic acid, i.e. palmitinic acid.Because the silica gel thin-layer chromatography behavior of Compound P M 22 is similar with PM 20, determines that by mass spectrum its molecular weight is 368, and NMR spectrum shape is similar substantially with PM 20, and authenticating compound PM 22 is Behenic acid thus, the Ji docosoic.
Pharmacological testing:
1. cell cultures:
Get the male SD rat in 40 day age, aseptic condition separates down prostate gland, subtracts brokenly after the nutrient solution rinsing, adds collagenase and DNA enzyme, 37 ℃ of water bath with thermostatic control vibration digestion, termination reaction after 18 hours is filtered, 1000rpm is collecting cell after centrifugal 5 minutes, and counting is inoculated in 24 well culture plates.
2. drug treating:
Prostatic cell is cultivated after 3 days and is begun drug treating.Each medicine is provided with 180umol/l and two dosage of 360umol/l respectively, and solvent control is 0.1%DMSO.Drug treating was measured cytoactive with mtt assay after 3 days, calculated each medicine to the outgrowth inhibiting rate of prostatic cell.This experiment is adopted and to be added in somatomedin and the nutrient solution two kinds of conditions of no somatomedin in the nutrient solution and respectively medicine is screened, and testing used somatomedin is Transferrins,iron complexes, Regular Insulin, Urogastron, PRL prolactin and testosterone.
3. inhibiting rate calculates:
Adopt following formula to calculate medicine to rat prostate cell inhibiting rate with the MTT measured value.
Inhibiting rate=(blank group cytoactive-administration group cytoactive)/blank group cytoactive * 100%
4. drug effect evaluation:
With reference to the inhibiting rate of positive control drug epristeride, press following standard evaluation medicine to the effect of rat prostate cell inhibiting.
Potent: two dosage inhibiting rates are all greater than 50%
Effectively: a dosage inhibiting rate is greater than 50% in two dosage, and another is less than＜50%
The weak effect: two dosage inhibiting rates are all less than 50%, but greater than 25%
Invalid: two dosage inhibiting rates are all less than 25%
Experimental result:
1, do not contain The selection result (table 1) under the somatomedin condition in the nutrient solution:
There is potent inhibiting compound to have to isolated culture rat prostate cell: PM7, PM15 and PM22
The effective inhibiting compound of isolated culture rat prostate cell is had: PM20
There is the inhibiting compound of weak effect to have to isolated culture rat prostate cell: PM12, PM14, PM16, PM11+2.
Compound to isolated culture rat prostate cell unrestraint effect has: PM1, PM2, PM4, PM5, PM6, PM9.
2, contain The selection result (table 2) under the somatomedin condition in the nutrient solution:
There is potent inhibiting compound to have to isolated culture rat prostate cell: PM7, PM8, PM15, PM20 and PM22
The effective inhibiting compound of isolated culture rat prostate cell is had: PM14
There is the inhibiting compound of weak effect to have to isolated culture rat prostate cell: PM12, PM16.
Compound to isolated culture rat prostate cell unrestraint effect has: PM1, PM2, PM4, PM5, PM6, PM9, PM11+2.
Table 1 compound is trained in the liquid the outgrowth inhibiting rate of prostatic cell (%) in no somatomedin
Table 2 compound is having somatomedin to train in the liquid the outgrowth inhibiting rate of prostatic cell (%)
Embodiment
1, dried Herba Pteridis Multifidae meal 1000 grams, with 40% extraction using alcohol three times, united extraction liquid concentrating under reduced pressure gets 395 gram medicinal extract, (comprise HPD100 by phenylethylene, HPD300, HPD400 etc.) macroporous resin or chromatography polyamide column, carbohydrate and inorganic salts compound are removed in elder generation's water flushing, use the water and the ethanol gradient elution of different concns again, collect 50% ethanol and contain flavonoid compound stream part, after concentrating, merging, uses methyl alcohol, the CHCl3 gradient elution again by the LH-20 gel filtration chromatography, be collected in stream part of single point on the polymeric amide thin layer plate, concentrate respectively thick PM-7 and PM-8, again with aqueous ethanol or aqueous acetone purifying, the pure PM-720mg and the PM-8 120mg of yellow or sundown powder;
2, dried Herba Pteridis Multifidae meal 1000 grams are used acetone extraction three times, and united extraction liquid concentrating under reduced pressure gets 256 gram medicinal extract, by silica gel column chromatography, Fractional Collections stream part is more respectively by the LH-20 gel filtration chromatography earlier, obtain PM-1515mg respectively, PM-2023mg, PM-22 18mg.
Claims (1)
1. plain derivative of foreign celery that structure is following
Application in the medicine of preparation prevention, treatment prostatitis and prostatomegaly.
Priority Applications (1)
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|---|---|---|---|
| CN2005100245546A CN1837226B (en) | 2005-03-23 | 2005-03-23 | Separation of medical derivatives from phoenix-tail fern and use thereof |
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|---|---|---|---|
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| CN1837226B true CN1837226B (en) | 2010-06-16 |
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|---|---|---|---|---|
| CN101574338B (en) * | 2008-05-05 | 2012-01-11 | 上海医药工业研究院 | Pharmaceutical composition for restraining activity of aromatizing enzyme and application thereof |
| CN101712667B (en) * | 2008-10-07 | 2012-05-30 | 上海医药工业研究院 | Flavonoid compound intermediate and preparation method and application thereof |
| CN102688261A (en) * | 2011-03-21 | 2012-09-26 | 苏州世林医药技术发展有限公司 | Pteris multifida extract, preparation method thereof and use thereof |
| CN104529977B (en) * | 2014-12-29 | 2016-09-21 | 贺州学院 | Corm Eleocharitis skin extracts the method for luteolin |
| IT201700075637A1 (en) | 2017-07-05 | 2019-01-05 | Vera Salus Ricerca S R L | COMPOSED FOR THE CARE OF MALIGNANT CANCERS |
| US20240024280A1 (en) * | 2020-12-01 | 2024-01-25 | Bionexa S.R.L | Senotherapeutic substance |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1196256A (en) * | 1998-05-06 | 1998-10-21 | 山东省蒙阴县第二人民医院 | Medicine for preventing and curing cardiovascular disease, treating prostatic hyperplasia and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1196256A (en) * | 1998-05-06 | 1998-10-21 | 山东省蒙阴县第二人民医院 | Medicine for preventing and curing cardiovascular disease, treating prostatic hyperplasia and preparation method thereof |
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