CN104059123A - Camellia saponin compound, preparation method and application thereof and antitumor drug prepared from same - Google Patents
Camellia saponin compound, preparation method and application thereof and antitumor drug prepared from same Download PDFInfo
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Abstract
The invention belongs to the technical field of medicinal chemistry and discloses a camellia saponin compound, a preparation method and an application thereof. A structure of the camellia saponin compound is represented as formula I. The camellia saponin compound has certain inhibitive effects on hepatoma cells, breast cancer cells, melanoma cells and lung cancer cells and can effectively inhibit generation of liver neoplasms. The camellia saponin compound is proved to have a significant antitumor activity and can be used for preparing an antitumor drug.
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), is called again tea subtree, tea oil tree, spends tea in vain, is the evergreen dungarunga of Theaceae (Theaceae) Camellia (Camellia Linn), is 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 ".With the seed oil expression of oil tea, obtain tea oil, 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; Compare 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 Materia Medica > >, records, and tea seed, the fragrant poison of bitter cold (saponin), cures mainly and breathes heavily anxious cough, removes disease dirt; Herbal > > is also on the books in < < China, that the root of oil tea and root leatherware thereof have is clearing heat and detoxicating, the 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, the separated chemical composition obtaining comprises 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 many caused Sasanguasaponin compounds different from array mode of its aglycon complex structure, sugar moieties kind are numerous, being further purified with separated 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 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, obtain.
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 separated through macroporous resin, with alcohol-water mixed solution wash-out, collected volume, than being the alcohol-water mixed solution elution fraction of 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 water or 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 0.1%～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 the oil tea root solution used with extraction of pulverizing in step 1 and mix, under 30 ℃～50 ℃ 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 water or alcohol-water mixing solutions, under 30 ℃～50 ℃ 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 1:5～20.
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 water or alcohol-water mixing solutions, under 30 ℃～50 ℃ 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 1:5～20 in mass ratio with water, 100 order～300 orders filter, and collect filtrate; After gained filtrate is centrifugal, get supernatant liquor separated 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, separated through decompression silica gel chromatographic column, with chloroform-methanol mixed solution wash-out, collected volume, than being the chloroform-methanol mixed solution elution fraction of 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 separated for the first time, with methanol-water mixture wash-out, collected volume, than being the methanol-water mixture elution fraction of 70:30～90:10, obtains purified for the second time;
Purified is separated through dynamic axial compression chromatographic column for the second time to get gained, and with methanol-water mixture wash-out, collected volume, than being the methanol-water mixture elution fraction of 75:25, obtains purified for the third time;
Purified is separated through half preparative high-performance liquid chromatographic post for the third time to get gained, 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, obtains; Octadecylsilane half preparative chromatography post (C18 half preparative chromatography post): 250mm * 10mm, 5 μ m.
Methanol-water-formic acid mixed solution is: the mixing solutions that first alcohol and water forms for 78:22 by volume 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 is separated, 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 separated 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 is separated, 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 separation provided by the invention, 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, obtain.
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, with chloroform-methanol system, carry out gradient elution, 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 separated in purified warp for the first time, with methanol-water system, carry out gradient elution, 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;
Purified is separated through dynamic axial compression chromatographic column for the second time to get gained, 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 being the methanol-water mixture elution fraction of 75:25, obtains purified for the third time;
Purified is separated through octadecylsilane chemically bonded silica half preparative high-performance liquid chromatographic post for the third time to get gained, 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, obtain;
Wherein, methanol-water-formic acid mixed solution is: the mixing solutions that first alcohol and water forms for 78:22 by volume 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, obtain.
The present invention also provides the application of a kind of Sasanguasaponin compound in preparing 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, obtain.
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, obtain.
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 prepare antitumor drug,
accompanying drawing explanation
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 DEPT spectrogram of the Sasanguasaponin compound that embodiment 1 makes;
Fig. 8 shows the hsqc spectrum figure of the Sasanguasaponin compound that embodiment 1 makes;
Fig. 9 shows the HMBC spectrogram of the Sasanguasaponin compound that embodiment 1 makes;
Figure 10 shows the H-H COSY spectrogram of the Sasanguasaponin compound that embodiment 1 makes;
Figure 11 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 or suitably change and combination within not departing from content of the present invention, spirit and scope, 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, U.S. Kromsil company.
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:
Embodiment 1 has the preparation of the Sasanguasaponin compound of structure shown in formula I
Extract:
Get dry oil tea root 2000g, with pulverizer, be ground into wood chip, adding 30.0L volume percent is 70% aqueous ethanolic solution, under 40 ℃ of conditions, soaks after 12h, be heated to reflux, extract 3.0h, collect extracting solution, by extracting liquid filtering, concentrating under reduced pressure, obtains fluid extract 618g.
Macroporous resin enrichment:
Get the fluid extract of extraction step gained, add 6.0L water, stirring and dissolving, with 100 order filter-cloth filterings, by the centrifugal 10min of filtrate 12000r/min, after centrifugal, obtain 5.8L supernatant liquor, get supernatant liquor further separated through D101 type macroporous resin, use successively 10.0L water, 15.0L30% aqueous ethanolic solution, 15.0L70% aqueous ethanolic solution carries out wash-out, collect 70% aqueous ethanolic solution wash-out component, concentrating under reduced pressure, , after testing, in gained concentrated solution, contain Sasanguasaponin compounds, through 100 ℃, baking oven, 48h is dry, obtain brown medicinal extract 51g, 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, through decompression silicagel column (silica gel: 60-100 order) separation, with chloroform-methanol system, carry out gradient elution, 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 1000mL; 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 following 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.The chloroform-methanol eluant solution component that merges 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 15.5g 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 carry out separation, with methanol-water system, carry out gradient elution, 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, through dynamic axial column chromatography, carry out separation (flow velocity 150mL/min, detect wavelength 203nm), in volume percent, with 60%, 75%, 80% and 90% methanol aqueous solution, carry out wash-out 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) carry out separation (flow velocity 2mL/min, detect wavelength 203nm), methanol-water-formic acid the mixed solution (mixing solutions that first alcohol and water forms as 78:22 by volume again with the mixed solution that accounts for the formic acid mixing gained that gained mixing solutions quality percentage composition is 0.2%) of take carries 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 48mg white.
Compound structure is identified:
The unsetting powdery substance of gained white is carried out to thin-layer chromatography, aceticanhydride-strong sulfuric acid response, Molish and react evaluation, qualification result is: thin-layer chromatography sulfuric acid ethanol colour developing result is red-purple spot, aceticanhydride-strong sulfuric acid response is 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/z1329.6129[M-H in this mass spectrogram]
-, with molecular formula C
64h
98o
29estimating of molecular weight value [M-H]
-, m/z1329.6116, basically identical, the molecular formula that shows this compound is C
64h
98o
29.With the complete acid hydrolysis of 2N TFA, obtain monose, gained monose is carried out after derivatize to GC analysis, the existence of D-Glucose aldehydic acid methyl esters, 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 and Fig. 6, and Fig. 6 is 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. 7.The hsqc spectrum figure of the unsetting powdery substance of this white is shown in Fig. 8.The HMBC spectrogram of the unsetting powdery substance of this white is shown in Fig. 9.Figure 10 is shown in by the H-H COSY collection of illustrative plates of the unsetting powdery substance of this white.Figure 11 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 powdery substance of table 1
| Position | δ H | δ C | Position | δ H | δ C |
| 1 | 0.99m,1.52m | 39.1 | 22-O-Ang | -- | -- |
| 2 | 1.91m,2.19m | 26.2 | 1″ | -- | 168.4 |
| 3 | 4.10dd(11.0,4.5) | 84.5 | 2″ | -- | 129.2 |
| 4 | -- | 43.6 | 3″ | 5.88dq(7.5,1.5) | 136.7 |
| 5 | 1.85m | 47.4 | 4″ | 2.05d(7.5) | 15.8 |
| 6 | 1.12m,1.50m | 18.7 | 5″ | 1.83s | 21.1 |
| 7 | 2.06m,2.10m | 36.5 | GlcA | -- | -- |
| 8 | -- | 41.7 | 1 | 4.84d(10.0) | 104.1 |
| 9 | 1.47m | 47.4 | 2 | 4.55m | 78.1 |
| 10 | -- | 37.1 | 3 | 4.35m | 85.0 |
| 11 | 1.85m,1.98m | 24.2 | 4 | 4.57m | 69.8 |
| 12 | 5.58brs | 125.6 | 5 | 4.18m | 77.2 |
| 13 | -- | 143.9 | 6 | -- | 170.0 |
| 14 | -- | 47.9 | OCH 3 | 3.78 | 52.3 |
| 15 | 4.27brs | 67.7 | Gal | -- | -- |
| 16 | 4.51brs | 73.2 | 1 | 5.74d(10.0) | 101.8 |
[0097]
| 17 | -- | 48.6 | 2 | 4.57m84.0 |
| 18 | 3.16m | 41.0 | 3 | 4.35m75.2 |
| 19 | 1.52m,3.16m | 46.9 | 4 | 4.59m70.7 |
| 20 | -- | 36.5 | 5 | 4.39m76.6 |
| 21 | 6.79d(10.5) | 78.8 | 6 | 4.43m4.45m61.9 |
| 22 | 6.40d(10.5) | 73.3 | Xyl | ,---- |
| 23 | 9.95brs | 64.4 | 1 | 5.12d(7.5)107.9 |
| 24 | 1.52s | 11.9 | 2 | 4.29m76.5 |
| 25 | 0.90s | 16.6 | 3 | 4.13m78.5 |
| 26 | 1.05s | 17.8 | 4 | 4.42m71.0 |
| 27 | 1.90s | 21.4 | 5 | 3.59m,4.53m67.8 |
| 28 | 3.60d(11.0) | 63.1 | Gal | ---- |
| -- | 3.84d(11.0) | -- | 1 | 5.86d(7.5)103.2 |
| 29 | 1.19s | 29.6 | 2 | 4.51m73.9 |
| 30 | 1.42s | 20.4 | 3 | 4.45m75.5 |
| 21-O-Ang | -- | -- | 4 | 4.46m70.6 |
| 1′ | -- | 167.5 | 5 | 4.53m76.5 |
| 2′ | -- | 129.0 | 6 | 4.55m,4.57m62.3 |
| 3′ | 6.05dq(7.5,1.5) | 137.6 | -- | ---- |
| 4′ | 2.17d(7.5) | 16.0 | -- | ---- |
| 5′ | 2.10s | 21.4 | -- | ---- |
Note: chemical shift YippmWei unit in table, coupling constant (J) YiHzWei unit.
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 data show 64 carbon signals altogether, analyze this compound and contain altogether 11 methyl carbon, 12 mesomethylene carbon, 31 methine carbons and 10 quaternary carbons by DEPT and hsqc spectrum.Further analyze
13c NMR spectrum, this spectrogram demonstrates four sugared end group carbon signal δ 104.1,101.8,107.9 and 103.2, is respectively the end group carbon of D-Glucose aldehydic acid methyl esters, 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 210.0.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), 1.05 (Me-26), 1.19 (Me-29), 1.42 (Me-30), 1.52 (Me-24) and 1.90 (Me-27); Even Oxymethylene δ 3.60 (H-28, d, J=11.0Hz) and 3.84 (H-28, d, J=11.0Hz); Five oxygen methyne δ 4.10 (1H, dd, J=11.0,4.5Hz, H-3) of company, 4.27 (1H, brs, H-15), 4.51 (1H, brs, H-16), 6.79 (1H, d, J=10.5Hz, H-21) and 6.40 (1H, d, J=10.5Hz, H-22); Alkene hydrogen proton δ 5.58 (1H, brs, H-12) and aldehyde radical hydrogen proton δ 9.95 (1H, brs, H-23).In addition this white unsetting powdery substance,
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 2.10 (3H, s, 21-O-Ang-5')]; δ [5.88 (1H, dq, J=7.5,1.5Hz, 22-O-Ang-3 "), 2.05 (3H, d, J=7.5Hz, 22-O-Ang-4 ") and 1.83 (3H, s, 22-O-Ang-5 ")].
In H-H COSY spectrogram, with the methyne δ that even oxygen methine protons is adjacent
h4.27 (1H, brs) and δ
h4.51 (1H, brs) are relevant, illustrate that they are in ortho position and δ
h4.51 is H-16 signal, δ
h4.27 is H-15 signal; δ
h6.79 (1H, d, J=10.5Hz) and δ
hthey are in ortho position and δ coherent signal explanation between 6.40 (1H, d, J=10.5Hz)
h6.79 is H-21 signal, δ
h6.40 is H-22 signal.
To the unsetting powdery substance HMBC of this white spectrogram analyze learn two angeloyl groups be connected in respectively 21,22 of this compound upper because in HMBC spectrogram δ
h6.79 (1H, d, J=10.5Hz, H-21) and δ
c167.5 (21-O-Ang-1') are relevant, δ
h6.40 (1H, d, J=10.5Hz, H-22) and δ
c168.4 (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, 15 α, 16 α; 28-tetrahydroxyolean-12-en-23-al(Helvetica Chimica Acta2013.Vol.96) nuclear magnetic data contrast; both data are basically identical, and the aglycon structure of determining this compound is 21 β, 22 α-O-, bis-angeloyl groups-15 α; 16 α, 28-trihydroxy-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, and result is as follows: δ
h4.84 (1H, d, J=10.0Hz), 5.74 (1H, d, J=10.0Hz), 5.12 (1H, d, J=7.5Hz) and 5.86 (1H, d, J=7.5Hz), be connected in respectively δ
c104.1 (glucuronic acid methyl ester-C-1), 101.8 (semi-lactosi-C-1), 107.9 (wood sugar-C-1), on 103.2 (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 spectrogram, the anomeric proton δ of glucuronic acid methyl ester
h4.84 with 3 δ of aglycon
c84.5 is relevant, further confirmed that sugar chain is connected in 3 of aglycon.Through By consulting literatures, find the camellioside H(Bioorganic & medicinal chemistry letters2010 reporting in this compound sugar chain and document, 20,7435-7439) sugar chain data are close, thereby 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 methyl esters.The order of connection of this sugar chain also can confirm by analyzing HMBC spectrogram: glucuronic acid methyl ester anomeric proton δ in HMBC spectrogram
h4.84 with 3 carbon δ of aglycon
c84.5 relevant, semi-lactosi anomeric proton δ
h5.74 with 3 carbon δ of glucuronic acid methyl ester
c85.0 relevant, wood sugar anomeric proton δ
h5.12 with 2 carbon δ of semi-lactosi
c84.0 relevant, 2 hydrogen δ of semi-lactosi
h4.57 with wood sugar end group carbon δ
c107.9 relevant, another semi-lactosi anomeric proton δ
h5.86 with 2 carbon δ of glucuronic acid methyl ester
c78.1 relevant, 2 hydrogen δ of glucuronic acid methyl ester
h4.55 with this semi-lactosi end group carbon δ
c103.2 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 spectrogram, δ
h6.79 (1H, d, J=10.5Hz, H-21) and δ
h1.19 (3H, s, H-29) are relevant, and prompting H-21 is α configuration; δ
h4.10 (1H, dd, J=4.5,11.0Hz, H-3) and δ
h9.95 (3H, brs, H-23) are relevant, and prompting H-3 is α configuration; δ
h4.51 (1H, brs, H-16) and δ
h3.60 (1H, d, J=11.0Hz, H-28), 3.84 (1H, d, J=11.0Hz, H-28) are relevant, and prompting H-16 is beta comfiguration, δ
h6.40 (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 called after 21 β; 22 α-O-, bis-angeloyl groups-15 α; 16 α, 28-trihydroxy-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 methyl esters glycosides.
Embodiment 2 has the preparation of the Sasanguasaponin compound of structure shown in formula I
Extract:
Get dry oil tea root 2000g, with pulverizer, be ground into wood chip, adding 40.0L volume percent is 0.1% aqueous ethanolic solution, under 50 ℃ of conditions, soaks after 12h, be heated to reflux, extract three times, each 0.5h, collects all extracting solutions, by extracting liquid filtering, concentrating under reduced pressure, obtains fluid extract 685g.
Macroporous resin enrichment:
Get the fluid extract that extraction step obtains, add 3.425L water, stirring and dissolving, with 300 order filter-cloth filterings, by the centrifugal 10min of filtrate 12000r/min, after centrifugal, obtain 5.9L supernatant liquor, get supernatant liquor further separated through D101 type macroporous resin, use successively 10.0L water, 15.0L60% aqueous ethanolic solution, 10.0L95% aqueous ethanolic solution carries out wash-out, collect 95% aqueous ethanolic solution wash-out component, concentrating under reduced pressure, after testing, in gained concentrated solution, contain Sasanguasaponin compounds, through 100 ℃, baking oven, 48h is dry, obtain brown medicinal extract 51g, 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, through decompression silicagel column (silica gel: 60-80 order) separation, with chloroform-methanol system, carry out gradient elution, 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 1000mL; 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 following 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.The chloroform-methanol eluant solution component that merges 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 12.2g 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 carry out separation, with methanol-water system, carry out gradient elution, 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, through dynamic axial column chromatography, carry out separation (flow velocity 150mL/min, detect wavelength 203nm), in volume percent, with 60%, 75%, 80% and 90% methanol aqueous solution, carry out wash-out 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) carry out separation (flow velocity 2mL/min, detect wavelength 203nm), methanol-water-formic acid the mixed solution (mixing solutions that first alcohol and water forms as 78:22 by volume again with the mixed solution that accounts for the formic acid mixing gained that gained mixing solutions quality percentage composition is 0.2%) of take carries 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 39mg 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
Embodiment 3 has the preparation of the Sasanguasaponin compound of structure shown in formula I
Extract:
Get dry oil tea root 2000g, with pulverizer, be ground into wood chip, adding 10.0L volume percent is under 30 ℃ of conditions, to soak after 10h in 95% aqueous ethanolic solution, is heated to reflux, extract twice, each 1.5h, collects all extracting solutions, by extracting liquid filtering, concentrating under reduced pressure, obtains fluid extract 585g.
Macroporous resin enrichment:
Get the fluid extract that extraction step obtains, add 11.7L water, stirring and dissolving, with 200 order filter-cloth filterings, by the centrifugal 10min of filtrate 12000r/min, after centrifugal, obtain 5.7mL supernatant liquor, get supernatant liquor further separated through D101 type macroporous resin, use successively 10.0L water, 15.0L40% aqueous ethanolic solution, 15.0L75% aqueous ethanolic solution carries out wash-out, collect 75% aqueous ethanolic solution wash-out component, concentrating under reduced pressure, after testing, in gained concentrated solution, contain Sasanguasaponin compounds, through 100 ℃, 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, through decompression silicagel column (silica gel: 60-100 order) separation, with chloroform-methanol system, carry out gradient elution, 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 1000mL; 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 following 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.The chloroform-methanol eluant solution component that merges 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 16.5g 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 carry out separation, with methanol-water system, carry out gradient elution, 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, through dynamic axial column chromatography, carry out separation (flow velocity 150mL/min, detect wavelength 203nm), in volume percent, with 60%, 75%, 80% and 90% methanol aqueous solution, carry out wash-out 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 15.0mL 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) carry out separation (flow velocity 2mL/min, detect wavelength 203nm), methanol-water-formic acid the mixed solution (mixing solutions that first alcohol and water forms as 78:22 by volume again with the mixed solution that accounts for the formic acid mixing gained that gained mixing solutions quality percentage composition is 0.2%) of take carries 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 53mg 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
Embodiment 4 has the preparation of the Sasanguasaponin compound of structure shown in formula I
Extract:
Get dry oil tea root 2000g, with pulverizer, be ground into wood chip, add 20.0L water, under 50 ℃ of conditions, soak after 6h, be heated to reflux, extract twice, each 2h, collects all extracting solutions, and by extracting liquid filtering, concentrating under reduced pressure, obtains fluid extract 652g.
Macroporous resin enrichment:
Get the fluid extract that extraction step obtains, add 6.0L water, stirring and dissolving, with 200 order filter-cloth filterings, by the centrifugal 10min of filtrate 12000r/min, centrifugal after, obtain 6.1L supernatant liquor, get supernatant liquor further separated through D101 type macroporous resin, with 10.0L water, 15.0L50% aqueous ethanolic solution, 15.0L70% aqueous ethanolic solution, carry out wash-out successively, collect 70% aqueous ethanolic solution wash-out component, concentrating under reduced pressure, after testing, in gained concentrated solution, contain Sasanguasaponin compounds, through 100 ℃, baking oven, 48h is dry, obtain brown medicinal extract 59g, 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, through decompression silicagel column (silica gel: 60-100 order) separation, with chloroform-methanol system, carry out gradient elution, 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 1000mL; 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 following 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.The chloroform-methanol eluant solution component that merges 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 11.4g 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 carry out separation, with methanol-water system, carry out gradient elution, 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, through dynamic axial column chromatography, carry out separation (flow velocity 150mL/min, detect wavelength 203nm), in volume percent, with 60%, 75%, 80% and 90% methanol aqueous solution, carry out wash-out 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), 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) carry out separation (flow velocity 2mL/min, detect wavelength 203nm), methanol-water-formic acid the mixed solution (mixing solutions that first alcohol and water forms as 78:22 by volume again with the mixed solution that accounts for the formic acid mixing gained that gained mixing solutions quality percentage composition is 0.2%) of take carries 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 32mg 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
Embodiment 5 has the preparation of the Sasanguasaponin compound of structure shown in formula I
Extract:
Get dry oil tea root 2000g, with pulverizer, be ground into wood chip, join in 16.0L aqueous ethanolic solution and extract, obtain extracting solution 15.0L, gained extracting solution is concentrated, obtain fluid extract 578g.
Macroporous resin enrichment:
In gained fluid extract, add water 6.0L, be stirred to dissolving, filter, obtain filtrate 5.6L; Gained filtrate is crossed to macroporous resin and carry out separation, with alcohol-water mixed solution wash-out, collected volume is than being the alcohol-water mixed solution elution fraction of 70:30～95:5, after concentrating under reduced pressure, through 100 ℃, baking oven, 48h is dry, obtain brown medicinal extract 61g, after testing, wherein contain Sasanguasaponin compounds, 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, separated through decompression silicagel column, with chloroform-methanol system, carry out gradient elution, 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 12.8g 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 carry out separation, with methanol-water system, carry out gradient elution, 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, the composition that contains in these three elution fractions similar (the efficient analysis liquid chromatography time is 30min, detected peaks is between 20min-28min), merge this three elution fractions, 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, through dynamic axial column chromatography, carry out separation, with methanol-water system, carry out wash-out, collected volume is than being the methanol-water mixture elution fraction of 75:25.Through efficient analysis liquid-phase chromatographic analysis, in 15.0min place, be there is to detected peaks in gained elution fraction, collect this elution fraction, obtain 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) carry out separation (flow velocity 2mL/min, detect wavelength 203nm), methanol-water-formic acid the mixed solution (mixing solutions that first alcohol and water forms as 78:22 by volume again with the mixed solution that accounts for the formic acid mixing gained that gained mixing solutions quality percentage composition is 0.2%) of take carries 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 42mg 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
Embodiment 6 In vitro cell experiments
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, Shanghai cell institute of the Jun Gouyu 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 ℃, 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.Mtt assay, claims again MTT colorimetry, is a kind of method that detects cell survival and growth.Its detection principle is that the succinodehydrogenase in viable cell plastosome can make exogenous MTT be reduced to water-insoluble bluish voilet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) (DMSO) energy dissolved cell, measures its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect viable cell quantity.Within the scope of certain cell count, the amount that MTT crystallization forms is directly proportional to cell count.The method has been widely used in the activity detection of some biologically active factorss, large-scale screening anti-tumor medicine, cell toxicity test and tumor radiosensitivity mensuration etc.Its feature is highly sensitive, economical.Therefore, in this experiment, we adopt mtt assay to screen tested 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.The solution that adds the Sasanguasaponin compound that the embodiment 1 that contains different concns makes in experimental group subject cell, 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, with perfect medium, adjusting each cell concn is 5 * 10
4/ mL, is inoculated in respectively in 96 well culture plates, mark one by one, 100 μ L/ holes, 37 ℃, 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 by microplate reader, detecting wavelength 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.
The formula of pressing below calculates growth of tumour cell inhibiting rate (Inhibition Rate):
Growth of tumour cell inhibiting rate (%)=[(A negative control group-A experimental group)/A negative control group] * 100%;
The concentration of Sasanguasaponin compound of take is X-coordinate, and 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） | 29.73±1.48 | 19.47±1.51 | 28.12±1.85 | 14.00±1.92 |
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 30 μ g/mL, shows that this Sasanguasaponin compound has good anti-tumor activity, especially obvious for the inhibition of MCF-7 cell, its IC
50value is only 14.00 μ g/mL.Experimental result shows, the Sasanguasaponin compound with structure shown in formula I that embodiment 1 makes has certain restraining effect to liver tumor, melanoma, breast tumor or lung tumor, can be applied to prepare antitumor drug.
Adopt identical experimental technique to investigate Sasanguasaponin compound that embodiment 2 to embodiment 5 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, obtained similar experimental result, illustrate that the Sasanguasaponin compound with structure shown in formula I that embodiment 2 to embodiment 5 makes has certain restraining effect to liver tumor, melanoma, breast tumor or lung tumor, can be applied to prepare antitumor drug.
Experiment in embodiment 7 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 ℃ 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, adjusts cell density to 5 * 10 with physiological saline
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:
With tumour inhibiting rate, investigate the inhibition situation of different group tumours, according to formula below, calculate tumour inhibiting rate:
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, compare 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, compares with blank group, and 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, significant difference; The inhibiting rate of comparison positive controls, experimental group 1 and experimental group 2, finds that the inhibiting rate of experimental group 2 will, higher than positive controls, illustrate that the Sasanguasaponin compound with structure shown in formula I that embodiment 1 makes can significantly suppress S
180the growth of meat cancer, this restraining effect is better than endoxan.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 49.7%, 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 61.8%, 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 prepare antitumor drug.
Adopt identical experimental technique to investigate Sasanguasaponin compound that embodiment 2 to embodiment 5 makes to mouse H
22liver cancer, S
180the restraining effect of meat cancer, has obtained similar experimental result, illustrates that the Sasanguasaponin compound that embodiment 2 to embodiment 5 makes can effectively suppress H
22liver cancer growth, S
180the growth of meat cancer.Illustrate that the Sasanguasaponin compound that embodiment 2 to embodiment 5 makes has good anti-tumor activity, can be applied to prepare antitumor drug.
The preparation of embodiment 8 tablets
Get the Sasanguasaponin compounds with structure shown in I that embodiment 1 makes and add conventional auxiliary material, according to ordinary method, make tablet.
The preparation of embodiment 9 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 10 capsules
Get the Sasanguasaponin compound with structure shown in I that embodiment 3 makes and add conventional auxiliary material, according to ordinary method, make capsule.
The preparation of embodiment 11 injections
Get the Sasanguasaponin compound with structure shown in I that embodiment 4 makes and add conventional auxiliary material, according to ordinary method, make injection.
The preparation of embodiment 12 emulsions
Get the Sasanguasaponin compound with structure shown in I that embodiment 5 makes and add conventional auxiliary material, according to ordinary method, make emulsion.
The preparation of embodiment 13 suspensoids
Get the Sasanguasaponin compound with structure shown in I that embodiment 1 makes and add conventional auxiliary material, according to ordinary method, make suspensoid.
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, obtain.
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 separated through macroporous resin, with alcohol-water mixed solution wash-out, collected volume, than being the alcohol-water mixed solution elution fraction of 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, separated through decompression silica gel chromatographic column, with chloroform-methanol mixed solution wash-out, collected volume, than being the chloroform-methanol mixed solution elution fraction of 80:20～60:40, obtains purified for the first time;
Described in getting, press anti-phase octadecylsilane chemically bonded silica chromatographic column separated for the first time in purified warp, with methanol-water mixture wash-out, collected volume, than being the methanol-water mixture elution fraction of 70:30～90:10, obtains purified for the second time;
Described in getting, purified is separated through dynamic axial compression chromatographic column for the second time, and with methanol-water mixture wash-out, collected volume, than being the methanol-water mixture elution fraction of 75:25, obtains purified for the third time;
Described in getting, purified is separated 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, obtains;
Described methanol-water-formic acid mixed solution is: the mixing solutions that first alcohol and water forms for 78:22 by volume 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 application of the Sasanguasaponin compound as described in claim 1 or 5 in preparing 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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| CN106008649A (en) * | 2016-05-10 | 2016-10-12 | 中国农业科学院茶叶研究所 | Method for quickly separating and purifying tea seed saponin through medium-pressure preparative chromatography |
| CN108840964A (en) * | 2018-05-24 | 2018-11-20 | 华南理工大学 | A kind of tea saponin Complex of Ge, tea saponin germanium nano particle and its preparation method and application |
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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 |
| CN102548405A (en) * | 2009-07-16 | 2012-07-04 | 太平洋艾瑞有限公司 | Inhibiting the invasion and metastasis of cancer cells |
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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 (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106008649A (en) * | 2016-05-10 | 2016-10-12 | 中国农业科学院茶叶研究所 | Method for quickly separating and purifying tea seed saponin through medium-pressure preparative chromatography |
| CN108840964A (en) * | 2018-05-24 | 2018-11-20 | 华南理工大学 | A kind of tea saponin Complex of Ge, tea saponin germanium nano particle and its preparation method and application |
| CN108840964B (en) * | 2018-05-24 | 2021-02-19 | 华南理工大学 | Tea saponin germanium complex, tea saponin germanium nanoparticles, and preparation method and application thereof |
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