CN106008548A - New clerodane diterpenoid compound and preparation method thereof - Google Patents
New clerodane diterpenoid compound and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a new clerodane diterpenoid compound and a preparation method thereof. The compound is reported for the first time, is a clerodane diterpenoid compound with a novel structure, and can be extracted, separated and purified from dry roots of scutellaria baicalensis. The compound (I) is proved to have a role of inducing SACC-M apoptosis through in vitro tests, the apoptosis rate is on the rise along with prolonging of the drug action time and increase of the drug concentration, and the compound (I) has the effect of treatment of adenoid cystic carcinoma. Moreover, when used as a drug, the compound (I) can be used directly, or is used in a form of a pharmaceutical composition, is applied to patients who need treatment through an oral or injectable form, can be made into various forms of drugs for use, and has a wide range of application.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to a kind of new Crow of isolated from the dry root of Radix Scutellariae
Alkane type diterpene-kind compound and preparation method thereof.
Background technology
Radix Scutellariae has another name called Radix Scutellariae, Radix Scutellariae, and head is loaded in Shennong's Herbal, for labiate Scutellaria
The dry root of baicalensis Georgi.Radix Scutellariae bitter in the mouth, cold in nature, there are heat clearing and damp drying, eliminating fire and detoxication, hemostasis, the effect such as antiabortive.
Cure mainly epidemic febrile disease, upper respiratory tract infection, cough due to lung-heat, damp and hot jaundice, pneumonia, dysentery, hemoptysis, conjunctival congestion, frequent fetal movement, high blood
The diseases such as pressure, carbuncle furuncle.Radix Scutellariae is one of China's tcm clinical practice conventional Chinese medicine kind, has long medication history, main product in
The provinces and regions such as Hebei, Shanxi, the Inner Mongol, Liaoning, Russia East Siberia, Mongolia, Korea, all there is distribution in Japan.
Radix Scutellariae is mainly containing flavone compound, and the flavone aglycone and the glycosides that separate from Radix Scutellariae at present have kind more than 40.In addition yellow
In a kind of reed mentioned in ancient books possibly together with substantial amounts of diterpene-kind compound and abundant trace element (wherein ferrum, copper, zinc, manganese content the highest).
Modern pharmacological research shows that Radix Scutellariae has antibacterial, antiviral, antiinflammatory, antitumor, antiallergic, removing free radical and resists
Oxidations etc. act on.Antibacterial and the antivirus action of Radix Scutellariae, with experience of tcm treatment " the hot disease of sky row ", " cough caused by pathogenic fire consumptive lung disease " and
" furuncle acute scleritis " is consistent.Radix Scutellariae antibacterial range is relatively wide, wherein the strongest to the inhibitory action of S. aureus L-forms, bacillus pyocyaneus, and to hook
Leptospiral also has certain inhibitory action.Baicalin can significantly inhibit the biosynthesis of intracellular leukotriene B4, leukotriene C,
Also can significantly inhibit Ca in the leukocyte that artificial tripeptides (fMLP) excites2+Raise, and promote that intracellular cAMP levels improves, show
The several functions of baicalin appreciable impact leukocyte also discloses its Anti-inflammatory Mechanism.Research display 5,7,2 '-trihydroxy is yellow
Ketone and 5,7,2 ', 3 '-kaempferol can significantly inhibit the generation of mouse skin tumor.Scutellaria baicalensis stem-leaf total flavonoid and open country in Radix Scutellariae
The Inhibition test of LA795 cell is shown by baicalin, and they have inhibitory action to LA795 cell under finite concentration;Meanwhile, no
Radix Scutellariae total flavones with dosage all can suppress the proliferation in vivo of transplanted sarcoma S180 tumor strain, and in dose-effect relationship, shows efficiently
The anti-tumor activity of low toxicity.4 kinds of Main Flavonoids compositions of Radix Scutellariae are respectively provided with elimination free radical in the different system of body and resist
Oxidation activity.Baicalin can prevent the one-tenth fiber finer that such as oxygen-derived free radicals such as hydroperoxidase, superoxide anion causes
The damage of born of the same parents.In the flavones ingredient of 4 kinds of Radix Scutellariaes, baicalin is maximally effective antioxidant.In ischemia-reperfusion injury model,
Baicalin can make cell avoid the damage of lethal dose oxidant.
Radix Scutellariae is a kind of more common Chinese medicine, and clinical practice is extensive, sick to its antioxidation, antitumor and AntiHIV1 RT activity in recent years
The research of poison is the most deep, and exploitation Radix Scutellariae and active component thereof are as blood pressure lowering, treatment coronary heart disease and anti-curing oncoma and AIDS
The prospect of medicine is the most wide.
Summary of the invention
It is an object of the invention to provide a kind of a kind of new Crow alkane type diterpene of isolated from the dry root of Radix Scutellariae
Compounds and preparation method thereof.
The above-mentioned purpose of the present invention is achieved by techniques below scheme:
A kind of new Crow alkane type diterpene-kind compound, has the compound (I) of following structural formula,
The preparation method of described compound (I), comprises following operating procedure: the dry root of Radix Scutellariae is pulverized by (a), with 75
~85% alcohol heat reflux extract, united extraction liquid, be concentrated into without alcohol taste, use petroleum ether, ethyl acetate and water saturated successively
N-butanol extraction, respectively obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract;Acetic acid in (b) step (a)
The macroporous resin remove impurity of ethyl ester extract, first with 8 column volumes of 15% ethanol elution, then with 12 cylinders of 75% ethanol elution
Long-pending, concentrating under reduced pressure obtains 75% ethanol elution thing extractum;C in () step (b), 75% ethanol elution extractum purification on normal-phase silica gel separates, depend on
Secondary volume ratio is that the methylene chloride-methanol gradient elution of 80:1,55:1,35:1,10:1 and 1:1 obtains 5 components;(d) step
Suddenly in (c), component 4 separates further by purification on normal-phase silica gel, is the methylene chloride-methanol of 15:1,10:1 and 5:1 by volume ratio successively
Gradient elution obtains 3 components;E reverse phase silica gel that in () step (d), component 2 is bonded by octadecylsilane separates, and uses volume
Percentage concentration is the methanol aqueous solution isocratic elution of 75%, collects 8~10 column volume eluents, and eluent is concentrated under reduced pressure to give
Pure compound (I).
Further, described macroporous resin is AB-8 type macroporous adsorbent resin.
Further, described alcohol heat reflux extracts the concentration of alcohol used is 80%.
A kind of pharmaceutical composition, wherein contains the described compound (I) of therapeutically effective amount and pharmaceutically acceptable load
Body.
When the compounds of this invention is used as medicine, can directly use, or use with the form of pharmaceutical composition.
This pharmaceutical composition contains the compounds of this invention (I) of therapeutically effective amount, and remaining is the most acceptable, right
Nontoxic and the inert pharmaceutically suitable carrier of humans and animals and/or excipient.
Described pharmaceutically suitable carrier or excipient are that one or more are selected from solid, semisolid and liquid diluent, filler
And pharmaceutical preparation adjuvant.The pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention can
It is applied to need the patient for the treatment of by oral or injection form.During for being administered orally, tablet, slow releasing tablet, control can be made into
Release sheet, capsule, drop pill, micropill, suspensoid, Emulsion, powder or granule, oral liquid etc.;During for injecting, can be made into sterilizing
Aqueous or oily solution, aseptic powder injection, liposome or Emulsion etc..
Compared with prior art, beneficial effects of the present invention is embodied in:
Through in vitro tests, the compound (I) of the present invention proves that compound (I) has the effect of induction SACC-M apoptosis, and wither
Rate of dying is in rising trend with the prolongation of drug treating time and the increase of Drug level, and compound (I) has treatment adenoid cystic
The effect of cancer.And the compounds of this invention (I) be used as medicine time, can directly use, or make with the form of pharmaceutical composition
With, can be applied to need the patient for the treatment of by oral or injection form, the medicine that i.e. can be made into various ways uses,
Its range of application is wider.
Accompanying drawing explanation
Fig. 1 is compound (I) structural formula;
Fig. 2 is that the theoretical ECD value of compound (I) compares with experiment ECD value.
Detailed description of the invention
Further illustrate the essentiality content of the present invention below in conjunction with embodiment, but do not limit the present invention with this and protect model
Enclose.Although the present invention being explained in detail with reference to preferred embodiment, it will be understood by those within the art that, can be right
Technical scheme is modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention.
Embodiment 1: compound (I) separates preparation and structural identification
Medical material and reagent source: ethanol, petroleum ether, ethyl acetate, n-butyl alcohol, dichloromethane are analytical pure, purchased from Shanghai
Ling Feng chemical reagent company limited, methanol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.The dry root of Radix Scutellariae is purchased
From Hui nationality's Chinese Medicinal Materials Markets.
Preparation method: the dry root (10kg) of (a) Radix Scutellariae is pulverized, extracts (25L × 3 time) with 80% alcohol heat reflux, closes
And extracting solution, be concentrated into without alcohol taste (6L), successively with petroleum ether (6L × 3 time), ethyl acetate (6L × 3 time) and water saturated just
Butanol (6L × 3 time) extracts, and respectively obtains petroleum ether extract, acetic acid ethyl ester extract (355g) and n-butyl alcohol extract;(b)
Acetic acid ethyl ester extract AB-8 type macroporous resin remove impurity in step (a), successively with 15% ethanol (8L) and 75% (12L) ethanol
Eluting, collects 75% ethanol elution, and concentrating under reduced pressure obtains 75% ethanol elution thing extractum (154g);75% second in (c) step (b)
Alcohol eluting extractum with purification on normal-phase silica gel separate, successively with volume ratio be 80:1 (8 column volumes), 55:1 (8 column volumes), 35:1 (6
Individual column volume), the methylene chloride-methanol gradient elution of 10:1 (8 column volumes) and 1:1 (5 column volumes) obtain 5 components;
D in () step (c), component 4 (46g) separates further by purification on normal-phase silica gel, be 15:1 (8 column volumes), 10:1 by volume ratio successively
The methylene chloride-methanol gradient elution of (10 column volumes) and 5:1 (6 column volumes) obtains 3 components;Group in (e) step (d)
2 (27g) reverse phase silica gel that octadecylsilane is bonded is divided to separate, isocratic with the methanol aqueous solution that concentration expressed in percentage by volume is 75%
Eluting, collects 8-10 column volume eluent, and eluent is concentrated under reduced pressure to give pure compound (I) (35mg).
Structural identification: HR-ESIMS shows [M+Na]+For m/z 443.1018, can obtain molecular formula in conjunction with nuclear-magnetism feature is
C20H20O10, degree of unsaturation is 11.Hydrogen nuclear magnetic resonance modal data δH(ppm, DMSO-d6, 500MHz): H-1 (3.53, t, J=
1.5), H-2 (4.51, dd, J=3.0,1.5), H-3 (6.54, dd, J=3.0,1.5), H-6 (1.93, m), H-6 (1.78, m),
H-7 (2.51, m), H-7 (1.82, m), H-12 (6.76, s), H-14 (6.32, dd, J=2.0,1.0), H-15 (7.31, t, J=
2.0), and H-16 (7.35, m), H-19 (4.81, d, J=7.5), H-19 (4.07, dd, J=7.5,2.0), H-20 (1.13, s);
Carbon-13 nmr spectra data δC(ppm, DMSO-d6, 150MHz): 68.2 (CH, 1-C), 63.1 (CH, 2-C), 128.1 (CH, 3-
C), 132.9 (C, 4-C), 42.6 (C, 5-C), 25.1 (CH2, 6-C), 27.3 (CH2, 7-C), 72.9 (C, 8-C), 42.7 (C, 9-
C), 75.2 (C, 10-C), 210.1 (C, 11-C), 86.3 (CH, 12-C), 123.1 (C, 13-C), 107.0 (CH, 14-C),
142.3 (CH, 15-C), 138.8 (CH, 16-C), 172.5 (C, 17-C), 167.9 (C, 18-C), 72.6 (CH2, 19-C), 19.3
(CH3, 20-C);Carbon atom labelling sees Fig. 1.IR spectrum shows that this compound contains hydroxyl (3470cm-1), lactone (1767 Hes
1726cm-1) and furan nucleus (876cm-1) group.13C H NMR spectroscopy shows 20 signals, including a methyl, three methylene
(one contain Oxymethylene), seven methines (three oxygen-containing methines, four alkene carbon) and nine quaternary carbons (three carbonyl carbon,
Two alkene carbon, two oxygen-containing quaternary carbons).In HMBC spectrum, H-3 (δ H6.54, dd, J=3.0,1.5Hz) and C-18 (δ C167.9)
Dependency show that this compound contains α, a β unsaturation-18,19-gamma lactone structure.Additionally, this compound is possibly together with one
Individual 12,17-delta-lactone (δ H6.76, δ C86.3, CH-12;δ C172.5, C-17).1H-1In H COSY spectrum, H-3 Yu H-2 (δ
H4.51, dd, J=3.0,1.5Hz) dependency show that C-2 (δ C63.1) position is connected with a hydroxyl.Additionally, H-2 Yu H-1 (δ
H3.53, t, J=1.5Hz) dependency, and the chemical shift of C-1 (δ C68.2) and C-10 (δ C75.2) shows C-1 and C-
10 are connected with a hydroxyl respectively.In HMBC spectrum, H-2 Yu C-4 (δ C132.9) and C-10 and H-1 and C-2 and C-3 (δ C128.1)
The relevance verification position of above-mentioned hydroxyl.?13In C H NMR spectroscopy, the signal of C-8 (δ C72.9) shows that this carbon is connected with a hydroxyl
Base group;H in HMBC spectrum2-6, H2-7 and H3-20 with the relevance verification of C-8 above-mentioned inference.H-12 (δ in composing according to HMBC
H6.76, s) with C-13 (δ C123.1), C-14 (δ C107.0) and the dependency of C-16 (δ C138.8), deducibility furan nucleus is positioned at
On C-12 position.In COSY spectrum, H3With the peak that intersects of H-14 and H-16 ,-20 show that furan nucleus is α-configuration.Comprehensive hydrogen is composed, carbon is composed,
HMBC spectrum and NOESY compose, and document is about correlation type nuclear magnetic data, can substantially determine this compound as it is shown in figure 1, solid
Configuration is determined by ECD test further, theoretical value and experiment value basically identical (Fig. 2).
Embodiment 2: compound (I) pharmacological action is tested
One, material and instrument
Salivary Adenoid Cystic Carcinoma Cell system SACC-M is purchased from Medical College, Shanghai Communication Univ.'s Oral and Maxillofacial Surgery laboratory.Compound
(I) self-control, HPLC normalization purity is more than 98%.RPMI-1640 culture medium, trypsin are purchased from SIGMA company of the U.S..Standard
Hyclone is purchased from Xingtai City Huifeng biotechnology research institute.Annexin-v-FITC/PI test kit is purchased from Beijing treasured match biology
Technology Co., Ltd..PBS is purchased from Tianjin recovery fine chemistry industry institute.MTT powder is purchased from Amresco company.Diethylamine tetrem
Acid disodium (EDTA) is purchased from Beijing Yili Fine Chemicals Co., Ltd..Penicillin, streptomycin are purchased from North China pharmaceutical Co. Ltd.
Dimethyl sulfoxide (DMSO) is purchased from Tianjin Standard Science company limited.Giemsa dye liquor is purchased from Zhuhai Bei Suo Technology Co., Ltd..
XYJ type medical treatment clean work station (Purifying Equipment Co., Ltd., Suzhou), BB5060/BB16 CO2 gas incubator
(Shanghai Heraeus company), CX21 optical microscope (Japan OLYMPUS), (south of the River is public for XD-10198101 inverted microscope
Department), flow cytometer (Bection Dickinson Facsca Libur), WeLLscan MK3 full-automatic microplate reader (Finland
LabsystemsDragon), BFX5-320 type low speed centrifuge (Chinese Shanghai Lu Nan scientific instrument related factory), GF-300 type electricity
Sub-balance (JapanA&DCompany, Limited), 725 type ultra cold storage freezers (Forma Scientific.Inc), trace adds
Sample device (EPPENDORF company).
Two, test method
1, cell is cultivated
1.1 cell recovery
Prepare 37 DEG C of constant water bath box, the SACC-M cell cryopreservation tube of Liquid nitrogen storage is taken out, direct plunges into water bath, gently
Jog moves, and after it melts rapidly, takes out cryopreservation tube from water tank, and this step is preferably fast, should complete within the 30-60 second.Afterwards with
Open cryopreservation tube after the 75% alcohol wipe sterilization mouth of pipe, by pipe inner cell suspension sucking-off, instill 50mL centrifuge tube and at centrifuge tube
More than 10 times RPMI-1640 culture fluid containing 10% serum of middle dropping, blow and beat so that it is become cell suspension, low-speed centrifugal gently
After (1000 revs/min) 5 minutes, suck supernatant, then washed once with the RPMI-1640 culture fluid of 10%, and recentrifuge, inhale
After removing supernatant, with the RPMI-1640 culture fluid containing 10% hyclone, cell is diluted to cell suspension, is inoculated in 50mL
In culture bottle, unscrew bottle cap gently, be then placed in CO2Cultivating in incubator, second day changes a culture fluid, is cultivating afterwards
Liquid color, by changing culture fluid after light red flavescence, when observation of cell climbs the 80% of full bottle diapire, carries out passage.
1.2 passage
After drawing original fluid first by elbow straw, repeatedly at the bottom of piping and druming bottle, remove old culture fluid in bottle afterwards, to bottle
Interior addition mixture slaking liquid (0.25% trypsin and 0.04%EDTA 1:1) (can be completely covered at the bottom of culture bottle is on a small quantity
Preferably), at the bottom of slowly shake body makes Digestive system soak bottle, then suck major part Digestive system, only stay and digest on a small quantity.To cultivate
Bottle is placed in CO2In incubator, (or under 25 DEG C of room temperatures) digest, and take out culture bottle after 2~5 minutes, put and see under inverted microscope
Examining, when finding that kytoplasm retraction, intercellular substance increase, part cell is become round by polygon, and when existing cell starts to suspend,
Digestion should be stopped immediately.The Digestive system of sucking-off remaining, adds the appropriate culture fluid without serum, rotates culture bottle gently, wash out
After the Digestive system of residual, by its sucking-off, add new culture fluid, after drawing culture in glassware liquid with elbow straw, repeatedly blow and beat bottle
The end, make patch.In the cell detachment at the bottom of bottle, form cell suspension, noticing that during piping and druming, action wants soft, in case overexerting, making thin
Born of the same parents are impaired.Draw cell suspension to be placed in centrifuge tube, after being centrifuged, remove supernatant, collect cell, add appropriate containing 10% hyclone
RPMI-1640 culture fluid, cell is blown and beaten into suspension, counting, with 1.0 × 105/ mL concentration is seeded in new culture bottle
In.
2, MTT colorimetric test
(1) inoculating cell: trophophase cell of taking the logarithm, making concentration with the RPMI-1640 containing 10% hyclone is
2×105The single cell suspension of/mL, is inoculated in 96 orifice plates, every hole 100 μ L.(2) cell is cultivated: above-mentioned 96 orifice plates are placed in CO2
Incubator, at 37 DEG C, 5%CO2And under the conditions of saturated humidity, continue to cultivate 24h.(3) dosing: the every hole of medication group adds dilution
After compound (I) medicinal liquid 100 μ L so that it is final concentration of 2.5,5.0,7.5,10,12.5mmol/L;The every hole of matched group adds 100 μ
The L RPMI-1640 without serum.Medication group and matched group often group is all provided with 6 multiple holes, and sets zeroing hole, continues training after dosing
Support.(4) colour generation: after continuing to cultivate 24h, 48h and 72h, medication group and the every hole of matched group add the MTT solution 20 that concentration is 5g/L
μ L, after continuing to cultivate 4h, sucks whole supernatant, adds the dimethyl sulfoxide (including hole of returning to zero) of 200 μ L, put and shake in each hole
Swing vibration on device to shake up 1 minute, make crystallization fully dissolve.(5) colorimetric: be placed in microplate reader by 96 orifice plates, is 490nm at wavelength
Place, measures each hole absorbance (A).Repeat to test 3 times, take its meansigma methods.(6) calculate and draw: cell proliferation inhibition rate=
(1-medication group average A-value/matched group average A-value) × 100%.The growth inhibition ratio of each concentration is calculated, so according to above formula
After with the time as transverse axis, suppression ratio is the longitudinal axis, draw 5 concentration suppression ratio curve.
3, Flow cytometry
The process of 3.1 cells
After cell inoculation, cultivation, trophophase cell of taking the logarithm, with 2 × 105/ mL concentration is inoculated in 6 orifice plates, then adds
Medicine, if time contrast groups, concentrations versus's group and matched group (normal apoptotic group): (1) time contrast groups: cell is inoculated in 6 holes
In plate, cultivate 24 hours, after cell attachment, with suction pipe along each hole wall by thorough for original fluid sucking-off, then in each hole addition etc.
The final concentration of 12.5mmol/L of the amount culture fluid (medication group) containing compound (I) medicinal liquid.Add the cultivation without compound (I)
Liquid as a control group, continues to cultivate 24h, 48h, 72h respectively.(2) concentrations versus's group: after cell inoculation adherent success, with suction
Pipe thoroughly exhausts original fluid, each hole is separately added into final concentration of 2.5,5.0,7.5,10,12.5mmol/L containing compound
(I) culture fluid (medication group).Matched group adds the culture fluid without compound (I), continues to cultivate 72h.(3) normal apoptotic group:
After inoculating cell adherent success, remove original fluid in each hole, add the culture fluid without compound (I), continue to cultivate
24h、48h、72h。
The preparation of 3.2 samples
(1) collect cell: draw 6 orifice plate each hole supernatant respectively, add in centrifuge tube, take PBS liquid 2mL and rinse 6 orifice plates
After each diapire, pour in same centrifuge tube, then 2mL mixture slaking drop is entered peptic cell in each hole, slow wave and culture
Plate makes Digestive system soak its diapire completely, after digestion 2min, Digestive system is sucked each centrifuge tube, adds the piping and druming of 2mL culture fluid each
Hole diapire, after cell thoroughly takes off wall, sucks in centrifuge tube.(2) centrifuge cell: centrifuge tube is placed in low speed centrifuge, in
After 1500rpm is centrifuged 5min, abandoning supernatant;Add 12mL PBS liquid re-suspended cell, be centrifuged 5min then at 1500rpm, abandon
Clear liquid;Cell suspension after re-suspended cell, is drawn to the special upper machine pipe of flow cytometer in centrifuge tube by the PBS liquid instilling 3mL again
In, after 1500rpm is centrifuged 5min, abandon supernatant.
3.3Annexin-V-FITC and PI double labelling living cells method detection apoptosis
(1) add the PI of Annexin-V-FITC and 5 μ L of 10 μ L in streaming pipe (two kinds of reagent are not with micro sample adding appliance
Mix mutually, do not contact with pipe floor cells).(2) add 200 μ L and combine buffer, Annexin-V-FITC and PI is rushed at the bottom of pipe
Mix mutually with cell, and blow and beat gently, form single cell suspension.(3) lucifuge is reacted 15min or puts into 4 DEG C of refrigerator 30min.(4) on
Add 300 μ L PBS before machine and combine buffer, and blow and beat gently, go up machine (flow cytometer) detection the most at once.Light source is
488nm argon ion laser, 10000, every part of specimen collection cell, FITC be stimulated after fluoresced green, the rubescent color of PI is glimmering
Light.(5) through flow cytometry analysis, it is thus achieved that the scatterplot being made up of four quadrants, on figure, each point represents a cell, all
There are two parameter values.Upper left Q1 quadrant represents the damaging cells (An-PI+) occurred during cell is collected, upper right Q2 quadrant
Representing non-viable apoptotic cell and non-viable non-apoptotic cell (An+PI+), lower-left Q3 quadrant represents normal cell (An-PI-), bottom right Q4 quadrant
Represent viable apoptotic cell (An+PI-).
4, statistical analysis
Statistics software SPSS10.0 is used to carry out statistical analysis.(1) variance analysis of Factorial Design is used, to identical medicine
The compound (I) life to SACC-M cell between substrate concentration different disposal time, each group of same treatment time different pharmaceutical concentration
Long suppression ratio compares two-by-two.(2) use simple correlation analysis, analyze growth inhibition ratio and between action time, suppression ratio
And the dependency between drug level, draws correlation coefficient r, and r value carries out t inspection, thus show that drug effect is in time with dense
The variation relation of degree.(3) each medication group and the apoptosis rate of matched group of the u-test flow cytometric art detection of binomial distribution are used
Carry out statistical analysis.
Three, result and conclusion
1, MTT colorimetric test
(1) experiment shows that compound (I) has an obvious inhibited proliferation to SACC-M cell, medicine effect 24,48,
After 72h, the highest suppression ratio is up to 80.4% (table 1).(2) suppression ratio between same treatment time, variable concentrations group compare have bright
Significant difference different (P < 0.01);Same concentrations group, suppression ratio between the different disposal time have notable difference (P < 0.01);Drug level
With process time non-interaction action (P=0.901).(3) compare two-by-two between group: between identical time, variable concentrations group, suppression ratio
There is notable difference (P < 0.01);Process after 24h, 48h, 72h, have between same concentration, different time group notable difference (P <
0.01).(3) drug effect (suppression ratio) is respectively provided with positive correlation with action time and drug level, and suppression ratio had with action time
Positive correlation (r=0.291, P=0.042);Suppression ratio and concentration have positive correlation (r=0.926, P < 0.001).
2, Flow cytometry
The apoptosis situation of 2.1 cellular control units
Matched group (normal apoptotic group) cell belongs to natural apoptosis, and during 24h, its apoptosis rate is 4.9%, apoptosis during 48h
Rate is 7.1%, and the apoptosis rate of 72h is 6.1% (table 2), and apoptosis rate is little with incubation time difference, not statistically significant.
2.2 medication group cells change over apoptosis situation
After 12.5mmol/L compound (I) processes 24h, its apoptosis rate is 25.5%;After processing 48h, apoptosis rate rises
To 40.9%, it mostly is early apoptosis;After processing 72h, apoptosis rate is 67.6%, mostly is late apoptic, and apoptosis rate is along with place
Manage time lengthening and substantially rise (table 2), apoptosis rate statistically significant (P < 0.01) between each time group.
2.3 medication group cells are with concentration change apoptosis situation
Process after 72h, 2.5,5.0,7.5,10.0, the apoptosis rate organized of 12.5mmol/L compound (I) is respectively
16%, 24.2%, 26.1%, 66.3%, 67.6%, and when drug level reaches 12.5mmol/L, late apoptic rate is obvious
Increase (table 2), apoptosis rate statistically significant (P < 0.01) between each drug level group.
Conclusion, the study show that compound (I) have induction SACC-M apoptosis effect, and apoptosis rate with medicine effect time
Between prolongation and the increase of Drug level in rising trend.Application compound (I) treatment adenoid cystic carcinoma is likely to become one
The most promising Therapeutic Method.
Table 1 variable concentrations difference suppression ratio action time (mean value ± SD)
Table 2 compound (I) induction SACC-M apoptosis situation
Concentration-time | Normal cell (%) | Early apoptosis (%) | End-stage apoptotic (%) | Damaging cells (%) |
0mmol/L—24h | 93.2 | 0.5 | 4.9 | 1.4 |
0mmol/L—48h | 90.4 | 2.3 | 7.1 | 0.3 |
0mmol/L—72h | 87.5 | 6.1 | 6.1 | 0.2 |
12.5mmol/L—24h | 74.4 | 11.8 | 13.7 | 0.1 |
12.5mmol/L—48h | 59 | 35.4 | 5.5 | 0 |
12.5mmol/L—72h | 32.4 | 21.3 | 46.3 | 0.1 |
2.5mmol/L—72h | 82.4 | 1.5 | 14.5 | 1.7 |
5.0mmol/L—72h | 75.7 | 19.3 | 4.9 | 0.1 |
7.5mmol/L—72h | 73.9 | 15.2 | 10.9 | 0 |
10.0mmol/L—72h | 33.6 | 58.7 | 7.6 | 0.1 |
Embodiment 3
The preparation of tablet: first prepare compound (I) by embodiment 1 method, and utilize organic acid such as tartaric acid or Fructus Citri Limoniae
The salt that acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, by its with excipient weight ratio for 1:9's
Ratio adds excipient, pelletizing press sheet.
Embodiment 4
The preparation of oral liquid: first prepare compound (I) by embodiment 1 method, and utilize organic acid such as tartaric acid or lemon
The salt that lemon acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, oral liquid preparation method makes mouth routinely
Take liquid.
Embodiment 5
Capsule or the preparation of granule: first prepare compound (I) by embodiment 1 method, and utilize organic acid such as wine
The salt that stone acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, by itself and excipient weight
Amount adds excipient than the ratio for 1:9, makes capsule or granule.
Embodiment 6
The preparation of injection: first prepare compound (I) by embodiment 1 method, and utilize organic acid such as tartaric acid or lemon
The salt that lemon acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, injects routinely and uses water, fine straining,
Injection is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection: first prepare compound (I) by embodiment 1 method, and utilize organic acid such as tartaric acid or
The salt that citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid are made, is dissolved in sterile water for injection
In, stirring makes molten, filters with aseptic suction funnel, more aseptic fine straining, is sub-packed in ampoule, aseptic sealing by fusing after frozen drying
Obtain injectable powder.
The effect of above-described embodiment indicates that the essentiality content of the present invention, but does not limit the protection of the present invention with this
Scope.It will be understood by those within the art that, technical scheme can be modified or equivalent,
Essence and protection domain without deviating from technical solution of the present invention.
Claims (5)
1. a new Crow alkane type diterpene-kind compound, it is characterised in that there is the compound (I) of following structural formula,
2. the preparation method of the compound (I) described in claim 1, it is characterised in that comprise following operating procedure: (a) is by Huang
The dry root of a kind of reed mentioned in ancient books is pulverized, with 75~85% alcohol heat reflux extract, united extraction liquid, be concentrated into without alcohol taste, use successively petroleum ether,
Ethyl acetate and water saturated n-butanol extraction, respectively obtain petroleum ether extract, acetic acid ethyl ester extract and n-butanol extraction
Thing;B acetic acid ethyl ester extract macroporous resin remove impurity in () step (a), first with 8 column volumes of 15% ethanol elution, then uses
75% ethanol elution 12 column volume, concentrating under reduced pressure obtains 75% ethanol elution thing extractum;75% ethanol elution in (c) step (b)
Extractum purification on normal-phase silica gel separates, successively by the methylene chloride-methanol gradient that volume ratio is 80:1,55:1,35:1,10:1 and 1:1
Afford 5 components;D in () step (c), component 4 separates further by purification on normal-phase silica gel, be 15:1,10:1 by volume ratio successively
3 components are obtained with the methylene chloride-methanol gradient elution of 5:1;E in () step (d), component 2 is bonded by octadecylsilane
Reverse phase silica gel separates, and with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collects 8~10 column volume eluting
Liquid, eluent is concentrated under reduced pressure to give pure compound (I).
The preparation method of compound the most according to claim 2 (I), it is characterised in that described macroporous resin is AB-8 type
Macroporous adsorbent resin.
The preparation method of compound the most according to claim 2 (I), it is characterised in that described alcohol heat reflux extracts
The concentration of alcohol used is 80%.
5. a pharmaceutical composition, it is characterised in that wherein contain the compound (I) described in the claim 1 of therapeutically effective amount
With pharmaceutically acceptable carrier.
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