CN106008548A - New clerodane diterpenoid compound and preparation method thereof - Google Patents

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

A kind of new Crow alkane type diterpene-kind compound and preparation method thereof

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 δ_H(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 δ_C(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.