CN107778318A - A kind of new diterpene compound and preparation method thereof and medical usage - Google Patents
A kind of new diterpene compound and preparation method thereof and medical usage Download PDFInfo
- Publication number
- CN107778318A CN107778318A CN201610738932.5A CN201610738932A CN107778318A CN 107778318 A CN107778318 A CN 107778318A CN 201610738932 A CN201610738932 A CN 201610738932A CN 107778318 A CN107778318 A CN 107778318A
- Authority
- CN
- China
- Prior art keywords
- compound
- preparation
- extract
- elution
- group
- 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
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of new diterpene compound and preparation method thereof and medical usage, there is provided the compound structure, containing its pharmaceutical composition and its preparation method and application.The compound is a kind of novel diterpene-kind compound of structure to report first, extracting and developing can purify to obtain from the ripening fruits of psoralea corylifolia drying.In vitro test proves that compound pretreatment can mitigate human liver cell ischemical reperfusion injury, plays hepatocytoprotection, can be used for developing into liver-protective medicine.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, and in particular to isolated a kind of tool from psoralea corylifolia dry mature fruit
There is diterpene compound of liver protection and preparation method thereof.
Background technology
Psoralea corylifolia is legumes psoraleae Psoralea corylifoliaL. dry mature fruit, first recorded in《Thunder
Public big gun moxibustion opinion》, have tonify the kidney and support yang, receive gas, antidiarrheal the effect of.The successive dynasties traditional Chinese medical science be used for kidney deficiency, it is cold rush down, lose involuntary emission, frequent urination,
Waist and knee crymodynia；Control outside for skin disease, leucoderma etc..Psoralea corylifolia clinical practice is extensive, version in 2010《People's Republic of China's medicine
Allusion quotation》Including the Chinese patent drug containing psoralea corylifolia has more than 20 to plant.
The research of the chemicalization composition of psoralea corylifolia is seen 1910 earliest.From 1933 isolated first from psoralea corylifolia
Sterling psoralea corylifolia (Psoralen), it is separated so far to identify more than 40 and plant composition.In recent years, many new perfume (or spice) and are therefrom got
Legumin, flavones and other compositions.At present, domestic and foreign scholars are more to its chemical constitution study, and main component is Coumarins, yellow
Ketone and diterpene-kind compound.
(1) Coumarins：There are furocoumarin such as psoralen (Psoralen) and Isopsoralen
(Isopsoralen).Coumestrol class such as psoralidine (Psoralidin) and double light isopsoralidines (Eorylidin).
(2) flavone compound, such as chalcones：Coryfolin (Ocrylioflni) and Corylifolinin
(Eorylioflinin).Flavanone：Corylifolin (Bavaehin), psoralea corylifolia dihydro methyl ether
And different Corylifolin (Isobavaehin) (Bavaehinin).Osajin：Corylin (Eorylin) and
Psoralen lipidol flavonoids.
(3) monoterpene phenols：Bakuchiol (Bakuehiol).
(4) Diterpenes.
(5) other constituents have stigmasterol, cupreol glucoside, melissane, glucose, gossypose, to light base benzene
The uncertain psoraldehyde of formic acid, resin, structure, two kinds of glucosides etc. more than 30 plants material.
The content of the invention
It is an object of the invention to provide one kind isolated in a kind of dry mature fruit from psoralea corylifolia to have liver guarantor
Diterpene compound of shield effect and preparation method thereof.
The above-mentioned purpose of the present invention is achieved by following technical scheme：
Compound (I) with following structural formula,
The preparation method of described compound (I), includes following operating procedure：(a) the ripening fruits powder that psoralea corylifolia is dried
It is broken, extracted with 85% alcohol heat reflux, merge extract solution, no alcohol taste is concentrated into, successively with petroleum ether, ethyl acetate and water saturation
Extracting n-butyl alcohol, respectively obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract；(b) second in step (a)
Acetoacetic ester extract is cleaned with macroreticular resin, successively with 15% ethanol and 75% ethanol elution, is collected 75% ethanol eluate, is subtracted
Pressure is concentrated to give 75% ethanol elution thing medicinal extract；(c) 75% ethanol elution medicinal extract is separated with purification on normal-phase silica gel in step (b), is used successively
Volume ratio is 80:1、50:1、30:1、15:1 and 1:1 methylene chloride-methanol gradient elution obtains 5 components；(d) step (c)
Middle component 4 is further separated with purification on normal-phase silica gel, is successively 20 with volume ratio:1、12:1 and 5:1 methylene chloride-methanol gradient is washed
It is de- to obtain 3 components；(e) reverse phase silica gel that component 2 is bonded with octadecylsilane in step (d) separates, dense with volume basis
The methanol aqueous solution isocratic elution for 70% is spent, collects 8-10 column volume eluent, eluent is concentrated under reduced pressure to give pure change
Compound (I).
Further, the macroreticular resin is AB-8 type macroporous absorbent resins.
Pharmaceutical composition, wherein the described compound (I) and pharmaceutically acceptable carrier containing therapeutically effective amount.
Application of the described compound (I) in the medicine of liver protecting is prepared.
Application of the described pharmaceutical composition in the medicine of liver protecting is prepared.
It when the compounds of this invention is used as medicine, can directly use, or be used in the form of pharmaceutical composition.
The pharmaceutical composition contains the compounds of this invention (I) of therapeutically effective amount, and remaining is pharmaceutically acceptable, right
The nontoxic and inert pharmaceutical acceptable carrier of humans and animals and/or excipient.
Described pharmaceutical acceptable carrier or excipient is one or more selected from solid, semisolid and liquid diluent, filler
And pharmaceutical preparation assistant agent.The pharmaceutical composition of the present invention is used in the form of per weight dose.Medicine of the present invention can
The patient for needing to treat is applied to by oral or injection form.For it is oral when, tablet, sustained release tablets, control can be made into
Release piece, capsule, dripping pill, micropill, supensoid agent, emulsion, powder or granule, oral liquid etc.；During for injecting, sterilizing can be made into
Water-based or oily solution, aseptic powder injection, liposome or emulsion etc..
Figure of description
Fig. 1 is compound (I) structural formula；
Fig. 2 is the theoretical ECD values of compound (I) compared with testing ECD values；
Fig. 3 is the two-dimentional NOESY spectrums of compound (I).
Embodiment
The essentiality content of the present invention is further illustrated with reference to embodiment, but present invention protection model is not limited with this
Enclose.Although being explained in detail with reference to preferred embodiment to the present invention, it will be understood by those within the art that, can be right
Technical scheme is modified or equivalent substitution, without departing from the spirit and scope of technical solution of the present invention.
Embodiment 1：Compound (I) separation prepares and structural identification
Reagent source：Ethanol, petroleum ether, ethyl acetate, n-butanol, dichloromethane are pure to analyze, and peaking is insulted purchased from Shanghai
Reagent Co., Ltd is learned, methanol, analysis is pure, purchased from Jiangsu Han Bang chemical reagent Co., Ltd.
Preparation method：(a) ripening fruits (8kg) that psoralea corylifolia is dried is crushed, and (25L × 3 are extracted with 85% alcohol heat reflux
It is secondary), merge extract solution, be concentrated into no alcohol taste (6L), satisfied successively with petroleum ether (6L × 3 time), ethyl acetate (6L × 3 time) and water
N-butanol (6L × 3 time) extraction of sum, respectively obtains petroleum ether extract, acetic acid ethyl ester extract (355g) and extracting n-butyl alcohol
Thing；(b) acetic acid ethyl ester extract is cleaned with AB-8 types macroreticular resin in step (a), successively with 15% ethanol (8L) and 75%
(12L) ethanol elution, 75% ethanol eluate is collected, be concentrated under reduced pressure to obtain 75% ethanol elution thing medicinal extract (136g)；(c) step
(b) 75% ethanol elution medicinal extract is separated with purification on normal-phase silica gel in, is successively 80 with volume ratio:1 (8 column volumes), 50:1 (8 posts
Volume), 30:1 (6 column volumes), 15:1 (8 column volumes) and 1:The methylene chloride-methanol gradient elution of 1 (5 column volumes)
Obtain 5 components；(d) component 4 (31g) is further separated with purification on normal-phase silica gel in step (c), is successively 20 with volume ratio:1 (8
Column volume), 12:1 (10 column volumes) and 5:The methylene chloride-methanol gradient elution of 1 (6 column volumes) obtains 3 components；
(e) reverse phase silica gel that component 2 (11g) is bonded with octadecylsilane in step (d) separates, and is 70% with concentration expressed in percentage by volume
Methanol aqueous solution isocratic elution, collects 8-10 column volume eluent, and eluent is concentrated under reduced pressure to give pure compound (I)
(23mg)。
Structural identification：White powder, it is soluble in chloroform, ethyl acetate, acetone and methanol；HR-ESIMS is shown [M+Na]+
For m/z 377.1042, it is C that can obtain molecular formula with reference to nuclear-magnetism feature20H18O6, degree of unsaturation 12.Hydrogen nuclear magnetic resonance modal data δH
(ppm,CDCl3,400MHz)：H-1 (2.92, d, J=11.8), H-1 (1.86, dd, J=1.5,11.8), H-3 (3.75, d, J
=11.0), H-5 (2.24, dd, J=1.3,5.8), H-6 (5.17), H-7 (4.05, dd, J=9.9,17.4), H-7 (2.98,
Dd, J=4.8,17.4), H-11 (6.52, s), H-15 (6.84, d, J=2.2), H-16 (7.52, d, J=2.2), H-18
(1.60, s), H-20 (4.25, dd, J=1.5,9.0), H-20 (3.76, dd, J=1.5,9.0), OMe (3.91, s)；Nuclear-magnetism is total to
Shake carbon modal data δC(ppm,CDCl3,100MHz)：40.3(CH2, 1-C), 103.9 (C, 2-C), 81.3 (CH, 3-C), 44.2 (C,
4-C), 54.5 (CH, 5-C), 74.7 (CH, 6-C), 24.7 (CH2, 7-C), 109.2 (C, 8-C), 133.8 (CH, 9-C), 48.0
(C, 10-C), 98.7 (CH, 11-C), 152.5 (C, 12-C), 117.0 (C, 13-C), 154.1 (C, 14-C), 143.9 (CH, 16-
C), 22.7 (CH3, 18-C), 180.1 (C, 19-C), 72.2 (CH2, 20-C), 55.7 (CH3, OMe)；Carbon atom is marked referring to figure
1.Infrared IR 1750cm-1Gamma lactone structure be present in prompting.1H-NMR shows alkene hydrogen H-15 (6.84, d, the J in the presence of 2 doublets
=2.2) and H-16 (7.52, d, J=2.2)；1 unimodal alkene hydrogen H-11 (6.52, s)；1 unimodal methoxyl group OMe
(3.91, s)；1 unimodal methyl H-18 (1.60, s).Pass through HSQC two-dimensional spectrums, it may be determined that related to these Hydrogen Protons
Carbon.CH3- 18 is related to connecting oxygen methine on C-3.H-1a (δ H 2.92) and H-20a (δ H 4.25) and 103.9 (C, 2-C) are remote
Cheng Xiangguan, prompt oxygen bridge be present between C-2 and C-20.The relative configuration of the compound can compose (Fig. 3) analysis by two-dimentional NOESY
It is determined that.Pass through CH3- 18 (δ H 1.60) are related to H-5 (δ H 2.24), H-6 (δ H 5.17), H-6 and H-5, H-7a (δ H
4.05), the information such as CH3-18 correlations, and data in literature, the compound can be determined substantially as shown in figure 1, simultaneously further passing through
ECD experiments determine that theoretical value and experiment value are basically identical (Fig. 2).Therefore, finally determine that the compound structure is as shown in Figure 1.
Embodiment 2：Compound (I) pharmacological action is tested
First, material and instrument
HL7702 liver cell lines are provided by Shanghai Life Sciences Research Institute, Chinese Academy Of Sciences.Compound (I) is self-control (purity
More than 98%).DMEM in high glucose culture medium, DMEM in high glucose/F12=1:1 culture medium, hyclone are purchased from HycLone.EDTA is purchased
In the factory of Shanghai reagent one.Trypan blue is purchased from Beijing Chemical Plant.Sugar-free DMEM culture mediums are purchased from Gibco.Pancreatin, MTT, DMSO are purchased
In Amresco.LDH method for releasing detection kits are purchased from GENMDE.ALT biochemistry detections kit, AST biochemistry detections kit,
LDH biochemistry detections kit is purchased from Beckman reagent Co., Ltd of the U.S..Western and IP cell pyrolysis liquids, PMSF, BCA
Determination of protein concentration kit, MDA detection kits are purchased from green skies biological study institute.
CO2Incubator, SheL-Lab 2300；Electric heating constant temperature incubator, Shanghai leap medical apparatus and instruments factory；Anaerobic culture box,
Changsha Chang Jin Science and Technology Ltd.s；ELIASA, U.S. BioTek Epoch；Full automatic biochemical apparatus, Beckman LX20；Centrifugation
Machine, German Eppendorf centrif μ ge 5415D.
2nd, test method
1st, cell is grouped
Cell is divided into 6 groups, every group of 6 multiple holes：(1) normal culture group (N groups)：Complete medium, train under normal condition
Support；(2) ischemia-reperfusion group (IR groups)：After cultivating 1h under complete medium normal condition, anoxic 8h, Reperfu- sion 4h are simulated；(3)
Compound (I) group：RE1 groups：Compound (I) 0.5ng/mL pre-processes 1h；RE2 groups：Compound (I) 5ng/mL pre-processes 1h；RE3
Group：Compound (I) 50ng/mL pre-processes 1h；Simulate anoxic 8h, Reperfu- sion 4h；(4) physiological saline group (NS groups)：With with chemical combination
The physiological saline pretreatment 1h of thing (I) equal volume, simulates anoxic 8h, Reperfu- sion 4h.
2nd, the pretreatment of compound (I)
(1) preparation of compound (I)：1mg compounds (I) are dissolved in 500mL physiological saline.Liquid-transfering gun is drawn
It is molten that 2.5mL is configured to the compound (I) that concentration is 500ng/mL in first centrifuge tube, with normal saline dilution to 10mL
Liquid.Drawn from first centrifuge tube in 1mL to second centrifuge tube, being configured to concentration with normal saline dilution to 10mL is
50ng/mL compound (I) solution.Drawn from second centrifuge tube in 1mL to the 3rd centrifuge tube, use normal saline dilution
Compound (I) solution that concentration is 5ng/mL is configured to 10mL.Perform mark.
(2) compound (I) pre-processes：Normal culture group and ischemia-reperfusion group are with complete culture fresh every μ L of hole 100
Base is changed.RE1 groups add the 5ng/mL μ L of compound (I) solution 10 and the every hole of fresh complete medium 90 μ L, RE2 group per hole
The μ L of compound (I) solution 10 and fresh complete medium 90 μ L, RE3 group for adding 50ng/mL add 500ng/mL's per hole
The μ L of compound (I) solution 10 and fresh μ L of complete medium 90.Physiological saline group adds μ L of physiological saline 10 and fresh per hole
The μ L of complete medium 90.96 orifice plates are gently shaken, decoction is fully drifted along or through even, is put into CO21h is incubated in incubator.
3rd, the foundation of the in-vitro simulated Ischemia-Reperfusion Injury Model of liver cell：
(1) in-vitro simulated ischemic period：After pretreatment time terminates, first piece of 96 orifice plate is taken out from cell culture incubator,
Normal culture group is changed per hole with 100 μ L complete mediums, is put into CO2Continue to cultivate 8h in incubator.From CO2Taken in incubator
Go out second piece of 96 orifice plate, ischemia-reperfusion group, compound (I) pretreated group and physiological saline group are per 100 μ L sugar-frees DMEM of hole
Culture medium replaces complete medium, is put into anaerobic culture box, cultivates and the sterile vials guarantor for filling 50mL aqua sterilisas is put into box
Hold saturated humidity.Anaerobic culture box is put into 37 DEG C of constant-temperature incubation casees, air inlet connection 94%N2- 5%CO2- 1%O2Mixing
Gas, gas outlet connection oxygen detection instrument.Mixed gas is passed through with 2L/min gas flow, when oxygen detection instrument shows outlet
Mouth oxygen concentration<When 1%, regulation mixed gas flow 300mL/min maintains gas outlet oxygen concentration<1%.With this simulated ischemia
Process 8h.
(2) in-vitro simulated refilling process：From CO2First piece of 96 orifice plate is taken out in incubator, the every hole of normal culture group is more
The fresh complete mediums of 100 μ L are changed, are put into CO2Continue to cultivate 4h in incubator.Second piece 96 is taken out from anaerobic culture box
Orifice plate, ischemia-reperfusion group, compound (I) pretreated group and physiological saline group are put per hole with the fresh complete mediums of 100 μ L
Sugar-free DMEM culture mediums are changed, are put into CO2(37 DEG C, 5%CO under normal condition in incubator2, 95% air, saturated humidity) culture
4h, simulate refilling process.
3rd, Indexs measure
3.1MTT methods detect Activity of hepatocytes
(1) MTT solution is prepared：250mgMTT is weighed with electronic micro balance, is put into small beaker, 50mLPBS is added and stirs
30min is mixed, is allowed to fully dissolve, as concentration is 5mg/mL MTT solution.With 0.22 μm of millipore filter in superclean bench
It is degerming, be packed as every 1mL, 4 DEG C be kept in dark place it is standby.
(2) cell prepares：Cell packet is carried out as stated above, and separately sets zeroing hole.And as stated above
Carry out compound (I) pretreatment and in-vitro simulated Ischemia-Reperfusion Injury.(3) colour generation：Last time point adds per hole eventually
Enter the 5mg/mL μ L of MTT solution 20, be put into 37 DEG C, 5%CO2, 95% air, the CO of saturated humidity2Continue to cultivate in incubator
4h.Culture is terminated, careful inhale abandons supernatant in hole, and 100 μ LDMSO solution are added per hole, 10min is vibrated on micro oscillator, makes
Crystal fully dissolves.(4) colorimetric：570nm wavelength is selected, each hole trap (A) is determined on ELIASA, records result.It is real
Test and be repeated 2 times.
3.2 LDH release cell proliferations and toxicity detection
Operated by GENMED LDH releases cell proliferation and toxicity immue quantitative detection reagent box specification, experiment weight
It is multiple 2 times.
(1) prepare cell to be measured as stated above, and separately set acellular nutrient solution (background empty map hole) and untreated
Follow-up cracking cell (sample maximum enzyme activity control wells), perform mark.
(2) by 1 hour before defined detection time point, from CO296 porocyte culture plates to be measured are taken out in incubator, are added
Enter 10 μ LGENMED lysates in the untreated cell hole subsequently cracked, at the same add 10 μ LGENMED replenishers to remaining
In all detection holes.Put back to CO2Continue to cultivate 1 hour i.e. regulation detection time in incubator.
(3) from CO296 porocyte culture plates to be measured are taken out in incubator, are put into orifice plate centrifuge 10min, speed
For 250g.
(4) 50 μ L of supernatant liquid are carefully pipetted respectively in the respective aperture of 96 new orifice plates, while perform mark.
(5) GENMED reaction solutions are mixed, 50 μ LGENMED reaction solutions are separately added into per hole, then add 10 μ per hole respectively
LGENMED nitrite ions, shake 96 orifice plates and mix.
(6) 30min is incubated at room temperature, avoids illumination.
(7) 10 μ LGENMED terminate liquids are separately added into per hole.
(8) put ELIASA measure absorbance (A) at once, select wavelength 570nm.
(9) cell mortality is calculated according to below equation：
It is empty to handle the actual extinction reading of sample=processing sample well extinction reading-sample controls hole extinction reading-background
The actual extinction reading of control wells extinction reading sample cell maximum enzyme activity=sample maximum enzyme activity control wells extinction is read
Number-sample controls hole extinction reading-background empty map hole extinction reading cell mortality=(the processing actual extinction reading of sample
The actual extinction reading of ÷ sample cell maximum enzyme activities) × 100%
The measure of 3.3 hepatocyte function indexs：
Cell packet, pretreatment and simulated ischemia reperfusion injury are carried out as stated above.Respectively will to last time point eventually
Each μ L of hole cell culture supernatant 100 are collected in Ep pipes, and often pipe is separately added into 100 μ L complete mediums and is diluted to 200 μ L, room
The lower centrifugation 5min, speed 1200r/min of temperature.Supernatant is taken to detect ASL, ALT and LDH level under full automatic biochemical apparatus.It is real
Test and be repeated 2 times.
3rd, result and conclusion
1st, compound (I) pre-processes the influence to ischemical reperfusion injury Activity of hepatocytes
IR group Activity of hepatocytes is decreased obviously (P than N group<0.05) in-vitro simulated Ischemia-Reperfusion Injury, is shown successfully
Cause liver cell ischemical reperfusion injury.Give 5ng/ml compounds (I) to pre-process 1 hour, ischemical reperfusion injury liver is thin
The vigor of born of the same parents is compared with ischemia-reperfusion group and physiological saline group enhancing (P<0.05), but fail to return to normal group Activity of hepatocytes water
Flat (P<0.05), physiological saline group then with ischemia-reperfusion group no significant difference (P>0.05).And 0.5ng/ml and
The compound (I) of 50ng/ml concentration pre-processes 1 hour, fails to strengthen the vigor (P of ischemical reperfusion injury liver cell>
0.05), the also not statistically significant (P of difference between its Activity of hepatocytes and physiological saline group>0.05).The results are shown in Table 1 (▲Represent
Compared with N groups, P<0.05, difference is statistically significant；★Represent compared with IR groups and NS groups, P<0.05, difference has statistics meaning
Justice).
2nd, compound (I) pretreatment is to the cell proliferation of liver cell ischemical reperfusion injury and the influence of toxicity
Compared with normal group, in-vitro simulated ischemical reperfusion injury makes cell mortality>90%.Give 5ng/ml compounds
(I) pre-process 1 hour, heptocellular death rate declines about 35%, the statistically significant (P of difference<, but its cell mortality 0.05)
It is still higher than normal group by about 50%, the statistically significant (P of difference<0.05)；And physiological saline group heptocellular death rate is than ischemic again
Though perfusion group is declined slightly (about 8%), no significant difference.The compound (I) of 0.5ng/ml and 50ng/ml concentration
Pretreatment 1 hour, heptocellular death rate reduces by 6% and 13%, but no significant difference (P respectively than ischemia-reperfusion group>
0.05)；And (P close with the heptocellular death rate of physiological saline group>0.05).The results are shown in Table 2 (▲Represent compared with N groups, P<
0.05, difference is statistically significant；★Represent compared with IR groups and NS groups, P<0.05, difference is statistically significant).
3rd, compound (I) pre-processes the influence to ischemical reperfusion injury liver cell liver function index
Compared with normal group, the ALT levels in ischemical reperfusion injury group cell culture supernatant have no significantly raised (P>
0.05)；And AST, LDH are horizontal and AST/ALT ratios significantly raise, the statistically significant (P of difference<0.05).Give 5ng/
Ml compounds (I) pre-process 1 hour, and AST, LDH level and AST/ALT ratios in hepatocyte cultures supernatant are compared with ischemic again
Perfusion group and physiological saline group reduce, but still compared with normal group height (P<0.05)；And ALT levels and ischemia-reperfusion group and physiology salt
Water group compares, no significant difference (P>0.05).ALT, AST, LDH in physiological saline group cell culture supernatant is horizontal
And no significant difference (P between AST/ALT ratios and ischemia-reperfusion group>0.05).0.5ng/ml and 50ng/mlization
Compound (I) pre-processes 1 hour, fails to make AST, LDH in hepatocyte cultures supernatant are horizontal to reduce (P>0.05).As a result see
Table 3 (▲Represent compared with N groups, P<0.05, difference is statistically significant；★Represent compared with IR groups and NS groups, P<0.05, difference
It is statistically significant).
This studies have shown that compound (I) (RE2 groups) makes liver cell to the tolerance of the ischemical reperfusion injury undergone afterwards
Enhancing, shows as Activity of hepatocytes enhancing, and cell mortality reduces.Result above is prompted, and compound (I) pretreatment can mitigate
Human liver cell ischemical reperfusion injury, plays hepatocytoprotection.
The mtt assay detection compound (I) of table 1 pre-processes the influence to ischemical reperfusion injury Activity of hepatocytes
| Group | Sample number n | A values |
| N groups | 6 | 2.222±0.135 |
| IR groups | 6 | 1.585±0.113▲ |
| RE1 groups | 6 | 1.509±0.073▲ |
| RE2 groups | 6 | 2.076±0.144▲★ |
| RE3 groups | 6 | 1.569±0.118▲ |
| NS groups | 6 | 1.484±0.085▲ |
The LDH method for releasing liver cell of table 2 breeds quantitatively detects cell mortality with toxicity
| Group | Sample number n | Cell mortality |
| N groups | 5 | 0.000±0.145 |
| IR groups | 5 | 0.915±0.076▲ |
| RE1 groups | 5 | 0.851±0.157▲ |
| RE2 groups | 5 | 0.531±0.058▲★ |
| RE3 groups | 5 | 0.791±0.065▲ |
| NS groups | 5 | 0.834±0.107▲ |
ALT, AST, LDH horizontal change (n=5) in the hepatocyte cultures supernatant of table 3
| Group | ALT | AST | AST/ALT | LDH |
| N groups | 14.0±1.58 | 21.1±0.84 | 1.53±0.13 | 100.8±9.52 |
| IR groups | 12.8±0.84 | 33.0±2.24▲ | 2.58±0.07▲ | 247.2±13.76▲ |
| RE1 groups | 14.0±1.58 | 33.0±1.58▲ | 2.37±0.16▲ | 260.0±11.77▲ |
| RE2 groups | 14.0±1.58 | 27.2±0.84▲★ | 1.96±0.24▲★ | 204.0±8.60▲★ |
| RE3 groups | 13.8±0.84 | 32.8±2.17▲ | 2.38±0.17▲ | 239.6±6.54▲ |
| NS groups | 12.8±0.84 | 31.2±0.84▲ | 2.45±0.22▲ | 249.2±8.87▲ |
Embodiment 3
The preparation of tablet：Compound (I) is first made by the method for embodiment 1, and utilizes organic acid such as tartaric acid or lemon
Acid or formic acid or ethanedioic acid etc., inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, it is 1 by itself and excipient weight ratio:9
Ratio adds excipient, pelletizing press sheet.
Embodiment 4
The preparation of oral liquid：Compound (I) is first made by the method for embodiment 1, and utilizes organic acid such as tartaric acid or lemon
Lemon acid or formic acid or ethanedioic acid etc., inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, routinely oral liquid preparation method mouth is made
Take liquid.
Embodiment 5
The preparation of capsule or granule：Compound (I) is first made by the method for embodiment 1, and utilizes organic acid such as wine
Stone acid or citric acid or formic acid or ethanedioic acid etc., the inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, by itself and excipient weight
Amount is than being 1:9 ratio adds excipient, and capsule or granule is made.
Embodiment 6
The preparation of parenteral solution：Compound (I) is first made by the method for embodiment 1, and utilizes organic acid such as tartaric acid or lemon
Lemon acid or formic acid or ethanedioic acid etc., inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, routinely plus water for injection, refined filtration,
Parenteral solution is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection：By the method for embodiment 1 first be made compound (I), and using organic acid for example tartaric acid or
Citric acid or formic acid or ethanedioic acid etc., the inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, are dissolved in sterile water for injection
In, stirring makes molten, is filtered with sterile suction funnel, then sterile refined filtration is sub-packed in ampoule, sterile sealing after frozen drying
Obtain powder-injection.
Claims (6)
1. the compound (I) with following structural formula,
2. the preparation method of the compound (I) described in claim 1, it is characterised in that include following operating procedure：(a) psoralea corylifolia
Dry ripening fruits crushes, and is extracted with 85% alcohol heat reflux, merges extract solution, is concentrated into no alcohol taste, successively with petroleum ether,
Ethyl acetate and water saturated extracting n-butyl alcohol, respectively obtain petroleum ether extract, acetic acid ethyl ester extract and extracting n-butyl alcohol
Thing；(b) acetic acid ethyl ester extract is cleaned with macroreticular resin in step (a), successively with 15% ethanol and 75% ethanol elution, is collected
75% ethanol eluate, be concentrated under reduced pressure to obtain 75% ethanol elution thing medicinal extract；(c) 75% ethanol elution medicinal extract is used just in step (b)
Phase silica gel separates, and is successively 80 with volume ratio:1、50:1、30:1、15:1 and 1:1 methylene chloride-methanol gradient elution obtains 5
Individual component；(d) component 4 is further separated with purification on normal-phase silica gel in step (c), is successively 20 with volume ratio:1、12:1 and 5:The two of 1
Chloromethanes-methanol elution gradient obtains 3 components；(e) reverse phase silica gel that component 2 is bonded with octadecylsilane in step (d)
Separation, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, 8-10 column volume eluent is collected, eluent subtracts
Pressure is concentrated to give pure compound (I).
3. preparation method according to claim 2, it is characterised in that：The macroreticular resin is AB-8 type macroporous absorption trees
Fat.
4. pharmaceutical composition, wherein compound (I) described in the claim 1 containing therapeutically effective amount and pharmaceutically acceptable
Carrier.
5. application of the compound (I) in the medicine of liver protecting is prepared described in claim 1.
6. application of the pharmaceutical composition described in claim 4 in the medicine of liver protecting is prepared.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610738932.5A CN107778318A (en) | 2016-08-28 | 2016-08-28 | A kind of new diterpene compound and preparation method thereof and medical usage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610738932.5A CN107778318A (en) | 2016-08-28 | 2016-08-28 | A kind of new diterpene compound and preparation method thereof and medical usage |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107778318A true CN107778318A (en) | 2018-03-09 |
Family
ID=61439549
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610738932.5A Pending CN107778318A (en) | 2016-08-28 | 2016-08-28 | A kind of new diterpene compound and preparation method thereof and medical usage |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107778318A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110437247A (en) * | 2018-05-03 | 2019-11-12 | 中国医学科学院药物研究所 | A kind of meroterpenoids compound and application thereof with liver protection function |
| CN111635380A (en) * | 2020-06-17 | 2020-09-08 | 中国科学院昆明植物研究所 | Sesquiterpene in mugwort, pharmaceutical composition thereof, preparation method and application thereof |
-
2016
- 2016-08-28 CN CN201610738932.5A patent/CN107778318A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| MING ZHAO,ET AL.,: "17-Norpimaranes and (9βH)-17-Norpimaranes from the Tuber of Icacina trichantha", 《J. NAT. PROD.》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110437247A (en) * | 2018-05-03 | 2019-11-12 | 中国医学科学院药物研究所 | A kind of meroterpenoids compound and application thereof with liver protection function |
| CN110437247B (en) * | 2018-05-03 | 2022-06-21 | 中国医学科学院药物研究所 | Mixed source terpenoid with liver protection function and application thereof |
| CN111635380A (en) * | 2020-06-17 | 2020-09-08 | 中国科学院昆明植物研究所 | Sesquiterpene in mugwort, pharmaceutical composition thereof, preparation method and application thereof |
| CN111635380B (en) * | 2020-06-17 | 2022-05-13 | 中国科学院昆明植物研究所 | Sesquiterpene in mugwort, pharmaceutical composition thereof, preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1972702B (en) | Composition comprising xanthoceras sorbifolia extracts, compounds isolated from same, methods for preparing same, function and uses thereof | |
| CN102138998B (en) | Honey tree fruit general flavone extract and application thereof in preparation of medicaments and food for resisting fatigue, hypoxia or plateau hypoxia | |
| CN104958393A (en) | Extract of potentilla anserina rhizome and medical application thereof | |
| CN105111269A (en) | Novel limonin compound as well as preparation method and medical application thereof | |
| CN106588947B (en) | Isoamylene radical chromocor class compound in jackfruit and its anti-osteoporosis new application | |
| CN108191931A (en) | Three kinds of noval chemical compounds and its health care and medical usage in root of Paeonia sinjiangensis | |
| CN103254259A (en) | Iridoid glycoside compound as well as preparation method and application thereof | |
| CN105943532A (en) | Application of diterpenoid compound to preparation of medicament for treating liver cancer | |
| CN107778318A (en) | A kind of new diterpene compound and preparation method thereof and medical usage | |
| CN101590128B (en) | Lipid regulating and antioxidant effect-based method for evaluating quality of Jiangzhining | |
| CN105111080A (en) | Novel diterpene compound and medical application thereof | |
| CN102824400A (en) | Composition containing eucommia ulmoides and teasel roots and application of composition for treating osteoporosis | |
| CN105294726A (en) | Novel flavanone compound, preparation method of novel flavanone compound and medical application of novel flavanone compound | |
| CN105524075A (en) | A novel diterpene compound, a preparing method thereof and medical uses of the diterpene compound | |
| CN105348223A (en) | Novel sesquiterpenoid and preparation method and medical application thereof | |
| CN105503896A (en) | New phloroglucin compounds, and preparation method and medical application thereof | |
| CN105503995A (en) | Novel triterpenoid compound as well as preparation method and medical application thereof | |
| CN105481876A (en) | Diterpene compound for treating ovarian cancer | |
| CN106749479A (en) | A kind of new triterpenoid and preparation method thereof and medical usage | |
| CN105481878A (en) | Novel limonin compound and preparation method and medical application thereof | |
| Souza et al. | Biotechnological potential of medicinal plant Erythrina velutina Willd: A systematic review | |
| CN105055490A (en) | Eucommia ulmoides oliv. extract and medical application of eucommia ulmoides oliv. extract for protecting liver | |
| CN105968161A (en) | Withanolides compound with liver protection function, and preparation method of withanolides compound | |
| CN105622443A (en) | Novel enamine type compound as well as preparation method and medical purpose thereof | |
| CN106008548A (en) | New clerodane diterpenoid compound and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180309 |