CN103833818A - Camellia oleifera Abel saponin compound, its preparation method, application and antitumor drug prepared from the same - Google Patents
Camellia oleifera Abel saponin compound, its preparation method, application and antitumor drug prepared from the same Download PDFInfo
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Abstract
Belonging to the technical field of pharmaceutical chemistry, the invention discloses a Camellia oleifera Abel saponin compound, its preparation method, application and an antitumor drug prepared from the same. The Camellia oleifera Abel saponin compound has a structure shown as formula I, and has certain inhibiting effect on liver cancer cells, breast cancer cells, melanoma cells and lung cancer cells, also can effectively inhibit the liver tumor growth. Thus, the Camellia oleifera Abel saponin compound has significant antitumor activity, and can be used for preparing anti-tumor drugs.
Description
Technical field
The invention belongs to pharmaceutical chemistry technical field, particularly the antitumor drug of a kind of Sasanguasaponin compound, its preparation method, application and preparation thereof.
Background technology
Oil tea (Camellia oleifera Abel), be called again tea subtree, tea oil tree, spend tea in vain, be the evergreen dungarunga of Theaceae (Theaceae) Camellia (Camellia Linn), be mainly distributed in the torrid zone and subtropical zone.In China, oil tea originates in the west and south and region of Southeast, spreads all over 17 provinces and regions.Oil tea is important woody edible oil material seeds, with Fructus oleae europaeae, oil palm, coconut title " the large woody oil tree species in the world four ".Extract oil and obtain tea oil with the seed of oil tea, the unsaturated fatty acid content of tea oil is up to 90%, and its content is far away higher than rape oil, peanut oil and soya-bean oil; Compared with sweet oil, the content of its vitamin-E doubles, and has high nutritive value.Oil tea also has higher pharmaceutical use, in Compendium of Material Medica, records, and tea seed, the fragrant poison of bitter cold (saponin), cures mainly and breathes heavily anxious cough, removes disease dirt; " China book on Chinese herbal medicine " is also on the books, and that the root of oil tea and root leatherware thereof have is clearing heat and detoxicating, effect of regulating QI to relieve pain, activating blood circulation and reducing swelling, cures mainly swelling and pain in the throat, stomachache, toothache, traumatic pain and burn due to hot liquid or fire.
Oil tea medicinal part mainly contains Semen Camelliae, tea oil, oil tea root, Root-bark of Oiltea Camellia and Camellia Leaves.From oil tea, separate the chemical composition obtaining and comprise saponins, flavonoid, tannin class, fragrant glycoside and alkaloids.Sasanguasaponin is called again oil tea saponin, is a kind of soap class extracting from tea seed grouts, is mainly present in Seed of Camellia oleifera, Camellia Leaves, oil tea root and Root-bark of Oiltea Camellia, is now used for the numerous areas such as daily-use chemical industry, agricultural chemicals, medicine, aquatic products, building materials.Research also finds, Sasanguasaponin has the general general character of Camellia Plants saponin(e, and bitter, pungent, foaming character is strong; Also have multiple good biological activity, performance in many aspects, has the effects such as hemolytic action, ichthyotoxin effect, sterilization anti-microbial activity, anti-inflammatory, hypertension, antitumor, protection cardiovascular systems, desinsection expelling parasite, Promoting plant growth.Sasanguasaponin mostly is the pentacyclic triterpene saponins of oleanane type, comprise aglycon, sugar, organic acid three parts, but how caused Sasanguasaponin compounds different from array mode are numerous for its aglycon complex structure, sugar moieties kind, and being further purified with separating of Sasanguasaponin proposed to challenge.So, although research at present finds that oil tea total saponin extracts has antineoplastic activity, the specifically wherein effect of which monomer saponin performance, or unknown, need to carry out deep research to it.
Summary of the invention
In view of this, goal of the invention of the present invention is to provide the antitumor drug of a kind of Sasanguasaponin compound, its preparation method, application and preparation thereof.This Sasanguasaponin compound has significant anti-tumor activity, can be applied to and prepare antitumor drug.
In order to realize goal of the invention of the present invention, the present invention adopts following technical scheme:
The invention provides a kind of Sasanguasaponin compound, it has structure shown in formula I:
The present invention also provides a kind of preparation method of Sasanguasaponin compound, comprises the following steps:
Step 1: obtain oil tea total saponin extracts;
Step 2: get step 1 gained oil tea total saponin extracts, through separation and purification, to obtain final product.
Preferably, in preparation method provided by the invention, step 1 is specially:
Get after the pulverizing of oil tea root, through extracting, obtain extracting solution;
Get gained extracting solution through concentrated, obtain fluid extract;
Get gained fluid extract and mix with water, filter, collect filtrate;
Get gained filtrate and separate through macroporous resin, with alcohol-water mixed solution wash-out, collected volume, than the alcohol-water mixed solution elution fraction for 70:30～95:5, obtains oil tea total saponin extracts.
In some embodiments of the invention, in preparation method's step 1 provided by the invention, extracting solution used is alcohol-water mixed solution.
In other embodiment of the present invention, in preparation method's step 1 provided by the invention, extract in alcohol-water mixed solution used, the volumn concentration of ethanol is 30%～95%.
In other embodiment of the present invention, in g/mL, the oil tea root of pulverizing in preparation method's step 1 provided by the invention is 1:5～20 with the mass volume ratio that extracts solution used.
In other embodiment of the present invention, before extracting in preparation method's step 1 provided by the invention, also comprise steeping process, be specially and get in step 1 the oil tea root solution used with extraction of pulverizing and mix, under 30 DEG C～50 DEG C conditions, soak 6h～12h.
In other embodiment of the present invention, in preparation method's step 1 provided by the invention, dipping, extraction are specially:
Get the oil tea root of pulverizing in step 1 and mix with alcohol-water mixing solutions, under 30 DEG C～50 DEG C conditions, soak 6h～12h, through reflux, filter, collect filtrate, obtain extracting solution.
Extraction in preparation method's step 1 provided by the invention, can extract by a reflux, filters, and collects filtrate, obtains extracting solution; Also can extract by 2～3 reflux, collect respectively each reflux gained extracting solution, merge the extracting solution of each reflux gained, filter, collect filtrate, obtain extracting solution.
In other embodiment of the present invention, in preparation method's step 1 provided by the invention, the mass ratio of fluid extract and water is 1:1～10.
In other embodiment of the present invention, preparation method's step 1 provided by the invention is specially:
Getting oil tea root pulverizes; In g/mL, the oil tea root of getting pulverizing mixes by mass volume ratio 1:5～20 with alcohol-water mixing solutions, under 30 DEG C～50 DEG C conditions, soaks 6h～12h, after reflux 1 time～3 times, merge the extracting solution of each reflux gained, filter, collect filtrate, obtain extracting solution.Get gained extracting solution through concentrated, obtain fluid extract.Get gained fluid extract and mix 1:1～10 in mass ratio with water, 100 order～300 orders filter, and collect filtrate; After centrifugal gained filtrate, getting supernatant liquor separates through D101 type macroporous resin, the alcohol-water mixed solution that water, ethanol volumn concentration are 30%～60% successively, the alcohol-water mixed solution that ethanol volumn concentration is 70%～95% carry out wash-out, collecting ethanol volumn concentration is 70%～95% alcohol-water mixed solution elution fraction, obtains oil tea total saponin extracts.
Preferably, in preparation method provided by the invention, step 2 is specially:
Get step 1 gained oil tea total saponin extracts, separate through decompression silica gel chromatographic column, with chloroform-methanol mixed solution wash-out, collected volume, than the chloroform-methanol mixed solution elution fraction for 80:20～60:40, obtains purified for the first time;
Get gained and in purified warp, press anti-phase octadecylsilane chemically bonded silica chromatographic column to separate for the first time, with methanol-water mixture wash-out, collected volume, than the methanol-water mixture elution fraction for 70:30～90:10, obtains purified for the second time;
Get gained for the second time purified separate through dynamic axial compression chromatographic column, with methanol-water mixture wash-out, collected volume is than being the methanol-water mixture elution fraction of 75:25, purified for the third time;
Get gained for the third time purified separate through half preparative high-performance liquid chromatographic post, with methanol-water-formic acid mixed solution wash-out, flow velocity 2mL/min, collect retention time be the corresponding elution fraction of 52.5min, to obtain final product;
This half preparative high-performance liquid chromatographic post is octadecylsilane half preparative chromatography post, and its model is 250mm × 10mm, 5 μ m;
Methanol-water-formic acid mixed solution is: first alcohol and water by volume for the mixing solutions of 72:28 composition again with the mixed solution that accounts for the formic acid mixing gained that gained mixing solutions quality percentage composition is 0.2%.
In other embodiment of the present invention, the particle diameter of the silica gel in preparation method's step 2 provided by the invention in decompression silica gel chromatographic column used is 60 order～100 orders.
In other embodiment of the present invention, when the silica gel chromatography that reduces pressure in preparation method's step 2 provided by the invention separates, chloroform-methanol mixing wash-out is gradient elution, and gradient is specially: the volume ratio of chloroform and methyl alcohol is 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 30:70 → 20:80.
In other embodiment of the present invention, in preparation method's step 2 provided by the invention, the flow velocity of the anti-phase octadecylsilane chemically bonded silica chromatographic column of middle pressure used is 25mL/min.
In other embodiment of the present invention, while pressing anti-phase octadecylsilane chemically bonded silica chromatographic column to separate in preparation method's step 2 provided by the invention, methanol-water mixture wash-out is gradient elution, and gradient is specially: the volume ratio of first alcohol and water is 50:50 → 60:40 → 70:30 → 80:20 → 90:10 → 100:0.
In other embodiment of the present invention, in preparation method's step 2 provided by the invention, dynamic axial compression chromatographic column used is octadecylsilane chemically bonded silica chromatographic column.
In other embodiment of the present invention, when in preparation method's step 2 provided by the invention, dynamic axial compression chromatographic column separates, methanol-water mixture wash-out is gradient elution, and gradient is specially: 60:40 → 75:25 → 80:20 → 90:10.
In other embodiment of the present invention, in preparation method's step 2 provided by the invention, the flow velocity of dynamic axial compression chromatographic column used is 150mL/min.
In other embodiment of the present invention, in preparation method provided by the invention, half preparative high-performance liquid chromatographic post used is octadecylsilane chemically bonded silica half preparative high-performance liquid chromatographic post.
Preferably, after preparation method's step 2 half preparative high-performance liquid chromatographic post provided by the invention separates, also comprise distillation, drying step, is specially: get the separating obtained elution fraction of half preparative high-performance liquid chromatographic post and distill, and dry, to obtain final product.
In other embodiment of the present invention, in preparation method provided by the invention, step 2 is specially:
Get step 1 gained oil tea total saponin extracts, through decompression silicagel column, carry out gradient elution with chloroform-methanol system, gradient is that the volume ratio of chloroform and methyl alcohol is 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 30:70 → 20:80, collect respectively, merging volume ratio is 80:20, and the chloroform-methanol mixed solution elution fraction of 70:30 and 60:40 obtains purified for the first time;
Getting gained presses anti-phase octadecylsilane chemically bonded silica chromatographic column to separate in purified warp for the first time, carry out gradient elution with methanol-water system, gradient is that the volume ratio of first alcohol and water is 50:50 → 60:40 → 70:30 → 80:20 → 90:10 → 100:0, flow velocity 25mL/min, collect respectively, merging volume ratio is 70:30,80:20, the methanol-water mixture elution fraction of 90:10, obtains purified for the second time;
Get gained for the second time purified separate through dynamic axial compression chromatographic column, methanol-water mixture with different volumes ratio carries out gradient elution, gradient is that the volume ratio of first alcohol and water is 60:40 → 75:25 → 80:20 → 90:10, flow velocity is 150mL/min, collected volume, than the methanol-water mixture elution fraction for 75:25, obtains purified for the third time;
Get gained for the third time purified through octadecylsilane chemically bonded silica half preparative high-performance liquid chromatographic post separate, with methanol-water-formic acid mixed solution wash-out, flow velocity 2mL/min, collecting retention time is the corresponding elution fraction of 52.5min, gained elution fraction is distilled, is dried, obtain white unsetting powdery substance, to obtain final product;
Wherein, methanol-water-formic acid mixed solution is: first alcohol and water by volume for the mixing solutions of 72:28 composition again with the mixed solution that accounts for the formic acid mixing gained that gained mixing solutions quality percentage composition is 0.2%.
The present invention also provides a kind of Sasanguasaponin compound, and the preparation method of this Sasanguasaponin compound comprises the following steps:
Step 1: obtain oil tea total saponin extracts;
Step 2: get step 1 gained oil tea total saponin extracts, through separation and purification, to obtain final product.
The present invention also provides a kind of Sasanguasaponin compound in the application of preparing in antitumor drug, and this Sasanguasaponin compound has structure shown in formula I,
The preparation method of this Sasanguasaponin compound comprises the following steps:
Step 1: obtain oil tea total saponin extracts;
Step 2: get step 1 gained oil tea total saponin extracts, through separation and purification, to obtain final product.
In some embodiments of the invention, application provided by the invention for tumour be liver tumor, melanoma, breast tumor or lung tumor.
The present invention also provides a kind of antitumor drug, and it comprises Sasanguasaponin compound provided by the invention and pharmaceutically acceptable auxiliary material, and this Sasanguasaponin compound has structure shown in formula I,
The preparation method of this Sasanguasaponin compound comprises the following steps:
Step 1: obtain oil tea total saponin extracts;
Step 2: get step 1 gained oil tea total saponin extracts, through separation and purification, to obtain final product.
Preferably, the formulation of a kind of antitumor drug provided by the invention is tablet, granule, capsule, injection, emulsion or suspensoid.
The invention provides the antitumor drug of a kind of Sasanguasaponin compound, its preparation method, application and preparation thereof.This Sasanguasaponin compound has structure shown in formula I.In vitro cell experiment result confirms that this Sasanguasaponin compound has certain restraining effect to liver cancer cell, breast cancer cell, melanoma cells, lung carcinoma cell; In body, experimental result confirms that this Sasanguasaponin compound has significant restraining effect to liver tumor, can effectively suppress the growth of liver tumor, illustrates that this Sasanguasaponin compound has significant anti-tumor activity, can be applied to and prepare antitumor drug,
Brief description of the drawings
Fig. 1 shows the high resolution mass spectrum spectrogram of the Sasanguasaponin compound that embodiment 1 makes;
Fig. 2 shows the hydrogen nuclear magnetic resonance spectrogram of the Sasanguasaponin compound that embodiment 1 makes;
Fig. 3 shows the hydrogen nuclear magnetic resonance spectrogram partial enlarged drawing of the Sasanguasaponin compound that embodiment 1 makes;
Fig. 4 shows the hydrogen nuclear magnetic resonance spectrogram partial enlarged drawing of the Sasanguasaponin compound that embodiment 1 makes;
Fig. 5 shows the carbon-13 nmr spectra figure of the Sasanguasaponin compound that embodiment 1 makes;
Fig. 6 shows the carbon-13 nmr spectra figure partial enlarged drawing of the Sasanguasaponin compound that embodiment 1 makes;
Fig. 7 shows the carbon-13 nmr spectra figure partial enlarged drawing of the Sasanguasaponin compound that embodiment 1 makes;
Fig. 8 shows the DEPT spectrogram of the Sasanguasaponin compound that embodiment 1 makes;
Fig. 9 shows the DEPT spectrogram partial enlarged drawing of the Sasanguasaponin compound that embodiment 1 makes;
Figure 10 shows the hsqc spectrum figure of the Sasanguasaponin compound that embodiment 1 makes;
Figure 11 shows the HMBC spectrogram of the Sasanguasaponin compound that embodiment 1 makes;
Figure 12 shows the H-H COSY spectrogram of the Sasanguasaponin compound that embodiment 1 makes;
Figure 13 shows the NOESY spectrogram of the Sasanguasaponin compound that embodiment 1 makes.
Embodiment
The invention discloses the antitumor drug of a kind of Sasanguasaponin compound, its preparation method, application and preparation thereof.Those skilled in the art can be with reference to this paper content, obtain this Sasanguasaponin compound, realize its application, special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Preparation method of the present invention and application are described by preferred embodiment, related personnel obviously can change this paper preparation method and application in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
In the antitumor drug of a kind of Sasanguasaponin compound provided by the invention, its preparation method, application and preparation thereof, reagent used and raw material all can be buied by market.In the embodiment of the present invention, the model of some instrument used and manufacturer are as follows:
Chemical reagent: analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group; Electronic balance: Mettler-Toledo Instrument (Shanghai) Co., Ltd.; Polarimeter 241PerkinElmer Inc., Waltham, MA, the U.S.; Rotary Evaporators: Tokyo physics and chemistry apparatus individual proprietorship factory; Thin-layer chromatography silica-gel plate: HSGF254, the yellow business of Yantai City's Zhifu silica gel development experiments factory produces; Various column chromatographys are Haiyang Chemical Plant, Qingdao with silica gel and produce; Nuclear magnetic resonance analyser 500Hz:Varian Inc., Palo Alto, CA, the U.S.; High resolution mass spectrum Q-TOF2:Micromass company, Britain; Half preparative high-performance liquid chromatographic instrument: LC-20AT, SPD-20A, Japanese Shimadzu company; The anti-phase octadecylsilane preparative liquid chromatography of middle pressure: B ü chi chromatographic system, C-650 pump, middle compression leg (460mm × 26mm i.d., B ü chi Corp., Flawil, Swiss); Dynamic axial column chromatography: NP7000 pump (Newstyle), NU3000UV-VIS detector (Newstyle) and octadecylsilane post (650mm × 100mm, 30 μ m, 1500g; Newstyle); Octadecylsilane half preparative chromatography post (C18 half preparative chromatography post): 250mm × 10mm, 5 μ m, Kromsil company of the U.S..
In order to make those skilled in the art can understand better technical scheme of the present invention, below in conjunction with embodiment, further set forth the present invention:
Extract:
Get dry oil tea root (picking up from Qichun, Hubei in November, 2011) 2000g, be ground into wood chip with pulverizer, adding 20.0L volume percent is 70% aqueous ethanolic solution, under 40 DEG C of conditions, soak after 10h, be heated to reflux, extract 3.0h, collect extracting solution, by extracting liquid filtering, concentrating under reduced pressure, obtains fluid extract 602g.
Macroporous resin enrichment:
Get the fluid extract of extraction step gained, add 6.02L water, stirring and dissolving, with 100 order filter-cloth filterings, by centrifugal filtrate 12000r/min 10min, centrifugal after, obtain 5.3L supernatant liquor, getting supernatant liquor further separates through D101 type macroporous resin, carry out wash-out with 10L water, 15L30% aqueous ethanolic solution, 15L70% aqueous ethanolic solution successively, collect 70% aqueous ethanolic solution wash-out component, concentrating under reduced pressure, after testing, in gained concentrated solution, contain Sasanguasaponin compounds, through 100 DEG C, baking oven, 48h is dry, obtain brown medicinal extract 45g, called after oil tea total saponin extracts.
The preparation of purified for the first time:
Get the oil tea total saponin extracts of macroporous resin enrichment gained, separate through decompression silicagel column (silica gel: 60-100 order), carry out gradient elution with chloroform-methanol system, gradient is that the volume ratio of chloroform and methyl alcohol is 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 30:70 → 20:80, each gradient 2.0L; Collect respectively each elution fraction, and label one by one.Gained elutriant is carried out to thin layer plate analysis: thin-layer chromatography silica-gel plate, BAW system, R
fvalue=0.4～0.7.After testing, the composition that below, elution fraction contains is similar: the chloroform-methanol eluant solution component of 80:20, the chloroform-methanol eluant solution component of 70:30, the chloroform-methanol eluant solution component of 60:40.Merge the chloroform-methanol eluant solution component of 80:20, the chloroform-methanol eluant solution component of the chloroform-methanol eluant solution component of 70:30 and 60:40, evaporated under reduced pressure,, obtain purified 12.6g for the first time.
The preparation of purified for the second time:
Get gained purified for the first time, in warp, press anti-phase octadecylsilane preparative liquid chromatography to separate, carry out gradient elution with methanol-water system, gradient is that the volume ratio of first alcohol and water is 50:50 → 60:40 → 70:30 → 80:20 → 90:10 → 100:0, each gradient 1000mL, flow velocity 25mL/min, detects wavelength 203nm.Fractional Collections elution fraction, obtains six elution fractions, one by one marks.Getting gradient is 60:40,70:30,80:20, the corresponding elution fraction of 90:10 carries out efficient analysis liquid-phase chromatographic analysis, result shows, gradient 70:30,80:20, the composition that contains in the corresponding elution fraction of 90:10 is similar, and (the efficient analysis liquid chromatography time is 30min, detected peaks is between 20min-28min), merge the corresponding elution fraction of gradient, after concentrating under reduced pressure, obtain purified 20mL for the second time.
The preparation of purified for the third time:
Get purified for the second time, separate (flow velocity 150mL/min through dynamic axial column chromatography, detect wavelength 203nm), in volume percent, carry out wash-out with 60%, 75%, 80% and 90% methanol aqueous solution successively, each gradient 3000mL, collects respectively, obtain five elution fractions, one by one marks.By gained elution fraction respectively through efficient analysis liquid-phase chromatographic analysis (detected peaks is between 13.5min-15.0min), collecting 75% elution fraction (is that volume ratio is the methanol-water mixture elution fraction of 75:25, high performance liquid phase detected peaks is positioned at 14.5min), concentrating under reduced pressure obtains purified 15mL for the third time.
The preparation of the unsetting powdery substance of white:
Get purified for the third time, through C18 half preparative chromatography post (250mm × 10mm, 5 μ m) separate (flow velocity 2mL/min, detect wavelength 203nm), taking methanol-water-formic acid mixed solution (first alcohol and water by volume as the mixing solutions of 72:28 composition again with the mixed solution that accounts for the formic acid mixing gained of gained mixing solutions quality percentage composition as 0.2%) carry out wash-out, in 52.5min place.Gained elution fraction is carried out to concentrating under reduced pressure, dry, obtain the unsetting powdery substance of 103mg white.
Compound structure qualification:
The unsetting powdery substance of gained white is carried out to thin-layer chromatography, aceticanhydride-strong sulfuric acid response, Molish and react qualification, qualification result is: thin-layer chromatography sulfuric acid ethanol colour developing result is red-purple spot, aceticanhydride-strong sulfuric acid response positive, Molish reacting positive, pointing out this material may be saponins compound.The high resolution mass spectrum spectrogram of this material is shown in Fig. 1, the accurate quasi-molecular ions m/z1299.5965[M-H in this mass spectrogram]-, with molecular formula be C
63h
96o
28estimating of molecular weight value [M-H]-, m/z1299.6010, basically identical, the molecular formula that shows this compound is C
63h
96o
28.Obtain monose with the complete acid hydrolysis of 2N TFA, sugar carries out GC analysis after derivatize, the existence of D-Glucose aldehydic acid, D-semi-lactosi, D-wood sugar detected.
The unsetting powdery substance of gained white is carried out to proton nmr spectra detection, carbon-13 nmr spectra detection and two dimensional NMR spectrum to be detected.The proton nmr spectra spectrogram of the unsetting powdery substance of this white is shown in Fig. 2, Fig. 3 and Fig. 4, and wherein Fig. 3 and Fig. 4 are the partial enlarged drawing of this material proton nmr spectra.The carbon-13 nmr spectra spectrogram of the unsetting powdery substance of this white is shown in Fig. 5, Fig. 6 and Fig. 7, and Fig. 6 and Fig. 7 are the partial enlarged drawing of this material carbon-13 nmr spectra.The DEPT spectrogram of the unsetting powdery substance of this white is shown in Fig. 8 and Fig. 9, the DEPT spectrogram partial enlarged drawing that wherein Fig. 9 is this material.The hsqc spectrum figure of the unsetting powdery substance of this white is shown in Figure 10.The HMBC spectrogram of the unsetting powdery substance of this white is shown in Figure 11.Figure 12 is shown in by the H-H COSY collection of illustrative plates of the unsetting powdery substance of this white.Figure 13 is shown in by the NOESY collection of illustrative plates of the unsetting powdery substance of this white.The nuclear magnetic resonance spectrum diagram data of the unsetting powdery substance of this white is in table 1.
The nuclear magnetic resonance data of the white unsetting powder compounds of table 1
| Position | δ H | δ C | Position | δ H | δ C |
| 1 | 0.97m,1.46m | 38.3 | 22-O-Ang | -- | -- |
| 2 | 1.94m,2.24m | 25.4 | 1″ | -- | 168.3 |
| 3 | 4.11dd(11.0,4.5) | 84.7 | 2″ | -- | 129.1 |
| 4 | -- | 55.3 | 3″ | 5.98dq(7.5,1.5) | 137.3 |
| 5 | 1.46m | 48.5 | 4″ | 2.13d(7.5) | 15.9 |
| 6 | 1.05m,1.42m | 20.4 | 5″ | 1.99s | 21.0 |
| 7 | 1.23m,1.60m | 32.5 | GlcA | -- | -- |
| 8 | -- | 41.8 | 1 | 4.81d(6.5) | 104.0 |
| 9 | 1.84m | 46.9 | 2 | 4.55m | 78.3 |
| 10 | -- | 36.2 | 3 | 4.39m | 85.1 |
| 11 | 1.88m,1.99m | 23.9 | 4 | 4.55m | 70.7 |
| 12 | 5.49brs | 123.7 | 5 | 4.44m | 77.2 |
| 13 | -- | 143.0 | 6 | -- | nd |
| 14 | -- | 40.4 | Gal | -- | -- |
| 15 | 1.63brs,1.88brs | 34.9 | 1 | 5.77d(7.5) | 101.8 |
| 16 | 4.55brs | 68.7 | 2 | 4.58m | 84.1 |
| 17 | -- | 48.2 | 3 | 4.37m | 75.1 |
| 18 | 3.18m | 40.2 | 4 | 4.44m | 71.0 |
| 19 | 1.50m,3.18m | 47.3 | 5 | 4.16m | 77.1 |
| 20 | -- | 36.5 | 6 | 4.42m | 62.0 |
| 21 | 6.77d(10.5) | 78.9 | Xyl | -- | -- |
| 22 | 6.39d(10.5) | 73.8 | 1 | 5.12d(7.5) | 107.8 |
| 23 | 10.0brs | 210.5 | 2 | 4.25m | 76.4 |
| 24 | 1.55s | 11.2 | 3 | 4.11m | 78.4 |
| 25 | 0.90s | 16.0 | 4 | 4.37m | 71.0 |
| 26 | 0.90s | 17.0 | 5 | 3.56m | 67.7 |
| 27 | 1.88s | 27.7 | -- | 4.53m | -- |
| 28 | 3.48d(11.0) | 63.8 | Gal | -- | -- |
| -- | 3.71d(10.0) | -- | 1 | 5.93d(7.5) | 103.0 |
| 29 | 1.18s | 29.7 | 2 | 4.51m | 73.9 |
| 30 | 1.42s | 20.4 | 3 | 4.42m | 75.5 |
| 21-O-Ang | -- | -- | 4 | 4.53m | 70.0 |
| 1′ | -- | 167.8 | 5 | 4.53m | 76.5 |
| 2′ | -- | 129.1 | 6 | 4.55m | 62.3 |
| 3′ | 6.05dq(7.5,1.5) | 137.3 | -- | -- | -- |
| 4′ | 2.17d(7.5) | 16.0 | -- | -- | -- |
| 5′ | 2.09s | 21.1 | -- | -- | -- |
Note: in table, chemical shift is taking ppm as unit, and coupling constant (J) is taking Hz as unit, and nd represents to exist but undetected signal.
According to gained nmr spectrum and data, the unsetting powdery substance of this white is carried out to further structure elucidation:
The unsetting powdery substance of this white
13c NMR spectrum shows 63 carbon signals altogether, analyzes this compound and contains altogether 10 methyl carbon, 11 mesomethylene carbon, 30 methine carbons and 12 quaternary carbons by DEPT and hsqc spectrum.Further analyze
13c NMR spectrum, this spectrogram demonstrates four sugared end group carbon signal δ 104.0,101.8,107.8 and 103.0, is respectively the end group carbon of D-Glucose aldehydic acid, D-semi-lactosi, D-wood sugar and another one D-semi-lactosi.In addition, also has an aldehyde radical carbon signal that is positioned at low place δ 210.5.The unsetting powdery substance of this white
1in H NMR spectrum, high field region has six obvious triterpenoid saponin angular methyl(group) hydrogen signal δ 0.90 (Me-25), 0.90 (Me-26), 1.18 (Me-29), 1.42 (Me-30), 1.55 (Me-24) and 1.88 (Me-27); Even Oxymethylene δ 3.48 (H-28, d, J=11.0Hz) and 3.71 (H-28, d, J=10.0Hz); Four oxygen methyne δ 4.11 (1H, dd, J=11.0,4.5Hz, H-3) of company, 4.55 (1H, brs, H-16), 6.77 (1H, d, J=10.5Hz, H-21) and 6.39 (1H, d, J=10.5Hz, H-22); Alkene hydrogen proton δ 5.49 (1H, brs, H-12) and aldehyde radical hydrogen proton δ 10.0 (1H, brs, H-23).In addition, the unsetting powdery substance of this white
1h NMR spectrum also demonstrates two groups of angeloyl groups signal δ [6.05 (1H, dq, J=7.5,1.5Hz, 21-O-Ang-3'), 2.17 (3H, d, J=7.5Hz, 21-O-Ang-4') and 2.09 (3H, s, 21-O-Ang-5')]; δ [5.98 (1H, dq, J=7.5,1.5Hz, 22-O-Ang-3 "), 2.13 (3H, d, J=7.5Hz, 22-O-Ang-4 ") and 1.99 (3H, s, 22-O-Ang-5 ")].
In H-H COSY, with the methylene radical δ that even oxygen methine protons is adjacent
h1.63 (1H, brs), 1.88 (1H, brs) and δ
h4.55 (1H, brs) are relevant, illustrate that they are in ortho position and δ
h4.55 is H-16 signal, δ
h1.63,1.88 is H-15 signal; δ
h6.77 (1H, d, J=10.5Hz) and δ
hthey are in ortho position and δ coherent signal explanation between 6.39 (1H, d, J=10.5Hz)
h6.77 is H-21 signal, δ
h6.39 is H-22 signal.
By the HMBC spectrum analysis of the unsetting powdery substance of this white learn two angeloyl groups be connected in respectively 21,22 of this compound upper because in HMBC δ
h6.77 (1H, d, J=10.5Hz, H-21) and δ
c167.8 (21-O-Ang-1') are relevant, δ
h6.39 (1H, d, J=10.5Hz, H-22) and δ
c168.3 (22-O-Ang-1'') are relevant.In conjunction with
13c NMR spectrum and
1h NMR spectrum data are comprehensively analyzed; and with known aglycon 21 β; 22 α-O-diangeloyl-3 β; 16 α, 28-trihydroxyolean-12-en-23-al[Sci.Pharm1982,50; 331] nuclear magnetic data contrast; the aglycon structure of determining the unsetting powdery substance of this white is 21 β, 22 α-O-, bis-angeloyl groups-16 α, 28-dihydroxyl olea-12-alkene-23-aldehyde.
Hsqc spectrum figure to the unsetting powdery substance of this white analyzes, and has belonged to sugared end group carbon, the hydrogen signal that on this saponin(e, connect, as follows: δ
h4.81 (1H, d, J=6.5Hz), 5.77 (1H, d, J=7.5Hz), 5.12 (1H, d, J=7.5Hz) and 5.93 (1H, d, J=7.5Hz), be connected in respectively δ
c104.0 (glucuronic acid-C-1), 101.8 (semi-lactosi-C-1), 107.8 (wood sugar-C-1), on 103.0 (semi-lactosi '-C-1).3 of this compound aglycons show that to low the about 12.6ppm in position sugar chain is connected in 3 of aglycons and goes up in addition, and in HMBC spectrum, glucuronic acid anomeric proton δ
h4.81 with 3 δ of aglycon
c84.7 is relevant, further confirmed that sugar chain is connected in 3 of aglycon.Further By consulting literatures is found close [the Bioorganic & medicinal chemistry letters2010 of the camellenodiol sugar chain data of reporting in this compound sugar chain and document, 20,7435-7439], thus infer that this compound sugar chain is β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid.The order of connection of this sugar chain also can confirm by analyzing HMBC spectrum, glucuronic acid anomeric proton δ in HMBC
h4.81 with 3 carbon δ of aglycon
c84.7 relevant, semi-lactosi anomeric proton δ
h5.77 with 3 carbon δ of glucuronic acid
c85.1 relevant, wood sugar anomeric proton δ
h5.12 with 2 carbon δ of semi-lactosi
c84.1 relevant, 2 hydrogen δ of semi-lactosi
h4.58 with wood sugar end group carbon δ
c107.8 relevant, another semi-lactosi anomeric proton δ
h5.93 with 2 carbon δ of glucuronic acid
c78.3 relevant, 2 hydrogen δ of glucuronic acid
h4.55 with this semi-lactosi end group carbon δ
c103.0 relevant.In addition four sugared anomeric proton coupling constants,
3j
h-1,
h-2larger, show that these four sugar are all beta configuration.
In NOESY spectrum, δ
h6.77 (1H, d, J=10.5Hz, H-21) and δ
h1.18 (3H, s, H-29) are relevant, and prompting H-21 is α configuration; δ
h4.11 (1H, dd, J=4.5,11.0Hz, H-3) and δ
h10.0 (1H, brs, H-23) are relevant, and prompting H-3 is α configuration; δ
h4.55 (1H, brs, H-16) and δ
hthe relevant prompting of 3.48 (1H, d, J=11.0Hz, H-28), 3.71 (1H, d, J=11.0Hz, H-28) H-16 is beta comfiguration, δ
h6.39 (1H, d, J=10.5Hz, H-22) and δ
h1.42 (3H, s, H-30) are relevant, and prompting H-22 is beta comfiguration.
Therefore, this compound has structure shown in formula I,
By this compound structure called after 21 β; 22 α-O-, bis-angeloyl groups-16 α, 28-dihydroxyl olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides.
Extract:
Get dry oil tea root (picking up from Qichun, Hubei in November, 2011) 2000g, be ground into wood chip with pulverizer, adding 40.0L volume percent is 30% aqueous ethanolic solution, under 30 DEG C of conditions, soak 12h post-heating to refluxing, extract three times, each 0.5h, collect extracting solution, by extracting liquid filtering, concentrating under reduced pressure, obtains fluid extract 628g.
Macroporous resin enrichment:
Get the fluid extract of extraction step gained, add 0.628L water, stirring and dissolving, with 300 order filter-cloth filterings, by centrifugal filtrate 12000r/min 10min, centrifugal after, obtain 5.8L supernatant liquor, getting supernatant liquor further separates through D101 type macroporous resin, carry out wash-out with 10L water, 15L60% aqueous ethanolic solution, 10L95% aqueous ethanolic solution successively, collect 95% aqueous ethanolic solution wash-out component, concentrating under reduced pressure, after testing, in gained concentrated solution, contain Sasanguasaponin compounds, through 100 DEG C, baking oven, 48h is dry, obtain brown medicinal extract 55g, called after oil tea total saponin extracts.
The preparation of purified for the first time:
Get the oil tea total saponin extracts of macroporous resin enrichment gained, separate through decompression silicagel column (silica gel: 60=100 order), carry out gradient elution with chloroform-methanol system, gradient is that the volume ratio of chloroform and methyl alcohol is 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 30:70 → 20:80, each gradient 1.0L; Collect respectively each elution fraction, and label one by one.Gained elutriant is carried out to thin layer plate analysis: thin-layer chromatography silica-gel plate, BAW system, R
fvalue=0.4～0.7.After testing, the composition that below, elution fraction contains is similar: the chloroform-methanol eluant solution component of 80:20, the chloroform-methanol eluant solution component of 70:30, the chloroform-methanol eluant solution component of 60:40.Merge the chloroform-methanol eluant solution component of 80:20, the chloroform-methanol eluant solution component of the chloroform-methanol eluant solution component of 70:30 and 60:40, evaporated under reduced pressure, obtains purified 15.2g for the first time.
The preparation of purified for the second time:
Get gained purified for the first time, in warp, press anti-phase octadecylsilane preparative liquid chromatography to separate, carry out gradient elution with methanol-water system, gradient is that the volume ratio of first alcohol and water is 50:50 → 60:40 → 70:30 → 80:20 → 90:10 → 100:0, each gradient 1000mL, flow velocity 25mL/min, detects wavelength 203nm.Fractional Collections elution fraction, obtains six elution fractions, one by one marks.Getting gradient is 60:40,70:30,80:20, the corresponding elution fraction of 90:10 carries out efficient analysis liquid-phase chromatographic analysis, result shows, gradient 70:30,80:20, the composition that contains in the corresponding elution fraction of 90:10 is similar, and (the efficient analysis liquid chromatography time is 30min, detected peaks is between 20min-28min), merge the corresponding elution fraction of gradient, concentrating under reduced pressure, obtains purified 20mL for the second time.
The preparation of purified for the third time:
Get purified for the second time, separate (flow velocity 150mL/min through dynamic axial column chromatography, detect wavelength 203nm), in volume percent, carry out wash-out with 60%, 75%, 80% and 90% methanol aqueous solution successively, each gradient 3000mL, collects respectively, obtain five elution fractions, one by one marks.By gained elution fraction respectively through efficient analysis liquid-phase chromatographic analysis (detected peaks is between 13.5min-15.0min), collecting 75% elution fraction (is that volume ratio is the methanol-water mixture elution fraction of 75:25, high performance liquid phase detected peaks is positioned at 13.5min), concentrating under reduced pressure, obtains purified 15mL for the third time.
The preparation of the unsetting powdery substance of white:
Get purified for the third time, through C18 half preparative chromatography post (250mm × 10mm, 5 μ m) separate (flow velocity 2mL/min, detect wavelength 203nm), taking methanol-water-formic acid mixed solution (first alcohol and water by volume as the mixing solutions of 72:28 composition again with the mixed solution that accounts for the formic acid mixing gained of gained mixing solutions quality percentage composition as 0.2%) carry out wash-out, collect 52.5min place chromatographic peak, gained elution fraction is carried out to concentrating under reduced pressure, dry, obtain the unsetting powdery substance of 125mg white.
The unsetting powdery substance of white that adopts the identical compound structure authentication method of embodiment 1 to make the present embodiment carries out Structural Identification, the Correlated Spectroscopy diagram data of high resolution mass spectrum spectrogram, proton nmr spectra, carbon-13 nmr spectra, DEPT spectrogram, hsqc spectrum figure, HMBC spectrogram, H-H COSY spectrogram, NOESY spectrogram and embodiment 1 gained obtaining is basically identical, result shows that the unsetting powdery substance of this white is the compound with structure shown in formula I
Extract:
Get dry oil tea root (picking up from Qichun, Hubei) 2000g, be ground into wood chip with pulverizer, adding 10.0L volume percent is 95% aqueous ethanolic solution, under 50 DEG C of conditions, soaks after 6h, is heated to reflux, extract twice, each 1h, collects extracting solution, by extracting liquid filtering, concentrating under reduced pressure, obtains fluid extract 638g.
Macroporous resin enrichment:
Get the fluid extract of extraction step gained, add 6.0L water, stirring and dissolving, with 200 order filter-cloth filterings, by centrifugal filtrate 12000r/min 10min, after centrifugal, obtain 5.5L supernatant liquor, getting supernatant liquor further separates through D101 type macroporous resin, use successively 10.0L water, 15.0L40% aqueous ethanolic solution, 15.0L L80% aqueous ethanolic solution carries out wash-out, collect 80% aqueous ethanolic solution wash-out component, concentrating under reduced pressure, after testing, in gained concentrated solution, contain Sasanguasaponin compounds, through 100 DEG C, baking oven, 48h is dry, obtain brown medicinal extract 52g, called after oil tea total saponin extracts.
The preparation of purified for the first time:
Get the oil tea total saponin extracts of macroporous resin enrichment gained, separate through decompression silicagel column (silica gel: 60-100 order), carry out gradient elution with chloroform-methanol system, gradient is that the volume ratio of chloroform and methyl alcohol is 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 30:70 → 20:80, each gradient 1.0L; Collect respectively each elution fraction, and label one by one.Gained elutriant is carried out to thin layer plate analysis: thin-layer chromatography silica-gel plate, BAW system, R
fvalue=0.4～0.7.After testing, the composition that below, elution fraction contains is similar: the chloroform-methanol eluant solution component of 80:20, the chloroform-methanol eluant solution component of 70:30, the chloroform-methanol eluant solution component of 60:40.Merge the chloroform-methanol eluant solution component of 80:20, the chloroform-methanol eluant solution component of the chloroform-methanol eluant solution component of 70:30 and 60:40, evaporated under reduced pressure, obtains purified 18.5g for the first time.
The preparation of purified for the second time:
Get gained purified for the first time, in warp, press anti-phase octadecylsilane preparative liquid chromatography to separate, carry out gradient elution with methanol-water system, gradient is that the volume ratio of first alcohol and water is 50:50 → 60:40 → 70:30 → 80:20 → 90:10 → 100:0, each gradient 1000mL, flow velocity 25mL/min, detects wavelength 203nm.Fractional Collections elution fraction, obtains six elution fractions, one by one marks.Getting gradient is 60:40,70:30,80:20, the corresponding elution fraction of 90:10 carries out efficient analysis liquid-phase chromatographic analysis, result shows, gradient 70:30,80:20, the composition that contains in the corresponding elution fraction of 90:10 is similar, and (the efficient analysis liquid chromatography time is 30min, detected peaks is between 20min-28min), merge the corresponding elution fraction of gradient, concentrating under reduced pressure, obtains purified 20mL for the second time.
The preparation of purified for the third time:
Get purified for the second time, separate (flow velocity 150mL/min through dynamic axial column chromatography, detect wavelength 203nm), in volume percent, carry out wash-out with 60%, 75%, 80% and 90% methanol aqueous solution successively, each gradient 3000mL, collects respectively, obtain five elution fractions, one by one marks.By gained elution fraction respectively through efficient analysis liquid-phase chromatographic analysis (detected peaks is between 13.5min-15.0min), collecting 75% elution fraction (is that volume ratio is the methanol-water mixture elution fraction of 75:25, high performance liquid phase detected peaks is positioned at 15.0min), obtain purified 15mL for the third time.
The preparation of the unsetting powdery substance of white:
Get purified for the third time, through C18 half preparative chromatography post (250mm × 10mm, 5 μ m) separate (flow velocity 2mL/min, detect wavelength 203nm), taking methanol-water-formic acid mixed solution (first alcohol and water by volume as the mixing solutions of 72:28 composition again with the mixed solution that accounts for the formic acid mixing gained of gained mixing solutions quality percentage composition as 0.2%) carry out wash-out, collect 52.5min place chromatographic peak, gained elution fraction is carried out to concentrating under reduced pressure, dry, obtain the unsetting powdery substance of 173mg white.
The unsetting powdery substance of white that adopts the identical compound structure authentication method of embodiment 1 to make the present embodiment carries out Structural Identification, the Correlated Spectroscopy diagram data of high resolution mass spectrum spectrogram, proton nmr spectra, carbon-13 nmr spectra, DEPT spectrogram, hsqc spectrum figure, HMBC spectrogram, H-H COSY spectrogram, NOESY spectrogram and embodiment 1 gained obtaining is basically identical, result shows that the unsetting powdery substance of this white is the compound with structure shown in formula I
Extract:
Get dry oil tea root 2000g, be ground into wood chip with pulverizer, join in 20.0L aqueous ethanolic solution and extract, gained extracting solution is concentrated, obtain fluid extract 655g.
Macroporous resin enrichment:
In gained fluid extract, add water 6.0L, be stirred to dissolving, filter, obtain filtrate 5.8L; Gained filtrate is crossed to macroporous resin to be separated, with alcohol-water mixed solution wash-out, collected volume is than the alcohol-water mixed solution elution fraction for 70:30～95:5, after testing, wherein contain Sasanguasaponin compounds, through 100 DEG C, baking oven, 48h is dry, obtain brown medicinal extract 55g, called after oil tea total saponin extracts.
The preparation of purified for the first time:
Get the oil tea total saponin extracts of macroporous resin enrichment gained, separate through decompression silicagel column, carry out gradient elution with chloroform-methanol system, each gradient 1000mL, collected volume is than being 80:20,70:30, the chloroform-methanol mixed solution elution fraction of 60:40 respectively.Three elution fractions of gained are carried out to thin layer plate analysis: thin-layer chromatography silica-gel plate, BAW system, R
fvalue=0.4～0.7.After testing, the composition that three elution fractions contain is similar, merges this three elution fractions, and evaporated under reduced pressure, obtains purified 15.9g for the first time.
The preparation of purified for the second time:
Get gained purified for the first time, in warp, press anti-phase octadecylsilane preparative liquid chromatography to separate, carry out gradient elution with methanol-water system, each gradient 1000mL, collected volume is than being 70:30,80:20, the methanol-water mixture elution fraction of 90:10 respectively.Three elution fractions of gained are carried out to efficient analysis Liquid Detection, obtain the composition similar (the efficient analysis liquid chromatography time is 30min, and detected peaks is between 20min-28min) containing in these three elution fractions, merge this three elution fractions, concentrating under reduced pressure, obtains purified 20mL for the second time.
The preparation of purified for the third time:
Get purified for the second time, separate through dynamic axial column chromatography, carry out wash-out with methanol-water system, collected volume is than the methanol-water mixture elution fraction 450mL for 75:25.Gained elution fraction, through efficient analysis liquid-phase chromatographic analysis, is occurred to detected peaks in 15.0min place, collect this elution fraction, concentrating under reduced pressure, obtains purified 15mL for the third time.
The preparation of the unsetting powdery substance of white:
Get purified for the third time, through half preparative high-performance liquid chromatographic post (250mm × 10mm, 5 μ m) separate (flow velocity 2mL/min, detect wavelength 203nm), taking methanol-water-formic acid mixed solution (first alcohol and water by volume as the mixing solutions of 72:28 composition again with the mixed solution that accounts for the formic acid mixing gained of gained mixing solutions quality percentage composition as 0.2%) carry out wash-out, collect 52.5min place chromatographic peak, gained elution fraction is carried out to concentrating under reduced pressure, dry, obtain the unsetting powdery substance of 152mg white.
The unsetting powdery substance of white that adopts the identical compound structure authentication method of embodiment 1 to make the present embodiment carries out Structural Identification, the Correlated Spectroscopy diagram data of high resolution mass spectrum spectrogram, proton nmr spectra, carbon-13 nmr spectra, DEPT spectrogram, hsqc spectrum figure, HMBC spectrogram, H-H COSY spectrogram, NOESY spectrogram and embodiment 1 gained obtaining is basically identical, result shows that the unsetting powdery substance of this white is the compound with structure shown in formula I
Experiment material:
People's lung cancer A549 cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell MCF-7, all purchase the Shanghai cell institute with the Chinese Academy of Sciences.These four kinds of cells all (separately add Glu0.03%, Hepes0.06%, NaHCO with the RPMI1640 substratum containing 10% deactivation NBS
30.2%) in 37 DEG C, 5%CO
2under condition, cultivate, within 3 days, go down to posterity.The vegetative period cell of taking the logarithm is tested.The Sasanguasaponin compound with structure shown in formula I that experiment used oil theasaponin compound makes for the preparation method who provides according to embodiment 1,
Experimental technique:
Adopt conventional mtt assay, measure the susceptibility of tumor cell line to medicine.Specific experiment arrangement is as follows:
Experiment is divided into negative control group, experimental group and positive controls.The solution adding in negative control group subject cell is perfect medium, establishes 3 multiple holes.In experimental group subject cell, add the solution of the Sasanguasaponin compound that the embodiment 1 that contains different concns makes, solvent is that volume ratio is the DMSO of 1:99 and the mixing solutions of PBS, experiment arranges 5 concentration, wherein the concentration of this Sasanguasaponin compound is respectively: 6.25 μ g/mL, 9.375 μ g/mL, 12.5 μ g/mL, 18.25 μ g/mL, 25.00 μ g/mL, all establish 3 multiple holes for every group.In positive controls subject cell, add containing 5-FU(5-Fluracil) solution, solvent is that volume ratio is the DMSO of 1:99 and the mixing solutions of PBS, the concentration of 5-FU is 50 μ g/mL, establishes 3 multiple holes.
According to experimental establishment, adjusting each cell concn with perfect medium is 5 × 10
4/ mL, is inoculated in respectively in 96 well culture plates, mark one by one, 100 μ L/ holes, 37 DEG C, 5%CO
2overnight incubation under condition.The Sasanguasaponin compound with structure shown in formula I (final concentration is respectively: 6.25 μ g/mL, 9.375 μ g/mL, 12.5 μ g/mL, 18.25 μ g/mL, 25.00 μ g/mL) that experimental group adds the embodiment 1 of different concns to make, 10 μ L/ holes; Positive controls adds 5-FU (50 μ g/mL), 10 μ L/ holes; Negative control group adds equal-volume perfect medium, 10 μ L/ holes; All establish 3 multiple holes, cultivate respectively 24h for every group.Before stopping cultivating, 4h adds respectively MTT (5mg/mL), 10 μ l/ holes, and after cultivation finishes, every hole adds DMSO100 μ L, places after shaking table vibration 10min, and detecting wavelength by microplate reader is the absorbance A value at 490nm place.
Processing for Data Analysis in Physics:
With IC
50value is evaluated the restraining effect of Sasanguasaponin compound to different tumour cells.
Calculate growth of tumour cell inhibiting rate (Inhibition Rate) by formula below:
Growth of tumour cell inhibiting rate (%)=[(A negative control group-A experimental group)/A negative control group] × 100%;
Taking the concentration of Sasanguasaponin compound as X-coordinate, growth of tumour cell inhibiting rate is ordinate zou mapping, obtains inhibiting rate-concentration curve, the concentration of medicine while then drawing 50% inhibiting rate, the i.e. IC of Sasanguasaponin compound to subject cell
50value (half-inhibition concentration), the IC of the Sasanguasaponin compound that embodiment 1 makes to different subject cells
50value is in table 2.
The IC of table 2 Sasanguasaponin compound to four kinds of different tumour cells
50value
| Subject cell | A549 | B16 | BEL-7402 | MCF-7 |
| IC 50（μg/mL） | 10.63±0.07 | 13.14±0.24 | 12.73±0.84 | 11.95±1.20 |
Experimental result shows, positive controls 5-FU, under 50 μ g/mL concentration, has certain restraining effect to all subject cells of the present embodiment.From experimental group data, the IC of the Sasanguasaponin compound that embodiment 1 makes to these four kinds of tumour cells
50value is all less than 15 μ g/mL, shows that this Sasanguasaponin compound has good anti-tumor activity, especially obvious for the inhibition of A549 cell, its IC
50value is only 10.63 μ g/mL.Experimental result shows, the Sasanguasaponin compound of structure shown in formula I that what embodiment 1 made have has certain restraining effect to liver tumor, melanoma, breast tumor or lung tumor, can be applied to and prepare antitumor drug.
Adopt identical experimental technique to investigate Sasanguasaponin compound that embodiment 2 to embodiment 4 makes to people's lung cancer A549 cell, B16 mouse black-in tumor cell, the restraining effect of human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell MCF-7, obtain similar experimental result, illustrate that the Sasanguasaponin compound with structure shown in formula I that embodiment 2 to embodiment 4 makes has certain restraining effect to liver tumor, melanoma, breast tumor or lung tumor, can be applied to and prepare antitumor drug.
Experiment in embodiment 6 bodies
Experiment material:
ICR mouse, clean level, male, body weight 18-22g, is provided by Shanghai Slac Experimental Animal Co., Ltd..H
22, S
180liver cancer cell is purchased from Chinese Academy of Sciences's Shanghai cell bank.The compound with structure shown in formula I that experiment used oil theasaponin compound makes for the preparation method who provides according to embodiment 1,
Experimental technique:
The effect of the Sasanguasaponin compound with structure shown in formula I that the present embodiment investigation embodiment 1 makes to liver cancer.Study subject is H
22transplanted tumor model mice, S
180transplanted tumor model mice.
Mouse H
22transplanted tumor model, mouse S
180the foundation of transplanted tumor model and experimental establishment:
Get H
22or S
180liver cancer (sarcoma) cell cryopreservation tube, is placed in 37 DEG C of waters bath with thermostatic control and thaws, centrifugal collecting cell, by PBS liquid washed twice, use again PBS liquid re-suspended cell, more than ICR mouse peritoneal passed for 3 generations, get the mouse that belly obviously expands, dislocation is put to death, to belly alcohol disinfecting, disposable sterilized injector extracts oyster white ascites, injects sterilized centrifuge tube, cell counter counting, with physiological saline adjustment cell density to 5 × 10
6/ mL, carries out modeling to every the right oxter inoculation of mouse 0.2mL cell suspension.At random be divided into blank group, experimental group 1, experimental group 2 and positive controls by mouse next day, and 12 every group, and carry out administration.The Sasanguasaponin compound with structure shown in formula I that experimental group 1 and experimental group 2 provide embodiment 1 to make, administering mode is abdominal injection, the dosage of two groups is: experimental group 1 for 1.5mg/kg(be every 1kg body weight administration 1.5mg), the physiological saline that solvent is 0.9%, once a day; Experimental group 2 is 3mg/kg, the physiological saline that solvent is 0.9%, once a day.Positive controls provides CTX (endoxan), and administering mode is abdominal injection, and dosage is: 20mg/kg, the physiological saline that solvent is 0.9%, the next day once.Blank group provides blank solvent, i.e. 0.9% physiological saline, and abdominal injection, injection volume is 0.2mL, once a day.All group mouse successive administrations 10 days, take Mouse Weight every day, record the clinical manifestation of mouse.After last administration 2h, peel off knurl body, take knurl weight, and calculate tumour inhibiting rate.
Processing for Data Analysis in Physics:
Investigate the inhibition situation of different group tumours with tumour inhibiting rate, calculate tumour inhibiting rate according to formula below:
Tumour inhibiting rate (%)=[(the average knurl weight of the average knurl weight-administration of blank group group) the average knurl weight of/blank group] × 100%
Heavy and the tumour inhibiting rate of the transplanted tumor knurl of each group is in table 3.
The transplanted tumor knurl of the different groups of table 3 weighs and tumour inhibiting rate
Note: compare with blank group, * P<0.05, * * P<0.01
Experimental result shows, for H
22transplanted tumor model, compared with blank group, the knurl of the transplanted tumor of positive controls, experimental group 1 and experimental group 2 is heavily significantly less than the knurl weight of the transplanted tumor of blank group, and significant difference illustrates that the Sasanguasaponin compound with structure shown in formula I that embodiment 1 makes can significantly suppress H
22the growth of liver cancer.For S
180transplanted tumor model, compared with blank group, the knurl of the transplanted tumor of positive controls, experimental group 1 and experimental group 2 is heavily significantly less than the knurl weight of the transplanted tumor of blank group, and significant difference illustrates that the Sasanguasaponin compound with structure shown in formula I that embodiment 1 makes can significantly suppress S
180the growth of sarcoma.Experimental result also shows, when the dosage of the Sasanguasaponin compound with structure shown in formula I making as embodiment 1 is 3.0mg/kg, to H
22the inhibiting rate of transplanted tumor reaches 42.2%, points out this Sasanguasaponin compound to have significant anti-mouse H
22the effect of liver cancer growth; When the dosage of the Sasanguasaponin compound with structure shown in formula I making as embodiment 1 is 3.0mg/kg, to S
180the inhibiting rate of transplanted tumor reaches 32.1%, points out this Sasanguasaponin compound to have stronger anti-mouse S
180the effect of liver cancer growth.Above result shows that the Sasanguasaponin compound with structure shown in formula I that embodiment 1 makes has good anti-tumor activity, can be applied to and prepare antitumor drug.
Adopt identical experimental technique to investigate Sasanguasaponin compound that embodiment 2 to embodiment 4 makes to mouse H
22liver cancer, S
180the restraining effect of liver cancer, has obtained similar experimental result, illustrates that the Sasanguasaponin compound that embodiment 2 to embodiment 4 makes can effectively suppress H
22liver cancer growth, S
180the growth of meat cancer.Illustrate that the Sasanguasaponin compound that embodiment 2 to embodiment 4 makes has good anti-tumor activity, can be applied to and prepare antitumor drug.
The preparation of embodiment 7 tablets
Get the Sasanguasaponin compounds with structure shown in I that embodiment 1 makes and add conventional auxiliary material, make tablet according to ordinary method.
The preparation of embodiment 8 granules
Get the Sasanguasaponin compound with structure shown in I that embodiment 2 makes and add conventional auxiliary material, according to the agent of ordinary method granulation.
The preparation of embodiment 9 capsules
Get the Sasanguasaponin compound with structure shown in I that embodiment 3 makes and add conventional auxiliary material, make capsule according to ordinary method.
The preparation of embodiment 10 injections
Get the Sasanguasaponin compound with structure shown in I that embodiment 4 makes and add conventional auxiliary material, make injection according to ordinary method.
The preparation of embodiment 11 emulsions
Get the Sasanguasaponin compound with structure shown in I that embodiment 1 makes and add conventional auxiliary material, make emulsion according to ordinary method.
The preparation of embodiment 12 suspensoids
Get the Sasanguasaponin compound with structure shown in I that embodiment 1 makes and add conventional auxiliary material, make suspensoid according to ordinary method.
Below be only the preferred embodiment of the present invention, it should be pointed out that above-mentioned preferred implementation should not be considered as limitation of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For those skilled in the art, without departing from the spirit and scope of the present invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. a Sasanguasaponin compound, is characterized in that, has structure shown in formula I:
2. a preparation method for Sasanguasaponin compound, is characterized in that, comprises the following steps:
Step 1: obtain oil tea total saponin extracts;
Step 2: get described oil tea total saponin extracts, through separation and purification, to obtain final product.
3. preparation method according to claim 2, is characterized in that, step 1 is specially:
Get after the pulverizing of oil tea root, through extracting, obtain extracting solution;
Get described extracting solution through concentrated, obtain fluid extract;
Get described fluid extract and mix with water, filter, collect filtrate;
Get described filtrate and separate through macroporous resin, with alcohol-water mixed solution wash-out, collected volume, than the alcohol-water mixed solution elution fraction for 70:30～95:5, obtains described oil tea total saponin extracts.
4. preparation method according to claim 2, is characterized in that, step 2 is specially:
Get described oil tea total saponin extracts, separate through decompression silica gel chromatographic column, with chloroform-methanol mixed solution wash-out, collected volume, than the chloroform-methanol mixed solution elution fraction for 80:20～60:40, obtains purified for the first time;
Described in getting, press anti-phase octadecylsilane chemically bonded silica chromatographic column to separate for the first time in purified warp, with methanol-water mixture wash-out, collected volume, than the methanol-water mixture elution fraction for 70:30～90:10, obtains purified for the second time;
Described in getting, purified separates through dynamic axial compression chromatographic column for the second time, and with methanol-water mixture wash-out, collected volume, than the methanol-water mixture elution fraction for 75:25, obtains purified for the third time;
Described in getting, purified separates through half preparative high-performance liquid chromatographic post for the third time, with methanol-water-formic acid mixed solution wash-out, and flow velocity 2mL/min, collecting retention time is the corresponding elution fraction of 52.5min, to obtain final product;
Described methanol-water-formic acid mixed solution is: first alcohol and water by volume for the mixing solutions of 72:28 composition again with the mixed solution that accounts for the formic acid mixing gained that described mixing solutions quality percentage composition is 0.2%.
5. the Sasanguasaponin compound that the preparation method as described in any one in claim 2 to 4 makes.
6. the Sasanguasaponin compound as described in claim 1 or 5 is in the application of preparing in antitumor drug.
7. application according to claim 6, is characterized in that, described tumour is liver tumor, melanoma, breast tumor or lung tumor.
8. an antitumor drug, is characterized in that, it comprises Sasanguasaponin compound and pharmaceutically acceptable auxiliary material as described in claim 1 or 5.
9. antitumor drug according to claim 8, is characterized in that, its formulation is tablet, granule, capsule, injection, emulsion or suspensoid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021109836A1 (en) * | 2019-12-06 | 2021-06-10 | 江苏一片叶高新科技有限公司 | Tea saponin compound, preparation method therefor, and application thereof |
| CN114886904A (en) * | 2022-05-07 | 2022-08-12 | 赣南医学院第一附属医院 | Hyaluronic acid-tea saponin, and its preparation method and anti-tumor effect |
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| CN101392015A (en) * | 2008-07-22 | 2009-03-25 | 沈阳药科大学 | Triterpene saponin in camellia seeds, preparation method and medical use thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021109836A1 (en) * | 2019-12-06 | 2021-06-10 | 江苏一片叶高新科技有限公司 | Tea saponin compound, preparation method therefor, and application thereof |
| CN114886904A (en) * | 2022-05-07 | 2022-08-12 | 赣南医学院第一附属医院 | Hyaluronic acid-tea saponin, and its preparation method and anti-tumor effect |
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