CN105669692B - A kind of extracting method and purposes of phthalide-type dimer compound - Google Patents
A kind of extracting method and purposes of phthalide-type dimer compound Download PDFInfo
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Abstract
The invention provides a kind of extracting method and purposes of phthalide-type dimer compound, the method for three kinds of phthalide-type dimer compounds of extraction separation specifically from Radix Angelicae Sinensis, wherein compound I is the noval chemical compound for having no report.Meanwhile this experiment has carried out antitumor activity detection to such compound using mtt assay, experimental result shows that such compound has and suppresses human ileocecal carcinoma cell HCT 8, Non-small cell lung carcinoma cell A549, human liver cancer cell HepG2The activity of growth, show that such compound can be applied in antineoplastic is prepared.
Description
Technical field：
The present invention relates to the method for separating and preparing of natural drug and application, and in particular to three kinds that separation is extracted from Radix Angelicae Sinensis
Phthalide-type dimer compound and application thereof.
Background technology：
Radix Angelicae Sinensis is one of the most frequently used Chinese medicine, from umbelliferae angelica Angelica sinensis (Oliv.)
Diels dry root.Radix Angelicae Sinensis contains polytype chemical composition such as volatile oil, organic acid, polysaccharide, flavones, its pharmacological action
Extensively, it is related to the multiple systems of body.Substantial amounts of pharmacological research, which is reported, to be shown, the hematopoiesis of Radix Angelicae Sinensis and its main chemical compositions to body
Multiple systems such as system, the circulatory system, nervous system, immune system have pharmacological action；Its primary bioactivity include hematopoiesis,
Platelet aggregation-against, anti-arrhythmia, radioresistance, antitumor, analgesia, regulation smooth muscle and the protective effect to internal organs.
Phthalide analog compound is primarily present in Umbelliferae as a major class native compound, the plant of the phthalide-type carried out at present
Thing research majority concentrates on celery, Jehol Ligusticum Rhizome (Ligusticum jeholense, Xinjiang Jehol Ligusticum Rhizome), Radix Angelicae Sinensis (ligusticum acutilobum, lovage), Ligusticum wallichii (tea rhizome of chuanxiong, day
This Ligusticum wallichii).Such compound mainly includes phthalide monomer and phthalide dimer class compound, the structure of phthalide monomer class compound
Parent nucleus is benzofuran lactone structure, has 12 carbon, is one of representative composition of Ligusticum Secondary metabolites；Benzene
Phthalein dimer is that phthalide monomer obtains through cycloaddition, and the difference of monomer structure, polymerization methodses and polymerization site generates its knot
The diversity of structure.Report at present on phthalide-type monomer reactivity is more, the activity report of rare phthalide dimer class compound
Road.
Natural plants with antitumor activity have become the important sources of people's developing new drug, therefore people are not
It is disconnected to find activity preferably, the novel new type natural compound of structure.The present invention three kinds of phthalide-type dimerization isolated from Radix Angelicae Sinensis
Body compound, pharmacological testing research show that such compound has significant antitumor activity, by literature search, so far
Have no the active reporter of three kinds of compounds in this regard.
The content of the invention：
It is an object of the invention to provide a kind of new phthalide-type dimer compound.
It is another object of the present invention to provide a kind of preparation method of phthalide-type dimer compound, and the side
The purposes for the compound that method is prepared.
A kind of phthalide-type dimer compound provided by the invention, it is characterised in that structural formula I, II or III：
The preparation method of a kind of phthalide-type dimer compound provided by the invention, it is characterised in that comprise the following steps：
(1) preparation of crude extract
Using supercritical CO2Extract angelica sinensis, extraction conditions：Temperature：50℃；Pressure：20MP；CO2Flow：15kg/h；
Raw meal particle size：20 mesh；Extraction time：3h；
(2) compound isolates and purifies
Silica gel mixed sample by above-mentioned crude extract with 200~300 mesh, silica gel column chromatography is carried out, with petroleum ether-ethyl acetate system
System gradient elution, flow point is merged according to thin-layer chromatography situation；
Petroleum ether：Ethyl acetate=1：The component at 9 elution positions is through silica gel column chromatography, gel filtration chromatography separation, Ran Houjing
Efficient liquid phase separation is prepared, mobile phase is methanol：Water=7：3, the material that appearance time is 45.59min is collected, obtains new chemical combination
Thing I (C24H28O4), Tokinolide C are named as, the material that appearance time is 61.15min is collected, obtains compound II
(Tokinolide A)；
Petroleum ether：Ethyl acetate=2：The component at 8 elution positions separates through silica gel column chromatography, then through Sephadex LH-
20 chromatographic purifyings, use chloroform：Methanol=2：The solvent elution of 1 (volume ratio), obtains compound III (Riligustilide).
Experimental result is shown：Phthalide-type dimer compound I, II or III made from methods described is in 1~80 μM of concentration model
In enclosing, there is certain inhibitory action to different tumour cells, and obvious concentration dependent be present.Therefore, it can make
Application in standby antineoplastic.
Beneficial effect of the present invention is：
Phthalide-type dimer compound provided by the invention can be isolated from the medicinal materials such as Radix Angelicae Sinensis, also can be through this area skill
It is chemically synthesized to obtain known to art personnel, there is good DEVELOPMENT PROSPECT；Phthalide-type dimer compound of the present invention is to more
The inhibitory action of kind tumor cell line also indicates that it can be further used as new antineoplastic and be researched and developed.
Brief description of the drawings：
Fig. 1 compounds I's1HNMR composes (hydrogen spectrum).
Fig. 2 compounds I's13CNMR composes (carbon spectrum).
Fig. 3 compounds I HMQC spectrums.
Fig. 4 compounds I HMBC spectrums.
Fig. 5 compounds I NOESY spectrums.
Fig. 6 compounds I HR-ESI-MS spectrums.
Fig. 7 .ChemDraw3D simulated compounds I Preferred conformations and NOESY compose main coherent signal
Fig. 8 separation process figures
Embodiment：
The extraction separation of 1. 3 kinds of phthalide-type dimer compounds of embodiment, is concretely comprised the following steps：
(1) supercritical CO27.3kg angelica sinensis is extracted, extraction conditions is -- temperature：50℃；Pressure：20MP；CO2Flow：
15kg/h；Raw meal particle size：20 mesh；Extraction time：3h.Obtain brownish red medicinal extract 81.2g.Medicinal extract 1kg normal-phase silica gel column chromatographies
Separation, successively using petroleum ether-ethyl acetate (1：99、2：98、5：95、7：93、1：9、3：17、2：8、3：7、5：5、0：1) it is terraced
Degree elution, is merged into 28 flow points, developer is 10% methanolic solution according to thin-layer chromatography situation.
(2) component Fr.14 (petroleum ethers are taken：Ethyl acetate=1:9 elution positions) silica gel column chromatography is carried out, collect oil
Ether：Ethyl acetate=1:19 elution position, then separates through gel filtration chromatography, uses chloroform：Methanol=2：1 (volume ratio) it is molten
Agent elutes, and concentration merges the component that blackening is purer under uviol lamp.The inverted C18 of gained medicinal extract prepares post separation, and mobile phase is first
Alcohol：Water=7：3, the material that appearance time is 45.59min is collected, obtains noval chemical compound I (C24H28O4) 30mg, it is named as
Tokinolide C, the material that appearance time is 61.15min is collected, obtains compound II (Tokinolide A) 35mg.
(3) component Fr.17 (petroleum ethers are taken：Ethyl acetate=2：8 elution positions) silica gel column chromatography is carried out, collect oil
Ether：Ethyl acetate=3:17 elution position, then through Sephadex LH-20 chromatographies, use chloroform：Methanol=2：1 (body
Product ratio) solvent elution, concentration merges the purer component of blackening under uviol lamp, obtains compound III (Riligustilide)
56mg。
Compound I-III physicochemical property and the Qualitative Identification of chemical constitution of the embodiment 2. to the gained of embodiment 1
Compound I：Yellow oil, chloroform is soluble in, is insoluble in methanol.High-resolution HR-ESI-MS provides quasi-molecular ions m/
z:783.3898[2M+Na]+,403.1881[M+Na]+,381.2057[M+H]+The molecular weight for prompting the compound is 380, point
Minor is C24H28O4.UV spectrum have strong absworption peak at 280.7nm.1HNMR spectrums provide two methyl signals δ 0.96 (3H, t, J
=7.4Hz, H-11), there are four alkene hydrogen signals in 0.92 (3H, t, J=7.4Hz, H-11'), low field area, respectively δ 6.11 (1H,
Ddd, J=9.9Hz, H-6'), 5.77 (1H, dd, J=1.7,9.9Hz, H-7'), 5.24 (1H, t, J=7.9Hz, H-8), 4.88
(1H, t, J=7.9Hz, H-8'), δ 6.11 and 5.77 liang of hydrogen coupling constants of δ are J=9.9Hz, prompt the compound suitable containing one
Formula double bond.The signal that δ 5.24 and δ 4.88 alkene hydrogen provides is triplet, and coupling constant J=7.9Hz, prompts two this alkene hydrogen
With key-CH2- be connected.13There are two carbonyl signals δ 168.73 and δ 176.92 in CNMR low fields area, eight olefinic carbon signals, illustrates this
4 double bonds be present in compound.HMBC spectrum displays, the protons of δ 6.11 (6') and δ 48.2 (7a') carbon phase close, and δ 5.77 is (7')
The carbon phase of proton and δ 48.4 (3a') is closed, and prompts double bond cis each other respectively in 6' and 7'.HMBC spectrums provide signal δ 1.77
(4') the proton on position closes with the carbon phase on δ 21.8 (5), δ 37.1 (6) position, and NOESY spectrums display that matter of the δ 1.77 (4') on position
Son is related to proton on δ 1.98 (5), δ 2.99 (6) position respectively, and it is 3a' that above evidence, which prompts the polymerization site of the dimer,
.6,7a'.7.(4') NOESY, which is composed, returns δ 1.77, δ 1.86 (5') the proton on position and δ 4.88 (8') on position double bond proton
Space correlation signal, it was demonstrated that 4', 5' carbon and the double bond of 8' positions are located at the same side.Matter on δ 1.98 (5), δ 2.99 (6) position
The space correlation of son and the (7') protons on position of δ 5.77, two hexatomic rings are prompted to be located at the same side.
All carbon atoms and the signals assignment of hydrogen atom and the structure of the compound are determined by HMQC and HMBC spectrums
(being shown in Table 1 and structural formula 1), and the relative configuration that compound is determined is composed by NOESY, related collection of illustrative plates is shown in accompanying drawing 1~6.Through
SciFinder is retrieved, and it is the new phthalide-type dimer compound without document report to find this compound.Pass through
Chemdraw3D U ltra softwares energy minimization calculates, and it is as shown in Figure 7 to obtain the most stable of molecular configuration of compound.
The compound I nuclear magnetic datas of table 1. (1HNMR 600MHZ,13CNMR 150MHZ, solvent：Deuterochloroform)
Compound II:Yellow oil, chloroform is soluble in, is insoluble in methanol.Through Structural Identification, the compound is
Tokinolide A, nuclear magnetic data are shown in Table 2 (reports that its nuclear magnetic data is had no through SCIFinder retrievals).
The compound II nuclear magnetic datas of table 2. (1HNMR 600MHZ,13CNMR 150MHZ, solvent：Deuterochloroform)
Compound III：Yellow oil, chloroform is soluble in, is insoluble in methanol.1H-NMR(600MHZ,CDCl3)δ:6.16
(1H, d, J=9.6Hz, H-7'), 5.93 (1H, dt, J=9.6,1.8Hz, H-6'), 5.23 (1H, t, J=7.9Hz, H-8),
3.47 (1H, d, J=7.8Hz, H-7), 2.96 (1H, dt, J=7.8Hz, H-8'), 2.71-2.78 (1H, m, H-4'), 2.54-
2.62(3H,m,H-4,4',5'),2.42-2.50(1H,m,H-4),2.32(2H,m,H-9),2.10-2.24(2H,m,H-5,
6), 2.00-2.04 (1H, m, H-5), 1.41-1.53 (4H, m, H-10,9'), 1.13 (2H, m, H-10'), 0.95 (3H, t, J=
7.4Hz, H-11), 0.86 (3H, t, J=7.4Hz, H-11')；13C-NMR(150MHZ,CDCl3)δ:170.2(C-1'),168.4
(C-1),160.2(C-3'a),154.5(C-3a),149.1(C-3),128.6(C-6'),123.2(C-7'a),122.2(C-
7a),116.8(C-7'),111.9(C-8),91.8(C-3'),43.8(C-7),34.8(C-6),32.2(C-8'),27.8(C-
9),26.1(C-5),22.4(C-10'),22.2(C-10),20.9(C-4'),20.5(C-5'),19.9(C-9'),19.5(C-
4),14.0(C-11),13.7(C-11').Through Structural Identification, the compound is Riligustilide.
The anti-tumor activity test of the phthalide-type dimer compound of embodiment 3.
1st, test method
Take the logarithm A549 (human lung carcinoma cell), HCT-8 (human ileocecal carcinoma cell) and the HepG in growth period2(human liver cancer is thin
Born of the same parents), concentration of cell suspension is adjusted, is added in 96 porocyte culture plates per the μ L of hole 100, bed board makes the cell concentration to be measured be
3000/ hole, and in 5%CO2, cultivate 24h in 37 DEG C of incubator；Sample to be measured sets 1,5,10,20,40,80 μM respectively
5 concentration gradients, each concentration set 3 secondary orifices.Parallel progress 3 times (n=3) is tested, while zeroing hole and control wells are set,
Sample liquid or each 100 μ L of blank solution are added per hole.10 μ LMTT solution (5mg/mL, PBS solutions are added per hole after cultivating 48h respectively
Configuration), continue after cultivating 4h, with liquid-transfering gun Aspirate supernatant；Then do not have in hole and be separately added into 100 μ LDMSO, in micro vibration
Vibration to crystallization is completely dissolved on device, determines the light absorption value (OD values) in each hole under 570nm with ELIASA, is calculated by OD values each dense
Proliferation inhibition rate under degree, then by the corresponding proliferation inhibition rate of each concentration, using Graphpad Prism
Software softwares carry out curve fitting, so as to obtain the IC50 values that each compound suppresses growth of tumour cell.
2nd, experimental result
The result that phthalide-type dimer compound suppresses tumour growth effect is as shown in table 3.
The antitumor action of the phthalide-type dimer compound of table 3
Result of the test is shown：In 1~80 μM of concentration range, three kinds of phthalide-type dimer compounds are thin to different tumours
Born of the same parents have certain inhibitory action, and obvious concentration dependent be present.Wherein, compound R iligustilide suppresses tumour
The IC50 values of cell growth are smaller, show that its inhibitory action to growth of tumour cell is most strong, and compound Tokinolide A
Antitumor action is also shown to a certain extent with noval chemical compound.
Claims (3)
1. a kind of phthalide-type dimer compound, it is characterised in that structural formula is as follows：
2. a kind of extracting method of phthalide-type dimer compound, it is characterised in that comprise the following steps：
(1) preparation of crude extract
Using supercritical CO2Extract angelica sinensis, extraction conditions：Temperature：50℃；Pressure：20MP；CO2Flow：15kg/h；Material
Granularity：20 mesh；Extraction time：3h；
(2) compound isolates and purifies
Silica gel mixed sample by above-mentioned crude extract with 200~300 mesh, silica gel column chromatography is carried out, with petroleum ether-ethyl acetate system ladder
Degree elution, flow point is merged according to thin-layer chromatography situation；
Petroleum ether: ethyl acetate=1: the component at 9 elution positions is through silica gel column chromatography, gel filtration chromatography separation, then through preparing
Efficient liquid phase separates, and mobile phase is methanol：Water=7: 3, the material that appearance time is 45.59min is collected, obtains compound I, is received
Integrate material of the appearance time as 61.15min, obtain compound II；
Petroleum ether: ethyl acetate=2: the component at 8 elution positions separates through silica gel column chromatography, then through Sephadex LH-20 layers
Analysis purifying, eluted with the chloroform-methanol solvent that volume ratio is 2: 1, obtain compound III；
Described compound I, II, III structural formula is：
3. application of the phthalide-type dimer compound as claimed in claim 1 in antineoplastic is prepared.
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