CN105622706A - Triterpene compound for liver protection and preparation method thereof - Google Patents
Triterpene compound for liver protection and preparation method thereof Download PDFInfo
- Publication number
- CN105622706A CN105622706A CN201511000781.5A CN201511000781A CN105622706A CN 105622706 A CN105622706 A CN 105622706A CN 201511000781 A CN201511000781 A CN 201511000781A CN 105622706 A CN105622706 A CN 105622706A
- Authority
- CN
- China
- Prior art keywords
- compound
- group
- extract
- preparation
- alcohol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/004—Expansion of ring B by one atom, e.g. B homo steroids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a triterpene compound for liver protection and a preparation method thereof. The triterpene compound is reported for the first time, is novel in structure, and can be obtained by being extracted from dried branches of shiny-leaved yellowhorn through separation and purification. It is proved through an in vitro test that the tolerance of liver cells to ischemia reperfusion injuries from which a patient suffers later on is enhanced through the compound, it is shown that the activity of the liver cells is enhanced, and the death rate of the cells is reduced. By means of pretreatment of the compound (I), the liver cell ischemia reperfusion injuries of people can be relieved, the function of protecting the liver cells is achieved, and the triterpene compound can be used for being further developed into a drug for liver protection.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, it is specifically related to from the dry branch of Wood of Shinyleaf Yellowhorn and is separated a kind of triterpene compound and its preparation method with liver protection obtained.
Background technology
Wood of Shinyleaf Yellowhorn XanthocerassorbifoliaBunge is Sapindaceae (Sapindaceae) Wood of Shinyleaf Yellowhorn platymiscium, singly belongs to single, another name Wen Dengge, Seng Dengmao road, precipice pawpaw, mountain pawpaw etc. It is distributed in the hillside, hills etc. on the ground such as China northeast, North China and Shaanxi, Gansu, Ningxia, Anhui, Henan, and also often there is cultivation various places, and Wood of Shinyleaf Yellowhorn begins to be loaded in " Herbal for Relief of Famines ", runs after fame with Wen Guanhua, also has record in Compendium of Material Medica.
Wood of Shinyleaf Yellowhorn is the distinctive rare traditional oil tree of China, has the title of northern oil tea, and because its kind of benevolence is rich in fatty oil, its massfraction, up to 52%, has very high economic worth. Except as except oil plant seeds, this plant also have good medicinal, eat, view and admire etc. and to be worth. The stem of Wood of Shinyleaf Yellowhorn, branch (literary composition crowntree) taste hardship sweet, micro-, cool in nature, there is swelling and pain relieving, wind-damp dispelling, effect of holding back dry yellow water, Mongolian medicine is usually used in the diseases such as treatment hot " Xieri Wusu Symptom ", scrofula, rheumatic arthritis. Among the people with its kind of benevolence treatment infantile enuresis, Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences is developed to the preparation for the treatment of bed-wetting, evident in efficacy.
From the positions such as Wood of Shinyleaf Yellowhorn husk, carpopodium, it is separated the chemical composition obtained mainly with triterpenes (especially in the majority based on beautiful stamen alcohol type triterpene compound), flavonoid, also has phenylpropyl alcohol element, steroid class, phenolic acid, alkaloid, single terpene and fatty acid compound in addition. Triterpene compound reports maximum constituents in Wood of Shinyleaf Yellowhorn, its structure parent nucleus is mainly the beautiful stamen alcohol type (A, B) of oleanane skeleton structure, also has lupinane type (C), root of gansui alkane type (D), cycloartane type, lanolin alkane type etc. in addition.
Wood of Shinyleaf Yellowhorn has many-sided pharmacologically active, as inflammation anti-oxidant, anti-, antitumor, antibacterial, suppress hiv protease, improve learning and memory function etc. Wherein Wood of Shinyleaf Yellowhorn kind benevolence Linoleic acid, linolenic acid amount are enriched, and often eat and can prevent cardiovascular and cerebrovascular diseases; Bark of ash glycosides in calyx sheet can be used for convulsion antipyretic, sleeping, anti-; Myricitroside in leaf can be used for sterilization, decreasing cholesterol etc. It is evident in efficacy that the water logging cream of Wood of Shinyleaf Yellowhorn kind benevolence, carpopodium and leaf is used for infantile enuresis, and can the memory dysfunction of chemical resistance drug-induced short of money.
Summary of the invention
It is an object of the invention to provide and a kind of from the dry branch of Wood of Shinyleaf Yellowhorn, it is separated a kind of triterpene compound and its preparation method with liver protection obtained.
The above-mentioned purpose of the present invention is achieved by technical scheme below:
The compound (I) with following structural formula,
The preparation method of described compound (I), comprise following operation steps: the dry branch of Wood of Shinyleaf Yellowhorn is pulverized by (a), extract with 70��80% alcohol heat reflux, united extraction liquid, concentrate to without alcohol taste, successively with the n-butanol extraction of sherwood oil, ethyl acetate and water saturation, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; B acetic acid ethyl ester extract macroporous resin removal of impurities in () step (a), first with 10% ethanol elution, 8 column volumes, then with 80% ethanol elution, 10 column volumes, collects 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract; C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, the methylene chloride-methanol gradient elution being 75:1,45:1,25:1,15:1 and 1:1 by volume ratio successively obtains 5 components; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, the methylene chloride-methanol gradient elution being 20:1,15:1 and 8:1 by volume ratio successively obtains 3 components; E reverse phase silica gel separation that in () step (d), component 2 is bonded by octadecylsilane, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collecting 10��12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
Further, described macroporous resin is D101 macroporous adsorbent resin.
Further, described alcohol heat reflux extracts the alcohol concn adopted is 75%.
A kind of pharmaceutical composition, wherein containing treating the described compound (I) of significant quantity and pharmaceutically acceptable carrier.
When the compounds of this invention is used as medicine, it is possible to directly use, 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, humans and animals is nontoxic 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 auxiliary dose of liquid diluent, filler and pharmaceutical preparation. The 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 can be made into, drip ball, micro-ball, suspensoid, emulsion, powder or granule, oral liquid etc.; During for injecting, can be made into the water-based of sterilizing or oily solution, aseptic powder injection, liposome or emulsion etc.
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.
Embodiment
The essentiality content of the present invention is described further below in conjunction with embodiment, but does not limit protection domain of the present invention with this. Although the present invention being explained in detail with reference to better embodiment, it will be understood by those within the art that, it is possible to the technical scheme of the present invention is modified or equivalent replacement, and do not depart from essence and the scope of technical solution of the present invention.
Embodiment 1: compound (I) separation preparation and structural identification
Reagent source: ethanol, sherwood oil, ethyl acetate, propyl carbinol, methylene dichloride are analytical pure, purchased from Shanghai Ling Feng chemical reagent company limited, methyl alcohol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Preparation method: the dry branch (10kg) of Wood of Shinyleaf Yellowhorn is pulverized by (a), (25L �� 3 time) are extracted with 75% alcohol heat reflux, united extraction liquid, concentrate to without alcohol taste (3L), extract with the propyl carbinol (3L �� 3 time) of sherwood oil (3L �� 3 time), ethyl acetate (3L �� 3 time) and water saturation successively, obtain petroleum ether extract, acetic acid ethyl ester extract (431g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract D101 macroporous resin removal of impurities in (b) step (a), first with 10% ethanol elution, 8 column volumes, again with 80% ethanol elution, 10 column volumes, collecting 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract (161g); C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, the methylene chloride-methanol gradient elution being 75:1 (8 column volumes), 45:1 (8 column volumes), 25:1 (8 column volumes), 15:1 (10 column volumes) and 1:1 (5 column volumes) by volume ratio successively obtains 5 components; D in () step (c), component 4 (52g) is separated further by purification on normal-phase silica gel, the methylene chloride-methanol gradient elution being 20:1 (8 column volumes), 15:1 (10 column volumes) and 8:1 (8 column volumes) by volume ratio successively obtains 3 components; E reverse phase silica gel separation that in () step (d), component 2 (26g) is bonded by octadecylsilane, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collecting 10-12 column volume elutriant, elutriant concentrating under reduced pressure obtains pure compound (I) (37mg).
Structural identification: HR-ESIMS shows [M+Na]+For m/z505.3326, it is C that syncaryon magnetic feature can obtain molecular formula31H46O4, degree of unsaturation is 9. Hydrogen nuclear magnetic resonance modal data ��H(ppm, DMSO-d6, 600MHz): H-2 (6.81, d, J=9.8), H-3 (6.26, d, J=9.8), H-6 (2.71, dd, J=14.4, 7.7), H-6 (2.42, t, J=14.4), H-7 (1.69, m), H-7 (1.33, m), H-8 (1.15, dd, J=12.1, 2.7), H-11 (3.27, d, J=6.3), H-12 (1.81, dd, J=15.0, 6.4), H-12 (1.78, d, J=15.0), H-15 (1.21, m), H-15 (1.46, m), H-16 (1.85, m), H-16 (1.46, m), H-17 (1.42, m), H-18 (0.93, s), H-19 (2.75, d, J=14.3), H-19 (2.38, d, J=14.3), H-20 (2.14, m), H-21 (1.01, d, J=6.6), H-23 (2.47, m), H-23 (2.26, m), H-24 (2.41, m), H-26 (5.01, s), H-26 (4.79, s), H-27 (1.68, s), H-28 (0.85, s), H-29 (1.32, s), H-30 (1.21, s), H-31 (0.93, d, J=7.0), carbon-13 nmr spectra data ��C(ppm, DMSO-d6, 150MHz): 185.1 (C, 1-C), 124.0 (CH, 2-C), 157.3 (CH, 3-C), 39.8 (C, 4-C), 167.1 (C, 5-C), 29.2 (CH2, 6-C), 24.5 (CH2, 7-C), 48.1 (CH, 8-C), 62.7 (C, 9-C), 131.6 (C, 10-C), 60.9 (CH, 11-C), 33.4 (CH2, 12-C), 44.8 (C, 13-C), 46.3 (C, 14-C), 33.6 (CH2, 15-C), 26.7 (CH2, 16-C), 40.0 (CH, 17-C), 14.2 (CH3, 18-C), 30.1 (CH2, 19-C), 45.3 (CH, 20-C), 11.7 (CH3, 21-C), 210.6 (C, 22-C), 45.9 (CH2, 23-C), 37.1 (CH, 24-C), 148.1 (C, 25-C), 109.4 (CH2, 26-C), 19.3 (CH3, 27-C), 17.7 (CH3, 28-C), 24.4 (CH3, 29-C), 24.7 (CH3, 30-C), 16.3 (CH3, 31-C); Carbon atom marks see Fig. 1.1HNMR spectrum display five methyl list peaks (�� H0.93, Me-18; 1.68, Me-27; 0.85, Me-28; 1.32, Me-29; 1.21, Me-30), two methyl bimodal (�� H1.01, J=6.6Hz, Me-21; 0.93, J=7.0Hz, Me-31), one to cis dibasic double bond Hydrogen Proton (�� H6.81, d, J=9.8Hz, H-2; 6.26, d, J=9.8Hz, H-3), terminal double link (�� H5.01, s, a H-26; 4.79, s, H-26), and one containing oxygen methyne (�� H3.27, d, J=6.3Hz, H-11).13CNMR spectrum display 31 carbon signals, are respectively seven methyl, eight methylene radical (is alkene carbon), seven methyne (two alkene carbon, one containing oxygen methyne) and nine quaternary carbons (containing oxygen quaternary carbon for a ketone carbonyl, three alkene carbon). During HMBC composes, CH3-29(CH3And C-3, C-4 and C-5 ,-30) and the dependency of H-2 and C-1, C-4 and C-10 show that ring A is 4,4-dimethyl-2,5-dienone part. During HMBC composes, H2-19 and C-1, C-10, C-5, C-9 and C-8, H2-6 and the dependency of C-4, C-5, C-7, C-8 and C-10, and CH2(6)-CH2(7)-CH (8) spin system, shows that B ring is the suberane replaced. CH in being composed by HMBC3-31 and the dependency of C-23, C-24 and C-25, it is seen that C-24 position is connected with a methyl. In addition, CH3-27 and the dependency of C-24, C-25 and C-26 show to exist between C-25 and C-26 double bond. The chemical shift of C-9 (�� C62.7) and C-11 (�� C60.9) shows that both are respectively connected with an oh group. Comprehensive hydrogen is composed, and carbon is composed, HMBC spectrum and NOESY spectrum, and document is about correlation type nuclear magnetic data, can substantially determine this compound as shown in Figure 1, and steric configuration is determined by ECD test further, theoretical value and experimental value basically identical (Fig. 2).
Embodiment 2: compound (I) pharmacological action is tested
One, material and instrument
HL7702 liver cell line is provided by Shanghai Life Sciences Research Institute, Chinese Academy Of Sciences. Compound (I) is made by oneself, and method is shown in that embodiment 1, HPLC normalization method purity is greater than 98%. DMEM in high glucose substratum, DMEM in high glucose/F12=1:1 substratum, foetal calf serum is all purchased from HycLone. EDTA is purchased from Shanghai reagent one factory. Trypan blue is purchased from Beijing Chemical Plant. It is purchased from Gibco without sugar DMEM substratum. Pancreatin, MTT, DMSO are all purchased from Amresco. LDH method for releasing detection kit is purchased from GENMDE. ALT biochemistry detection test kit, AST biochemistry detection test kit, LDH biochemistry detection test kit are all purchased from the graceful reagent company limited in U.S. Bake. Western and IP cell pyrolysis liquid, PMSF, BCA determination of protein concentration test kit, MDA detection kit are all purchased from green skies biotechnology research institute.
CO2Incubator (SheL-Lab2300), electric heating constant temperature incubator (Shanghai leap medical apparatus and instruments factory), anaerobic culture box (Changsha Chang Jin Science and Technology Ltd.), GasALertExtreme oxygen detection instrument (Canada BWTechnoLogies), microplate reader (U.S. BioTekEpoch), full automatic biochemical apparatus (BeckmanLX20), whizzer (Germany Eppendorfcentrif �� ge5415D), orifice plate whizzer (Germany Eppendorfcentrif �� ge5430R), electronic micro balance (METTLERTOLEDOAL104), PH instrument (ThermoORION3STAR).
Two, test method
1, cell grouping
Cell is divided into 6 groups, often organizes 6 multiple holes:
(1) normally cultivation group (N group): perfect medium, cultivates under normal condition;
(2) ischemia-reperfusion group (IR group): after the normal CMC model 1h of perfect medium, simulation anoxic 8h, Reperfu-sion 4h;
(3) compound (I) group:
RE1 group: compound (I) 0.5ng/mL pre-treatment 1h;
RE2 group: compound (I) 5ng/mL pre-treatment 1h;
RE3 group: compound (I) 50ng/mL pre-treatment 1h; Simulation anoxic 8h, Reperfu-sion 4h;
(4) physiological saline group (NS group): with the physiological saline pre-treatment 1h with compound (I) equal volume, simulation anoxic 8h, Reperfu-sion 4h.
2, the pre-treatment of compound (I)
(1) preparation of compound (I): 1mg compound (I) is dissolved in 500mL physiological saline. Move liquid rifle and draw 2.5mL in first centrifuge tube, be mixed with, to 10mL, compound (I) solution that concentration is 500ng/mL with normal saline dilution. 1mL is drawn to, in the 2nd centrifuge tube, being mixed with, to 10mL, compound (I) solution that concentration is 50ng/mL with normal saline dilution from first centrifuge tube. 1mL is drawn to, in the 3rd centrifuge tube, being mixed with, to 10mL, compound (I) solution that concentration is 5ng/mL with normal saline dilution from the 2nd centrifuge tube. Perform mark.
(2) compound (I) pre-treatment: normal cultivation group and ischemia-reperfusion group are changed with the perfect medium that every hole 100 �� L is fresh. The every hole of RE1 group adds compound (I) the solution 10 �� L and fresh perfect medium 90 �� L of 5ng/mL, the every hole of RE2 group adds compound (I) the solution 10 �� L and fresh perfect medium 90 �� L of 50ng/mL, and the every hole of RE3 group adds compound (I) the solution 10 �� L and fresh perfect medium 90 �� L of 500ng/mL. The every hole of physiological saline group adds physiological saline 10 �� L and fresh perfect medium 90 �� L. Shake 96 orifice plates gently, liquid is fully drifted along or through even, put into CO2Incubator is hatched 1h.
3, the foundation of the in-vitro simulated ischemical reperfusion injury model of liver cell:
(1) in-vitro simulated ischemic period: after pretreatment time terminates, takes out first piece of 96 orifice plate from cell culture incubator, and the normal every hole of cultivation group is changed with 100 �� L perfect mediums, puts into CO2Incubator resume cultivates 8h. From CO2Incubator takes out the 2nd piece of 96 orifice plates, ischemia-reperfusion group, compound (I) pretreated group and the every hole of physiological saline group replace perfect medium with 100 �� L without sugar DMEM substratum, put into anaerobic culture box, cultivate the sterile vials maintenance saturated humidity put in box and fill 50mL aqua sterilisa. Anaerobic culture box is put into 37 DEG C of constant temperature incubators, and inlet mouth connects 94%N2-5%CO2-1%O2Mixed gas, air outlet connects oxygen detection instrument. Lead to into mixed gas with the gas flow of 2L/min, < when 1%, regulate mixed gas flow 300mL/min to maintain air outlet oxygen concentration < 1% when oxygen detection instrument demonstrates gas port oxygen concentration. With this simulated ischemia process 8h.
(2) in-vitro simulated refilling process: from CO2Taking out first piece of 96 orifice plate in incubator, the fresh perfect medium of 100 �� L is changed in the normal every hole of cultivation group, puts into CO2Incubator resume cultivates 4h. Taking out the 2nd piece of 96 orifice plates from anaerobic culture box, ischemia-reperfusion group, compound (I) pretreated group and the every hole of the physiological saline group perfect medium that 100 �� L are fresh is replaced without sugar DMEM substratum, puts into CO2(37 DEG C, 5%CO under normal condition in incubator2, 95% air, saturated humidity) cultivate 4h, simulation refilling process.
3, Indexs measure
3.1MTT method detection Activity of hepatocytes
(1) MTT solution preparation: take 250mgMTT with electronics microbalance, put into small beaker, adds 50mLPBS and stirs 30min, make it abundant dissolving, be the MTT solution that concentration is 5mg/mL. In Bechtop, millipore filter with 0.22 ��m is degerming, is packed as and often props up 1mL, and 4 DEG C keep in Dark Place for subsequent use.
(2) cell prepares: carry out cell grouping as stated above, and separately establishes zeroing hole. And carry out compound (I) pre-treatment and in-vitro simulated ischemia-reperfusion process as stated above.
(3) in look: the every hole of time point, end adds the MTT solution 20 �� L of 5mg/mL eventually, puts into 37 DEG C, 5%CO2, 95% air, saturated humidity CO2Incubator resume cultivates 4h. Terminating cultivating, careful suction abandons supernatant liquor in hole, and every hole adds 100 �� LDMSO solution, and micro oscillator vibrates 10min, and crystallisate is fully dissolved.
(4) colorimetric: select 570nm wavelength, measure each hole absorbance (A) in microplate reader, record result. Experiment repeats 2 times.
3.2 LDH release cell proliferation and toxicity detection
Operate by GENMED LDH release cell proliferation and toxicity detection by quantitative test kit specification sheets, repeat 2 times.
(1) prepare cell to be measured as stated above, and separately establish acellular nutrient solution (background empty map hole) and the cell (sample maximum enzyme activity control wells) of untreated follow-up cracking, perform mark.
(2) put first 1 hour to the detection time specified, from CO2Incubator takes out 96 porocyte culture plates to be measured, adds 10 �� LGENMED lysates to, in the cell hole of untreated follow-up cracking, adding 10 �� LGENMED replenishers in all the other all detect aperture simultaneously. Put back to CO2Incubator resume is cultivated and is namely specified detection time in 1 hour.
(3) from CO2Incubator takes out 96 porocyte culture plates to be measured, puts into the centrifugal 10min of orifice plate whizzer, and speed is 250g.
(4) carefully pipette 50 �� L of supernatant liquid respectively in the respective aperture of 96 new orifice plates, perform mark simultaneously.
(5) mixed even GENMED reaction solution, every hole adds 50 �� LGENMED reaction solutions respectively, more every hole adds 10 �� LGENMED colour developing liquid respectively, shakes 96 orifice plates and mixes even.
(6) at room temperature hatch 30min, avoid illumination.
(7) every hole adds 10 �� LGENMED stop buffers respectively.
(8) at once put microplate reader into and measure absorbance (A), select wavelength 570nm.
(9) according to following formulae discovery cell mortality:
The actual suction photoreading of processing sample=treatment samples sample wells is inhaled photoreading-sample controls hole and is inhaled photoreading-background empty map hole suction photoreading
The actual suction photoreading of sample cell maximum enzyme activity=sample maximum enzyme activity control wells is inhaled photoreading-sample controls hole and is inhaled photoreading-background empty map hole suction photoreading
Cell mortality=(the actual suction photoreading of processing sample actual suction photoreading �� sample cell maximum enzyme activity) �� 100%
The mensuration of 3.3 hepatocyte function indexs:
Carry out cell grouping, pre-treatment and simulated ischemia reperfusion injury as stated above. Being collected in Ep pipe by each porocyte culture supernatant 100 �� L respectively to eventually end time point, often pipe adds 100 �� L perfect mediums respectively and is diluted to 200 �� L, centrifugal 5min under room temperature, and speed is 1200r/min. Get supernatant liquor under full automatic biochemical apparatus, detect ASL, ALT and LDH level. Experiment repeats 2 times.
4, statistical procedures
SPSS13.0 software package is used to carry out statistical procedures. Measurement data adopts and all counts �� standard deviation (�� �� s) expression, compares employing one-way analysis of variance between group, and P < 0.05 difference has statistical significance.
Three, result and conclusion
1, compound (I) pre-treatment is on the impact of ischemical reperfusion injury Activity of hepatocytes
IR group Activity of hepatocytes decline (P<0.05) more obvious than N group, shows that in-vitro simulated ischemia-reperfusion process successfully causes the ischemical reperfusion injury of liver cell. Give 5ng/mL compound (I) pre-treatment 1 hour, relatively ischemia-reperfusion group and physiological saline group strengthen (P<0.05) to the vigor of ischemical reperfusion injury liver cell, but fail to return to normal group Activity of hepatocytes level (P<0.05), physiological saline group then with ischemia-reperfusion group difference not statistically significant (P>0.05). And the compound of 0.5ng/mL and 50ng/mL concentration (I) pre-treatment 1 hour, all fail to strengthen the vigor (P>0.05) of ischemical reperfusion injury liver cell, difference also not statistically significant (P>0.05) between its Activity of hepatocytes and physiological saline group. The results are shown in Table 1 (��Representing and compare with N group, P < 0.05, difference has statistical significance;��Representing and compare with IR group and NS group, P < 0.05, difference has statistical significance, lower same).
2, compound (I) pre-treatment is on the impact of the cell proliferation of liver cell ischemical reperfusion injury and toxicity
Compared with normal group, in-vitro simulated ischemical reperfusion injury makes cell mortality>90%. Give 5ng/mL compound (I) pre-treatment 1 hour, liver cell mortality ratio decline about 35%, difference has statistical significance (P<0.05), but its cell mortality is still high than normal group by about 50%, and difference has statistical significance (P<0.05); And though physiological saline group liver cell mortality ratio slightly declines (being about 8%) than ischemia-reperfusion group, but difference not statistically significant. Compound (I) pre-treatment of 0.5ng/mL and 50ng/mL concentration 1 hour, liver cell mortality ratio reduces by 6% and 13% respectively than ischemia-reperfusion group, but difference not statistically significant (P>0.05); And close with the liver cell mortality ratio of physiological saline group (P>0.05). The results are shown in Table 2.
3, compound (I) pre-treatment is on the impact of ischemical reperfusion injury liver cell liver function index
Comparing with normal group, the ALT level in ischemical reperfusion injury group cell culture supernatant has no obvious rising (P>0.05); And AST, LDH level and AST/ALT ratio significantly raise, difference has statistical significance (P<0.05). Give 5ng/mL compound (I) pre-treatment 1 hour, AST, LDH level in liver cell culture supernatant liquor and AST/ALT ratio relatively ischemia-reperfusion group and physiological saline group reduce, but still relatively normal group height (P<0.05); And ALT level compares with ischemia-reperfusion group and physiological saline group, difference not statistically significant (P>0.05). ALT, AST, LDH level in physiological saline group cell culture supernatant and difference not statistically significant (P>0.05) between AST/ALT ratio and ischemia-reperfusion group. 0.5ng/mL and 50ng/mL compound (I) pre-treatment 1 hour, all fails to make AST, LDH level in liver cell culture supernatant liquor to reduce (P>0.05). The results are shown in Table 3.
This research shows, and compound (I) (RE2 group) makes liver cell be strengthened by the tolerance of the ischemical reperfusion injury experienced afterwards, shows as Activity of hepatocytes and strengthens, and cell mortality reduces. Above result is pointed out, and compound (I) pre-treatment can alleviate human liver cell ischemical reperfusion injury, plays hepatocytoprotection.
Table 1MTT method detection compound (I) pre-treatment is on the impact of ischemical reperfusion injury Activity of hepatocytes
| Group | Sample number n | A value |
| N group | 6 | 2.222��0.135 |
| IR group | 6 | 1.585��0.113�� |
| RE1 group | 6 | 1.509��0.073�� |
| RE2 group | 6 | 2.076��0.144���� |
| RE3 group | 6 | 1.569��0.118�� |
| NS group | 6 | 1.484��0.085�� |
The breeding of table 2LDH method for releasing liver cell and toxicity detection by quantitative cell mortality
| Group | Sample number n | Cell mortality |
| N group | 5 | 0.000��0.145 |
| IR group | 5 | 0.915��0.076�� |
| RE1 group | 5 | 0.851��0.157�� |
| RE2 group | 5 | 0.531��0.058���� |
| RE3 group | 5 | 0.791��0.065�� |
| NS group | 5 | 0.834��0.107�� |
The change (n=5) of ALT, AST, LDH level in table 3 liver cell culture supernatant liquor
| Group | ALT | AST | AST/ALT | LDH |
| N group | 14.0��1.58 | 21.1��0.84 | 1.53��0.13 | 100.8��9.52 |
| IR group | 12.8��0.84 | 33.0��2.24�� | 2.58��0.07�� | 247.2��13.76�� |
| RE1 group | 14.0��1.58 | 33.0��1.58�� | 2.37��0.16�� | 260.0��11.77�� |
| RE2 group | 14.0��1.58 | 27.2��0.84���� | 1.96��0.24���� | 204.0��8.60���� |
| RE3 group | 13.8��0.84 | 32.8��2.17�� | 2.38��0.17�� | 239.6��6.54�� |
| NS group | 12.8��0.84 | 31.2��0.84�� | 2.45��0.22�� | 249.2��8.87�� |
Embodiment 3
The preparation of tablet: by embodiment 1 method first obtained compound (I), and utilize the salt that organic acid makes such as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, the ratio being 1:7 in itself and vehicle weight ratio adds vehicle, pelletizing press sheet.
Embodiment 4
Prepared by oral liquid: by embodiment 1 method first obtained compound (I), and utilizing the salt that organic acid makes such 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 utilize the salt that organic acid makes such as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, the ratio being 1:7 in itself and vehicle weight ratio adds vehicle, makes capsule or granule.
Embodiment 6
The preparation of injection liquid: by embodiment 1 method first obtained compound (I), and utilize the salt that organic acid makes such as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, injecting 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 utilize the salt that organic acid makes such as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, it is dissolved in sterile water for injection, stirring makes molten, filter with aseptic filter funnel of taking out, aseptic essence filter, is sub-packed in ampoule again, aseptic after frozen drying molten seals to obtain powder injection.
The effect of above-described embodiment is to illustrate the essentiality content of the present invention, but does not limit protection scope of the present invention with this. It will be understood by those within the art that, it is possible to the technical scheme of the present invention is modified or equivalent replacement, and do not depart from essence and the protection domain of technical solution of the present invention.
Claims (5)
1. the triterpenoid for liver protecting, it is characterised in that: it has 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 branch of Wood of Shinyleaf Yellowhorn is pulverized by (a), extract with 70��80% alcohol heat reflux, united extraction liquid, concentrate to without alcohol taste, successively with the n-butanol extraction of sherwood oil, ethyl acetate and water saturation, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; B acetic acid ethyl ester extract macroporous resin removal of impurities in () step (a), first with 10% ethanol elution, 8 column volumes, then with 80% ethanol elution, 10 column volumes, collects 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract; C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, the methylene chloride-methanol gradient elution being 75:1,45:1,25:1,15:1 and 1:1 by volume ratio successively obtains 5 components; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, the methylene chloride-methanol gradient elution being 20:1,15:1 and 8:1 by volume ratio successively obtains 3 components; E reverse phase silica gel separation that in () step (d), component 2 is bonded by octadecylsilane, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collecting 10��12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
3. the preparation method of compound according to claim 2 (I), it is characterised in that: described macroporous resin is D101 macroporous adsorbent resin.
4. the preparation method of compound according to claim 2 (I), it is characterised in that: extracting, with alcohol heat reflux, the alcohol concn adopted in step a is 75%.
5. a pharmaceutical composition, it is characterised in that: wherein containing treating the compound according to claim 1 (I) of significant quantity and pharmaceutically acceptable carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511000781.5A CN105622706A (en) | 2015-12-27 | 2015-12-27 | Triterpene compound for liver protection and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511000781.5A CN105622706A (en) | 2015-12-27 | 2015-12-27 | Triterpene compound for liver protection and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105622706A true CN105622706A (en) | 2016-06-01 |
Family
ID=56038089
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201511000781.5A Pending CN105622706A (en) | 2015-12-27 | 2015-12-27 | Triterpene compound for liver protection and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105622706A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105152873A (en) * | 2015-10-16 | 2015-12-16 | 温州泓呈祥科技有限公司 | New lignan compounds, and preparation method and medical application thereof |
| CN105152895A (en) * | 2015-10-09 | 2015-12-16 | 富阳鸿祥技术服务有限公司 | New meroterpenoid as well as preparation method and medical application thereof |
-
2015
- 2015-12-27 CN CN201511000781.5A patent/CN105622706A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105152895A (en) * | 2015-10-09 | 2015-12-16 | 富阳鸿祥技术服务有限公司 | New meroterpenoid as well as preparation method and medical application thereof |
| CN105152873A (en) * | 2015-10-16 | 2015-12-16 | 温州泓呈祥科技有限公司 | New lignan compounds, and preparation method and medical application thereof |
Non-Patent Citations (1)
| Title |
|---|
| BO WANG ET AL.: "Cytotoxic Clerodane Diterpenoids from the Leaves and Twigs of Casearia balansae", 《J. NAT. PROD.》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105111269A (en) | Novel limonin compound as well as preparation method and medical application thereof | |
| CN105330717A (en) | Novel triterpenoid and preparation method and medical application thereof | |
| CN105153187A (en) | New diterpenoid as well as preparation method and medical application thereof | |
| CN105330716A (en) | New withanolides compound, and preparation method and medical application thereof | |
| CN105153084A (en) | Novel diterpene compound as well as preparation method and medicinal application thereof | |
| CN105943532A (en) | Application of diterpenoid compound to preparation of medicament for treating liver cancer | |
| CN105218617A (en) | A kind of new triterpenoid and preparation method thereof and medicinal use | |
| CN106008543A (en) | Novel diterpenoid compound and preparation method thereof | |
| CN105237380A (en) | Triterpene compound used for treating ovarian cancer and preparation method of triterpene compound | |
| CN105153263A (en) | New limonin compound as well as preparation method and medical application thereof | |
| CN105418729A (en) | Limonin compound for treating chronic granulocytic leukemia and preparation method thereof | |
| CN105367536A (en) | Novel iridoid and preparation method and medical application thereof | |
| CN105566427A (en) | Lanostane triterpene compound, and preparation method and medicinal use thereof | |
| CN105503896A (en) | New phloroglucin compounds, and preparation method and medical application thereof | |
| CN105524075A (en) | A novel diterpene compound, a preparing method thereof and medical uses of the diterpene compound | |
| CN105503995A (en) | Novel triterpenoid compound as well as preparation method and medical application thereof | |
| CN105254497A (en) | Novel diterpenoid compound and preparation method and medical application thereof | |
| CN106397369A (en) | Novel labdane-type diterpenoid compound, preparation method and application thereof, pharmaceutical composition and application of pharmaceutical composition | |
| CN105481876A (en) | Diterpene compound for treating ovarian cancer | |
| CN105198844A (en) | Novel limonin compound as well as preparation method and medical application thereof | |
| CN105461732A (en) | Novel diterpenoid compound and preparation method and medical application thereof | |
| CN105198897A (en) | New kaurane diterpenoid compound, preparation method and medical application of compound in treating liver cancer | |
| CN105622629A (en) | Kaurane type diterpenoid compound for treating prostatic cancer | |
| CN105968161A (en) | Withanolides compound with liver protection function, and preparation method of withanolides compound | |
| CN105481875A (en) | New limonin compound as well as preparation method and medical application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160601 |
|
| RJ01 | Rejection of invention patent application after publication |