CN105777772B - Compound with insect antifeedant activity and growth inhibitory activity and preparation method thereof - Google Patents
Compound with insect antifeedant activity and growth inhibitory activity and preparation method thereof Download PDFInfo
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- CN105777772B CN105777772B CN201610238969.1A CN201610238969A CN105777772B CN 105777772 B CN105777772 B CN 105777772B CN 201610238969 A CN201610238969 A CN 201610238969A CN 105777772 B CN105777772 B CN 105777772B
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- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
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Abstract
The invention belongs to pharmaceutical technology fields, disclose the activity and structure of a variety of auspicious promise alkane diterpene-kind compounds of separation and Extraction from plant Chinese cassia tree, provide the method for separating and preparing of Xin Ruinuo alkane diterpene compounds.Present invention is disclosed such compounds to have immunosuppressive activity, insect antifeedant activity and growth inhibitory activity, wherein the compound with immunosuppressive activity shows as significantly inhibiting effect to the T cell proliferation that ConA is induced, and insect antifeedant activity and growth inhibitory activity show as having beet exigua larvae antifeedant activity, have cotton bollworm larvae antifeedant activity and having growth inhibition effect to cotton bollworm larvae.These compounds can be used for preparing immunosupress, have antifeedant activity and the drug of growth inhibitory activity to insect.
Description
The application is the divisional application of No. 2014103695394 patent applications.
The applying date of original application is：On July 30th, 2014.
The application number of original application is：201410369539.4.
The invention and created name of original application is：Compound and its preparation with insect antifeedant activity and growth inhibitory activity
Method.
Technical field
The invention belongs to pharmaceutical technology fields, are related to the method for separating and preparing and purposes of auspicious promise alkane diterpene compound 1-11.
More particularly to the process that isolates and purifies, structural identification, immunosupress, insect antifeedant and growth inhibition effect etc..
Background technology
Autoimmune disease is to be only second to the third-largest disease of angiocardiopathy and cancer, has been developed at present a large amount of
Immunosuppressive drug, the rejection for being mainly used for preventing in organ transplant and the progress for inhibiting certain autoimmune diseases
Deng.Though wherein many drugs have immunosuppressive action, toxic side effect is strong, be easy to cause systemic immunity inhibition.It finds simultaneously
The immunosupress lead compound for developing high-efficiency low-toxicity is a key subjects of medicine sector of the world.Newest research hair
Existing, autoimmune disease and T cell relationship are especially close.ConA can stimulate the receptor on T cell surface in vitro, and selectivity swashs
Therefore T cell hyper-proliferative living, immunosupress can be fast and efficiently screened using the multiplicative model of the ConA splenic t-cells established
Compound.
Beet armyworm and bollworm are all omnivorousness agricultural pests widely distributed in world wide, main harm cotton,
The crops such as corn and soybean and vegetables.Prevention beet armyworm and bollworm still depend on chemical pesticide at present, but largely not
The pollution of soil, water, air is reasonably caused using chemical pesticide, plant drug resistance generates and harmful to human animal health
Etc. disastrous drawback.The thus research hotspot being developed into recent years of botanical pesticide, the insect killing substance in plant are mainly
Secondary metabolites does not pollute the environment, comparatively safe to crops and ecological environment, and insect is not easy to produce drug resistance,
Therefore the development and application of botanical pesticide play increasingly important role in current " pollution-free food " production.
Cassia is the dry bark of canella xylocinnamomum (Cinnamomum cassia Presl (Lauraceae)),
Basyleaves are the drying leaf of Chinese cassia tree.Chinese cassia tree can adjust body immunity, and Chinese cassia tree and its different auspicious promise alkane diterpene have anticomplement
Activity, Chinese cassia tree tool can expelling parasite, purification air, have the active constituent that can make insect antifeedant in Chinese cassia tree.
Invention content：
The task of the present invention is provide biologically active compound and its method for separating and preparing and application in Chinese cassia tree.
Realize the technical scheme is that：
The method of isolated auspicious promise alkane diterpene-kind compound includes the following steps from Chinese cassia tree：
Dry cassia is crushed, is extracted with ethyl alcohol room temperature, merges ethanol extract and is recovered under reduced pressure and be condensed into medicinal extract, is obtained
To total cream, total cream is dispersed to suspension shape by hot water, is extracted successively with the incremental petroleum ether of polarity, chloroform, ethyl acetate, n-butanol
It takes, ethyl acetate extract, n-butanol portion, petroleum ether and chloroform extract removal is concentrated under reduced pressure to obtain in recycling extract liquor.
Ethyl acetate extract is subjected to silica gel column chromatography, chloroform to methanol elution gradient (chloroform/methanol 10:1-3:1), chlorine
Imitative/methanol 6:One section of Fr.1 is obtained when 1, Fr.1 passes through reversed-phase silica gel column chromatography (water/methanol 9:1) position is obtained, this position passes through again
Gel, preparation liquid phase purify to obtain compound 1；
N-butanol portion is subjected to silica gel column chromatography, chloroform to methanol elution gradient (chloroform/methanol 10:1-3:1), chlorine
Imitative/methanol 8:Fr.2, chloroform/methanol 6 are obtained when 1:Fr.3, chloroform/methanol 5 are obtained when 1:Fr.4, chloroform/methanol 4 are obtained when 1:It is obtained when 1
Fr.5；Fr.2 passes through reversed-phase silica gel column chromatography (water/methanol 9:1) position is obtained, this position purifies again through gel, preparation liquid phase
To compound 4,5；Fr.3 passes through reversed-phase silica gel column chromatography (water/methanol 9:1-7:3) two positions be respectively Fr.3.1,
Fr.3.2.Fr.3.1 purifies to obtain compound 2,6,7 through silica gel, gel, preparation liquid phase；Fr.3.2 is through gel, preparation liquid-phase pure
Change obtains compound 8,9；Fr.4 passes through reversed-phase silica gel column chromatography (water/methanol 9:1), then through silica gel, gel, preparation liquid phase purifying
Obtain compound 3,11；Fr.5 passes through reversed-phase silica gel column chromatography (water/methanol 8:2) a position, this position is again through silica gel, solidifying
Glue, preparation liquid phase purify to obtain compound 10.The structural formula of the isolated auspicious promise alkane diterpene-kind compound 1-11 of the present invention and
Title is shown in Table 1, and separation process is shown in attached drawing 1.
1. present invention of table auspicious promise alkane diterpene-kind compound 1-11 structural formulas isolated from Chinese cassia tree
Wherein compound Chinese cassia tree terpene F, Chinese cassia tree terpene G be New skeleton compound, 19- deoxidations Chinese cassia tree terpene G 16- glucosides and
18-hydroxyperseanol is noval chemical compound.The single crystal diffraction structure chart of compound 1 is shown in attached drawing 2, and the monocrystalline of compound 2 spreads out
It penetrates structure chart and sees attached drawing 3.
Under the guidance that the present invention is screened in immunosuppressive activity, system research has been carried out to Chinese cassia tree active constituent.Pass through this
The research of T cell inhibited proliferation that 11 compounds induce ConA, present invention discover that compound Chinese cassia tree terpene G and
Cinnacaslo has significant inhibiting effect to the ConA T cells induced, represents that it has significant immunosuppressive action.By right
Compound 1-7 carries out the research of insect antifeedant and growth inhibitory activity, finds compound Chinese cassia tree terpene F, Chinese cassia tree terpene G and 18-
Hydroxyperseanol has beet exigua larvae an antifeedant activity, compound Chinese cassia tree terpene F and 18-hydroxyperseanol pairs
Cotton bollworm larvae has antifeedant activity；Compound 1-7 has growth inhibition effect to cotton bollworm larvae.
The present invention provides the plant origin of compound 1-11, isolation and purification method, immunosuppressive activity, insect antifeedant and
Growth inhibitory activity and application.Under the guidance that the present invention is screened in immunosuppressive activity, to Chinese cassia tree immunosuppressive activity ingredient into
System research is gone, isolated compound 1-11, wherein compound 1,2 is New skeleton compound, and compound 3 and 4 is new
Compound.Structural formula is as shown in table 1.The research of T cell inhibited proliferation induced by this 11 compounds ConA, hair
Existing compound 2 and the T cell of 11 pairs of ConA inductions have significant inhibiting effect, represent that it has significant immunosuppressive action.It is logical
The research that insect antifeedant and growth inhibitory activity are carried out to compound 1,2,4,5,6,8,9 is crossed, finds 1,2 and 4 pair of sweet tea of compound
Dish exigua larvae has antifeedant activity, and compound 2 and 4 pairs of cotton bollworm larvaes have antifeedant activity；Compound 1,2,4,5,6,8,9 pairs of cottons
Earworm larva has growth inhibition effect.
Immunosuppressive activity provided by the present invention, insect antifeedant activity and growth inhibitory activity compound 1,2,3,4 and 5
Structural formula it is as follows：
1,2,3,4 method for separating and preparing of the compounds of this invention is：By Chinese cassia tree ethyl acetate extract and n-butanol portion by
Normal phase silicagel column (mobile phase：Chloroform/methanol 10:1-3:1 gradient elution) draw section；Then reverse phase silicon is passed through into obtained part
Glue (mobile phase：Water/methanol 10:0-7:3 gradient elutions) it carries out drawing section again；By isolated segment further across solidifying
The isolated compound 1,2,3,4 such as glue, HPLC.
Present patent application embodiment 1 set forth the specific of the present invention auspicious promise alkane diterpene-kind compound isolated from Chinese cassia tree
Method.
Description of the drawings
Fig. 1：1 flow chart of embodiment；
Fig. 2：The single crystal diffraction structure chart of compound 1；
Fig. 3：The single crystal diffraction structure chart of compound 2.
Specific embodiment
Embodiment 1：It is prepared by the separation of compound.
50Kg drying cassias crush, and are extracted 3 times with ethyl alcohol room temperature, 7 days every time, merge ethanol extract and be recovered under reduced pressure
Be condensed into medicinal extract, obtain total cream 5.1Kg, total cream is dispersed to suspension shape by hot water, successively with the incremental petroleum ether of polarity, chloroform,
Ethyl acetate extract 70g, n-butanol portion 320g, petroleum ether is concentrated under reduced pressure to obtain in ethyl acetate, extracting n-butyl alcohol, recycling extract liquor
And chloroform extract removal.
Compound 1 (22mg)：Ethyl acetate extract 70g progress silica gel column chromatographies, chloroform to methanol elution gradient (chloroform/
Methanol 10:1-3:1), chloroform/methanol 6:One section of Fr.1 is obtained when 1, Fr.1 passes through reversed-phase silica gel column chromatography (water/methanol 9:1) one is obtained
Position, this position purify to obtain compound 1 through gel, preparation liquid phase again.
Compound 4 (3mg), 5 (6mg)：N-butanol portion 320g carries out silica gel column chromatography, chloroform to methanol elution gradient
(chloroform/methanol 10:1-3:1), chloroform/methanol 8:Fr.2, chloroform/methanol 6 are obtained when 1:Fr.3, chloroform/methanol 5 are obtained when 1:When 1
Obtain Fr.4, chloroform/methanol 4:Fr.5 is obtained when 1.Fr.2 passes through reversed-phase silica gel column chromatography (water/methanol 9:1) position, this position are obtained
It purifies to obtain compound 4,5 through gel, preparation liquid phase again.
Compound 2 (4mg), 6 (21mg), 7 (42mg)：Fr.3 passes through reversed-phase silica gel column chromatography (water/methanol 9:1-7:3)
Two positions are respectively Fr.3.1, Fr.3.2.Fr.3.1 purifies to obtain compound 2,6,7 through silica gel, gel, preparation liquid phase.
Compound 8 (12mg), 9 (28mg)：Fr.3.2 purifies to obtain compound 8,9 through gel, preparation liquid phase.
Compound 3,11：Fr.4 passes through reversed-phase silica gel column chromatography (water/methanol 9:1), then through silica gel, gel, preparation liquid phase
Purifying obtains compound 3 (3mg), 11 (57mg).
Compound 10：Fr.5 passes through reversed-phase silica gel column chromatography (water/methanol 8:2) a position, this position is again through silica gel, solidifying
Glue, preparation liquid phase purify to obtain compound 10.
Separation preparation flow figure is shown in Fig. 1.
By NMR, X-Ray and chemical reaction test parsing have obtained the absolute configuration of compound 1,2,3,4.
Compound 1, Chinese cassia tree terpene F (cinncassiol F) are colorless prismatic crystal,UV
(MeOH)λmax(logε):204(3.78)nm；IR(KBr)Vmax 3394,2968,2881,1709,1445,1388,1367,
1331,1257,1086,1039,999,851,787,758,683cm–1；The quasi-molecular ion peak [M+Na] that HRESIMS is provided+m/
z 421.1822.Its1HNMR and13C H NMR spectroscopy diagram datas are shown in Table 1 and Table 2.
Compound 2, Chinese cassia tree terpene G (cinncassiol G) are water white transparency column crystal,UV(MeOH)λmax(3.7) nm of (log ε)=202；IR(KBr)Vmax 3418,2969,
2886,2115,1641,1581,1412,1388,1269,1103,1026,833cm–1；The quasi-molecular ion peak that HRESIMS is provided
[M+H]+M/z 383.2066 determines that its molecular formula is C with reference to hydrogen spectrum and carbon modal data20H30O7(calculated value C20H32O7H[M+H
]+, 383.2065), 195-196 DEG C of fusing point.Its1H NMR and13C H NMR spectroscopy diagram datas are shown in Table 1 and Table 2.
Compound 3,19- deoxidation Chinese cassia tree terpene G 16- glucosides (19-deoxycinncassiol G 16-O- β-D-
Glucopyranoside it is) colourless oil liquid,UV(MeOH)λmax(log ε)=204
(3.7)nm；IR(KBr)Vmax 3391,2960,2931,1716,1629,1459,1376,1240,1214,1165,1081,
1027,943,869,774,693cm–1；The quasi-molecular ion peak [M+Na] that HRESIMS is provided+(calculated value is m/z 567.2385
C26H40O12[M+Na]+,567.2412).Its1H NMR and13C H NMR spectroscopy diagram datas are shown in Table 1 and Table 2.
Compound 4,18-hydroxyperseanol are colourless oil liquid,UV
(MeOH)λmax(3.4) nm of (log ε)=206；IR(KBr)Vmax3388,2963,2879,1602,1465,1387,1177,
1110,1029,962,892,829,699cm–1；The quasi-molecular ion peak [M+Na] that HRESIMS is provided+M/z423.1979 (is calculated
It is worth for C20H32O8H[M+Na]+,423.1989).Its1H NMR and13C H NMR spectroscopy diagram datas are shown in Table 1 and Table 2.
1 compound 1-4's of Table1H NMR datas
Table2 compounds 1-4's13C NMR datas
Embodiment 2
Compound 2 and the T cell proliferation of 11 pairs of ConA inductions significantly inhibit effect, compound 1-11 equal nothings to splenocyte
Toxic side effect, splenocyte survival rate are all higher than 90%.Compound 1-11 ion vitro immunizations inhibitory activity lures ConA by CCK-8 methods
The influence of T cell proliferation led is evaluated, as a result as shown in following table Table 3：
The inhibiting rate that 3 compounds of Table are proliferated the T cell that ConA is induced
Experiment conclusion：Compound 2 has the ConA mouse T cells induced the inhibiting effect of highly significant, inhibiting effect
In concentration correlation, the inhibiting rate of various concentration is all higher than 85%, and inhibition can compare with positive control CsA.Compound 11
Also there is the inhibiting effect of very strong highly significant to the mouse boosting cell of ConA inductions, its inhibiting rate is all higher than under experimental concentration
50%.
Embodiment 3：1,2 and 4 pair of beet exigua larvae of compound has the antifeedant activity of antifeedant activity and cotton bollworm larvae；Table
In 7 compounds have growth inhibition effect to cotton bollworm larvae.Antifeedant activity observes compound in interior table 4 for 24 hours by leaf dish method
Its antifeedant activity is evaluated to beet armyworm and cotton bollworm larvae feeding blade area.Growth inhibitory activity is mixed malicious method by feed and is seen
Influence of the compound in interior following table for 24 hours (50 μ g/ml) to cotton bollworm larvae weight is examined, its growth inhibition to larva is evaluated and lives
Property.As a result as shown in following table Table 4.
4. compounds of Table are to concentration and growth inhibition ratio in the food refusal of larva
Experiment conclusion：Except 5 most diterpene of compound has beet armyworm or bollworm different degrees of food refusal to live
Property.1,2 and 4 pair of beet exigua larvae of compound has notable antifeedant activity, and compound 1 and 4 pairs of cotton bollworm larvaes have notable food refusal
Activity；Compound is chosen in all experiments has cotton bollworm larvae growth inhibition effect, and compound 2 even makes the cotton boll in the growth stage
Worm weight loss.
Claims (1)
1. a kind of method of following auspicious promise alkane diterpene-kind compound 11 isolated from Chinese cassia tree, includes the following steps：
Step 1：50Kg drying cassias crush, and are extracted 3 times with ethyl alcohol room temperature, 7 days every time, merge ethanol extract and depressurize
Recycling is condensed into medicinal extract, obtains total cream 5.1Kg, total cream is dispersed to suspension shape by hot water, successively with the incremental petroleum ether of polarity, chlorine
N-butanol portion 320g is concentrated under reduced pressure to obtain in imitative, ethyl acetate, extracting n-butyl alcohol, recycling extract liquor；
Step 2：N-butanol portion 320g carries out silica gel column chromatography, chloroform to methanol elution gradient, and gradient is：Chloroform/first
Alcohol 10:1-3:1, chloroform/methanol 5:Fr.4 is obtained when 1；
Step 3：Fr.4 passes through reversed-phase silica gel column chromatography, and mobile phase is：Water/methanol 9:1, then through silica gel, gel, preparation liquid phase
Purifying obtains 57mg compounds 11.
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| CN201610238969.1A CN105777772B (en) | 2014-07-30 | 2014-07-30 | Compound with insect antifeedant activity and growth inhibitory activity and preparation method thereof |
| CN201410369539.4A CN104151323B (en) | 2014-07-30 | 2014-07-30 | There is compound of insect antifeedant activity and growth inhibitory activity and preparation method thereof |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102170893A (en) * | 2008-08-04 | 2011-08-31 | 酚醛塑料有限公司 | Novel compositions containing isolated tetrameric type A proanthocyanadin and methods of use and manufacture |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55136283A (en) * | 1979-03-29 | 1980-10-23 | Itsuo Nishioka | Novel diterpene derivative |
| JPS61112080A (en) * | 1985-10-31 | 1986-05-30 | Nippon Shinyaku Co Ltd | Novel tannin |
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2014
- 2014-07-30 CN CN201610238969.1A patent/CN105777772B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102170893A (en) * | 2008-08-04 | 2011-08-31 | 酚醛塑料有限公司 | Novel compositions containing isolated tetrameric type A proanthocyanadin and methods of use and manufacture |
Non-Patent Citations (3)
| Title |
|---|
| Evaluation of the quality of Chinese and Vietnamese cassia using LC-MS and multivariate analysis;Tanaka, Ken等;《Natural Product Communications》;20131231;第8卷(第1期);第75-78页，参见全文 * |
| The constituents of Cinnamomi Cortex. I. Structures of cinncassiol A and its glucoside;Yagi, Akira等;《Chemical & Pharmaceutical Bulletin》;19801231;第28卷(第5期);chart1，摘要、formula2 * |
| Two New Diterpenes from the Twigs of Cinnamomum cassia;Ngoc, Tran Minh等;《Helvetica Chimica Acta》;20091231;第92卷(第10期);第2058页摘要，第2059页figure1，第2062页实验部分 * |
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