CN103755766A - Novel compound separated out from guava leaves as well as preparation method and application thereof - Google Patents
Novel compound separated out from guava leaves as well as preparation method and application thereof Download PDFInfo
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- CN103755766A CN103755766A CN201310736918.8A CN201310736918A CN103755766A CN 103755766 A CN103755766 A CN 103755766A CN 201310736918 A CN201310736918 A CN 201310736918A CN 103755766 A CN103755766 A CN 103755766A
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Abstract
The invention relates to a novel compound separated out from guava leaves as well as a preparation method and an application thereof. The chemical name of the novel compound is 3a, 22a-dihydroxyolean-12-en-30-oic acid. The novel compound is obtained through repeated silica-gel column chromatography, thin layer chromatography and crystal separation of the guava leaves; the structural formula of the novel compound is determined by a wave spectrum analysis method; and activity experiments prove that the compound has a very good glucose uptake promotion effect.
Description
Technical field
The present invention relates to medicine, functional food field, particularly one isolated new compound and preparation method and purposes from Guava Leaf.
Background technology
Diabetes B is that multiple virulence factor acts on a series of metabolism disorder syndromes that body causes hypoinsulinism, insulin resistant etc. and cause.The World Health Organization points out, worldwide diabetes situation is still very severe, and the situation of developing country is especially troubling.
Guava Leaf is used for preventing as medication among the people and the existing long history for the treatment of diabetes, there is a large amount of bibliographical informations in type-II diabetes patient disease's the process of preventing and treating about Guava Leaf, and a large amount of clinical trials proves its determined curative effect, therefore domestic and international many scholars have launched a large amount of research to its hypoglycemic effect, but up to the present, in Guava Leaf, blood-sugar decreasing active and mechanism of action are not yet definitely confirmed.Guava Leaf resource is very abundant in China, if Guava Leaf is goed deep into systematic research, determine its blood sugar reducing function basic substance, identify the chemical structure of its hypoglycemic monomer, bright its mechanism of action of analysing, and develop on this basis and prevent and treat ofhypoglycemic medicine or the functional food that diabetes B generation develops, not only can make full use of Guava Leaf resource, but also can reduce diabetics misery, create larger economic benefit and social benefit.
Summary of the invention
The object of the present invention is to provide one isolated new compound and preparation method and purposes from Guava Leaf.
One of the present invention is isolated new compound from Guava Leaf, and the chemistry of new compound is by name: 3 α, and neat honest fruit alkane-12-alkene-30 acid of 22 α dihydroxyl (3 α, 22 α-dihydroxyolean-12-en-30-oic acid), chemical structural formula is:
Of the present invention a kind of from Guava Leaf the preparation method of isolated new compound, comprise the following steps: 1) take the Guava Leaf powder of pulverizing, add the sherwood oil refluxing extraction 1h of 10 times of volumes, filter, filter residue adds the sherwood oil refluxing extraction 40min of 10 times of volumes again, filters, and gets filter residue.Filter residue volatilizes after solvent, adds respectively the chloroform refluxing extraction 3 times of 10,10,8 times of volumes, is 1h at every turn, filters, merges three chloroform extracted solutions, and reclaim under reduced pressure chloroform also obtains chloroform extract through drying under reduced pressure, standby;
2) chloroform extract carries out silica gel column chromatography separation, adopts petroleum ether-ethyl acetate to carry out gradient elution, through thin-layer chromatography, detects, and colour developing, merges same composition, must this compound through recrystallizing methanol.
While carrying out gradient elution, the volume ratio of sherwood oil and ethyl acetate is followed successively by 50:1,20:1,10:1,5:1,1:1,0:1.
A kind of from Guava Leaf isolated new compound as ofhypoglycemic medicine.
One isolated new compound from Guava Leaf, as blood-sugar lowering type health-care food or functional food additive.
The novel substance providing in the present invention, in experiment, shown that it has definite short sugared assimilation effect in vitro, itself plant origin is nontoxic, and the diabetes B prevention and control situation that the present invention can be current sternness provides valuable novel chemical structure, promotes health of people.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation:
Fig. 1 is that the hydrocarbon multikey that new compound HMBC(hydrogen of the present invention detects is correlated with) correlogram.
Embodiment
New compound of the present invention is to separate through the method such as silica gel column chromatography, thin-layer chromatography and crystallization repeatedly from Guava Leaf.Below in conjunction with practical situation, the specific embodiment of the present invention is elaborated:
Embodiment:
1. extract: take the Guava Leaf powder 3.2kg of pulverizing, add the sherwood oil of 10 times of volumes, 90 ℃ of refluxing extraction 1h, filter, and filter residue adds the sherwood oil of 10 times of volumes again, and 90 ℃ of refluxing extraction 40min filter, and get filter residue.Filter residue volatilizes after solvent, and the chloroform that adds respectively 10,10,8 times of volumes, 60 ℃ of refluxing extraction 3 times, is 1h at every turn, filters, merges three chloroform extracted solutions, and reclaim under reduced pressure chloroform also obtains chloroform extract 21.8g through drying under reduced pressure, standby.
2. column chromatography for separation and solvent recrystallization: 21.8g chloroform extract carries out silica gel column chromatography separation (4.5cm × 120cm), adopt petroleum ether-ethyl acetate to carry out gradient elution, the volume ratio of sherwood oil and ethyl acetate is followed successively by 50:1,20:1,10:1,5:1,1:1,0:1, thin-layer chromatography detects, colour developing, merges same composition, is divided into obtain 10 components.The 4th component 1.2g is again through silica gel column chromatography (2.5cm × 70cm), petroleum ether-ethyl acetate 5:1, and 2:1 gradient elution, must this compound 12.7mg through recrystallizing methanol from petroleum ether-ethyl acetate 5:1 component.
Experimental example:
1, compound structure is identified:
Compound is colourless crystallization powder, after thin-layer chromatography launches, and sulfuric acid baking displaing amaranth, aceticanhydride-strong sulfuric acid response positive, finally shows red-purple in addition, illustrates that this compound is triterpene compound.
Electron impact mass spectra provides molecular ion peak 472, and atmospheric pressure chemical ionization mass spectrum provides quasi-molecular ion peak 471.2 ([M-H]-) and illustrates that the molecular weight of compound is 472amu.Carbon spectrum and the DEPT spectrum of binding compounds show that it contains 7 methyl (δ 15.1,16.4,22.2 altogether, 24.7,25.8,28.2and28.6), 9 methylene radical, 6 methynes (wherein, 2 oxygen δ 73.7and74.5 of company, 1 is olefinic carbon, its excess-three is sp
3hydridization) and 8 quaternary carbons.Hydrogen spectrum carbon spectrum clearly discloses compound for honest alkane type triterpenoid compounds really simultaneously, and molecular formula is C
30h
48o
4(degree of unsaturation Ω=7), contain two hydroxyls, a carboxyl and a two key (in 12,13 positions) in molecule.
As seen from Figure 1, hydrogen on 24 methyl (δ 0.77 (3H, s)) and C-3, C-4, C-5 is relevant with C-23; Hydrogen on 28 methyl (δ 0.81 (3H, s)) and C-16, C-17, C-18 and C-22 distant relation; Hydrogen and C-20 on 29 methyl simultaneously, C-21 is relevant with C-30, and above result has clearly disclosed two hydroxyls and has been positioned at respectively 3,22.
With relatively carbon spectrum data of known compound " 3 β; 22 α-dihydroxyolean-12-en-30-oic acid ", both carbon spectrum data on pentacyclic triterpene parent nucleus C, D, E ring are in full accord, and therefore the hydroxyl of known 22 is in α configuration, and carboxyl is positioned on 30 simultaneously.In addition, because 24 carbon geochemistry shift values of new compound are at δ 22.2, with respect to 24 carbon geochemistry shift value δ 15.6 of 3 β-OH, obviously mobile to low, illustrate that 3 hydroxyls are also in α configuration, the structure of to sum up determining new compound is 3 α, 22 α-dihydroxyolean-12-en-30-oic acid.
Hydrogen spectrum attribution data is as follows: δ 5.16 (1H, dd, J=3.0,3.6, H-12), 3.14-.3.20 (2H, m, H-3,22), 0.85 (3H, s, H-23), 0.77 (3H, s, H-24), 0.89 (3H, s, H-25), 0.91 (3H, s, H-26), 1.14 (3H, s, H-27), 0.81 (3H, s, H-28), 1.08 (3H, s, H-29).
Carbon spectrum attribution data is as follows: δ 32.7 (t, C-1), 25.1 (t, C-2), 73.7 (d, C-3), 36.9 (s, C-4), 48.1 (d, C-5), 17.7 (t, C-6), 32.0 (t, C-7), 39.9 (s, C-8), 46.8 (d, C-9), 36.4 (s, C-10), 25.1 (t, C-11), 121.9 (d, C-12), 143.7 (s, C-13), 41.7 (s, C-14), 22.9 (t, C-15), 19.5 (t, C-16), 37.5 (s, C-17), 48.1 (d, C-18), 42.4 (t, C-19), 43.4 (s, C-20), 38.6 (t, C-21), 76.2 (d, C-22), 27.5 (q, C-23), 14.8 (q, C-24), 15.1 (q, C-25), 16.2 (q, C-26), 25.4 (q, C-27), 24.1 (q, C-28), 27.9 (q, C-29), 179.9 (s, C-30).
2, in the present invention, new compound hypoglycemic activity is measured:
2.1 glucose consumption hypoglycemic fat cell models
The disconnected neck of male SD rat is put to death, and the aseptic abdomen of opening is got the outer white adipose tissue of epididymis, rejects subsidiary blood vessel, shreds to about 1mm
3size, adds NTx enzymic digestion 1h, collects upper strata adipocyte, adds appropriate Hanks damping fluid washed cell to obtain Primary adipocyte three times.In 96 orifice plates, set up 200 μ L reaction systems: concentration is 2 × 10
6cells/mL cell suspension 100 μ L foetal calf serum+40, μ L+30 μ L6mg/mL glucose+30 μ L sample solutions (comprising each extract or each column chromatography elution fraction), with saturated humidity, 37 ℃, 5%CO
2cultivate reaction 30min, every hole adds 1 μ L2mmol/L epinephrine solution again, then cultivates 3h, ice bath termination reaction with similarity condition.Adopt micro-glucose oxidase method colour developing, reading in microplate reader, measures and calculates each hole glucose concn, and and control group relatively calculate the consumption concentration of each group of glucose, the ingestion of glucose ability of evaluating thus each extract or each column chromatography component, specific formula for calculation is as follows:
glucose-uptake?level=C
ctrl－C
compound(mmol/L)
Wherein, C
ctrlfor the concentration of control group glucose, C
compoundthe effective constituent glucose concn in residual substratum after 3 hours cultivate obtaining for sample or separation.
2.2 new compound hypoglycemic experiments
Under gnotobasis, monomer is dissolved in to the Hanks damping fluid (DMSO final concentration is 0.1%) containing DMSO, cross 0.22 μ m millipore filtration, being configured to final concentration is each monomer sample solution of 10,30,100 μ mol/L, according to cell model, carry out the analysis of monomer hypoglycemic activity, and take 0.1%DMSO as blank group, the positive hypoglycemic control group of 10nmol/L Regular Insulin, result is as shown in table 1 below.
The impact (X ± SD, n=6) of table 1. new compound of the present invention on adipocyte glucose uptake amount
Take the benchmark value of control group glucose uptake amount as 0, table 1 is the numerical value that monomer absorbs at the short glucose uptake of different concns, and its result shows, positive ofhypoglycemic medicine Regular Insulin can significantly promote the picked-up of glucose to absorb, and the feasibility of this detection model is described.New compound of the present invention can promote the picked-up utilization of glucose, and shows as dose-dependence, and in 10-100 μ mol/L amount ranges, the concentration of compound is higher, and hypoglycemic effect is more obvious.
Claims (5)
1. an isolated new compound from Guava Leaf, is characterized by: the chemistry of new compound is by name: 3 α, and neat honest fruit alkane-12-alkene-30 acid of 22 α dihydroxyl (3 α, 22 α-dihydroxyolean-12-en-30-oic acid), chemical structural formula is:
2. a preparation method for isolated new compound from Guava Leaf, is characterized in that, comprises the following steps:
1), take the Guava Leaf powder of pulverizing, add the sherwood oil refluxing extraction 1h of 10 times of volumes, filter, filter residue adds the sherwood oil refluxing extraction 40min of 10 times of volumes again, filters, and gets filter residue.Filter residue volatilizes after solvent, adds respectively the chloroform refluxing extraction 3 times of 10,10,8 times of volumes, is 1h at every turn, filters, merges three chloroform extracted solutions, and reclaim under reduced pressure chloroform also obtains chloroform extract through drying under reduced pressure, standby;
2), chloroform extract carries out silica gel column chromatography separation, adopts petroleum ether-ethyl acetate to carry out gradient elution, through thin-layer chromatography, detects, colour developing, merges same composition, through recrystallizing methanol, obtains new compound.
According to claim 2 a kind of from Guava Leaf the preparation method of isolated new compound, while it is characterized in that carrying out gradient elution, the volume ratio of sherwood oil and ethyl acetate is followed successively by 50:1,20:1,10:1,5:1,1:1,0:1.
4. one according to claim 1 isolated new compound from Guava Leaf, is characterized in that: as ofhypoglycemic medicine.
5. one according to claim 1 isolated new compound from Guava Leaf, is characterized in that: as blood-sugar lowering type health-care food or functional food additive.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09169648A (en) * | 1995-12-22 | 1997-06-30 | Nippon Shinyaku Co Ltd | Medicine composition |
| CN102178741A (en) * | 2011-04-11 | 2011-09-14 | 暨南大学 | Guava leaf extract with function of reducing blood sugar as well as preparation method and application thereof |
| CN102286057A (en) * | 2011-07-07 | 2011-12-21 | 北华大学 | Oleanane-type triterpenoid compounds and preparation method and medicinal use thereof |
| CN102924557A (en) * | 2012-05-14 | 2013-02-13 | 深圳清华大学研究院 | Triterpene compounds, preparation methods and applications thereof |
-
2013
- 2013-12-27 CN CN201310736918.8A patent/CN103755766A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09169648A (en) * | 1995-12-22 | 1997-06-30 | Nippon Shinyaku Co Ltd | Medicine composition |
| CN102178741A (en) * | 2011-04-11 | 2011-09-14 | 暨南大学 | Guava leaf extract with function of reducing blood sugar as well as preparation method and application thereof |
| CN102286057A (en) * | 2011-07-07 | 2011-12-21 | 北华大学 | Oleanane-type triterpenoid compounds and preparation method and medicinal use thereof |
| CN102924557A (en) * | 2012-05-14 | 2013-02-13 | 深圳清华大学研究院 | Triterpene compounds, preparation methods and applications thereof |
Non-Patent Citations (2)
| Title |
|---|
| 付辉政等: "番石榴叶化学成分研究", 《中国中药杂志》 * |
| 邵萌等: "番石榴叶的化学成分及药理活性研究进展", 《天然产物研究与开发》 * |
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