CN103739657B - A kind of Sasanguasaponin compound, its preparation method, the antitumor drug applying and prepare - Google Patents
A kind of Sasanguasaponin compound, its preparation method, the antitumor drug applying and prepare Download PDFInfo
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Abstract
The invention belongs to field of pharmaceutical chemistry technology, disclose a kind of Sasanguasaponin compound, its preparation method, the antitumor drug applying and prepare.This Sasanguasaponin compound has structure shown in Formulas I.This Sasanguasaponin compound has certain inhibitory action to hepatoma carcinoma cell, breast cancer cell, melanoma cell, lung carcinoma cell, and can effectively suppress the growth of liver tumor, illustrate that this Sasanguasaponin compound has significant anti-tumor activity, it is possible to be applied to prepare antitumor drug
Description
Technical field
The invention belongs to field of pharmaceutical chemistry technology, particularly to a kind of Sasanguasaponin compound, it prepares
Method, the antitumor drug applied and prepare.
Background technology
Oil tea (Camellia oleifera Abel), is also called Fructus Camelliae sinensis tree, tea oil tree, spends tea in vain, be Flos Camelliae Japonicae
Section (Theaceae) Camellia (Camellia Linn) evergreen dungarunga, is distributed mainly on the torrid zone and ground, subtropical zone
District.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, Elaeis guineensis Jacq., Cortex cocois radicis claim " the big woody oil tree species in the world four ".
Extracting oil with the seed of oil tea and i.e. obtain Oleum Camelliae, the unsaturated fatty acid content of Oleum Camelliae is up to 90%, its content
It is significantly larger than vegetable oil, Oleum Arachidis hypogaeae semen and Oleum Glycines；Compared with olive oil, the content of its vitamin E doubles,
There is high nutritive value.Oil tea also has higher medical value, described in Compendium of Material Medica, and tea
Seed, bitter cold perfume (or spice) poison (Saponin), cure mainly dyspnea with rapid respiration cough, remove disease dirt；" China's book on Chinese herbal medicine " is the most on the books, oil tea
Root and root bark there is effect of heat-clearing and toxic substances removing, regulating QI to relieve pain, promoting blood circulation and detumescence, cure mainly laryngopharynx swelling and pain,
Stomachache, toothache, traumatic pain and burn due to hot liquid or fire.
Oil tea medicinal part mainly has Semen Camelliae, Oleum Camelliae, oil tea root, Cortex Camelliae Oleiferae Radicis and Camellia Leaves.From oil
In tea, the chemical composition of isolated includes saponins, flavonoid, tannin class, fragrance glycoside and alkaloid
Class.Sasanguasaponin is also called oil tea saponin, is a kind of soaps extracted from Semen Camelliae grouts, mainly
Be present in Seed of Camellia oleifera, Camellia Leaves, oil tea root and Cortex Camelliae Oleiferae Radicis, be now used for daily-use chemical industry, pesticide,
The numerous areas such as medicine, Aquatic product, building materials.Research also finds, Sasanguasaponin has Camellia Plants saponin
The general general character, bitter in the mouth, pungent, foam characteristics is strong；Also there is multiple good biological activity, performance
In many aspects, have haemolysis, ichthyotoxin effect, sterilization antibacterial activity, antiinflammatory, resisting hypertension,
The effects such as antitumor, protection cardiovascular system, parasite killing anthelmintic, promotion plant growing.Sasanguasaponin mostly is
The pentacyclic triterpene saponins of oleanane type, including aglycon, sugar, organic acid three part, but its aglycon knot
Structure is complicated, many different with compound mode to cause Sasanguasaponin compounds numerous, to oil for sugar moieties kind
The further purification and isolation of theasaponin proposes challenge.So, although research finds the total soap of oil tea at present
It is active that glucoside extract has antineoplastic, but the effect that wherein which monomer saponin plays, also
It is unknown, needs it is carried out in-depth study.
Summary of the invention
In view of this, the goal of the invention of the present invention is to provide a kind of Sasanguasaponin compound, its preparation side
Method, the antitumor drug applied and prepare.This Sasanguasaponin compound has significant anti-tumor activity,
Can apply to prepare antitumor drug.
In order to realize the goal of the invention of the present invention, the present invention adopts the following technical scheme that
The invention provides a kind of Sasanguasaponin compound, it has a structure shown in Formulas I:
Present invention also offers the preparation method of a kind of Sasanguasaponin compound, comprise the following steps:
Step 1: obtain Total oil tea saponins extract；
Step 2: take step 1 gained Total oil tea saponins extract, separated purification, to obtain final product.
Preferably, the present invention provide preparation method in, step 1 particularly as follows:
After taking the pulverizing of oil tea root, extracted, obtain extracting solution；
Take gained extracting solution concentrated, obtain fluid extract；
Take gained fluid extract to mix with water, filter, collect filtrate；
Taking gained filtrate to separate through macroporous resin, with ethanol-water mixture eluting, collected volume ratio is
The ethanol-water mixture elution fraction of 70:30～95:5, obtains Total oil tea saponins extract.
In some embodiments of the invention, used by the preparation method step 1 that the present invention provides is extracted
Solution is water or ethanol-water mixture.
In the other embodiment of the present invention, the preparation method step 1 that the present invention provides is extracted institute
Ethanol-water mixture in, the volumn concentration of ethanol is 0.1%～95%.
In the other embodiment of the present invention, in terms of g/mL, the preparation method step that the present invention provides
The mass volume ratio of the oil tea root pulverized in rapid 1 solution used with extraction is 1:5～20.
In the other embodiment of the present invention, the preparation method step 1 that the present invention provides extracts it
Before also include dipping process, specially take in step 1 pulverize oil tea root with extraction used by solution mix,
6h～12h is soaked under the conditions of 30 DEG C～50 DEG C.
In the other embodiment of the present invention, the preparation method step 1 that the present invention provides impregnates,
Extract particularly as follows:
Take the oil tea root pulverized in step 1 to mix with water or alcohol-water mixed solution, in 30 DEG C～50 DEG C of bars
Soak 6h～12h, heated backflow under part, filter, collect filtrate, obtain extracting solution.
Extraction in the preparation method step 1 that the present invention provides, can be by being once heated to reflux carrying
Take, filter, collect filtrate, obtain extracting solution；Can also divide by being heated to reflux extracting for 2～3 times
Do not collect and be heated to reflux gained extracting solution every time, merge the extracting solution being every time heated to reflux gained, filter,
Collect filtrate, obtain extracting solution.
In the other embodiment of the present invention, fluid extract in the preparation method step 1 that the present invention provides
It is 1:5～20 with the mass ratio of water.
In the other embodiment of the present invention, the present invention provide preparation method step 1 particularly as follows:
Take oil tea root to pulverize；In terms of g/mL, take the oil tea root of pulverizing and water or alcohol-water mixed solution by
Mass volume ratio 1:5～20 mixing, soaks 6h～12h, heated backflow 1 under the conditions of 30 DEG C～50 DEG C
Secondary～3 times after, merge and be heated to reflux the extracting solution of gained every time, filter, collect filtrate, obtain extracting solution.
Take gained extracting solution concentrated, obtain fluid extract.Take gained fluid extract to mix with water 1:5 in mass ratio～20,
100 mesh～300 mesh filter, and collect filtrate；After centrifugal for gained filtrate, take supernatant big through D101 type
Hole resin separates, successively with the ethanol-water mixture that water, ethanol volumn concentration are 30%～60%,
Ethanol volumn concentration be 70%～95% ethanol-water mixture carry out eluting, collect ethanol volume hundred
Dividing content is 70%～95% ethanol-water mixture elution fraction, obtains Total oil tea saponins extract.
Preferably, the present invention provide preparation method in, step 2 particularly as follows:
Take step 1 gained Total oil tea saponins extract, separate, with chloroform-methanol through decompression silica gel chromatographic column
Mixed liquor eluting, collected volume, than the chloroform-methanol mixed liquor elution fraction for 80:20～60:40, obtains
Purified；
Taking in gained purified warp for the first time presses anti-phase octadecylsilane chemically bonded silica chromatographic column to separate, with
Methanol-water mixture eluting, collected volume than the methanol-water mixture elution fraction for 70:30～90:10,
Obtain second time purified；
Take gained second time purified to separate through dynamic axial compression chromatographic column, wash with methanol-water mixture
De-, collected volume, than the methanol-water mixture elution fraction for 75:25, obtains third time purified；
Take gained third time purified to separate through half preparative high-performance liquid chromatographic post, mix with methanol-water-formic acid
Closing liquid eluting, flow velocity 2mL/min, collecting retention time is the elution fraction corresponding to 44.5min, i.e.
?；
This half preparative high-performance liquid chromatographic post is octadecylsilane half preparative hplc post, and its model is 250
Mm × 10mm, 5 μm；
Methanol-water-formic acid mixed liquor is: first alcohol and water by volume for 78:22 composition mixed solution again with
Account for the mixed liquor of the formic acid mixing gained that gained mixed solution weight/mass percentage composition is 0.2%.
In the other embodiment of the present invention, used in the preparation method step 2 that the present invention provides
Decompression silica gel chromatographic column in the particle diameter of silica gel be 60 mesh～100 mesh.
In the other embodiment of the present invention, reduce pressure in the preparation method step 2 that the present invention provides silicon
During glue chromatographic isolation, chloroform-methanol mixing eluting is gradient elution, and gradient is particularly as follows: chloroform and first
The volume ratio of alcohol is 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 30:70 → 20:80.
In the other embodiment of the present invention, used in the preparation method step 2 that the present invention provides
The flow velocity of middle pressure anti-phase octadecylsilane chemically bonded silica chromatographic column be 25mL/min.
In the other embodiment of the present invention, in the preparation method step 2 that the present invention provides, middle pressure is anti-
When phase octadecylsilane chemically bonded silica chromatographic column separates, methanol-water mixture eluting is gradient elution, washes
De-gradient particularly as follows: the volume ratio of first alcohol and water be 50:50 → 60:40 → 70:30 → 80:20 →
90:10→100:0。
In the other embodiment of the present invention, used in the preparation method step 2 that the present invention provides
Dynamic axial compression chromatographic column be octadecylsilane chemically bonded silica chromatographic column.
In the other embodiment of the present invention, dynamic shaft in the preparation method step 2 that the present invention provides
When compression of chromatography columns separates, methanol-water mixture eluting is gradient elution, gradient particularly as follows:
60:40→75:25→80:20→90:10。
In the other embodiment of the present invention, used in the preparation method step 2 that the present invention provides
The flow velocity of dynamic axial compression chromatographic column be 150mL/min.
In the other embodiment of the present invention, half system used in the preparation method that the present invention provides
Standby performance liquid chromatographic column is octadecylsilane chemically bonded silica half preparative high-performance liquid chromatographic post.
Preferably, the preparation method step 2 half preparative high-performance liquid chromatographic post that the present invention provides is gone back after separating
Including distillation, drying steps, particularly as follows: take the half separating obtained elution fraction of preparative high-performance liquid chromatographic post
Distill, be dried, to obtain final product.
In the other embodiment of the present invention, the present invention provide preparation method in step 2 particularly as follows:
Take step 1 gained Total oil tea saponins extract, through decompression silicagel column, carry out with chloroform-methanol system
Gradient elution, gradient is that the volume ratio of chloroform and methanol is
90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 30:70 → 20:80, collects respectively, closes
And the chloroform-methanol mixed liquor elution fraction that volume ratio is 80:20,70:30 and 60:40, obtain the purest
Compound；
Taking in gained purified warp for the first time presses anti-phase octadecylsilane chemically bonded silica chromatographic column to separate, with
Methanol-water system carries out gradient elution, and 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, closes
And volume ratio is 70:30, the methanol-water mixture elution fraction of 80:20,90:10, obtain second time purification
Thing；
Take gained second time purified to separate through dynamic axial compression chromatographic column, with the methanol of different volumes ratio-
Water mixed liquid carries out gradient elution, and 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, and collected volume is than the first for 75:25
Alcohol-water mixtures elution fraction, obtains third time purified；
Take gained third time purified to divide through octadecylsilane chemically bonded silica half preparative high-performance liquid chromatographic post
From, with methanol-water-formic acid mixed liquor eluting, flow velocity 2mL/min, collection retention time is 44.5min
Corresponding elution fraction, carries out gained elution fraction distilling, being dried, obtains white unsetting powder
Material, to obtain final product；
Wherein, methanol-water-formic acid mixed liquor is: the mixing that first alcohol and water forms for 78:22 by volume is molten
Liquid again with the mixed liquor accounting for the formic acid mixing gained that gained mixed solution weight/mass percentage composition is 0.2%.
Present invention also offers a kind of Sasanguasaponin compound, the preparation method bag of this Sasanguasaponin compound
Include following steps:
Step 1: obtain Total oil tea saponins extract；
Step 2: take step 1 gained Total oil tea saponins extract, separated purification, to obtain final product.
Present invention also offers the application in preparing antitumor drug of a kind of Sasanguasaponin compound, this oil
Theasaponin compound has structure shown in Formulas I,
The preparation method of this Sasanguasaponin compound comprises the following steps:
Step 1: obtain Total oil tea saponins extract；
Step 2: take step 1 gained Total oil tea saponins extract, separated purification, to obtain final product.
In some embodiments of the invention, the present invention provide apply targeted tumor be liver tumor,
Melanoma, breast tumor or lung tumor.
Present invention also offers a kind of antitumor drug, it includes the Sasanguasaponin compound that the present invention provides
With pharmaceutically acceptable adjuvant, this Sasanguasaponin compound has structure shown in Formulas I,
The preparation method of this Sasanguasaponin compound comprises the following steps:
Step 1: obtain Total oil tea saponins extract；
Step 2: take step 1 gained Total oil tea saponins extract, separated purification, to obtain final product.
Preferably, the dosage form of a kind of antitumor drug that the present invention provides be tablet, granule, capsule,
Injection, Emulsion or suspensoid.
The invention provides a kind of Sasanguasaponin compound, its preparation method, apply and prepare anti-swollen
Tumor medicine.This Sasanguasaponin compound has structure shown in Formulas I.In vitro cell experiment result confirms this oil
Hepatoma carcinoma cell, breast cancer cell, melanoma cells, lung carcinoma cell are had certain by theasaponin compound
Inhibitory action；Experiment in vivo result confirms that liver tumor is had by this Sasanguasaponin compound and significantly suppresses to make
With, can effectively suppress the growth of liver tumor, illustrate that this Sasanguasaponin compound has significant antitumor
Activity, can apply to prepare antitumor drug,
Accompanying drawing explanation
Fig. 1 shows the high resolution mass spectrum spectrogram of the prepared Sasanguasaponin compound of embodiment 1；
Fig. 2 shows the hydrogen nuclear magnetic resonance spectrogram of the prepared Sasanguasaponin compound of embodiment 1；
Fig. 3 shows the hydrogen nuclear magnetic resonance spectrogram partial enlarged drawing of the prepared Sasanguasaponin compound of embodiment 1；
Fig. 4 shows the carbon-13 nmr spectra figure of the prepared Sasanguasaponin compound of embodiment 1；
Fig. 5 shows the carbon-13 nmr spectra figure partial enlarged drawing of the prepared Sasanguasaponin compound of embodiment 1；
Fig. 6 shows the DEPT spectrogram of the prepared Sasanguasaponin compound of embodiment 1；
Fig. 7 shows the hsqc spectrum figure of the prepared Sasanguasaponin compound of embodiment 1；
Fig. 8 shows the HMBC spectrogram of the prepared Sasanguasaponin compound of embodiment 1；
Fig. 9 shows the H-H COSY spectrogram of the prepared Sasanguasaponin compound of embodiment 1；
Figure 10 shows the NOESY spectrogram of the prepared Sasanguasaponin compound of embodiment 1.
Detailed description of the invention
The invention discloses a kind of Sasanguasaponin compound, its preparation method, apply and prepare anti-swollen
Tumor medicine.Those skilled in the art are referred to present disclosure, it is thus achieved that this Sasanguasaponin compound, it is achieved
Its application, it is accordingly required in particular to it is noted that all similar replacements and change are for a person skilled in the art
Being apparent from, they are considered as being included in the present invention.The preparation method and application of the present invention is
Through being described by preferred embodiment, related personnel substantially can be without departing from present invention, essence
In god and scope, this paper preparation method and application it is modified or suitably changes and combine, realize and answer
Use the technology of the present invention.
A kind of Sasanguasaponin compound that the present invention provides, its preparation method, apply and prepare anti-swollen
Reagent used in tumor medicine and raw material all can be buied by market.Certain used in the embodiment of the present invention
Model and the manufacturer of a little instruments are as follows:
Chemical reagent: analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group；Electronic balance: prunus mume (sieb.) sieb.et zucc. Teller-
Torr benefit instrument (Shanghai) Co., Ltd.；Polariscope 241PerkinElmer Inc., Waltham, MA are beautiful
State；Rotary Evaporators: Tokyo physics and chemistry apparatus individual proprietorship factory；Thin layer chromatography silica gel plate: HSGF254, cigarette
Platform city Zhifu Huang business silica gel development experiments factory produces；Various column chromatography silica gel are Haiyang Chemical Plant, Qingdao
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, Shimadzu Corporation of Japan；Middle pressure anti-phase octadecylsilane preparative liquid chromatography: B ü chi color
Spectra 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)
With octadecylsilane post (650mm × 100mm, 30 μm, 1500g;Newstyle)；Octadecylsilane
Half preparative hplc post (C18 half preparative hplc post): 250mm × 10mm, 5 μm, U.S. Kromsil is public
Department.
In order to make those skilled in the art better understood when technical scheme, below
In conjunction with the embodiments, the present invention is expanded on further:
Embodiment 1 has the preparation of the Sasanguasaponin compound of structure shown in Formulas I
Extract:
Taking dry oil tea root 2000g, be ground into wood flour with pulverizer, adding 16L percent by volume is
The ethanol water of 70%, after soaking 12h, is heated to backflow, extracts 3.0h, collect under the conditions of 40 DEG C
Extracting solution, filters extracting solution, concentrating under reduced pressure, obtains fluid extract 575g.
Macroporous resin enrichment:
Taking the fluid extract of extraction step gained, add 6.0L water, stirring and dissolving, by 100 mesh filter cloth mistakes
Filter, is centrifuged 10min by filtrate 12000r/min, after being centrifuged, obtains 5.9L supernatant, takes supernatant
Separate further through D101 type macroporous resin, successively with 10.0L water, 15.0L30% ethanol water,
15.0L70% ethanol water carries out eluting, collects 70% ethanol water eluting component, concentrating under reduced pressure,
After testing, containing Sasanguasaponin compounds in gained concentrated solution, through 100 DEG C of baking oven, 48h is dried,
Brown extractum 57g, named Total oil tea saponins extract.
The preparation of purified for the first time:
Take the Total oil tea saponins extract of macroporous resin enrichment gained, through decompression silicagel column (silica gel: 60-100
Mesh) separate, carry out gradient elution with chloroform-methanol system, gradient is the volume ratio of chloroform and methanol
For 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 30:70 → 20:80, Mei Geti
Degree 1000mL；Collect each elution fraction, and label one by one respectively.Gained eluent is carried out thin layer
Plate analysis: thin layer chromatography silica gel plate, BAW system, RfValue=0.4～0.7.After testing, following eluting group
Divide the composition contained similar: the chloroform-methanol elution fraction of 80:20, the chloroform-methanol of 70:30 is molten
Liquid elution fraction, the chloroform-methanol elution fraction of 60:40.Merge the chloroform-methanol of 80:20
Elution fraction, the chloroform-methanol elution fraction of 70:30 and the chloroform-methanol eluting group of 60:40
Point, evaporated under reduced pressure, obtain purified 9.8g for the first time.
The preparation of purified for the second time:
Take gained purified for the first time, warp press anti-phase octadecylsilane preparative liquid chromatography to separate,
Carrying 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, marks one by one
Note.Taking gradient is 60:40, and the elution fraction corresponding to 70:30,80:20,90:10 is carried out efficiently
Analyzing liquid-phase chromatographic analysis, result shows, gradient 70:30, the eluting corresponding to 80:20,90:10
The composition that contains in component is similar, and (efficient analysis LC time is 30min, and detection peak is positioned at
Between 20min-28min), merge elution fraction corresponding to gradient, concentrating under reduced pressure, obtain the
Secondarily purified thing 20mL.
The preparation of purified for the third time:
Take second time purified, carry out separating (flow velocity 150mL/min, detection ripple through dynamic axial column chromatography
Long 203nm), with volume percentage, enter with 60%, 75%, 80% and 90% methanol aqueous solution successively
Row eluting, each gradient 3000mL, collect respectively, obtain five elution fractions, one by one labellings.By institute
Elution fraction respectively through efficient analysis liquid-phase chromatographic analysis (detection peak be positioned at 13.5min-15.0min it
Between), (i.e. volume ratio is the methanol-water mixture elution fraction of 75:25, high to collect 75% elution fraction
Effect Liquid Detection peak is positioned at 13.5min), obtain third time purified 15mL.
The preparation of the unsetting powdered substance of white:
Take third time purified, carry out through C18 half preparative hplc post (250mm × 10mm, 5 μm)
Separate (flow velocity 2mL/min, detect wavelength 203nm), with methanol-water-formic acid mixed liquor (methanol and
Water by volume for 78:22 composition mixed solution again with account for gained mixed solution weight/mass percentage composition and be
The mixed liquor of the formic acid mixing gained of 0.2%) carry out eluting, collect the chromatographic peak at 44.5min, by institute
Obtain elution fraction to carry out distilling, being dried, obtain the 57mg unsetting powdered substance of white.
Compound structure is identified:
The gained unsetting powdered substance of white is carried out thin layer chromatography, acetic anhydride-strong sulfuric acid response, Molish
Reaction is identified, qualification result is: thin layer chromatography sulphuric acid ethanol colour developing result is aubergine speckle, acetic anhydride-
Strong sulfuric acid response is positive, Molish reacting positive, and pointing out this material may be saponins compound.This change
The HR-ESI-MS(high resolution mass spectrum spectrogram of compound) see Fig. 1, the quasi-quasi-molecular ions m/z in this mass spectrogram
995.5257[M-H]-, with molecular formula C51H80O19Estimating of molecular weight value [M-H]-, m/z995.5216,
Basically identical, show that the molecular formula of this compound is C51H80O19.Obtain with the complete acid hydrolysis of 2N TFA
And monosaccharide, sugar carries out GC analysis after derivatization, D-Glucose aldehydic acid, L-arabinose detected
Exist.
The gained unsetting powdered substance of white is carried out proton nmr spectra detection, carbon-13 nmr spectra inspection
Survey and ID NMR speetna detects.The proton nmr spectra spectrogram of this white unsetting powdered substance is shown in
Fig. 2 and Fig. 3, wherein Fig. 2 is the partial enlarged drawing of this material proton nmr spectra.This white is unsetting
The carbon-13 nmr spectra spectrogram of powdered substance is shown in that Fig. 4 and Fig. 5, Fig. 5 are this material carbon-13 nmr spectra
Partial enlarged drawing.The DEPT spectrogram of this white unsetting powdered substance is shown in Fig. 6.This white is indefinite
The hsqc spectrum figure of shape powdered substance is shown in Fig. 7.The HMBC spectrogram of this white unsetting powdered substance
See Fig. 8.Fig. 9 is shown in by the H-H COSY collection of illustrative plates of this white unsetting powdered substance.This white is unsetting
Figure 10 is shown in by the NOESY collection of illustrative plates of powdered substance.The nuclear magnetic resoance spectrum of this white unsetting powdered substance
Diagram data is shown in Table 1.
The nuclear magnetic resoance spectrum diagram data of the white unsetting powdered substance of table 1
Note: in table, chemical shift is in units of ppm, coupling constant (J) is in units of Hz
According to gained nmr spectrum and data, this white unsetting powdered substance is carried out further
Structure elucidation:
The unsetting powdered substance of white13C H NMR spectroscopy shows 51 carbon signals altogether, passes through DEPT
Analyze this compound with hsqc spectrum and contain 10 methyl carbon, 10 mesomethylene carbons, 12 methines altogether
Carbon and 11 quaternary carbons.Analyze further13C H NMR spectroscopy, this spectrogram demonstrates two sugared end group carbon letters
Number δ 105.7 and 105.4, is the end group carbon of D-Glucose aldehydic acid and L-arabinose respectively.White is indefinite
Shape powdered substance1In H H NMR spectroscopy, high field region has six obvious triterpene saponin angular methyl hydrogen signal δ
1.09(Me-25),1.15(Me-26),1.15(Me-29),1.39(Me-30),1.04(Me-24)and1.90
(Me-27)；Two companies Oxymethylene δ 3.56 (1H, d, J=11.0Hz, H-28), 3.87 (1H, d, J=11.0
Hz, H-28) and 3.77 (1H, d, J=11.0Hz, H-23), 4.38 (1H, d, J=11.0Hz, H-23)；Five
Individual even oxygen methine δ 4.38 (1H, dd, J=11.0,4.5Hz, H-3), 4.26 (1H, brs, H-15), 4.46
(1H, brs, H-16), 6.71 (1H, d, J=10.5Hz, H-21) and 6.32 (1H, d, J=10.5Hz,
And an alkene Hydrogen Proton δ 5.60 (1H, brs, H-12) H-22).Additionally, white unsetting powdered substance
's1H H NMR spectroscopy also demonstrate one group of angeloyl groups signal δ [6.15 (1H, dq, J=5.5,1.5Hz,
21-O-Ang-3'), 2.24 (3H, d, J=7.5Hz, 21-O-Ang-4') and 2.12 (3H, s,
21-O-Ang-5')]；And one group of 2-methylbutyryl base signal δ [2.16 (1H, m, 22-O-MB-2''), 1.31
(1H,m,22-O-MB-3''a),1.67(1H,m,22-O-MB-3''b),0.77(3H,t,J=7.5Hz,
22-O-MB-4'') He 1.04 (3H, d, J=6.5Hz, 22-O-MB-5'')].
In H-H COSY, the methine δ adjacent with even oxygen methine protonsH4.26 (1H, brs) and δH
4.46 (1H, brs) are correlated with, and illustrate that they are in ortho position and δH4.46 is H-16 signal, δH4.26 be H-15
Signal；δH6.71 (1H, d, J=10.5Hz) and δHCoherent signal between 6.32 (1H, d, J=10.5Hz) is said
Bright they be in ortho position and δH6.71 is H-21 signal, δH6.32 is H-22 signal.
Learn that this angeloyl groups is connected in this by the HMBC spectrum analysis of white unsetting powdered substance
On 21 of compound, and 2-methylbutyryl base is connected on this compound 22, because at HMBC
Middle δH6.71 (1H, d, J=10.5Hz, H-21) and δC167.8 (21-O-Ang-1') are correlated with, δH6.32
(1H, d, J=10.5Hz, H-22) and δC176.8 (22-O-MB-1'') are correlated with.The present embodiment system of thereby determining that
The aglycone structure of the compound obtained is 21 β-O-angeloyl groups-22 α-O-(2-methylbutyryl base)-15 α, 16 α,
23 α, 28-tetrahydroxy olive-12-alkene.
The hsqc spectrum figure of white unsetting powdered substance is analyzed, has belonged to and connected on this saponin
Sugar end group carbon, hydrogen signal, as follows: δH5.34 (1H, d, J=7.0Hz) and 5.38 (1H, d, J=6.5Hz),
It is connected in respectively on 105.7 (glucuronic acid-C-1) and 105.4 (arabinose-C-1).Additionally this compound
3 carbon potentials of aglycon are in low field δC82.3 show that sugar chain is connected on aglycon 3, and in HMBC composes,
Glucuronic acid anomeric proton δH5.34 and 3 δ of aglyconC81.3 are correlated with, and further demonstrate that sugar chain is even
At aglycon 3.Consulting literatures finds this compound sugar chain and report in document further
Gordonoside A sugar chain data close [J.Nat.Prod.2009,72,866-870], thus infer
This compound sugar chain is α-L-arabinose-(1 → 3)-β-D-Glucose aldehydic acid.The order of connection of this sugar chain is also
Can confirm by analyzing HMBC spectrum, glucuronic acid anomeric proton δ in HMBCH5.34 with aglycon
3 carbon δC81.3 relevant, 3 hydrogen δ of aglyconH4.38 and glucal acid end group carbon δC105.7 are correlated with,
Arabinose anomeric proton δH5.38 and 3 carbon δ of glucuronic acidC85.8 are correlated with, glucuronic acid 3
Position hydrogen δH4.30 and arabinose end group carbon δC105.4 it is relevant.Additionally, the anomeric proton of glucuronic acid
Coupling constant3JH-1,H-2Relatively big, show that it is beta configuration；And arabinose terminal hydrogen δ in NOESYH5.38
Respectively with 3 hydrogen δ of arabinoseH4.58,4 hydrogen δ of arabinoseH4.38 be correlated with, show its be α-
Configuration.
In NOESY spectrum, δH6.71 (1H, d, J=10.5Hz, H-21) and δH1.15 (3H, s, H-29) phase
Closing, prompting H-21 is α configuration；δH4.38 (1H, dd, J=4.5,11.0Hz, H-3) and δH3.77(1H,d,
J=11.0Hz, H-23), 4.38 (1H, d, J=11.0Hz, H-23) are correlated with, and prompting H-3 is α configuration；δH
4.46 (1H, brs, H-16) and δH3.56(1H,d,J=11.0Hz,H-28)、3.87(1H,d,J=11.0Hz,
H-28) relevant prompting prompting H-16 is beta comfiguration, δH6.32 (1H, d, J=10.5Hz, H-22) and δH1.39
(3H, s, H-30) is correlated with, and prompting H-22 is beta comfiguration.
Therefore, this compound has structure shown in Formulas I,
Named 21 β-O-angeloyl groups-22 α-O-(2-methylbutyryl base)-15 α, 16 α of this compound structure,
23 α, 28-tetrahydroxy olive-12-alkene 3 β-O-α-L-arabinose-(1 → 3)-β-D-Glucose aldehydic acid glycosides.
Embodiment 2 has the preparation of the Sasanguasaponin compound of structure shown in Formulas I
Extract:
Take dry oil tea root 2000g, be ground into wood flour with pulverizer, add 40.0L percent by volume
It is the ethanol water of 0.1%, soaks after 6h under the conditions of 50 DEG C, be heated to backflow, extract three times, often
Secondary 0.5h, collects all extracting solution, is filtered by extracting solution, concentrating under reduced pressure, obtains fluid extract 653g.
Macroporous resin enrichment:
Taking the fluid extract that extraction step obtains, add 13.06L water, stirring and dissolving, with 300 mesh filter clothes
Filter, filtrate 12000r/min is centrifuged 10min, after being centrifuged, obtain 6.1L supernatant, take supernatant
Liquid separates further through D101 type macroporous resin, uses 10.0L water, 15.0L60% ethanol water-soluble successively
Liquid, 10.0L95% ethanol water carry out eluting, collect 95% ethanol water eluting component, decompression
Concentrating, after testing, containing Sasanguasaponin compounds in gained concentrated solution, through 100 DEG C of baking oven, 48h does
Dry, obtain brown extractum 45g, named Total oil tea saponins extract.
The preparation of purified for the first time:
Take the Total oil tea saponins extract of macroporous resin enrichment gained, through decompression silicagel column (silica gel: 80 mesh)
Separating, carry out gradient elution with chloroform-methanol system, gradient is that the volume ratio of chloroform and methanol is
90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 30:70 → 20:80, each gradient
1000mL；Collect each elution fraction, and label one by one respectively.Gained eluent is carried out lamellae divide
Analysis: thin layer chromatography silica gel plate, BAW system, RfValue=0.4～0.7.After testing, following elution fraction contains
Some compositions are similar: the chloroform-methanol elution fraction of 80:20, and the chloroform-methanol of 70:30 is washed
De-component, the chloroform-methanol elution fraction of 60:40.Merge the chloroform-methanol eluting of 80:20
Component, the chloroform-methanol elution fraction of 70:30 and the chloroform-methanol elution fraction of 60:40,
Evaporated under reduced pressure, obtains purified 7.6g for the first time.
Take gained purified for the first time, warp press anti-phase octadecylsilane preparative liquid chromatography to separate,
Carrying 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, marks one by one
Note.Taking gradient is 60:40, and the elution fraction corresponding to 70:30,80:20,90:10 is carried out efficiently
Analyzing liquid-phase chromatographic analysis, result shows, gradient 70:30, the eluting corresponding to 80:20,90:10
The composition that contains in component is similar, and (efficient analysis LC time is 30min, and detection peak is positioned at
Between 20min-28min), merge elution fraction corresponding to gradient, concentrating under reduced pressure, obtain the
Secondarily purified thing 20mL.
The preparation of purified for the third time:
Take second time purified, carry out separating (flow velocity 150mL/min, detection ripple through dynamic axial column chromatography
Long 203nm), with volume percentage, enter with 60%, 75%, 80% and 90% methanol aqueous solution successively
Row eluting, each gradient 3000mL, collect respectively, obtain five elution fractions, one by one labellings.By institute
Elution fraction respectively through efficient analysis liquid-phase chromatographic analysis (detection peak be positioned at 13.5min-15.0min it
Between), (i.e. volume ratio is the methanol-water mixture elution fraction of 75:25, high to collect 75% elution fraction
Effect Liquid Detection peak is positioned at 15.0min), obtain third time purified 15mL.
The preparation of the unsetting powdered substance of white:
Take third time purified, carry out through C18 half preparative hplc post (250mm × 10mm, 5 μm)
Separate (flow velocity 2mL/min, detect wavelength 203nm), with methanol-water-formic acid mixed liquor (methanol and
Water by volume for the mixed solution of 78:22 composition again with to account for gained mixed solution percentage composition be 0.2%
The mixed liquor of formic acid mixing gained) carry out eluting, collect chromatographic peak at 44.5min, by gained eluting group
Divide and carry out distilling, being dried, obtain the 48mg unsetting powdered substance of white.
The white using the compound structure authentication method that embodiment 1 is identical to prepare the present embodiment is unsetting
Powdered substance carries out Structural Identification, it is thus achieved that high resolution mass spectrum spectrogram, proton nmr spectra, nuclear-magnetism common
The carbon that shakes spectrum, DEPT spectrogram, hsqc spectrum figure, HMBC spectrogram, H-H COSY spectrogram, NOESY
Spectrogram is basically identical with the Correlated Spectroscopy diagram data of embodiment 1 gained, and result shows this white unsetting powder
Shape material is the compound with structure shown in Formulas I,
Embodiment 3 has the preparation of the Sasanguasaponin compound of structure shown in Formulas I
Extract:
Take dry oil tea root 2000g, be ground into wood flour with pulverizer, add 10L percent by volume
It is that the ethanol water of 95% soaks after 8h under the conditions of 30 DEG C, is heated to backflow, extract twice, often
Secondary 1.5h, collects all extracting solution, is filtered by extracting solution, concentrating under reduced pressure, obtains fluid extract 595g.
Macroporous resin enrichment:
Taking the fluid extract that extraction step obtains, add 2.975L water, stirring and dissolving, with 200 mesh filter clothes
Filter, filtrate 12000r/min is centrifuged 10min, after being centrifuged, obtain 5.8mL supernatant, take supernatant
Liquid separates further through D101 type macroporous resin, successively with 10L water, 15L40% ethanol water,
15L75% ethanol water carries out eluting, collects 75% ethanol water eluting component, concentrating under reduced pressure,
After testing, containing Sasanguasaponin compounds in gained concentrated solution, through 100 DEG C of baking oven, 48h is dried,
Brown extractum 52g, named Total oil tea saponins extract.
The preparation of purified for the first time:
Take the Total oil tea saponins extract of macroporous resin enrichment gained, through decompression silicagel column (silica gel: 60-100
Mesh) separate, carry out gradient elution with chloroform-methanol system, gradient is the volume ratio of chloroform and methanol
For 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 30:70 → 20:80, Mei Geti
Degree 1000mL；Collect each elution fraction, and label one by one respectively.Gained eluent is carried out thin
Laminate is analyzed: thin layer chromatography silica gel plate, BAW system, RfValue=0.4～0.7.After testing, following eluting
The composition that component contains is similar: the chloroform-methanol elution fraction of 80:20, the chloroform-methanol of 70:30
Eluant solution component, the chloroform-methanol elution fraction of 60:40.The chloroform-methanol merging 80:20 is molten
Liquid elution fraction, the chloroform-methanol elution fraction of 70:30 and the chloroform-methanol eluting of 60:40
Component, evaporated under reduced pressure, obtain purified 8.1g for the first time.
The preparation of purified for the second time:
Take gained purified for the first time, warp press anti-phase octadecylsilane preparative liquid chromatography to separate,
Carrying 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, marks one by one
Note.Taking gradient is 60:40, and the elution fraction corresponding to 70:30,80:20,90:10 is carried out efficiently
Analyzing liquid-phase chromatographic analysis, result shows, gradient 70:30, the eluting corresponding to 80:20,90:10
The composition that contains in component is similar, and (efficient analysis LC time is 30min, and detection peak is positioned at
Between 20min-28min), merge elution fraction corresponding to gradient, concentrating under reduced pressure, obtain the
Secondarily purified thing 20mL.
The preparation of purified for the third time:
Take second time purified, carry out separating (flow velocity 150mL/min, detection ripple through dynamic axial column chromatography
Long 203nm), with volume percentage, enter with 60%, 75%, 80% and 90% methanol aqueous solution successively
Row eluting, each gradient 3000mL, collect respectively, obtain five elution fractions, one by one labellings.By institute
Elution fraction respectively through efficient analysis liquid-phase chromatographic analysis (detection peak be positioned at 13.5min-15.0min it
Between), (i.e. volume ratio is the methanol-water mixture elution fraction of 75:25, high to collect 75% elution fraction
Effect Liquid Detection peak is positioned at 14.5min), concentrating under reduced pressure, obtain third time purified 15mL.
The preparation of the unsetting powdered substance of white:
Take third time purified, carry out through C18 half preparative hplc post (250mm × 10mm, 5 μm)
Separate (flow velocity 2mL/min, detect wavelength 203nm), with methanol-water-formic acid mixed liquor (methanol and
Water by volume for 78:22 composition mixed solution again with account for gained mixed solution weight/mass percentage composition and be
The mixed liquor of the formic acid mixing gained of 0.2%) carry out eluting, collect chromatographic peak at 44.5min, by gained
Elution fraction carries out distilling, being dried, and obtains the 61mg unsetting powdered substance of white.
The white using the compound structure authentication method that embodiment 1 is identical to prepare the present embodiment is unsetting
Powdered substance carries out Structural Identification, it is thus achieved that high resolution mass spectrum spectrogram, proton nmr spectra, nuclear-magnetism common
The carbon that shakes spectrum, DEPT spectrogram, hsqc spectrum figure, HMBC spectrogram, H-H COSY spectrogram, NOESY
Spectrogram is basically identical with the Correlated Spectroscopy diagram data of embodiment 1 gained, and result shows this white unsetting powder
Shape material is the compound with structure shown in Formulas I,
Embodiment 4 has the preparation of the Sasanguasaponin compound of structure shown in Formulas I
Extract:
Take dry oil tea root 2000g, be ground into wood flour with pulverizer, add 20L water, 40 DEG C of bars
After soaking 12h under part, it is heated to backflow, extracts twice, each 2h, collect all extracting solution, will carry
Take liquid to filter, concentrating under reduced pressure, obtain fluid extract 612g.
Macroporous resin enrichment:
Taking the fluid extract that extraction step obtains, add 6.0L water, stirring and dissolving, by 200 mesh filter cloth mistakes
Filter, is centrifuged 10min by filtrate 12000r/min, after being centrifuged, obtains 6.0L supernatant, takes supernatant
Separate further through D101 type macroporous resin, successively with 10L water, 15L50% ethanol water, 15
L70% ethanol water carries out eluting, collects 70% ethanol water eluting component, concentrating under reduced pressure, warp
Detection, containing Sasanguasaponin compounds in gained concentrated solution, through 100 DEG C of baking oven, 48h is dried, and obtains brown
Color extractum 59g, named Total oil tea saponins extract.
The preparation of purified for the first time:
Take the Total oil tea saponins extract of macroporous resin enrichment gained, through decompression silicagel column (silica gel: 60-100
Mesh) separate, carry out gradient elution with chloroform-methanol system, gradient is the volume ratio of chloroform and methanol
For 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 30:70 → 20:80, Mei Geti
Degree 1000mL；Collect each elution fraction, and label one by one respectively.Gained eluent is carried out thin layer
Plate analysis: thin layer chromatography silica gel plate, BAW system, RfValue=0.4～0.7.After testing, following eluting group
Divide the composition contained similar: the chloroform-methanol elution fraction of 80:20, the chloroform-methanol of 70:30 is molten
Liquid elution fraction, the chloroform-methanol elution fraction of 60:40.Merge the chloroform-methanol of 80:20
Elution fraction, the chloroform-methanol elution fraction of 70:30 and the chloroform-methanol eluting group of 60:40
Point, evaporated under reduced pressure, obtain purified 8.6g for the first time.
The preparation of purified for the second time:
Take gained purified for the first time, warp press anti-phase octadecylsilane preparative liquid chromatography to separate,
Carrying 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, marks one by one
Note.Taking gradient is 60:40, and the elution fraction corresponding to 70:30,80:20,90:10 is carried out efficiently
Analyzing liquid-phase chromatographic analysis, result shows, gradient 70:30, the eluting corresponding to 80:20,90:10
The composition that contains in component is similar, and (efficient analysis LC time is 30min, and detection peak is positioned at
Between 20min-28min), merge elution fraction corresponding to gradient, concentrating under reduced pressure, obtain the
Secondarily purified thing 20mL.
The preparation of purified for the third time:
Take second time purified, carry out separating (flow velocity 150mL/min, detection ripple through dynamic axial column chromatography
Long 203nm), with volume percentage, enter with 60%, 75%, 80% and 90% methanol aqueous solution successively
Row eluting, each gradient 3000mL, collect respectively, obtain five elution fractions, one by one labellings.By institute
Elution fraction respectively through efficient analysis liquid-phase chromatographic analysis (detection peak be positioned at 13.5min-15.0min it
Between), (i.e. volume ratio is the methanol-water mixture elution fraction of 75:25, high to collect 75% elution fraction
Effect Liquid Detection peak is positioned at 15.0min), concentrating under reduced pressure, obtain third time purified 15mL.
The preparation of the unsetting powdered substance of white:
Take third time purified, carry out through C18 half preparative hplc post (250mm × 10mm, 5 μm)
Separate (flow velocity 2mL/min, detect wavelength 203nm), with methanol-water-formic acid mixed liquor (methanol and
Water by volume for 78:22 composition mixed solution again with account for gained mixed solution weight/mass percentage composition and be
The mixed liquor of the formic acid mixing gained of 0.2%) carry out eluting, collect chromatographic peak at 44.5min, by gained
Elution fraction carries out distilling, being dried, and obtains the 52mg unsetting powdered substance of white.
The white using the compound structure authentication method that embodiment 1 is identical to prepare the present embodiment is unsetting
Powdered substance carries out Structural Identification, it is thus achieved that high resolution mass spectrum spectrogram, proton nmr spectra, nuclear-magnetism common
The carbon that shakes spectrum, DEPT spectrogram, hsqc spectrum figure, HMBC spectrogram, H-H COSY spectrogram, NOESY
Spectrogram is basically identical with the Correlated Spectroscopy diagram data of embodiment 1 gained, and result shows this white unsetting powder
Shape material is the compound with structure shown in Formulas I,
Embodiment 5 has the preparation of the Sasanguasaponin compound of structure shown in Formulas I
Extract:
Take dry oil tea root 2000g, be ground into wood flour with pulverizer, join 20L ethanol water-soluble
Liquid extracts, gained extracting solution is concentrated, obtain fluid extract 585g.
Macroporous resin enrichment:
Adding water 6.0L in gained fluid extract, stirring, to dissolving, is filtered, is obtained filtrate 5.9L；By institute
Filtrate is crossed macroporous resin and is separated, with ethanol-water mixture eluting, collected volume ratio is
The ethanol-water mixture elution fraction of 70:30～95:5, after testing, wherein contains Sasanguasaponin compounds,
Through 100 DEG C of baking oven, 48h is dried, and obtains brown extractum 58g, named Total oil tea saponins extract.
The preparation of purified for the first time:
Take the Total oil tea saponins extract of macroporous resin enrichment gained, through decompression silicagel column separate, with chloroform-
Methanol system carries out gradient elution, each gradient 1000mL, and collected volume ratio is for 80:20,70:30 respectively,
The chloroform-methanol mixed liquor elution fraction of 60:40.Three elution fractions of gained are carried out lamellae analysis:
Thin layer chromatography silica gel plate, BAW system, RfValue=0.4～0.7.After testing, three elution fractions contain
Composition is similar, merges these three elution fraction, evaporated under reduced pressure, obtains purified 9.2g for the first time.
The preparation of purified for the second time:
Take gained purified for the first time, warp press anti-phase octadecylsilane preparative liquid chromatography to separate,
Gradient elution is carried out with methanol-water system, each gradient 1000mL, collected volume ratio is for 70:30 respectively,
The methanol-water mixture elution fraction of 80:20,90:10.Three elution fractions of gained are carried out efficient analysis
Liquid Detection, obtaining the composition contained in these three elution fraction, similar (efficient analysis LC time is
30min, detection peak is positioned between 20min-28min), merging these three elution fraction, concentrating under reduced pressure,
Obtain second time purified 20mL.
The preparation of purified for the third time:
Take second time purified, separate through dynamic axial column chromatography, carry out eluting with methanol-water system,
Collected volume is than the methanol-water mixture elution fraction for 75:25.By gained elution fraction through efficient analysis
Liquid-phase chromatographic analysis, occurs detection peak at 15.0min, collects this elution fraction, concentrating under reduced pressure, i.e.
Obtain third time purified 15mL.
The preparation of the unsetting powdered substance of white:
Take third time purified, through half preparative high-performance liquid chromatographic post (C18 half preparative hplc post: 250
Mm × 10mm, 5 μm) carry out separating (flow velocity 2mL/min detects wavelength 203nm), with methanol-
Water-formic acid mixed liquor (first alcohol and water by volume for 78:22 composition mixed solution again with account for gained and mix
Solution quality percentage composition is the mixed liquor of the formic acid mixing gained of 0.2%) carry out eluting, collect 44.5min
Place's chromatographic peak, carries out gained elution fraction distilling, being dried, and obtains the 55mg unsetting powder thing of white
Matter.
The white using the compound structure authentication method that embodiment 1 is identical to prepare the present embodiment is unsetting
Powdered substance carries out Structural Identification, it is thus achieved that high resolution mass spectrum spectrogram, proton nmr spectra, nuclear-magnetism common
The carbon that shakes spectrum, DEPT spectrogram, hsqc spectrum figure, HMBC spectrogram, H-H COSY spectrogram, NOESY
Spectrogram is basically identical with the Correlated Spectroscopy diagram data of embodiment 1 gained, and result shows this white unsetting powder
Shape material is the compound with structure shown in Formulas I,
Embodiment 6 In vitro cell experiment
Experiment material:
TypeⅡ pneumocyte, B16 mouse melanin tumor cell, human hepatocellular carcinoma BEL-7402 cell and people
Breast cancer cell MCF-7, is all purchased from Shanghai cell institute of the Chinese Academy of Sciences.These four cell is all with containing 10%
The RPMI1640 culture medium of inactivation NBS (separately adds Glu0.03%, Hepes0.06%, NaHCO30.2%)
In 37 DEG C, 5%CO2Under the conditions of cultivate, within 3 days, pass on.Trophophase cell of taking the logarithm is tested.Real
Test Sasanguasaponin compound used to have shown in Formulas I for what the preparation method provided according to embodiment 1 prepared
The Sasanguasaponin compound of structure,
Experimental technique:
Use conventional mtt assay, measure the tumor cell line sensitivity to medicine.Mtt assay, also known as
MTT colorimetry, is a kind of method detecting cell survival and growth.Its Cleaning Principle is living cells line grain
Succinate dehydrogenase in body can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation
(Formazan) and be deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO)
The first a ceremonial jade-ladle, used in libation in cell can be dissolved, at 490nm wavelength, measure its absorbance value with enzyme-linked immunosorbent assay instrument,
Can indirectly reflect living cells quantity.In the range of certain cell number, the amount of MTT crystallization formation and cell number
It is directly proportional.The method is widely used in the Activity determination of some bioactie agents, large-scale antitumor
Drug screening, cell toxicity test and tumor radiosensitivity mensuration etc..Its feature be highly sensitive,
Economical.Therefore, in this experiment, we use mtt assay to screen test medicine.Specific experiment is pacified
Arrange as follows:
Experiment is divided into negative control group, experimental group and positive controls.Negative control group subject cell adds
The solution entered is complete medium, if 3 multiple holes.Experimental group subject cell adds containing variable concentrations
The solution of Sasanguasaponin compound for preparing of embodiment 1, solvent be volume ratio be the DMSO of 1:99
With the mixed solution of PBS, 5 concentration of Setup Experiments, wherein the concentration of this Sasanguasaponin compound is respectively
For: 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, often
Group is all provided with 3 multiple holes.In positive controls subject cell add the fluorouracil Han 5-FU(5-) solution,
Solvent be volume ratio be the mixed solution of DMSO Yu PBS of 1:99, the concentration of 5-FU is 50 μ g/mL,
If 3 multiple holes.
According to experimental establishment, adjusting each cell concentration with complete medium is 5 × 104/ mL, connects respectively
Plant in 96 well culture plates, labelling one by one, 100 μ L/ holes, 37 DEG C, 5%CO2Under the conditions of overnight incubation.
Experimental group adds the Sasanguasaponin compound with structure shown in Formulas I that the embodiment 1 of variable concentrations prepares
(final concentration be respectively as follows: 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), 10 μ L/ holes；Positive controls adds 5-FU (50 μ g/mL), 10 μ L/ holes；Negative control group
Add equal-volume complete medium, 10 μ L/ holes；Often group is all provided with 3 multiple holes, cultivates 24h respectively.Terminate training
Before supporting, 4h is separately added into MTT (5mg/mL), 10 μ L/ holes, and after cultivation terminates, every hole adds DMSO
100 μ L, after placing shaking table vibration 10min, are the absorbance at 490nm with microplate reader detection wavelength
A value.
Processing for Data Analysis in Physics:
With IC50Value evaluates the Sasanguasaponin compound inhibitory action to different tumor cells.
By equation below calculating growth of tumour cell suppression ratio (Inhibition Rate):
Growth of tumour cell suppression ratio (%)=[(A negative control group-A experimental group)/A negative control
Group] × 100%；
With the concentration of Sasanguasaponin compound as abscissa, growth of tumour cell suppression ratio is vertical coordinate mapping,
Obtain suppression ratio-concentration curve, the concentration of medicine when then drawing 50% suppression ratio, i.e. Sasanguasaponin compound
IC to subject cell50Value (half-inhibition concentration), the Sasanguasaponin compound pair that embodiment 1 prepares
The IC of different subject cells50Value is shown in Table 2.
The table 2 Sasanguasaponin compound IC to four kinds of different tumor cells50Value
| Subject cell | A549 | B16 | BEL-7402 | MCF-7 |
| IC50(μ g/mL) | 33.27±0.28 | 31.80±2.15 | 35.11±2.31 | 26.98±2.79 |
From experimental group data, the Sasanguasaponin compound that embodiment 1 prepares is to these four tumor cell
IC50Value both less than 40 μ g/mL, show that this Sasanguasaponin compound has preferable anti-tumor activity,
Inhibition especially for MCF-7 cell is the most obvious, its IC50Value is only 26.98 μ g/mL.
Test result indicate that, liver is swollen by the Sasanguasaponin compound with structure shown in Formulas I that embodiment 1 prepares
Tumor, melanoma, breast tumor or lung tumor have certain inhibitory action, can apply to preparation anti-
Tumour medicine.
Identical experimental technique is used to investigate the Sasanguasaponin compound pair that embodiment 2 to embodiment 5 prepares
TypeⅡ pneumocyte, B16 mouse melanin tumor cell, human hepatocellular carcinoma BEL-7402 cell and people's mammary gland
The inhibitory action of cancerous cell MCF-7, it is thus achieved that similar experimental result, illustrates embodiment 2 to embodiment
Liver tumor, melanoma, mammary gland are swollen by the 5 Sasanguasaponin compounds with structure shown in Formulas I prepared
Tumor or lung tumor have certain inhibitory action, can apply to prepare antitumor drug.
Embodiment 7 experiment in vivo
Experiment material:
ICR mice, cleaning grade, male, body weight 18-22g, by the Shanghai limited public affairs of Si Laike laboratory animal
Department provides.H22Hepatocarcinoma, S180Sarcoma is purchased from Chinese Academy of Sciences's Shanghai cell bank.Experiment Sasanguasaponin chemical combination used
The compound with structure shown in Formulas I that thing prepares for the preparation method provided according to embodiment 1,
Experimental technique:
The present embodiment investigates the prepared Sasanguasaponin compound with structure shown in Formulas I of embodiment 1 to liver
The effect of cancer.Study subject is H22Transplanted tumor model mice, S180Transplanted tumor model mice.
Mice H22Transplanted tumor model, mice S180The foundation of Transplanted tumor model and experimental establishment:
Take H22Hepatocarcinoma or S180Sarcoma cell cryopreservation tube, is placed in 37 DEG C of waters bath with thermostatic control defrosting, centrifugal receipts
Collection cell, washes twice with PBS liquid, then with PBS liquid re-suspended cell, passes 3 through ICR mouse peritoneal
More than Dai, taking the mice that abdominal part substantially expands, dislocation is put to death, to abdominal part alcohol disinfecting, disposable sterilized
Syringe extraction milky ascites, injects in sterilized centrifuge tube, and cell counter counts, and uses physiology
Saline adjusts cell density to 5 × 106/ mL, enters to every mice right oxter inoculation 0.2mL cell suspension
Row modeling.Mice is randomly divided into blank group, experimental group and positive controls by next day, often organizes 12
, and be administered.Experimental group provides the Sasanguasaponin with structure shown in Formulas I that embodiment 1 prepares
Compound, administering mode is lumbar injection, and dosage is: 3mg/kg(the most every 1kg body weight is administered 3mg),
Solvent is the normal saline of 0.9%, once a day.Positive controls provides CTX (cyclophosphamide), is administered
Mode is lumbar injection, and dosage is: 20mg/kg, and solvent is the normal saline of 0.9%, the next day one
Secondary.Blank group provides blank solvent, and the normal saline of i.e. 0.9%, lumbar injection, injection volume is
0.2mL, once a day.All group mice successive administrations 10 days, weigh Mouse Weight, record every day
The clinical manifestation of mice.After last administration 2h, peel off tumor body, weigh tumor weight, and calculate tumor suppression
Rate.
Processing for Data Analysis in Physics:
The suppression situation of different group tumor is investigated, according to equation below calculating tumour inhibiting rate with tumour inhibiting rate:
Tumour inhibiting rate (%)=[(blank group average tumor weight-administration group average tumor weight)/blank group is put down
All tumor weights] × 100%
The transplanted tumor tumor weight of each group and tumour inhibiting rate are shown in Table 3.
The transplanted tumor tumor weight of the different group of table 3 and tumour inhibiting rate
Note: compared with blank group, * P < 0.05, * * P < 0.01
Test result indicate that, for H22Transplanted tumor model, compared with blank group, positive controls
The tumor weight of the transplanted tumor of blank group heavily it is significantly less than with the tumor of the transplanted tumor of experimental group, significant difference,
Illustrate that the Sasanguasaponin compound with structure shown in Formulas I that embodiment 1 prepares can effectively suppress H22
The growth of hepatocarcinoma.For S180Transplanted tumor model, compared with blank group, positive controls and experiment
The tumor of the transplanted tumor of group is heavily significantly less than the tumor weight of the transplanted tumor of blank group, significant difference, illustrates real
Execute the prepared Sasanguasaponin compound with structure shown in Formulas I of example 1 and can significantly inhibit S180Hepatocarcinoma
Growth.Experimental result is it is also shown that work as the Sasanguasaponin with structure shown in Formulas I that embodiment 1 prepares
When the dosage of compound is 3.0mg/kg, to H22The suppression ratio of transplanted tumor reaches 25.5%, points out this oil
Theasaponin compound has significant anti-mouse H22The effect of liver cancer growth；When the tool that embodiment 1 prepares
When to have the dosage of the Sasanguasaponin compound of structure shown in Formulas I be 3.0mg/kg, to S180Transplanted tumor
Suppression ratio reaches 32.0%, points out this Sasanguasaponin compound to have stronger anti-mouse S180Liver cancer growth
Effect.Result above shows the Sasanguasaponin compound with structure shown in Formulas I that embodiment 1 prepares
There is preferable anti-tumor activity, can apply to prepare antitumor drug.
Identical experimental technique is used to investigate the Sasanguasaponin compound pair that embodiment 2 to embodiment 5 prepares
Mice H22Hepatocarcinoma, S180The inhibitory action of hepatocarcinoma, it is thus achieved that similar experimental result, illustrates embodiment 2
The Sasanguasaponin compound prepared to embodiment 5 can effectively suppress H22Liver cancer growth, S180Liver cancer growth.
Illustrate that the Sasanguasaponin compound that embodiment 2 to embodiment 5 prepares has preferable anti-tumor activity, can
To be applied to prepare antitumor drug.
The preparation of embodiment 8 tablet
The Sasanguasaponin compounds with structure shown in I that Example 1 prepares adds customary adjuvant,
Conventionally make tablet.
The preparation of embodiment 9 granule
The Sasanguasaponin compound with structure shown in I that Example 2 prepares adds customary adjuvant, presses
More solito makes granule.
The preparation of embodiment 10 capsule
The Sasanguasaponin compound with structure shown in I that Example 3 prepares adds customary adjuvant, presses
More solito makes capsule.
The preparation of embodiment 11 injection
The Sasanguasaponin compound with structure shown in I that Example 4 prepares adds customary adjuvant, presses
More solito makes injection.
The preparation of embodiment 12 Emulsion
The Sasanguasaponin compound with structure shown in I that Example 5 prepares adds customary adjuvant, presses
More solito makes Emulsion.
The preparation of embodiment 13 suspensoid
The Sasanguasaponin compound with structure shown in I that Example 1 prepares adds customary adjuvant, presses
More solito makes suspensoid.
Below it is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred implementation is not
Should be regarded as limitation of the present invention, protection scope of the present invention should be with claim limited range
Accurate.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 regarded as protection scope of the present invention.
Claims (1)
1. the preparation method of the Sasanguasaponin compound of structure shown in a Formulas I, it is characterised in that
Comprise the following steps:
Step 1: after taking the pulverizing of oil tea root, extracted, obtains extracting solution；
Take described extracting solution concentrated, obtain fluid extract；
Take described fluid extract to mix with water, filter, collect filtrate；
Taking described filtrate to separate through macroporous resin, with ethanol-water mixture eluting, collected volume ratio is
The ethanol-water mixture elution fraction of 70:30～95:5, obtains described Total oil tea saponins extract；
Step 2: take described Total oil tea saponins extract, separates through decompression silica gel chromatographic column, with chloroform
-methyl alcohol mixed liquor eluting, collected volume is than the chloroform-methanol mixed liquor eluting group for 80:20～60:40
Point, obtain purified for the first time；
Taking in described first time purified warp presses anti-phase octadecylsilane chemically bonded silica chromatographic column to divide
From, with methanol-water mixture eluting, collected volume is than the methanol-water mixture for 70:30～90:10
Elution fraction, obtains second time purified；
Take described second time purified to separate through dynamic axial compression chromatographic column, with methanol-water mixture
Eluting, collected volume, than the methanol-water mixture elution fraction for 75:25, obtains third time purified；
Take described third time purified to separate through half preparative high-performance liquid chromatographic post, with methanol-water-formic acid
Mixed liquor eluting, flow velocity 2mL/min, collecting retention time is the elution fraction corresponding to 44.5min,
Obtain；
Described methanol-water-formic acid mixed liquor is: first alcohol and water is by volume for the mixing of 78:22 composition
Solution again with the mixing accounting for the formic acid mixing gained that described mixed solution weight/mass percentage composition is 0.2%
Liquid；
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Citations (1)
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| 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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| KR101886350B1 (en) * | 2012-03-26 | 2018-09-11 | (주)아모레퍼시픽 | Composition containing triterpenoid saponin extracted from the root of Camellia sinensis |
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Non-Patent Citations (1)
| Title |
|---|
| Tea Triterpenoidal Saponins from the Roots of Camellia sinensis Have Inhibitory Effects against Alcohol Dehydrogenase;Titto Varughese, et al.;《Planta Medica》;20110722;第77卷;第2029-2036页 * |
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