CN103864876A - Xylogranatumin G and preparation method and application thereof - Google Patents
Xylogranatumin G and preparation method and application thereof Download PDFInfo
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- CN103864876A CN103864876A CN201210552780.1A CN201210552780A CN103864876A CN 103864876 A CN103864876 A CN 103864876A CN 201210552780 A CN201210552780 A CN 201210552780A CN 103864876 A CN103864876 A CN 103864876A
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- heptan
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- xylocarpus granatum
- triterpene compound
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Abstract
The invention relates to a new triterpenoid xylogranatumin G obtained by separating from xylocarpus granatum koenig, a preparation method and an application thereof in preparation of medicines for treating tumor. The xylogranatumin G has the following structural formula as shown in the description. Multi-time in-vitro antitumor activity experiments show that the compound has obvious activity of inhibiting tumor cells and is expected to be applied in the preparation of the medicines for treating tumor.
Description
Technical field
The present invention relates to medical technical field, extract first and separate new triterpene compound xylocarpus granatum element in heptan, the Its Preparation Method And Use that obtain (Xylocarpusgranatum Koenig) in particular to one from xylocarpus granatum.Biological activity test shows, this compound has remarkable inhibiting activity to A-549 human lung adenocarcinoma cell line, can be used as a class and develop the lead compound of new antitumor drug, also can be used as the medicine of the various clinical common multiple cancers for the treatment of.
Background technology
Xylocarpus granatum is Meliaceae Xylocarpus Koenig plant, and normal mixed being born in mangrove forest, is distributed widely in the coastlands such as South East Asia, Australia, East Africa, the Indian Ocean.Xylocarpus granatum as medication among the people, is used for the treatment of diarrhoea, cholera, dysentery etc. in South East Asia and India, also has insecticidal action.Its chemical composition mainly comprises triterpene, alkaloid, phenolic acid, flavones, steroidal, monoterpene etc.
Summary of the invention
The present invention extracts first and separates the new triterpene compound xylocarpus granatum element in heptan obtaining from xylocarpus granatum.Show through pharmacological testing research, this compound has remarkable inhibiting activity to A-549 human lung adenocarcinoma cell line.
Therefore, an object of the present invention is to provide new triterpene compound xylocarpus granatum element in heptan.
Another object of the present invention is to provide the preparation method of described xylocarpus granatum element in heptan.
A further object of the present invention is to provide described xylocarpus granatum element in heptan in the application in the medicine of preparation treatment tumour, particularly the application aspect treatment lung cancer, mammary cancer.
According to first object of the present invention, the present invention has found a new triterpene compound xylocarpus granatum element in heptan first from xylocarpus granatum, and its chemical structure is as follows:
According to second object of the present invention, the invention provides the preparation method of xylocarpus granatum element in heptan, it extracts to separate and obtains from xylocarpus granatum, and concrete steps are as follows:
1) extract: by xylocarpus granatum branches and leaves methanol extraction, after gained extracting solution is concentrated, obtain crude extract; By water-soluble this crude extract, after suspendible is even, be extracted with ethyl acetate, after gained extraction liquid is concentrated, obtain ethyl acetate extract;
2) separate: described ethyl acetate extract is carried out to Sephadex LH-20 gel filtration chromatography, with chloroform/methanol volume ratio 1:1 wash-out, monitor and be divided into four parts with thin-layer chromatography (TLC), wherein, second section is again through MCI column chromatography, with methanol/water gradient elution; Wherein, methanol/water volume ratio is that the wash-out part of 50:50 is further through silica gel column chromatography, with sherwood oil/acetone gradient elution, wherein, sherwood oil/acetone volume ratio 70:30 wash-out part with acetonitrile/water volume ratio 85:15 wash-out, obtains xylocarpus granatum element in heptan finally by preparation HPLC.
In above-mentioned preparation method, in extraction step, the method that described extraction adopts is that methyl alcohol diacolation extracts.
In above-mentioned preparation method, in separating step, the concentration of methanol/water gradient elution is followed successively by volume ratio 30:70,50:50,80:20,100:0.
In above-mentioned preparation method, in separating step, the concentration of sherwood oil/acetone gradient elution is followed successively by volume ratio 90:10,80:20,70:30 and 50:50.
In above-mentioned preparation method, in separating step, the filler of described preparation HPLC is anti-phase octadecyl silane.
According to the 3rd object of the present invention, the invention provides the purposes of xylocarpus granatum element in heptan at the medicine of preparation treatment tumour.
Preferred described tumour is mammary cancer or lung cancer.
Xylocarpus granatum element in heptan of the present invention can obtain by separation and purification from plant; Also can be through the synthetic acquisition of chemical modification method well known to those skilled in the art.
Embodiment
In following embodiment, the chemical structural formula of the xylocarpus granatum element in heptan of indication is as follows: (Arabic numerals in structural formula are marks of carbon atom in chemical structure):
Agents useful for same is all analytical pure, and wherein, sherwood oil heavily steams rear use, and acetonitrile is chromatographically pure.Thin-layer chromatography used silica gel plate is prefabricated silica-gel plate HSGF254 (Chemical Reagent Co., Ltd., Sinopharm Group's production), gel is Sephadex LH-20 (Amersham Biosciences), MCI silica gel is that (75 ~ 150 μ are (Mitsubishi chemical company) m) for CHP-20P, liquid phase is Agilent 1100 series, UV-detector is VWD G1314A, detection wavelength is 210nm, chromatographic column is that (250mm × 9.4mm, particle size 5 μ m) for Agilent semipreparative column ZORBAX ODS post.Polarimetry instrument is Perkin-Elmer polarimeter341, and carbon spectrum and hydrogen spectrum are measured and obtained by Bruker DRX 400 and Varian Mercury 300 respectively.High resolution mass spectrum is recorded by Q-TOF Micro LC-MS-MS mass spectrograph, is infraredly measured by infrared instrument Nicolet-MagnaFT-IR750.
The preparation of embodiment 1 xylocarpus granatum element in heptan
1) extract: the xylocarpus granatum branches and leaves 2kg methyl alcohol that picks up from Hainan Province of China is carried out to diacolation and extract three times, after gained extracting solution is concentrated, obtain crude extract; By water-soluble this crude extract, after suspendible is even, be extracted with ethyl acetate three times, after gained extraction liquid is concentrated, obtain ethyl acetate extract.
2) separate: ethyl acetate extract is carried out to Sephadex LH-20 gel filtration chromatography, with chloroform/methanol volume ratio 1:1 wash-out, monitor and be divided into four parts with thin-layer chromatography (TLC); By its second section again through MCI column chromatography, with methanol/water gradient elution (volume ratio is followed successively by 30:70,50:50,80:20,100:0); By the wash-out part that wherein volume ratio of methanol/water is 50:50 again through silica gel column chromatography, with sherwood oil/acetone, (volume ratio is followed successively by 90:10,80:20,70:30,50:50) carry out gradient elution, by the wash-out part that wherein sherwood oil/acetone volume ratio is 70:30 again through preparation HPLC with acetonitrile/water volume ratio 85:15 wash-out, obtain element 2.1mg in xylocarpus granatum heptan.
The xylocarpus granatum element in heptan of the present invention that separation and purification obtains is colourless jelly, and molecular formula is defined as C by HR-ESI (high resolution mass spectrum)
36h
58o
7.
1h and
13c data are as shown in table 1 below.
Table 1 xylocarpus granatum element in heptan
1h and
13c NMR (ppm, CDCl
3)
| no. | δ H(mult,J，Hz) | δ C |
| 1 | 1.46,m | 33.5 |
| 2α | 1.62,m | 22.6 |
| 2β | 1.91,m | - |
| 3 | 4.69,s | 78.1 |
| 4 | - | 36.4 |
| 5 | 2.01,m | 41.8 |
| 6 | 1.66-1.77 | 23.6 |
| 7 | 3.91,s | 72.0 |
| 8 | - | 44.4 |
| 9 | 2.01,m | 41.7 |
| 10 | - | 37.5 |
| 11α | 1.71,m | 16.2 |
| 11β | 1.49,m | - |
| 12α | 1.80,m | 32.7 |
| 12β | 1.46,m | - |
| 13 | - | 46.9 |
| 14 | - | 162.2 |
| 15 | 5.45,s | 119.3 |
| 16α | 2.08,m | 34.7 |
| 16β | 2.22,m | - |
| 17 | 1.68,m | 57.4 |
| 18 | 1.07,s | 19.3 |
| 19 | 0.92,s | 15.2 |
| 20 | 2.33，m | 45.8 |
| 21 | 4.81,d(3.6) | 109.5 |
| 22 | 1.93,m | 33.7 |
| 23 | 4.26,dd(11.1,4.7) | 77.0 |
| 24 | 3.25,s | 75.3 |
| 25 | - | 73.1 |
| 26 | 1.27,s | 26.4 |
| 27 | 1.30,s | 26.5 |
| 28 | 0.87,s | 27.7 |
| 29 | 0.92,s | 21.7 |
| 30 | 1.06,s | 27.8 |
| 1' | - | 167.6 |
| 2' | - | 129.2 |
| 3' | 6.85,q(7.2) | 136.7 |
| 4' | 1.78,d(6.9) | 12.2 |
| 5' | 1.84,s | 14.4 |
| 21-OMe | 3.36,s | 55.7 |
Xylocarpus granatum element in heptan:
-46 (c 0.185, MeOH); IR (KBr) v
max3440,2930,1645,1460,1382,1229,1150,1034cm
-1; HRESIMS m/z625.4072[M+Na]
+(calcd fprC
36h
58o
7na 625.4080)
The test of embodiment 2 anti-tumor activities
1, laboratory sample and experimental technique
The preparation of sample solution: test sample is the sterling compound xylocarpus granatum element in heptan of preparation in above-described embodiment 1.Accurately take appropriate sample, be mixed with the solution of desired concn with DMSO, for active testing.
Anti-tumor activity test:
The anti tumor activity in vitro of A-549 human breast cancer cell is measured, adopted sulphonyl rhodamine B (SRB) method.To be inoculated in 96 hole microtest plates by 90 μ L/ holes in the A-549 of logarithmic growth human breast cancer cell, add the solution 10 μ L/ holes of xylocarpus granatum element in heptan of the present invention after cultivation 24h, each concentration is three multiple holes, separately establishes acellular zeroing hole.Tumour cell is at 37 ° of C, 5%CO
2under condition, cultivate after 72h, discard training liquid, add 100 μ L/ hole 10%TCA in the fixing 1h of 4 ° of C, distilled water wash three times, seasoning.Add 4mg/mL SRB (Sigma) staining fluid 100 μ L/ holes, room temperature dyeing 15 minutes, 1% aqueous acetic acid washing three times, seasoning.Add the 100 μ L/ hole 10mM Tris-base aqueous solution.Then be marked on 560nm wavelength with enzyme and measure optical density(OD) (OD
560) value.Calculate the inhibiting rate of analyte to growth of tumour cell by (control group OD value-administration group OD value)/control group OD value × 100%.
2, experimental result
To the inhibiting rate % of A-549 growth of tumour cell
3, conclusion
Xylocarpus granatum element in heptan has certain inhibition activity to A-549 human breast cancer cell under above-mentioned concentration.Therefore, xylocarpus granatum element in heptan of the present invention can be used for medicine or the lead compound of preparation treatment tumour, especially for medicine and the lead compound for the treatment of mammary cancer, lung cancer.
Claims (9)
1. the following triterpene compound xylocarpus granatum element in heptan of chemical structural formula:
2. a preparation method for triterpene compound xylocarpus granatum element in heptan claimed in claim 1, is characterized in that, described triterpene compound xylocarpus granatum element in heptan extracts first to separate and obtains from xylocarpus granatum Xylocarpus granatum Koenig.
3. the preparation method of triterpene compound xylocarpus granatum element in heptan according to claim 2, is characterized in that, the method comprises the following steps:
1) extract: by xylocarpus granatum branches and leaves methanol extraction, after gained extracting solution is concentrated, obtain crude extract; By water-soluble this crude extract, after suspendible is even, be extracted with ethyl acetate, after gained extraction liquid is concentrated, obtain ethyl acetate extract;
2) separate: above-mentioned ethyl acetate extract is carried out to Sephadex LH-20 gel filtration chromatography, with chloroform/methanol volume ratio 1:1 wash-out, monitor and be divided into four parts with thin-layer chromatography (TLC), wherein, second section is again through MCI column chromatography, with methanol/water gradient elution; Wherein, methanol/water volume ratio 50:50 wash-out part is further through silica gel column chromatography, with sherwood oil/acetone gradient elution, wherein, sherwood oil/acetone volume ratio 70:30 wash-out part with acetonitrile/water volume ratio 85:15 wash-out, obtains xylocarpus granatum element in heptan finally by preparation HPLC.
4. the preparation method of triterpene compound xylocarpus granatum element in heptan according to claim 3, is characterized in that, 1) in extraction step, the method that described extraction adopts is that methyl alcohol diacolation extracts.
5. the preparation method of triterpene compound xylocarpus granatum element in heptan according to claim 3, is characterized in that, 2) in separating step, the concentration of methanol/water gradient elution is followed successively by volume ratio 30:70,50:50,80:20,100:0.
6. the preparation method of triterpene compound xylocarpus granatum element in heptan according to claim 3, is characterized in that, 2) in separating step, the concentration of sherwood oil/acetone gradient elution is followed successively by volume ratio 90:10,80:20,70:30,50:50.
7. the preparation method of triterpene compound xylocarpus granatum element in heptan according to claim 3, is characterized in that, 2) in separating step, the filler of described preparation HPLC is anti-phase octadecyl silane.
8. the purposes of triterpene compound xylocarpus granatum element in heptan claimed in claim 1 in the medicine of preparation treatment tumour.
9. purposes claimed in claim 8, wherein, described tumour is mammary cancer or lung cancer.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107955060A (en) * | 2016-11-07 | 2018-04-24 | 暨南大学 | Limonin compound and its dimer |
| US11291647B2 (en) | 2018-08-30 | 2022-04-05 | Nisarga Biotech Private Limited | Process for preparation of CO2 extract of Azadirachta indica and herbal compositions thereof for treatment of cancers |
| EP4342482A1 (en) | 2022-09-23 | 2024-03-27 | Laurenz Vorderwülbecke | Neem tree leaves extract |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093187A (en) * | 2010-12-03 | 2011-06-15 | 沈阳药科大学 | Polyketone compounds and application thereof |
| JP2012184195A (en) * | 2011-03-04 | 2012-09-27 | Chiba Univ | Xylogranin a and xylogranin b |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093187A (en) * | 2010-12-03 | 2011-06-15 | 沈阳药科大学 | Polyketone compounds and application thereof |
| JP2012184195A (en) * | 2011-03-04 | 2012-09-27 | Chiba Univ | Xylogranin a and xylogranin b |
Non-Patent Citations (2)
| Title |
|---|
| JIANXIN CUI等: "Protolimonoids and Limonoids from the Chinese Mangrove Plant Xylocarpus granatum", 《HELVETICA CHIMICA ACTA》 * |
| KHANITHA PUDHOM 等: "Protoxylocarpins F-H, Protolimonoids from Seed Kernels of Xylocarpus granatum", 《JNOURNAL OF NATURAL PRODUCTS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107955060A (en) * | 2016-11-07 | 2018-04-24 | 暨南大学 | Limonin compound and its dimer |
| CN107955060B (en) * | 2016-11-07 | 2018-11-30 | 暨南大学 | Limonin compound and its dimer |
| US11291647B2 (en) | 2018-08-30 | 2022-04-05 | Nisarga Biotech Private Limited | Process for preparation of CO2 extract of Azadirachta indica and herbal compositions thereof for treatment of cancers |
| EP4342482A1 (en) | 2022-09-23 | 2024-03-27 | Laurenz Vorderwülbecke | Neem tree leaves extract |
| WO2024062023A1 (en) | 2022-09-23 | 2024-03-28 | Vorderwuelbecke Laurenz | Neem tree leaves extract |
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