CN102796113B - Xanthone compound, and preparation method and application thereof - Google Patents
Xanthone compound, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a xanthone compound, and a preparation method and application thereof. The xanthone compound is separated from garcinia frutex and named oliganthoneA; the molecular formula is C30H34O7; and the structure of the xanthone compound is disclosed in the specification. The preparation method of the xanthone compound is implemented by extractum extraction, silica gel column chromatography and high pressure liquid chromatography separation by using the garcinia frutex as the raw material. The method concretely comprises the following steps: pulverizing the garcinia frutex sample, extracting, merging the extracting solutions, filtering, and concentrating under reduced pressure to obtain an extractum; extracting the extractum with an organic solvent, filling silica gel in a column, and carrying out silica gel column chromatography on the extract phase; carrying out gradient elution with a solution of n-hexane and acetone in a volume ratio of 1:0-0:1; and separating and purifying the 1:0 part by reverse-phase column chromatography, gel column chromatography and high pressure liquid chromatography to obtain the xanthone compound. The test proves that the xanthone compound has high apoptosis and cell toxicant induction activities and has the advantages of novel structure and high activity; and the xanthone compound can be used as a precursor compound for antineoplastic drugs.
Description
Technical field
The invention belongs to effective ingredients in plant extractive technique field, be specifically related to a kind of xanthone compounds and preparation method thereof and application.
Background technology
Apoptosis is a kind of procedural necrocytosis, is a kind of basic biological phenomena of cell, plays a part necessary when multicellular organism is removed unwanted or abnormal cell.Apoptosis is different from necrocytosis, apoptosis is not a passive process, but active process, it relates to the effect of activation, expression and the regulation and control etc. of series of genes, it is not a kind of phenomenon of autologous damage under pathological conditions, but a kind of death process of initiatively striving for for adapting to better living environment.Apoptosis is the strict process of controlling of polygene.These genes are very conservative between kind, if Bcl-2 family, caspase family, oncogene are as C-myc, cancer suppressor gene P53 etc., the disorder of apoptotic process may be direct or indirect with having of numerous disease relation, as tumour, autoimmune disorder etc.Can bring out apoptotic factor a lot, as ray, medicine etc.Therefore the apoptosis of inducing cancer cell is considered to effective cancer treatment method.Caspase family gene plays very important effect in the process of mediating apoptosis, wherein caspase-3 is crucial execution molecule, it brings into play function in many approach of apoptosis signal transduction, the activation of caspase-3 gene means the event that apoptotic most critical occurs, be used to monitor the degree that apoptosis occurs, the natural product that therefore can activate caspase-3 gene may be the very potential primer that is developed as cancer therapy drug.
Research shows, garcinia plant is rich in isopentene group xanthone and the benzophenone with activity inducing apoptosis and cytotoxic activity.Cortex Garciniae oliganthae belongs to the shrub of guttiferae garcinia, and high 1 – 3m is distributed in the woods of Hainan and Guangdong two province's height above sea level 200 – 1200 m.Among the people, this kind of plant has anti-inflammatory, heat-clearing toxin-expelling functions, is used to have a toothache, der Halsschmerz, en and scald.This plant is at Laos's medicinal plant that is also used as among the people.The present invention is separated from Cortex Garciniae oliganthae obtains a novel xanthone compound, and this compound has significant activity inducing apoptosis and cytotoxic activity.
Summary of the invention
The first object of the present invention is to provide a kind of xanthone compounds; The second object is to provide the preparation method of described xanthone compounds; The 3rd object is to provide the application of described xanthone compounds in preparing antitumor drug.
The first object of the present invention is achieved in that described xanthone compounds is that separation obtains from garcinia shrub, called after oliganthone A, and its molecular formula is C
30h
34o
7, there is following structure:
The second object of the present invention is to realize like this, described xanthone compounds preparation method, to take garcinia shrub as raw material, through through medicinal extract extraction, organic solvent extraction, the decolouring of MCI post, silica gel column chromatography, reversed phase column chromatography, gel filtration chromatography, high pressure liquid chromatography separating step, specifically comprise:
A, medicinal extract extract: garcinia shrub sample is crushed to 20 ~ 40 orders, and with organic solvent supersound extraction 2 ~ 4 times, each 30 ~ 60min, extracting solution merges, and filters, during the volume of concentrating under reduced pressure extracting solution to 1/4 ~ 1/2, standing rear filtering throw out, is then condensed into medicinal extract;
B, organic solvent extraction: it is the water of 1.5 ~ 3 times that A step gained medicinal extract adds weight ratio, and the organic solvent extraction of 1 ~ 2 times of volume of water 3 ~ 5 times, merges the organic solvent extraction of each separation mutually, and concentrating under reduced pressure becomes medicinal extract;
C, MCI post decolouring B step gained medicinal extract carry out chromatography with the reversed material MCI of 4 ~ 5 times of weight ratios, and the methyl alcohol with 90 ~ 99% carries out wash-out;
D, silica gel column chromatography: the medicinal extract after decolouring carries out silica gel column chromatography with 160-200 order silica gel dress posts of 6 ~ 8 times of amounts of weight ratio; Normal hexane-acetone soln that the volume proportion of take is 1:0 ~ 0:1 carries out gradient elution, collects each several part elutriant concentrated, merges identical part;
E, reversed phase column chromatography: the 1:0 part of D step elutriant is further carried out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part;
F, gel filtration chromatography: the methanol aqueous solution wash-out part of the 85:15 of E step elutriant is further carried out chromatography with gel column Sephadex LH-20, with organic solvent, rushes post, collect containing the elutriant of target compound also concentrated;
G, high pressure liquid chromatography are separated: the xanthone compounds described in further the obtaining with high pressure liquid chromatography separation and purification of F step elutriant.
The structure of the xanthone compounds of preparing with aforesaid method is to measure out by the following method:
The compounds of this invention is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030); UV spectrum (solvent is methyl alcohol),
λ max(log ε): 269 (4.08), 228 (2.68), 205 (2.34) nm; Infrared spectra (pressing potassium bromide troche)
ν max3447,3377,3,133 2286,1656,1597,1439,1266,1122,1064,861,817,621,569 cm
-1; HRESIMS shows the compounds of this invention quasi-molecular ion peak
m/z[507.2384 M+H]
+(calculated value is 507.2384), in conjunction with
13c and
1h NMR spectrum (figure-1 and figure-2, attribution data is in Table-1) provides its molecular formula C
30h
34o
7, degree of unsaturation is 14.
Carbon spectrum (
table 1) and DEPT spectrum in show 7 methyl, 3 methylene radical, 6 methynes (wherein 5 for unsaturated) and 14 quaternary carbons (9 unsaturated, 1 oxidations, 3 carbonyls).Infrared absorption shows there is hydroxyl (3447 cm
-1) and xanthone carbonyl (1656 cm
-1).Hydrogen spectrum signal
δ h4.89 – 4.91 and 4.85 – 4.89 (2H, m, H-17, H-22), 2.60 – 2.66 (m) and 2.70 – 2.75 (m) (each 2H, m, H-16 and H-21), 1.56,1.64,1.53 and 1.58 (each 3H, s, H-19, H-20, H-24, and H-25) illustrate in this compound and exist a gem bis (3-methylbut-2-enyl) group to be connected to a sp
3on carbon atom
δ c58.5 (C-1).In carbon spectrum except xanthone carbonyl signal
δ coutside 180.1 (C-9), also has a carbonyl signal
δ c212.2 (C-2) illustrate that this compound should be the skeleton of xanthenedione.From
1h and
13this compound of C NMR spectrum judgement contains three isopentene groups.In two-dimensional spectrum, C-6 is relevant to the HMBC of 8-OH, H-11 and H-12, the HMBC related description of H-12 and oxidized quaternary carbon C-13 and two methyl carbon C-14 and C-15, two isopentene groups in gem bis (3-methylbut-2-enyl) group, also have an isopentene group formed dimethyl pyrans be connected to C-6 (
δ c162.0) position.Two isopentene groups of the relevant susceptible of proof of HMBC of H-16 and C-1 and H-21 and C-2 are in C-1 position.In hydrogen spectrum, also have a fragrant proton and C-6, C-7, C-8a, has HMBC relevant with C-10a, illustrate its be H-5 (
fig. 3).
Compare with conventional xanthone, C-3 in the compounds of this invention carbon spectrum and hydrogen spectrum, C-26, C-27, and the chemical shift of C-28 has larger variation.By carefully analyzing its two-dimensional spectrum, find,
δ h5.15 (br) (OH-3) with
δ c212.2 (C-2), 164.0 (C-4a), have HMBC relevant with 42.8 (C-26), and deducibility C-4a is connected with C-3 accordingly.In the carbon spectrum of the compounds of this invention, do not find C-4 signal, illustrate that this is the xanthone of the new skeleton of a class.
δ h2.77 – 2.82 (m) (H-26) with
δ c212.2 (C-2), 75.9 (C-3), 208.4 (C-27), 31.9 (C-28), and
δ h2.26 (s) (H-28) with
δ c42.8 (C-26), the HMBC related description C-3 of 208.4 (C-27) also has an acetonyl to replace.So far the structure of this compound is determined, and called after oliganthone A.
the compound of table-1. 1h NMR and
13(solvent is CDCl to C NMR data
3)
| Atom | δ H | δ C | Atom | δ H | δ C |
| 1 | ? | 58.5 s | 15 | 1.81 (s) | 28.2 q |
| 2 | ? | 212.2 s | 16 | 2.60–2.66 (m) | 33.6 t |
| 3 | ? | 75.9 s | 17 | 4.89–4.91 (m) | 119.7 d |
| 5 | 6.30 (s) | 100.7 d | 18 | ? | 135.9 s |
| 6 | ? | 162.0 s | 19 | 1.56 (s) | 17.7 q |
| 7 | ? | 101.7 s | 20 | 1.64 (s) | 25.9 q |
| 8 | ? | 159.7 s | 21 | 2.70–2.75 (m) | 34.6 t |
| 9 | ? | 180.1 s | 22 | 4.85–4.89 (m) | 118.9 d |
| 4a | ? | 164.0 s | 23 | ? | 135.6 s |
| 8a | ? | 106.0 s | 24 | 1.53 (s) | 17.7 q |
| 9a | ? | 119.8 s | 25 | 1.58 (s) | 25.8 q |
| 10a | ? | 152.2 s | 26 | 2.77–2.82 (m) | 42.8 t |
| 11 | 6.63 (d, 10.0) | 114.4 d | 27 | ? | 208.4 s |
| 12 | 5.61 (d, 10.0) | 127.8 d | 28 | 2.26 (s) | 31.9 q |
| 13 | ? | 78.3 s | 3-OH | 5.15 (br) | ? |
| 14 | 1.49 (s) | 28.4 q | 8-OH | 12.66 (s) | ? |
The 3rd object of the present invention is achieved in that and is about to the preparation that described xanthone compounds is applied to antitumor drug.
Xanthone compounds of the present invention is separated first, has determined for xanthone compounds, and characterized its concrete structure by nucleus magnetic resonance and measuring method of mass spectrum.Apoptosis-induced experiment through to HeLa-C3 cell can effectively reduce below the YFP/CFP ratio to 3 of HeLa-C3 cell when lower concentration 25 μ M in 72 hours, had good cell death inducing effect.Through the anti-cell cytotoxic activity of HeLa cell is detected to test, test-results clearly illustrates that, IC
50value is 7.27 ± 0.23 μ M.Above result has disclosed compound of the present invention good application prospect in preparing antitumor drug.The compounds of this invention novel structure activity is good, can be used as the guiding compound of antitumor drug.
Accompanying drawing explanation
Fig. 1 be compound carbon-13 nmr spectra (
13c NMR);
Fig. 2 be compound proton nmr spectra (
1h NMR);
Fig. 3 is that the main HMBC of compound is relevant.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is further illustrated, but never in any form the present invention is limited, and any conversion or the improvement based on training centre of the present invention, done, all fall into protection scope of the present invention.
Xanthone compounds of the present invention is that separation obtains from garcinia shrub, called after oliganthone A, and molecular formula is C
30h
34o
7, its preparation method is to take garcinia shrub as raw material, through medicinal extract extraction, organic solvent extraction, the decolouring of MCI post, silica gel column chromatography, reversed phase column chromatography, gel filtration chromatography, high pressure liquid chromatography separating step, specifically comprises:
It is that garcinia shrub sample is crushed to 20 ~ 40 orders that described medicinal extract extracts, with organic solvent supersound extraction 2 ~ 4 times, and each 30 ~ 60min, extracting solution merges, and filters, during the volume of concentrating under reduced pressure extracting solution to 1/4 ~ 1/2, standing rear filtering throw out, is then condensed into medicinal extract;
Described organic solvent extraction is that medicinal extract extraction step gained medicinal extract is added to weight ratio is the water of 1.5 ~ 3 times, and the organic solvent extraction of 1 ~ 2 times of volume of water 3 ~ 5 times, merges the organic solvent extraction of each separation mutually, and concentrating under reduced pressure becomes medicinal extract;
Described MCI post decolouring is that organic solvent extraction step gained medicinal extract is carried out to chromatography with the reversed material MCI of 4 ~ 5 times of weight ratios, and the methyl alcohol with 90 ~ 99% carries out wash-out;
Described silica gel column chromatography is that the medicinal extract after decolouring is carried out to silica gel column chromatography with 160-200 order silica gel dress posts of 6 ~ 8 times of amounts of weight ratio; Normal hexane-acetone soln that the volume proportion of take is 1:0 ~ 0:1 carries out gradient elution, collects each several part elutriant concentrated, merges identical part;
Described reversed phase column chromatography is that the 1:0 part of silica gel column chromatography step elutriant is further carried out to chromatography with reversed material C-18 dress post, the methanol-water solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collect each several part elutriant concentrated, merge identical part;
Described gel filtration chromatography is that the methanol-water eluant solution part of the 85:15 of reversed phase column chromatography step elutriant is further carried out to chromatography with gel column Sephadex LH-20, with organic solvent, rushes post, collects containing the elutriant of target compound also concentrated;
Described high pressure liquid chromatography separation is that gel filtration chromatography step elutriant further obtained to described xanthone compounds with high pressure liquid chromatography separation and purification.
In described medicinal extract extraction step, solvent can adopt any one in the acetone, ethanol, methyl alcohol of 70%-100%.
The organic solvent extracting in described organic solvent extraction step can adopt any one in ethyl acetate, chloroform, methylene dichloride, ether, sherwood oil, benzene.
In described MCI post decolouring step, medicinal extract is before silica gel column chromatography rough segmentation, with using 80 ~ 100 order silica gel silica gel mixed samples of 0.8 ~ 1.2 times of medicinal extract weight ratio after the acetone solution of 1.5 ~ 3 times of amounts of weight ratio.
In described silica gel column chromatography step, normal hexane-acetone soln volume proportion is 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1.
In described reversed phase column chromatography step, methanol-water liquor capacity proportioning is 80:20,85:15,90:10,95:5,100:0.
In described gel filtration chromatography step, organic solvent can adopt any one in chloroform, methyl alcohol, acetone.
The separation and purification of described high pressure liquid chromatography separating step mesohigh liquid chromatography is that the methyl alcohol of employing 75 ~ 90% is moving phase, flow velocity 2.5 ~ 4mL/min, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 45 ~ 55 μ L collect the chromatographic peak of 9.1 ~ 39.5 min, repeatedly cumulative after evaporate to dryness.
Described high pressure liquid chromatography separation and purification is that the methyl alcohol of employing 80 ~ 85% is moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 47 ~ 49 μ L collect the chromatographic peak of 16.5 ~ 27.3min, repeatedly cumulative after evaporate to dryness.
The application of xanthone compounds of the present invention is its application in preparing antitumor drug.
Raw materials used area and the kind of not being subject to of Cortex Garciniae oliganthae of the present invention limits, and is also not limited only to garcinia shrub, all can realize the present invention, and to derive from the Cortex Garciniae oliganthae in Hainan, the present invention will be further described below:
Embodiment 1
Get dry Cortex Garciniae oliganthae branch and leaf sample 5.5 kg are crushed to 40 orders, the ethanol ultrasonic extraction with 70% 2 times, each 60min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/4, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 436 g.After the water dissolution of medicinal extract by 1.5 times of amounts of weight ratio, use and the dichloromethane extraction of 1.5 times of volumes of water 3 times, dichloromethane extraction is the concentrated medicinal extract 242g that to obtain mutually.Dichloromethane extraction is mutually again with the decolouring of MCI reversed-phase column, and with 90% methanol-eluted fractions, elutriant concentrates to obtain 130g study.Sample after decolouring adds the 100 order silica gel mixed samples of 156g for 3 times of acetone solutions, the 200 order silica gel dress posts of 1.04Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 5.05g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 80% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 48 μ L, and the chromatographic peak of collection 27.3min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 2
Get dry Cortex Garciniae oliganthae branch and leaf sample 3 kg are crushed to 20 orders, supersound extraction 4 times for the ethanol with 99%, each 30min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/2, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 240 g.After the water dissolution of medicinal extract by 2 times of amounts of weight ratio, use and the dichloromethane extraction of 2 times of volumes of water 4 times, dichloromethane extraction is the concentrated medicinal extract 135g that to obtain mutually.Dichloromethane extraction is mutually again with the decolouring of MCI reversed-phase column, and with 95% methanol-eluted fractions, elutriant concentrates to obtain 70g study.Sample after decolouring adds the 100 order silica gel mixed samples of 56g for 1.5 times of acetone solutions, the 200 order silica gel dress posts of 0.42Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 3.12g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 85% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 49 μ L, and the chromatographic peak of collection 16.5min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 3
Get dry Cortex Garciniae oliganthae branch and leaf sample 4 kg are crushed to 30 orders, supersound extraction 3 times for the ethanol with 80%, each 40min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/3, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 336 g.After the water dissolution of medicinal extract by 3 times of amounts of weight ratio, use with the ethyl acetate of 1 times of volume of water and extract 5 times, the concentrated medicinal extract 187g that to obtain of ethyl acetate extraction phase.Ethyl acetate extraction phase is again with the decolouring of MCI reversed-phase column, and with 99% methanol-eluted fractions, elutriant concentrates to obtain 95g study.Sample after decolouring adds the 90 order silica gel mixed samples of 95g for 2 times of acetone solutions, the 180 order silica gel dress posts of 0.67Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 4.2g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 83% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 49 μ L, and the chromatographic peak of collection 22.1min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 4
Get dry Cortex Garciniae oliganthae branch and leaf sample 3 kg are crushed to 20 orders, supersound extraction 3 times for the methyl alcohol with 70%, each 60min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/4, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 245g.The chloroform extraction of 2 times of volumes of water after the water dissolution of weight ratio 1.5 times of amounts 5 times for medicinal extract, chloroform extraction is the concentrated medicinal extract 119g that to obtain mutually.Chloroform extraction is mutually again with the decolouring of MCI reversed-phase column, and with 97% methanol-eluted fractions, elutriant concentrates to obtain 61g study.Sample after decolouring adds the 90 order silica gel mixed samples of 156g for 3 times of acetone solutions, the 180 order silica gel dress posts of 1.04Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 2.96g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 75% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 45 μ L, and the chromatographic peak of collection 38.1min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 5
Get dry Cortex Garciniae oliganthae branch and leaf sample 6 kg are crushed to 40 orders, supersound extraction 2 times for the methyl alcohol with 99%, each 60min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/3, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 497 g.The extracted with diethyl ether of 1.5 times of volumes of water after the water dissolution of weight ratio 3 times of amounts 4 times for medicinal extract, extracted with diethyl ether is the concentrated medicinal extract 265g that to obtain mutually.Extracted with diethyl ether is mutually again with the decolouring of MCI reversed-phase column, and with 98% methanol-eluted fractions, elutriant concentrates to obtain 147g study.Sample after decolouring adds the 90 order silica gel mixed samples of 117.6g for 2 times of acetone solutions, the 200 order silica gel dress posts of 0.882Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 6.1g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 80% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 47 μ L, and the chromatographic peak of collection 27.3min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 6
Get dry Cortex Garciniae oliganthae branch and leaf sample 3.5 kg are crushed to 40 orders, supersound extraction 4 times for the methyl alcohol with 85%, each 45min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/4, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 293 g.The petroleum ether extraction of 2 times of volumes of water after the water dissolution of weight ratio 2 times of amounts 3 times for medicinal extract, petroleum ether extraction is the concentrated medicinal extract 162g that to obtain mutually.Petroleum ether extraction is mutually again with the decolouring of MCI reversed-phase column, and with 92% methanol-eluted fractions, elutriant concentrates to obtain 89g study.Sample after decolouring adds the 100 order silica gel mixed samples of 106.8g for 1.5 times of acetone solutions, the 170 order silica gel dress posts of 0.712Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 3.23g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 85% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 49 μ L, and the chromatographic peak of collection 16.5min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 7
Get dry Cortex Garciniae oliganthae branch and leaf sample 4.3kg is crushed to 30 orders, supersound extraction 4 times for the acetone with 99%, each 30min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/2, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 368 g.Medicinal extract extracts 3 times with the benzene of 2 times of volumes of water after the water dissolution of 2.5 times of amounts of weight ratio, the concentrated medicinal extract 179g that to obtain of benzene extraction phase.Benzene extraction phase is again with the decolouring of MCI reversed-phase column, and with 95% methanol-eluted fractions, elutriant concentrates to obtain 102g study.Sample after decolouring adds the 100 order silica gel mixed samples of 101g for 3 times of acetone solutions, the 180 order silica gel dress posts of 0.835Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 4.5g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part and obtains 14 components; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 90% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 55 μ L, and the chromatographic peak of collection 9.6min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 8
Get dry Cortex Garciniae oliganthae branch and leaf sample 5.6 kg are crushed to 20 orders, supersound extraction 2 times for the acetone with 80%, each 50min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/2, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 453 g.Medicinal extract extracts 5 times with the benzene of 1 times of volume of water after the water dissolution of 2 times of amounts of weight ratio, the concentrated medicinal extract 261g that to obtain of benzene extraction phase.Benzene extraction phase is again with the decolouring of MCI reversed-phase column, and with 99% methanol-eluted fractions, elutriant concentrates to obtain 142g study.Sample after decolouring adds the 80 order silica gel mixed samples of 150g for 2 times of acetone solutions, the 200 order silica gel dress posts of 1.14Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 5.05g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 85% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 45 μ L, and the chromatographic peak of collection 16.5min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 9
Get dry Cortex Garciniae oliganthae branch and leaf sample 3.8kg is crushed to 30 orders, supersound extraction 3 times for the acetone with 70%, each 35min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/4, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 367g.Medicinal extract extracts 4 times by the ethyl acetate of 1.5 times of volumes of water after the water dissolution of 3 times of amounts of weight ratio, the concentrated medicinal extract 186g that to obtain of ethyl acetate extraction phase.Ethyl acetate extraction phase is again with the decolouring of MCI reversed-phase column, and with 98% methanol-eluted fractions, elutriant concentrates to obtain 130g study.Sample after decolouring adds the 100 order silica gel mixed samples of 141g for 2 times of acetone solutions, the 200 order silica gel dress posts of 0.92Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 4.05g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 90% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 47 μ L, and the chromatographic peak of collection 9.6min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 10
Get dry Cortex Garciniae oliganthae branch and leaf sample 3.6kg is crushed to 20 orders, supersound extraction 2 times for the ethanol with 79%, each 55min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/3, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 348 g.The petroleum ether extraction of 2 times of volumes of water after the water dissolution of weight ratio 1.5 times of amounts 4 times for medicinal extract, petroleum ether extraction is the concentrated medicinal extract 159g that to obtain mutually.Petroleum ether extraction is mutually again with the decolouring of MCI reversed-phase column, and with 92% methanol-eluted fractions, elutriant concentrates to obtain 100g study.Sample after decolouring adds the 80 order silica gel mixed samples of 90g for 3 times of acetone solutions, the 160 order silica gel dress posts of 0.78Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 3.6g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 85% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 55 μ L, and the chromatographic peak of collection 16.5min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 11
Get dry Cortex Garciniae oliganthae branch and leaf sample 6.5 kg are crushed to 40 orders, supersound extraction 3 times for the methyl alcohol with 83%, each 45min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/3, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 588g.The extracted with diethyl ether of 1 times of volume of water after the water dissolution of weight ratio 3 times of amounts 5 times for medicinal extract, extracted with diethyl ether is the concentrated medicinal extract 298g that to obtain mutually.Extracted with diethyl ether is mutually again with the decolouring of MCI reversed-phase column, and with 95% methanol-eluted fractions, elutriant concentrates to obtain 223g study.Sample after decolouring adds the 100 order silica gel mixed samples of 178.4g for 2 times of acetone solutions, the 160 order silica gel dress posts of 1.338Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 5.98g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 82% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 50 μ L, and the chromatographic peak of collection 26.3min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 12
Get dry Cortex Garciniae oliganthae branch and leaf sample 5 kg are crushed to 20 orders, supersound extraction 4 times for the acetone with 75%, each 50min, extracting solution merges, and filters concentrating under reduced pressure extracting solution to 1/4, standing rear filtering throw out, is then condensed into medicinal extract, obtains medicinal extract 396g.The chloroform extraction of 1 times of volume of water after the water dissolution of weight ratio 2 times of amounts 4 times for medicinal extract, chloroform extraction is the concentrated medicinal extract 198g that to obtain mutually.Chloroform extraction is mutually again with the decolouring of MCI reversed-phase column, and with 99% methanol-eluted fractions, elutriant concentrates to obtain 130g study.Sample after decolouring adds the 80 order silica gel mixed samples of 0.83g for 3 times of acetone solutions, the 200 order silica gel dress posts of 1.04Kg carry out silica gel column chromatography, it by volume ratio, is normal hexane-acetone soln gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1, TLC monitoring merges identical part, obtains 8 parts; The 1:0 part 5.05g of elutriant carries out chromatography with reversed material C-18 dress post, and the methanol aqueous solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated, merges identical part; 85% the methanol aqueous solution wash-out part of reversed phase chromatography elutriant is further carried out chromatography with Sephadex LH-20 gel column, with pure methyl alcohol, rushes post, collects containing the elutriant of target compound and concentrates; With the elutriant of gel filtration chromatography, with 80% methyl alcohol, be further moving phase, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 48 μ L, and the chromatographic peak of collection 28.5min, evaporate to dryness after repeatedly adding up, can this new compound.
Embodiment 13
The compound of getting embodiment 1 preparation is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030);
Measuring method is: with nucleus magnetic resonance, in conjunction with other spectroscopic technique, identify structure.
1) UV spectrum (solvent is methyl alcohol),
λ max(log ε): 269 (4.08), 228 (2.68), 205 (2.34) nm;
2) infrared spectra (pressing potassium bromide troche)
ν max3447,3377,3,133 2286,1656,1597,1439,1266,1122,1064,861,817,621,569 cm
-1;
3) HRESIMS shows the compounds of this invention quasi-molecular ion peak
m/z[507.2384 M+H]
+(calculated value is 507.2384), in conjunction with
13c and
1h NMR spectrum (figure-1 and figure-2, attribution data is in Table-1) provides its molecular formula C
30h
34o
7, degree of unsaturation is 14.
Carbon spectrum (
table 1) and DEPT spectrum in show 7 methyl, 3 methylene radical, 6 methynes (wherein 5 for unsaturated) and 14 quaternary carbons (9 unsaturated, 1 oxidations, 3 carbonyls).Infrared absorption shows there is hydroxyl (3447 cm
-1) and xanthone carbonyl (1656 cm
-1).Hydrogen spectrum signal
δ h4.89 – 4.91 and 4.85 – 4.89 (2H, m, H-17, H-22), 2.60 – 2.66 (m) and 2.70 – 2.75 (m) (each 2H, m, H-16 and H-21), 1.56,1.64,1.53 and 1.58 (each 3H, s, H-19, H-20, H-24, and H-25) illustrate in this compound and exist a gem bis (3-methylbut-2-enyl) group to be connected to a sp
3on carbon atom
δ c58.5 (C-1).In carbon spectrum except xanthone carbonyl signal
δ coutside 180.1 (C-9), also has a carbonyl signal
δ c212.2 (C-2) illustrate that this compound should be the skeleton of xanthenedione.From
1h and
13this compound of C NMR spectrum judgement contains three isopentene groups.In two-dimensional spectrum, C-6 is relevant to the HMBC of 8-OH, H-11 and H-12, the HMBC related description of H-12 and oxidized quaternary carbon C-13 and two methyl carbon C-14 and C-15, two isopentene groups in gem bis (3-methylbut-2-enyl) group, also have an isopentene group formed dimethyl pyrans be connected to C-6 (
δ c162.0) position.Two isopentene groups of the relevant susceptible of proof of HMBC of H-16 and C-1 and H-21 and C-2 are in C-1 position.In hydrogen spectrum, also have a fragrant proton and C-6, C-7, C-8a, has HMBC relevant with C-10a, illustrate its be H-5 (
fig. 3).
Compare with conventional xanthone, C-3 in the compounds of this invention carbon spectrum and hydrogen spectrum, C-26, C-27, and the chemical shift of C-28 has larger variation.By carefully analyzing its two-dimensional spectrum, find,
δ h5.15 (br) (OH-3) with
δ c212.2 (C-2), 164.0 (C-4a), have HMBC relevant with 42.8 (C-26), and deducibility C-4a is connected with C-3 accordingly.In the carbon spectrum of the compounds of this invention, do not find C-4 signal, illustrate that this is the xanthone of the new skeleton of a class.
δ h2.77 – 2.82 (m) (H-26) with
δ c212.2 (C-2), 75.9 (C-3), 208.4 (C-27), 31.9 (C-28), and
δ h2.26 (s) (H-28) with
δ c42.8 (C-26), the HMBC related description C-3 of 208.4 (C-27) also has an acetonyl to replace.So far the structure of this compound is determined, and called after oliganthone A.
Embodiment 14
The compound of getting embodiment 2 preparations is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030).Measuring method is identical with embodiment 13, confirms that the compound of embodiment 2 preparations is described xanthone compounds---oliganthone A.
Embodiment 15
The compound of getting embodiment 3 preparations is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030).Measuring method is identical with embodiment 13, confirms that the compound of embodiment 3 preparations is described xanthone compounds---oliganthone A.
Embodiment 16
The compound of getting embodiment 4 preparations is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030).Measuring method is identical with embodiment 13, confirms that the compound of embodiment 4 preparations is described xanthone compounds---oliganthone A.
Embodiment 17
The compound of getting embodiment 5 preparations is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030).Measuring method is identical with embodiment 13, confirms that the compound of embodiment 5 preparations is described xanthone compounds---oliganthone A.
Embodiment 18
The compound of getting embodiment 6 preparations is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030).Measuring method is identical with embodiment 13, confirms that the compound of embodiment 6 preparations is described xanthone compounds---oliganthone A.
Embodiment 19
The compound of getting embodiment 7 preparations is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030).Measuring method is identical with embodiment 13, confirms that the compound of embodiment 7 preparations is described xanthone compounds---oliganthone A.
Embodiment 20
The compound of getting embodiment 8 preparations is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030).Measuring method is identical with embodiment 13, confirms that the compound of embodiment 8 preparations is described xanthone compounds---oliganthone A.
Embodiment 21
The compound of getting embodiment 9 preparations is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030).Measuring method is identical with embodiment 13, confirms that the compound of embodiment 9 preparations is described xanthone compounds---oliganthone A.
Embodiment 22
The compound of getting embodiment 10 preparations is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030).Measuring method is identical with embodiment 13, confirms that the compound of embodiment 10 preparations is described xanthone compounds---oliganthone A.
Embodiment 23
The compound of getting embodiment 11 preparations is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030).Measuring method is identical with embodiment 13, confirms that the compound of embodiment 11 preparations is described xanthone compounds---oliganthone A.
Embodiment 24
The compound of getting embodiment 12 preparations is yellow amorphous powder; Optical value [α] 25.3 D – 3.6 (solvent is methyl alcohol c 0.030).Measuring method is identical with embodiment 13, confirms that the compound of embodiment 12 preparations is described xanthone compounds---oliganthone A.
Embodiment 25
The xanthone compounds of getting embodiment 1 ~ 12 preparation carries out cytotoxic activity test, and test situation is as follows:
1) induction HeLa-C3 apoptosis is active
All detected samples are all dissolved in DMSO as stock solutions.The concentration of each stock is at least 1000 times of working concentration.For monitoring minimum essential medium (MEM) substratum for HeLa-C3 cell whether caspase be activated, (contain 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin), at 5% CO that contains of 37 ℃
2humidified incubator in cultivate.Each sample well for detection of apoptosis activity adds the 100 μ L substratum that are suspended with 7,500 HeLa-C3 cells.Through 12 – 16 h, cultivate, old substratum is outwelled, and added the substratum of the sample that contains working concentration that 100 μ L are fresh, in background, only add fresh substratum.The substratum that contains 0.1% DMSO is used as negative control, adds the substratum of 500 nM taxols as positive control.Then use Perkin-Elmer Victor reader with excitation wavelength at 440 ± 10 nm and emission wavelength at 486 ± 8 nm for cyan fluorescent protein (CFP) and 535 ± 8 nm for yellow fluorescent protein (YFP) in each experimental period point reading of data.The time of data acquisition was over 72 hours.And the ratio of YFP/CFP is calculated.If YFP/CFP ratio is reduced to below 3, and observe cellular atrophy, just can think under working concentration effectively cell death inducing of this sample.The experiment of all samples is all carried out three times and is repeated.
2) cytotoxicity assay of sample to HeLa cell
With MTT assay, determine cell viability.MTT powder dissolution is in PBS, and concentration is 5 mg/mL.Cell adds sample after 72 hours, adds the MTT solution of 10 μ L in each hole of 96 orifice plates.At 37 ℃, place after 2 hours, add 100 μ L containing the 0.01 M HCl solution of 10% SDS, purple crystals is dissolved.Cultivate after 24 hours, measure the OD value (optical density) of its 595 nm.The vigor of the cell of crossing by compound treatment equals measured OD value divided by the OD value of negative control.(this value represents by the form of percentage ratio.)
Also by MTT method, measured the IC of this new compound
50value (this compound suppresses the concentration of half Growth of Cells).First, in each hole of 96 orifice plates by 2,500 HeLa cell suspensions in 100 μ L MEM substratum.After the cultivation of 24 h, add each hole to replace old substratum the fresh culture that contains different concns compound.The concentration of compound is 100 μ M to 1.5625 μ M, and these concentration obtain through six twice dilutions.Control group OD at 0 h and 72 h
595the OD that is added with compound group of value and 72 h
595value is measured with plate reader.
6) test-results
Apoptosis-induced experiment through to HeLa-C3 cell can effectively reduce below the YFP/CFP ratio to 3 of HeLa-C3 cell when lower concentration 25 μ M in 72 hours, had good cell death inducing effect.Through the anti-cell cytotoxic activity of HeLa cell is detected to test, test-results clearly illustrates that, IC
50value is 7.27 ± 0.23 μ M.Above result has disclosed compound of the present invention good application prospect in preparing antitumor drug.The compounds of this invention novel structure activity is good, can be used as the guiding compound of antitumor drug.
Claims (10)
1. an xanthone compounds, is characterized in that described xanthone compounds is that separation obtains from garcinia shrub, called after oliganthone A, and its molecular formula is C
30h
34o
7, there is following structure:
。
2. an xanthone compounds preparation method claimed in claim 1, it is characterized in that take that garcinia shrub is as raw material, through medicinal extract extraction, organic solvent extraction, the decolouring of MCI post, silica gel column chromatography, reversed phase column chromatography, gel filtration chromatography, high pressure liquid chromatography separating step, specifically comprise:
A, medicinal extract extract: garcinia shrub sample is crushed to 20 ~ 40 orders, and with organic solvent supersound extraction 2 ~ 4 times, each 30 ~ 60min, extracting solution merges, and filters, during the volume of concentrating under reduced pressure extracting solution to 1/4 ~ 1/2, standing rear filtering throw out, is then condensed into medicinal extract; Described garcinia shrub sample is Cortex Garciniae oliganthae branch and the leaf that derives from Hainan;
B, organic solvent extraction: it is the water of 1.5 ~ 3 times that A step gained medicinal extract adds weight ratio, and the organic solvent extraction of 1 ~ 2 times of volume of water 3 ~ 5 times, merges the organic solvent extraction of each separation mutually, and concentrating under reduced pressure becomes medicinal extract;
C, the decolouring of MCI post: B step gained medicinal extract carries out chromatography with the reversed material MCI of 4 ~ 5 times of weight ratios, and the methyl alcohol with 90 ~ 99% carries out wash-out;
D, silica gel column chromatography: the medicinal extract after decolouring carries out silica gel column chromatography with 160-200 order silica gel dress posts of 6 ~ 8 times of amounts of weight ratio; Normal hexane-acetone soln that the volume proportion of take is 1:0 ~ 0:1 carries out gradient elution, collects each several part elutriant concentrated, merges identical part;
E, reversed phase column chromatography: the 1:0 part of D step elutriant is further carried out chromatography with reversed material C-18 dress post, and the methanol-water solution that the volume proportion of take is 4:1 ~ 1:0 carries out gradient elution, collects each several part elutriant concentrated;
F, gel filtration chromatography: the methanol-water eluant solution part of the 85:15 of E step elutriant is further carried out chromatography with gel column Sephadex LH-20, with organic solvent, rushes post, collects elutriant concentrated;
G, high pressure liquid chromatography separation: F step elutriant further obtains described xanthone compounds with high pressure liquid chromatography separation and purification.
3. the preparation method of xanthone compounds according to claim 2, is characterized in that solvent in described A step can adopt any one in the acetone, ethanol, methyl alcohol of 70%-99%.
4. the preparation method of xanthone compounds according to claim 2, is characterized in that the organic solvent extracting in described B step can adopt any one in ethyl acetate, chloroform, methylene dichloride, ether, sherwood oil, benzene.
5. the preparation method of xanthone compounds according to claim 2, it is characterized in that in described C step that medicinal extract is before silica gel column chromatography rough segmentation, with using 80 ~ 100 order silica gel mixed samples of 0.8 ~ 1.2 times of medicinal extract weight ratio after the acetone solution of 1.5 ~ 3 times of amounts of weight ratio.
6. the preparation method of xanthone compounds according to claim 2, is characterized in that in described D step, normal hexane-acetone soln volume proportion is 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1.
7. the preparation method of xanthone compounds according to claim 2, is characterized in that in described E step, methanol-water liquor capacity proportioning is 80:20,85:15,90:10,95:5,100:0.
8. the preparation method of xanthone compounds according to claim 2, is characterized in that organic solvent in described F step can adopt any one in chloroform, methyl alcohol, acetone.
9. the preparation method of xanthone compounds according to claim 2, it is characterized in that the separation and purification of described G step mesohigh liquid chromatography is that to adopt 75 ~ 90% methyl alcohol be moving phase, flow velocity 2.5 ~ 4mL/min, 9.4 * 250 mm, the Alltima C of 5 μ m
18anti-phase preparative column is stationary phase, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 45 ~ 55 μ L collect the chromatographic peak of 9.1 ~ 39.5 min, repeatedly cumulative after evaporate to dryness.
10. the application of xanthone compounds in preparing antitumor drug described in a claim 1.
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| CN113004299B (en) * | 2021-02-19 | 2022-11-11 | 中国人民解放军北部战区总医院 | Xanthone compound in mangosteen bark for reducing postprandial blood sugar, and extraction method and application thereof |
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