CN105481799A - Highly-oxidized sesquiterpene compound as well as preparation method and medical application thereof - Google Patents
Highly-oxidized sesquiterpene compound as well as preparation method and medical application thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D301/00—Preparation of oxiranes
- C07D301/32—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
- C07D303/02—Compounds containing oxirane rings
- C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
- C07D303/32—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by aldehydo- or ketonic radicals
Abstract
The invention discloses a highly-oxidized sesquiterpene compound as well as a preparation method and a medical application thereof, and belongs to the technical field of medicines. The compound is reported for the first time, is a sesquiterpene compound with a novel structure, and can be obtained by extracting dried leaves of guavas and performing separation and purification. An in-vitro test proves that the compound has an effect of inducing SACC-M apoptosis, and the apoptosis rate is on the rise with the prolongation of the acting time and the increase of the concentration of a drug. The application of the compound (I) in the treatment of adenoid cystic carcinoma may become a promising treatment method, and the compound can be used for developing a medicine for treating adenoid cystocarcinoma of salivary gland.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to from the dry leave of piscidia, be separated a kind of sesquiterpenoids obtained, containing its pharmaceutical composition and its preparation method and application.
Background technology
Plant piscidia (PsidiumguajavaLinn.) is Myrtaceae (Myrtaceae) Psidium plant, is deciduous tree, high 5-10m.Bark light tan, spray square, tool white undercoat, then comes off always; Bud is close by white undercoat.Single leaf alternate, rare verticillate, square round shape is oval to oval, long 5-12cm, wide 3-5cm, that rubs has fragrance, keratin, tip circle or short point, base portion is blunt to circular, Quan Yuan, above deep green, vein nick or smooth, dredges raw undercoat time tender, below light green, dredge raw glandula, close by pubescence, master pulse swells, lateral vein 7-11 couple, also swells, and nearly tiltedly goes out leaf margin and bends; Cry handle long 4mm.Flower both sexes, the raw 1-4 piece of armpit; Calyx 5, green, oval; Petal white, avette, long 2-2.5cm; Stamen is most, isometric with petal, filigree white, and flower pesticide is light yellow, lobe; Gynoecium 1, style is longer than filigree, and column cap is circular, and ovary is the next, Room 3, and ovule is most.Berry is spherical, oval or foreign pears shape, and long 2.5-8cm, footpath 3-5cm, pulp is usually yellow, also adularescent or kermes.Seed oval, pale.The florescence 5-8 month.The fruit phase 8-11 month.Guava Leaf is dry leave and the band leaf spray of piscidia, and another name chicken vows tea, kind peach leaf, that pulls out leaf etc.Guava Leaf is flat, sweet-puckery flavor, and entering spleen, stomach, large intestine, Liver Channel, have drying damp and strengthening spleen, clearing heat and detoxicating effect, is folk therapy diabetes, enteritis and dysentery, the common drug of wound hemorrhage.
Guava Leaf chemical composition is various, mainly comprises phenolic acids, flavonoid, triterpenes and sesquiterpenoids.In recent years, the sesquiterpenoids composition in Guava Leaf attracts wide attention because of its good biological activity.Sesquiterpenoids (sesquiterpenoids) is made up of 3 isoprene units, containing the compound monoid of 15 carbon atoms.Sesquiterpene is extensively present in plant, microorganism, marine organisms and some insect, much there is important biological function and physiologically active, particularly sesquiterpene lactones, there are antibacterial, antitumor, antiviral, cell toxicant, immunosuppression, vegetable poison, insect hormone, antifeedant for insect isoreactivity, also have some to have nervous system activity.
Summary of the invention
The object of this invention is to provide and a kind ofly from the dry leave of piscidia, be separated a kind of sesquiterpenoids obtained, containing its pharmaceutical composition and its preparation method and application.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
There is the compound (I) of following structural formula:
The preparation method of described compound (I), comprise following operation steps: the dry leave of piscidia is pulverized by (a), extract with 80 ~ 90% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; B in () step (a), the removal of impurities of thing macroporous resin got by propyl carbinol, first use 5% ethanol elution, 6 column volumes, then use 75% ethanol elution, 8 column volumes, collect 75% elutriant, concentrating under reduced pressure obtains 75% ethanol elution enriched material; C in () step (b), 75% ethanol elution enriched material purification on normal-phase silica gel is separated, obtain 4 components successively with the methylene chloride-methanol gradient elution that volume ratio is 65:1,30:1,15:1 and 8:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 15:1,8:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 8 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
Further, in step (a), extract with 85% alcohol heat reflux, united extraction liquid.
Further, described macroporous resin is AB-8 type macroporous adsorbent resin.
Pharmaceutical composition, the described compound (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
The application of described compound (I) in the medicine of preparation treatment adenoid cystocarcinoma of salivary gland.
The application of described pharmaceutical composition in the medicine of preparation treatment adenoid cystocarcinoma of salivary gland.
When the compounds of this invention is used as medicine, directly can uses, or use with the form of pharmaceutical composition.
This pharmaceutical composition contains the compounds of this invention (I) for the treatment of significant quantity, and all the other are acceptable on pharmacology, nontoxic to humans and animals and pharmaceutically acceptable carrier of inertia and/or vehicle.
Described pharmaceutically acceptable carrier or vehicle are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, dripping pill, micropill, suspensoid, emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into water-based or oily solution, aseptic powder injection, liposome or the emulsion etc. of sterilizing.
Accompanying drawing explanation
Fig. 1 is compound (I) structural formula;
Fig. 2 is that compound (I) calculates ECD and experiment ECD figure.
Embodiment
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
Main raw, reagent source and instrument type:
Ethanol, sherwood oil, ethyl acetate, propyl carbinol, methylene dichloride are analytical pure, and purchased from Shanghai Ling Feng chemical reagent company limited, methyl alcohol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Embodiment 1: compound (I) is separated preparation and structural identification
A the dry leave (8kg) of piscidia is pulverized by (), (30L × 3 time) are extracted with 85% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste (6L), use sherwood oil (6L × 3 time), ethyl acetate (6L × 3 time) and water saturated propyl carbinol (6L × 3 time) to extract successively, obtain petroleum ether extract, acetic acid ethyl ester extract (375g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract AB-8 macroporous resin removal of impurities in (b) step (a), first use 5% ethanol elution, 6 column volumes, use 75% ethanol elution, 8 column volumes again, collect 75% elutriant, concentrating under reduced pressure obtains 75% ethanol elution enriched material (129g); C in () step (b), 75% ethanol elution enriched material purification on normal-phase silica gel is separated, successively with volume ratio be 65:1 (8 column volumes), the methylene chloride-methanol gradient elution of 30:1 (8 column volumes), 15:1 (8 column volumes) and 8:1 (10 column volumes) obtains 4 components; D component 4 (27g) is separated further by purification on normal-phase silica gel in () step (c), successively with volume ratio be 12:1 (8 column volumes), the methylene chloride-methanol gradient elution of 8:1 (10 column volumes) and 5:1 (8 column volumes) obtains 3 components; E in () step (d), component 2 (15g) reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 8 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I) (40mg).
Structural identification: colorless oil; HR-ESIMS shows [M+Na]
+for m/z331.1528, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
17h
24o
5, degree of unsaturation is 6.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 500MHz): H-1 (3.22, s), H-3 (3.13, m), H-3 (3.16, m), H-5 (4.19, br, s), H-6 (1.81, dt, J=15.0, 2.3), H-6 (2.15, ddd, J=5.2, 9.5, 15.0), H-7 (2.35, m), H-8 (1.64, m, 2H), H-9 (1.23, m), H-9 (1.97, ddd, J=3.2, 7.0, 14.0), H-12 (4.52, d, J=14.0), H-12 (4.60, d, J=14.0), H-13 (4.98, s), H-13 (5.04, s), H-14 (1.26, s), H-15 (5.17, s), H-15 (5.25, s), 12-OAc (2.11, s), carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 125MHz): 68.9 (CH, 1-C), 208.7 (C, 2-C), 37.1 (CH
2, 3-C), 150.2 (C, 4-C), 75.8 (CH, 5-C), 28.7 (CH
2, 6-C), 32.8 (CH, 7-C), 30.4 (CH
2, 8-C), 34.2 (CH
2, 9-C), 61.1 (C, 10-C), 149.8 (C, 11-C), 65.5 (CH
2, 12-C), 110.9 (CH
2, 13-C), 18.4 (CH
3, 14-C), 116.8 (CH
2, 15-C), 171.1 (C, 12-OAc), 20.7 (CH
3, 12-OAc), carbon atom mark is see Fig. 1.
1h-NMR modal data shows a methyl signals [δ H1.26 (s, Me-14)], a methoxyl group signal [δ H2.11 (s, 12-OAc)], one containing Oxymethylene signal [δ H4.52 (d, J=14.0Hz, H-12), 4.60 (d, J=14.0Hz, H-12)], two olefinic methylene signals [δ H4.98 (s, H-13), 5.04 (s, H-13), 5.17 (s, H-15), 5.25 (s, H-15)], two containing oxygen methine protons [δ H3.22 (s, H-1), 4.19 (br, s, H-5)].
13cNMR spectrum shows 17 carbon signals, comprise two methyl [a methoxyl group δ C20.7 (12-OAc)], [one containing Oxymethylene δ C65.5 (C-12) for seven methylene radical, two olefinic methylene radical δ C110.9 (C-13) and 116.8 (C-15)], three methynes [two containing oxygen methyne δ C68.9 (C-1) and 75.8 (C-5)], five quaternary carbons [two olefinic quaternary carbon δ C150.2 (C-4) and 149.8 (C-11), two carbonyl carbon δ C208.7 (C-2) and 171.1 (12-OAc), one containing oxygen quaternary carbon δ C61.1 (C-10)].According to nuclear magnetic signal δ
h/C3.22/68.9 and δ
h/C1.26/61.1 this compound known contains 1,10-epoxide group.In HMBC spectrum, H
2-12 (δ H4.52, d, J=14.0Hz and 4.60, d, J=14.0Hz) and the dependency of carbonyl carbon (δ C171.1), show that C-12 (δ C65.5) position is connected with an acetoxyl group.In NOESY spectrum, the dependency of H-7 and Me-14 and H-15 (δ H5.17) shows that they are α configuration.In addition, H-1 and H-3 (δ H3.13), H-1 and H-6 (δ H1.81), and the dependency of H-5 and H-3 (δ H3.13) shows that they are beta comfiguration.Comprehensive hydrogen spectrum, carbon spectrum, HMBC spectrum and NOESY spectrum, and document is about correlation type nuclear magnetic data, can substantially determine this compound as shown in Figure 1, steric configuration is determined further by ECD test, theoretical value and experimental 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) is made by oneself, and HPLC normalization method purity is greater than 98%.RPMI-1640 substratum, trypsinase are purchased from SIGMA company of the U.S..Standard foetal calf serum is purchased from Xingtai City Huifeng biotechnology research institute.Annexin-v-FITC/PI test kit is purchased from Beijing Bao Sai Bioisystech Co., Ltd.PBS is purchased from Tianjin recovery fine chemistry industry institute.MTT powder is purchased from Amresco company.Na2EDTA (EDTA) is purchased from Beijing Yili Fine Chemicals Co., Ltd..Penicillin, Streptomycin sulphate are purchased from North China pharmaceutical Co. Ltd.Dimethyl sulfoxide (DMSO) (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 CO2gas incubator (Shanghai Heraeus company), CX21 opticmicroscope (Japanese OLYMPUS), XD-10198101 inverted microscope (south of the River company), flow cytometer (BectionDickinsonFacscaLibur), the full-automatic microplate reader of WeLLscanMK3 (Finland LabsystemsDragon), BFX5-320 type low speed centrifuge (Chinese Shanghai Lu Nan scientific instrument related factory), GF-300 type electronic balance (JapanA & DCompany, Limited), 725 type Ultralow Temperature Freezers (FormaScientific.Inc), micro sample adding appliance (EPPENDORF company).
Two, test method
1, cell cultures
1.1 cell recovery
Prepare 37 DEG C of constant water bath box, taken out by the SACC-M cell cryopreservation tube of Liquid nitrogen storage, directly drop into water bath, shake gently, after it melts rapidly, from water tank, take out cryopreservation tube, this step should be fast, should complete within 30-60 second.Afterwards to open cryopreservation tube after the 75% alcohol wipe sterilization mouth of pipe, by the sucking-off of pipe inner cell suspension, instillation 50mL centrifuge tube also drips more than the 10 times RPMI-1640 nutrient solutions containing 10% serum in centrifuge tube, blow and beat gently, become cell suspension, low-speed centrifugal (1000 revs/min) is after 5 minutes, suck supernatant liquor, wash once with the RPMI-1640 nutrient solution of 10% again, and recentrifuge, after sucking supernatant liquor, with the RPMI-1640 nutrient solution containing 10% foetal calf serum, cell dilution is become cell suspension, be inoculated in 50mL culturing bottle, unscrew bottle cap gently, then CO is put into
2cultivate in incubator, second day changes a nutrient solution, changes nutrient solution afterwards after nutrient solution color is by light red flavescence, when observation of cell climbs 80% of full bottle diapire, carries out passage.
1.2 passage
First after using elbow straw to draw original fluid, repeatedly blow and beat at the bottom of bottle, remove old nutrient solution in bottle afterwards, mixture slaking liquid (0.25% trypsinase and 0.04%EDTA1:1) a small amount of (being advisable covering completely at the bottom of culturing bottle) is added in bottle, at the bottom of slow shake body makes Digestive system soak bottle, then suck most of Digestive system, only stay and digest on a small quantity.Culturing bottle is placed in CO
2in incubator, (or under 25 DEG C of room temperatures) digest, and take out culturing bottle, fell and put basis of microscopic observation after 2 ~ 5 minutes, when discovery kytoplasm retraction, intercellular substance increase, part cell becomes circle from polygon, and when existing cell starts to suspend, should stop digestion immediately.The Digestive system that sucking-off is remaining, adds the appropriate nutrient solution not containing serum, rotates culturing bottle gently, after washing out residual Digestive system, by its sucking-off, then add new nutrient solution, repeatedly blows and beats at the bottom of bottle, make subsides with elbow straw after drawing culture in glassware liquid.Cell detachment at the bottom of bottle, forms cell suspension, and when noting blowing and beating, action wants soft, in case overexert, make cell damage.Drawing cell suspension is placed in centrifuge tube, after centrifugal, removes supernatant, collecting cell, and add the appropriate RPMI-1640 nutrient solution containing 10% foetal calf serum, cell is blown and beaten into suspension, counting, with 1.0 × 10
5/ mL concentration is seeded in new culturing bottle.
2, MTT colorimetric test
(1) inoculating cell: cell in vegetative period of taking the logarithm, making concentration with the RPMI-1640 containing 10% foetal calf serum is 2 × 10
5the single cell suspension of/mL, is inoculated in 96 orifice plates, every hole 100 μ L.(2) culturing cell: above-mentioned 96 orifice plates are placed in CO
2incubator, at 37 DEG C, 5%CO
2and under saturated humidity condition, continue to cultivate 24h.(3) dosing: the every hole of medication group adds compound (I) the liquid 100 μ L after dilution, make its final concentration be 2.5,5.0,7.5,10,12.5mmol/L; The every hole of control group adds 100 μ L not containing the RPMI-1640 of serum.Medication group and control group are often organized and are all established 6 multiple holes, and establish zeroing hole, continue to cultivate after dosing.(4) colour generation: after continuing to cultivate 24h, 48h and 72h, medication group and the every hole of control group add the MTT solution 20 μ L that concentration is 5g/L, after continuing to cultivate 4h, suck whole supernatant liquor, the dimethyl sulfoxide (DMSO) (comprising zeroing hole) of 200 μ L is added in each hole, put vibration on vibrator and shake up 1 minute, crystallization is fully dissolved.(5) colorimetric: be placed in microplate reader by 96 orifice plates is 490nm place at wavelength, measures each hole absorbance (A).Repeat experiment 3 times, get its mean value.(6) calculate and draw: cell proliferation inhibition rate=(1-medication group average A-value/control group average A-value) × 100%.According to the growth inhibition ratio of each concentration of above formulae discovery, be then transverse axis with time, inhibiting rate is the longitudinal axis, draws the inhibiting rate curve of 5 concentration.
3, Flow cytometry
The process of 3.1 cells
After cell inoculation, cultivation, cell in vegetative period of taking the logarithm, with 2 × 10
5/ mL concentration is inoculated in 6 orifice plates, then dosing, if time comparative group, concentrations versus's group and control group (normal apoptotic group): (1) time comparative group: cell is inoculated in 6 orifice plates, cultivate 24 hours, after cell attachment, with suction pipe along each hole wall by thorough for original fluid sucking-off, the final concentration then adding equivalent in each hole is the nutrient solution (medication group) that 12.5mmol/L contains compound (I) liquid.Add not containing compound (I) nutrient solution as a control group, respectively continue cultivate 24h, 48h, 72h.(2) concentrations versus's group: cell inoculation and after adherent success, thoroughly to exhaust original fluid with suction pipe, add respectively in each hole final concentration be 2.5,5.0,7.5,10,12.5mmol/L containing compound (I) nutrient solution (medication group).Control group adds not containing the nutrient solution of compound (I), continues to cultivate 72h.(3) normal apoptotic group: inoculating cell also, after adherent success, removes original fluid in each hole, adds not containing the nutrient solution of compound (I), continues to cultivate 24h, 48h, 72h.
The preparation of 3.2 samples
(1) collecting cell: draw 6 orifice plate each hole supernatant liquors respectively, add in centrifuge tube, after getting each diapire of PBS liquid 2mL rinsing 6 orifice plate, 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, add 2mL nutrient solution again and blow and beat each hole diapire, after cell thoroughly takes off wall, suck in centrifuge tube.(2) eccentric cell: centrifuge tube is placed in low speed centrifuge, after the centrifugal 5min of 1500rpm, abandoning supernatant; Add 12mLPBS liquid re-suspended cell, then in the centrifugal 5min of 1500rpm, abandon supernatant liquor; Cell suspension after re-suspended cell, is drawn in the special upper machine pipe of flow cytometer, after the centrifugal 5min of 1500rpm, abandons supernatant liquor by the PBS liquid instilling 3mL again in centrifuge tube.
3.3Annexin-V-FITC and PI double-tagging viable cell method detects apoptosis
(1) in streaming pipe, the Annexin-V-FITC of 10 μ L and the PI (two kinds of reagent do not mix mutually, do not contact with pipe floor cells) of 5 μ L is added with micro sample adding appliance.(2) add 200 μ L binding buffer liquid, Annexin-V-FITC with PI punching is mixed mutually with cell at the bottom of pipe, and blows and beats gently, form single cell suspension.(3) lucifuge is reacted 15min or is put into 4 DEG C of refrigerator 30min.(4) add 300 μ LPBS binding buffer liquid before upper machine, and blow and beat gently, then go up machine (flow cytometer) at once and detect.Light source is 488nm Argon ion laser, and 10000, every part of specimen collection cell, fluoresced green after FITC is stimulated, PI sends out red fluorescence.(5) through flow cytometry analysis, obtain the scatter diagram be made up of four quadrants, on figure, each point represents a cell, all has two parameter values.Upper left Q1 quadrant represents the damaging cells (An-PI+) occurred in cell harvesting process, upper right Q2 quadrant represents non-viable apoptotic cell and non-viable non-apoptotic cell (An+PI+), lower-left Q3 quadrant represents normal cell (An-PI-), and bottom right Q4 quadrant represents viable apoptotic cell (An+PI-).
4, statistical analysis
Using statistics software SPSS10.0 carries out statistical study.(1) use the variance analysis of Factorial Design, compound (I) growth inhibition ratio to SACC-M cell between same medicine concentration different treatment time, each group of same treatment time different pharmaceutical concentration is compared between two.(2) use simple correlation analysis, analyze growth inhibition ratio and the dependency between action time, between inhibiting rate and drug level, draw correlation coefficient r, and t inspection is carried out to r value, thus draw drug effect in time with the variation relation of concentration.(3) each medication group using the u-test flow cytometric art of binominal distribution to detect and the apoptosis rate of control group carry out statistical study.
Three, result and conclusion
1, MTT colorimetric test
(1) experiment shows that compound (I) has obvious inhibited proliferation to SACC-M cell, drug effect 24,48, the highest inhibiting rate can reach 80.4% (table 1) after 72h.(2) inhibiting rate between same treatment time, different concns group compares notable difference (P<0.01); Same concentrations group, inhibiting rate between the different treatment time have notable difference (P<0.01); Drug level and treatment time non-interaction action (P=0.901).(3) compare between two between group: between same time, different concns group, inhibiting rate has notable difference (P<0.01); After process 24h, 48h, 72h, between same concentration, different time group, there is notable difference (P<0.01).(3) drug effect (inhibiting rate) all has positive correlation with action time and drug level, and inhibiting rate and action time have positive correlation (r=0.291, P=0.042); Inhibiting rate and concentration have positive correlation (r=0.926, P<0.001).
2, Flow cytometry
The apoptosis situation of 2.1 cellular control units
Control group (normal apoptotic group) cell all belongs to natural apoptosis, apoptosis rate when its apoptosis rate is 4.9%, 48h during 24h is the apoptosis rate of 7.1%, 72h is 6.1% (table 2), apoptosis rate is little with incubation time difference, not statistically significant.
2.2 medication group cells change apoptosis situation in time
After 12.5mmol/L compound (I) process 24h, its apoptosis rate is 25.5%; After process 48h, apoptosis rate rises to 40.9%, mostly is early apoptosis; After process 72h, apoptosis rate is 67.6%, mostly is late apoptic, and apoptosis rate extends along with the treatment time and obviously rises (table 2), and between each time group, apoptosis rate has statistical significance (P<0.01).
2.3 medication group cells are with change in concentration apoptosis situation
After process 72h, 2.5,5.0 the apoptosis rate that, 7.5,10.0,12.5mmol/L compound (I) is organized is respectively 16%, 24.2%, 26.1%, 66.3%, 67.6%, and when drug level reaches 12.5mmol/L, late apoptic rate obviously increases (table 2), and between each drug level group, apoptosis rate has statistical significance (P<0.01).
Conclusion, this research shows that compound (I) has the effect of induction SACC-M apoptosis, and apoptosis rate with the prolongation of drug treating time and the increase of Drug level in rising trend.Application compound (I) treatment adenoid cystic carcinoma likely becomes the very promising methods for the treatment of of one.
The different inhibiting rate action time (mean value ± SD) of table 1 different concns
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: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, vehicle is added, pelletizing press sheet than the ratio for 1:7 in itself and excipient weight.
Embodiment 4
The preparation of oral liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, oral liquid method for making makes oral liquid routinely.
Embodiment 5
The preparation of capsule or granule: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, add vehicle in itself and excipient weight than the ratio for 1:9, make capsule or granule.
Embodiment 6
The preparation of injection liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, inject with water routinely, essence filter, injection liquid is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, be dissolved in sterile water for injection, stirring makes molten, filter with aseptic suction funnel, aseptic essence filter again, be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains powder injection.
The effect of above-described embodiment is essentiality content of the present invention is described, but does not limit protection scope of the present invention with this.Those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the protection domain of technical solution of the present invention.
Claims (7)
1. there is the compound (I) of following structural formula:
2. the preparation method of compound according to claim 1 (I), it is characterized in that comprising following operation steps: the dry leave of piscidia is pulverized by (a), extract with 80 ~ 90% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; B in () step (a), the removal of impurities of thing macroporous resin got by propyl carbinol, first use 5% ethanol elution, 6 column volumes, then use 75% ethanol elution, 8 column volumes, collect 75% elutriant, concentrating under reduced pressure obtains 75% ethanol elution enriched material; C in () step (b), 75% ethanol elution enriched material purification on normal-phase silica gel is separated, obtain 4 components successively with the methylene chloride-methanol gradient elution that volume ratio is 65:1,30:1,15:1 and 8:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 15:1,8:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 8 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
3. the preparation method of compound according to claim 2 (I), is characterized in that: in step (a), extracts, united extraction liquid with 85% alcohol heat reflux.
4. the preparation method of compound according to claim 2 (I), is characterized in that: described macroporous resin is AB-8 type macroporous adsorbent resin.
5. a pharmaceutical composition, is characterized in that: the compound according to claim 1 (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
6. the application of compound according to claim 1 (I) in the medicine of preparation treatment adenoid cystocarcinoma of salivary gland.
7. the application of pharmaceutical composition according to claim 5 in the medicine of preparation treatment adenoid cystocarcinoma of salivary gland.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511016287.8A CN105481799A (en) | 2015-12-29 | 2015-12-29 | Highly-oxidized sesquiterpene compound as well as preparation method and medical application thereof |
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| CN201511016287.8A CN105481799A (en) | 2015-12-29 | 2015-12-29 | Highly-oxidized sesquiterpene compound as well as preparation method and medical application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294817A (en) * | 2015-11-25 | 2016-02-03 | 吕涛 | Novel triterpenoid, preparation method and medical application thereof |
| CN114796319A (en) * | 2022-03-22 | 2022-07-29 | 山东第一医科大学(山东省医学科学院) | Preparation method and application of guava leaf extract |
-
2015
- 2015-12-29 CN CN201511016287.8A patent/CN105481799A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294817A (en) * | 2015-11-25 | 2016-02-03 | 吕涛 | Novel triterpenoid, preparation method and medical application thereof |
| CN114796319A (en) * | 2022-03-22 | 2022-07-29 | 山东第一医科大学(山东省医学科学院) | Preparation method and application of guava leaf extract |
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Address after: 810016 Qinghai University, 251 Ning Ning Road, Chengbei District, Qinghai, Xining Applicant after: Wu Jinfeng Address before: 213003 Jiangsu province Changzhou Yanling West Road Zhonglou District Mingjun homes 2 building room 2407 Applicant before: Wu Jinfeng |
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Application publication date: 20160413 |