CN105669790A - Bibenzil compounds and extraction method thereof - Google Patents
Bibenzil compounds and extraction method thereof Download PDFInfo
- Publication number
- CN105669790A CN105669790A CN201610129936.3A CN201610129936A CN105669790A CN 105669790 A CN105669790 A CN 105669790A CN 201610129936 A CN201610129936 A CN 201610129936A CN 105669790 A CN105669790 A CN 105669790A
- Authority
- CN
- China
- Prior art keywords
- silica gel
- compound
- elutriant
- bibenzyl
- extracting method
- 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.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides bibenzil compounds of which the chemical name is dihydroresveratrol-4'-O-[6''-O-(4''-methylgalloyl)]-beta-D-glucopyranoside. The extraction method comprises the following steps: after finishing D101 macroporous resin adsorption, eluting sequentially with distilled water and an ethanol solution; carrying out silica gel chromatography on the eluate obtained by eluting with the 75-80 wt% ethanol solution, collecting the eluate by using 400-500mL as one unit, and carrying out separation and extraction on the 10th-unit eluate to obtain a multi-compound extracting solution; and carrying out silica gel chromatography on the multi-compound extracting solution, collecting the eluate by using 40-50mL as one unit, and carrying out separation and extraction on the 3rd-unit eluate to obtain the target compounds. The bibenzil compounds provided by the embodiment of the invention are novel single compounds, have the advantages of high purity, definite component and no impurity, and are obtained by extraction and purification for the first time.
Description
Technical field
The present invention relates to bibenzyl material in oil tea leaf and extract field, specifically, it relates to Bibenzyl compound in a kind of oil tea leaf extract and extracting method thereof.
Background technology
Oil tea (CamelliaoleiferaAbel) calls tea seed tree, tea oil tree, for Camellia tea section (Theaceae) plant, it it is one of big woody oleiferous plants in the world four, mainly it is distributed in the provinces such as Chinese yunnan, Guangxi, Guangdong, it is the Chinese senior oil crops of distinctive pure natural. Oil tea research is conceived to oil tea benevolence, the oil tea dregs of rice, tea-seed oil, oil tea shell or its germ plasm resource mostly, and the comprehensive utilizating research of oil tea leaf is less, and the large quantities of fallen leaves playing leaf generation between annual four Mays cause the very big wasting of resources. The exploitation of oil tea leaf can make full use of waste resource, it is to increase the comprehensive utilization ratio of oil tea, it is to increase the income of oil tea main producing region peasant. Oil tea leaf contains the compositions such as fat, protein, reducing sugar, acid, amino acid, tea saponin, chlorophyll and Bibenzyl compound, its trace element and VCContent is higher than some common fruit.
Bibenzyl compound is the material that a kind of biological activity is very strong, there is anti-oxygen, Green Tea Extract, bacteriostasis, preservation, anti-cancer, preventing bone rarefaction, cleaning nitrites etc. act on, along with the development of Natural products research, oil tea leaf Bibenzyl compound group part, its efficacy effect and utility value have been carried out deep research by people, but the bibenzyl material that prior art is extracted from oil tea leaf belongs to mixture more, multiple compound is also deposited, the indefinite impurity of composition is also many, impure, it is unfavorable for extensive utilization and the marketization of oil tea leaf extract.
In view of this, special proposition the present invention.
Summary of the invention
First object of the present invention is to provide a kind of Bibenzyl compound, overcoming in prior art the many technical problems existed, it is a kind of novel single compound, purity height, definite ingredients, without any impurity, belong to first from oil tea leaf extract and purifying obtain, without any relevant record in prior art, there is ground-breaking meaning, improve the added value of bibenzyl material, it is more conducive to carry out marketization application targetedly, there is market using value widely.
2nd object of the present invention is to provide the extracting method of this Bibenzyl compound a kind of, this extracting method has easy operation, simple and convenient, it is not necessary to be equipped with professional's operation and also can realize, save manpower and materials, front and back step is connected closely, extraction operational condition is gentle, and required chemical reagent is cheaply easy to get, by specifically extract operational condition grope obtain a kind of novel cpd, for subsequent extracted preparation provide can the operational path of reference, have very much to use for reference and be worth.
In order to realize the above-mentioned purpose of the present invention, spy by the following technical solutions:
Embodiments provide the Bibenzyl compound in a kind of oil tea leaf extract; the chemical name of this Bibenzyl compound is two hydrogen trans-resveratrols-4 '-O-[6 " '-O-(4 " "-methyl Nutgalls acyl group)]-β-D-glucopyranoside; being a kind of derivative of two hydrogen polydatins; molecular weight is 558, chemical structural formula is:
This kind of compound that the embodiment of the present invention discloses is that from oil tea leaf, separation and Extraction out, did not also have any relevant report, has ground-breaking meaning in prior art first. Why so concerned extraction research about oil tea leaf is, is because containing nutritive ingredients such as fat, protein, reducing sugar, acid, amino acid, tea saponin, chlorophyll and Bibenzyl compounds in oil tea leaf, its trace element and VCContent is also higher than some common fruit, further the bibenzyl substances content in oil tea leaf enriches, Bibenzyl compound is the material that a kind of biological activity is very strong, there is anti-oxygen, Green Tea Extract, bacteriostasis, preservation, anti-cancer, preventing bone rarefaction, cleaning nitrites etc. act on, can more strengthen its health care after extracting directly is concentrated to be worth, especially this kind that the present invention extracts has the Bibenzyl compound of ad hoc structure, its using value can have more commercial value through further development research, is worth developing widely.
In the present invention, extract in the extract obtained, the purity of this novel Bibenzyl compound can reach more than 95%, more excellent can reach more than 98%, only a small amount of impurity, the material obtained is very pure, is not the mixture of multiple bibenzyl material formation as major part in prior art, the high one matter of purity, compared to mixture, can improve its using value more.
The present invention is chemically confirmed the aspect such as title, chemical structural formula, molecular weight except the Bibenzyl compound novel for this, additionally provides the extracting method of this compound, comprises the steps:
(A) by oil tea leaf through overheated extraction, concentrated obtain concentrated solution, cross after D101 macroporous resin adsorption completes, successively with distilled water, 20-30wt% ethanol, 75-80wt% ethanolic soln wash-out;
(B) elutriant adopting 75-80wt% ethanolic soln wash-out to obtain is carried out silica gel column chromatography, moving phase adopts methylene chloride-methanol, every 400-500mL is that unit collects elutriant, the elutriant of the 10th unit is carried out continue separation and Extraction and obtains polyvoltine compound extracting solution;
(C) polyvoltine compound extracting solution being carried out silica gel column chromatography, moving phase adopts methylene chloride-methanol, and every 40-50mL is that unit collects elutriant, the elutriant of the 3rd unit is carried out continue separation and Extraction and namely obtains target compound.
The special specific compound for the present invention of the extracting method of the embodiment of the present invention designs, each step all needs to operate according to the solution of the present invention, any one step can not be lacked, the order of front and back step can not be put upside down, other working method more can not be adopted to replace any operation step of the present invention, especially each operating parameter related in the extracting method of the present invention, need to control in suitable scope, in such as (A) step, need to adopt different solvents to carry out wash-out, and owing to target compound is present in the elutriant that 75-80wt% ethanolic soln wash-out obtains, therefore extraction operations a series of further is carried out for this part elutriant, visible contriver is all that purposive carrying out step by step operates in the whole extracting method of design, it is need to pay a large amount of creative works, final could extract and obtain target compound.
In step (A), oil tea leaf is cross before pillar first will through overheated extraction, the pretreatment operation such as concentrated, the temperature that oil tea leaf heat is extracted preferably controls at 70-80 DEG C, more excellent is 75 DEG C, if the too high nutritive ingredient may destroyed in oil tea leaf of temperature, therefore it is noted that the control of temperature, oil tea leaf is first pulverized into the powdery of more than 60 orders by heat before extracting in addition;Preferably, it is that solvent extracts that heat extracts the ethanol adopting more than 50wt%, and that pulverizes that powdering is conducive to that extraction operates carries out smoothly.
In the step (B) of the present invention, after the elutriant that 75-80wt% ethanolic soln wash-out obtains first carries out concentrate drying, it is dissolved in methylene chloride-methanol, carry out silica gel column chromatography again, silica gel order Task-size Controlling wherein used in silica gel column chromatography process is at 200-300 order, to improve extraction efficiency, the volume ratio of methylene chloride-methanol preferably controls at 10:1-1:3, during layer analysis, every 400-500mL is that a unit collects elutriant, it is generally that every 500mL is collected as a stream part, collect general 24 group parts, wherein three components are segmented, respectively be row number be the 2nd, 10, 23 these three components, owing to the target compound of the present invention is present in the elutriant of the 10th unit, for the 10th further separation and Extraction of component, actual when operating, it is generally after the elutriant to the 10th unit carries out liquid chromatographic detection, adopt methanol-water solution to carry out wash-out according to detected result and obtain polyvoltine compound extracting solution, liquid chromatographic detection is that DAC preparative chromatography is separated and obtains color atlas, the constant wash-out of methanol-water obtains three monomeric compounds and a complex component containing multiple compound, this complex component is dissolved in a certain proportion of methylene chloride-methanol, continue silica gel column chromatography. wherein, the mass percent concentration of methanol-water solution is 60-65%.
Preferably, in step (C), after the elutriant of the 3rd unit utilizes semi-preparative liquid chromatography to detect, adopt acetonitrile-aqueous solution to carry out wash-out separation according to detected result and obtain target compound. Wherein, the mass percent concentration of acetonitrile-aqueous solution is preferably 20-30%, it is preferable to 25%.
Preferred, silica gel order Task-size Controlling used in the silica gel column chromatography process in step (C), at 200-300 order, to improve extraction efficiency, obtains purer material.
Prior art is compared, and the useful effect of the present invention is:
(1) Bibenzyl compound in novel oil tea leaf extract provided by the invention, purity height, definite ingredients, without any impurity, belong to first from oil tea leaf extract and purifying obtain, without any relevant record in prior art, there is ground-breaking meaning, improve the added value of bibenzyl material, it is more conducive to carry out marketization application targetedly, there is market using value widely;
(2) for above-mentioned Bibenzyl compound extracting method provided by the invention, there is easy operation; simple and convenient; also can realize without the need to being equipped with professional's operation; save manpower and materials; before and after step be connected closely, extract the advantage of operational condition gentleness, certain extracting method provided by the invention just preferably one method; as long as obtaining having the compound of ad hoc structure of the present invention for a kind of extracting method any in prior art, all in protection scope of the present invention.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, it is briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 is the HPLC collection of illustrative plates of the 80wt% ethanol eluate in the embodiment of the present invention one;
Fig. 2 is the DAC preparative chromatography separating spectrum of the elutriant of the 10th in the embodiment of the present invention one unit;
Fig. 3 is the liquid chromatogram of the 3rd in the embodiment of the present invention one stream part;
Fig. 4 is the ion figure of the target compound in the embodiment of the present invention one, color atlas and mass spectrum;
Fig. 5 is the target compound in the embodiment of the present invention one1H-NMR collection of illustrative plates;
Fig. 6 is the target compound in the embodiment of the present invention two13C-NMR collection of illustrative plates;
Fig. 7 is the H-HCOSY collection of illustrative plates of the target compound in the embodiment of the present invention two;
Fig. 8 is the hsqc spectrum figure collection of illustrative plates of the target compound in embodiment two;
Fig. 9 is the HMBC collection of illustrative plates of the target compound in the embodiment of the present invention two.
Embodiment
Embodiment of the present invention being described in detail below in conjunction with embodiment, but it will be understood to those of skill in the art that, the following example is only for illustration of the present invention, and should not be considered as limiting the scope of the invention. Unreceipted concrete condition person in embodiment, conveniently the condition of condition or manufacturers's suggestion carries out. Agents useful for same or the unreceipted production firm person of instrument, be and can buy, by commercially available, the conventional products obtained.
Embodiment 1
The extracting method following steps operation of Bibenzyl compound:
1) test apparatus and reagent prepare:
Green oil tealeaves: pick up from National Forest park, ridge, horizon, Hunan Province, be accredited as C. olelfera (CamelliaoleiferaAbel.) leaf by Sino-South African Forestry University of Science and Technology professor Tan Xiaofeng;
Methyl alcohol, acetonitrile: Fluka company of the U.S.;
Ethanol: traditional Chinese medicines group;
Methylene dichloride: traditional Chinese medicines group;
Dimethyl sulfoxide (DMSO) (DMSO): traditional Chinese medicines group;
Ultrasonic cleaner, AS series, Tianjin Ao Tesaiensi instrument company limited.
Rotary Evaporators, R-205B, Shanghai Shen Sheng instrument company;
D-101 macroporous resin: Tianjin Fu Bang Chemical Industry Science Co., Ltd;
Column chromatography silica gel: Qingdao Haiyang chemical plant produces;
Thin-layer chromatography silica-gel plate: Qingdao Haiyang chemical plant produces;
ZFB type ultraviolet analysis instrument for three purposed: Shanghai Kang Hua biochemical instrument Manufacturing Co., Ltd;
Thermostat water bath: Beijing medical electronics instrument factory;
Ultrasonic cleaner (JK-500B): Hefei Jin Nike Machinery Manufacturing Co., Ltd.;
Nitrogen evaporator: the excellent bright associating Science and Technology Ltd. in Beijing;
Analysis liquid chromatography: according to sharp extra-high effect liquid phase chromatogram, join UV1201 detector;
Analyze chromatographic column: UnitaryC184.6mm × 250mm, 5 μm, Zhejiang Hua Puxinchuan Science and Technology Ltd.;
Preparative chromatography post: C18,10mm × 250mm, 5 μm, Zhejiang Hua Puxinchuan Science and Technology Ltd.;
Semi-preparative liquid chromatography: the logical permanent LC3000 type of innovation;
DAC preparative chromatography: innovation Tong Heng company LC6000 high performance liquid chromatography;
2) concrete extracting method: the powder that the oil tea dry leave naturally dried in the shade is pulverized into more than 60 orders, getting 20kg divides two batches of use 50% ethanol to repeat to extract 2 times by solid-liquid ratio 1: 3 reflux, extract 2h every time, the temperature that heating is extracted controls at 70 DEG C, merge gained supernatant liquor, filter and obtain supernatant liquor, be evaporated to without alcohol taste, obtain the crude extract sample of original volume half, for subsequent use;
3) oil tea 50% alcohol extract sample is through mass spectroscopy, identifies 18 components, anthocyanidin wherein, and a large amount of polysaccharide in extracting solution, protein, the hydrophilic compositions such as tannin, complicated and be difficult to separation, not as the emphasis of the present invention's research. therefore need with D101 type purification with macroreticular resin, method is: the D101 type macroporous adsorbent resin being about 10L with industry ethanol postincubation, wash with water without alcohol taste, get half extracting solution and divide twice loading, successively with water, 20% ethanol, 80% ethanol elution, each gradient elution 4 times of column volumes, according to TLC and HPLC analytical results, each position merges concentrated, wherein the 80% total sample in ethanol position-silica gel column chromatography merges component color atlas as shown in Figure 1, three positions obtained by D101 macroporous resin wash-out carry out analyzing display, a large amount of polysaccharide and protein etc. are contained in washing position, 20% ethanol position may containing middle polarity compounds such as theasaponins, but this position has a large amount of coloring matter to disturb, and the uv-absorbing of sample is more weak, separating difficulty is also bigger.Bibenzyl and phenol glycosides constituents are rich in 80% ethanol position, therefore as the target of emphasis separation. Wherein, mobile phase A is methyl alcohol, and B is water; Elution program: 0-10min50%A, 10-20min80%A, 20-21min100%A; Determined wavelength 254nm, sample size 10 μ L;
4) 80% ethanol elution will be adopted to obtain eluate sample concentrate drying (about 150g), it is dissolved in a certain proportion of methylene chloride-methanol, admix 300g tlc silica gel (200-300 order), water-bath volatilizes, divide twice dress post, wherein blanket layer filling silica gel is about 800g every time, moving phase is methylene chloride-methanol (10:1-1:3 gradient elution), every 500mL is collected as a stream part, it is 1-2+2 ' through TLC combining data detection, 3-7+3 '-7 ', 8-10+8 '-10 ', 11-13+11 ', 14-16+12 '-13 ', 17-19+14 ', 20-22+15 '-18 ', 19 '-21 ', 23-26+22 '-27 ', 27-40, 28 '-34 ', 35 '-44 ', 41-47, 48-64, 65, 66-67, 68-70, 71, 72-78, 45 '-51 ', 52 '-63 ', 64 '-67 ', 68 '-71 ', 72 '-84 ' (stream part sequence number of twice silicagel column symbol below is distinguished to amount to 24 components, partial stream part carries out cross-combining, it is connected with plus sige),
5) through Liquid Detection, the elutriant 27-40 choosing the 10th wherein unit segments, DAC preparative chromatography is utilized to be separated, as shown in Figure 2, the 60% constant wash-out of methanol-water, obtains three monomeric compounds and a complex component fr.A (865mg) containing multiple compound to color atlas, this flow point is dissolved in a certain proportion of methylene chloride-methanol, admixing 2.5g tlc silica gel (200-300 order), water-bath volatilizes, and blanket layer filling silica gel is about 40g. Flow phase system is methylene chloride-methanol-water (the constant wash-out of 100:12:1), and every 40mL is collected as a stream part, is 1,2,3,4-12 through TLC combining data detection. 3rd stream part utilizes semi-preparative liquid chromatography to be separated, and the 25% constant wash-out of acetonitrile-water, color atlas (adds figure) as shown in Figure 3, obtains target compound, i.e. CO-9 in figure.
The target compound obtained is carried out structure qualification below, instrument used and reagent: NMR: all H spectrums of nucleus magnetic resonance are 400MHz entirely, all13C spectrum is 100MHz. Magnetic resonance detection condition: nucleus magnetic resonance one peacekeeping two dimension pulse sequence adopts the respective standard program of instrument, 1H-NMR spectrum sampling pulse width 90 °, accumulative frequency 32; 13C-NMR spectrum sampling pulse width 90 °, accumulative frequency, 2000, H-HCOSY, HSQC, HMBC tri-kinds test sampling data battle array is F2×F1=2048 × 256, zero is filled to 2048 × 1024 carries out FT conversion, window function process spectrogram.
Concrete qualification process carries out according to following operation:
Compound C O-9, white amorphous powder, iron trichloride reaction is positive, and Molish reaction is positive, and shows phenolic hydroxy group and sugar base in structure, HRESIMS (Positive): m/z559.1829 [M+H] in Fig. 4+, (CalcdforC28H31O12, 559.17), 329.0870 [M+H-dihydroreveratrol]+, 887.2605 [2M+H-dihydroreveratrol], in conjunction with1H-NMR and13The molecular formula of C-NMR deterministic compound CO-9 is C28H30O12。
1H-NMR collection of illustrative plates as shown in Figure 5, it may be seen that δ 6.91 (2H from spectrogram, d, J=8.5Hz), δ 6.97 (2H, d, J=8.5Hz), δ 6.11 (2H, d, J=1.9Hz), δ 7.09 (2H, s), 6.06 (1H, brs) are the proton signal on phenyl ring, and there are four groups of symmetrical structures, δ 3.86 (3H, s) for connecting oxygen methyl proton signal, δ 4.81 (1H, d, J=7.2Hz) it is sugar end group proton signal, owing to J1,2 value is greater than 7.0, it is β configuration according to the known glycosidic link of experience judge methods.
The compound resemblance 1-(3 ' that will report in the structure of Compound C O-9 supposition and document, 5 ' dihydroxyl) and phenyl 2-(4 "-O-β-D-glucopyranosyl) phenylethane contrast; part carbon modal data height is consistent, can speculate that Compound C O-9 is the derivative of two hydrogen polydatins.
In conjunction with all nuclear magnetic spectrograms and analysis, Compound C O-9 is accredited as two hydrogen trans-resveratrols-4 '-O-[6 " '-O-(4 " "-methyl Nutgalls acyl group)]-β-D-glucopyranoside. Looking into Scifinder database, this compound is novel substance, and molecular weight is 558, and purity can reach more than 95wt%, and chemical structural formula is:
Embodiment 2
The extracting method following steps operation of Bibenzyl compound:
1) test apparatus used and reagent prepare substantially the same manner as Example 1;
2) concrete extracting method: the powder that the oil tea dry leave naturally dried in the shade is pulverized into more than 70 orders, getting 20kg divides two batches of use 60% ethanol to repeat to extract 2 times by solid-liquid ratio 1: 3 reflux, extract 2h every time, the temperature that heating is extracted controls at 80 DEG C, merge gained supernatant liquor, filter and obtain supernatant liquor, be evaporated to without alcohol taste, obtain the crude extract sample of original volume half, for subsequent use;
3) oil tea 50% alcohol extract sample is through mass spectroscopy, identifies 18 components, anthocyanidin wherein, and a large amount of polysaccharide in extracting solution, protein, the hydrophilic compositions such as tannin, complicated and be difficult to separation, not as the emphasis of the present invention's research. Therefore need with D101 type purification with macroreticular resin, method is: the D101 type macroporous adsorbent resin being about 10L with industry ethanol postincubation, wash with water without alcohol taste, get half extracting solution and divide twice loading, successively with water, 30% ethanol, 75% ethanol elution, each gradient elution 4 times of column volumes, according to TLC and HPLC analytical results, each position merges concentrated;
4) 75% ethanol elution will be adopted to obtain eluate sample concentrate drying (about 150g), it is dissolved in a certain proportion of methylene chloride-methanol, admix 300g tlc silica gel (200-300 order), water-bath volatilizes, divide twice dress post, wherein blanket layer filling silica gel is about 800g every time, moving phase is methylene chloride-methanol (10:1-1:3 gradient elution), every 400mL is collected as a stream part, it is 1-2+2 ' through TLC combining data detection, 3-7+3 '-7 ', 8-10+8 '-10 ', 11-13+11 ', 14-16+12 '-13 ', 17-19+14 ', 20-22+15 '-18 ', 19 '-21 ', 23-26+22 '-27 ', 27-40, 28 '-34 ', 35 '-44 ', 41-47, 48-64, 65, 66-67, 68-70, 71, 72-78, 45 '-51 ', 52 '-63 ', 64 '-67 ', 68 '-71 ', 72 '-84 ' (stream part sequence number of twice silicagel column symbol below is distinguished to amount to 24 components, partial stream part carries out cross-combining, it is connected with plus sige),
5) through Liquid Detection, the elutriant 27-40 choosing the 10th wherein unit segments, DAC preparative chromatography is utilized to be separated, the 65% constant wash-out of methanol-water, obtain three monomeric compounds and a complex component fr.A (865mg) containing multiple compound, this flow point is dissolved in a certain proportion of methylene chloride-methanol, admixes 2.5g tlc silica gel (200-300 order), water-bath volatilizes, and blanket layer filling silica gel is about 40g. Flow phase system is methylene chloride-methanol-water (the constant wash-out of 100:12:1), and every 50mL is collected as a stream part, is 1,2,3,4-12 through TLC combining data detection.3rd stream part utilizes semi-preparative liquid chromatography to be separated, and the 30% constant wash-out of acetonitrile-water, obtains target compound, and the process that this compound carries out structure qualification is as follows:
13C-NMR collection of illustrative plates is as shown in Figure 6, as can be seen from spectrogram, δ 166.24 is fat carbonyl characteristic signal, comprise a series of unsaturated carbon signal within the scope of 95.00-160.00ppm, wherein have four groups of signals overlapping between two, prompting compound exists symmetrical structure, δ 59.43 connects oxygen-methyl carbon signal, δ 101.03 is sugar end group signal, has even oxygen carbon signal within the scope of 60.00-80.00ppm, by determining that this sugar base is glucosyl group with document contrast.
As shown in Figure 7, from spectrogram, (H-2 ', 6 ') and δ 6.97 (H-3 ', 5 ') form the AA ＇ BB ＇ spin system of typical para-orientation benzene to the H-HCOSY of target compound it may be seen that δ 6.91; δ 6.06 (H-4 ") with 6.11 (H-2 ", 6 ") formed typical between trisubstituted benzene AX2Spin system; δ 7.09 (H-2 " ", 6 " ") form the spin system of typical 1,3,4,5-tetra-substituted benzene; δ 2.73 (H-1) and 2.64 (H-2) form one group of A2B2Spin system, i.e.-CH2CH2-unit. Therefore, preliminary infer in compound containing a para-orientation benzene, trisubstituted benzene, 1,3,4,5-tetra-substituted benzene and one group of-CH between one2CH2-unit.
By HSQC (Fig. 8) by hydrocarbon signal one_to_one corresponding, structure fragment is stitched together by recycling HMBC. Carry out resolving as follows to the HMBC figure of target compound: as shown in Figure 9, δ 2.73 (H-1) and δ 135.83 (C-1 '), 128.90 (C-2 ', 6’), δ 2.64 (H-2) and δ 144.08 (C-1 "), 106.59 (C-2 ", 6 ") are relevant, will between trisubstituted benzene, one group of-CH2CH2-unit and para-orientation benzene three structure fragments connect to get up, in conjunction with the Compound C O-12 identified and according to deduction before, the structure of this compound also has a glucosyl group, a tetra-substituted phenyl, a fat carbonyl and company's oxygen methyl, utilizing HMBC to be connected with parent nucleus by these groups, wherein δ 4.81 (Glc-H-1) and δ 155.66 (C-4 ') are relevant, and prompting glucosyl group becomes glycosides with 4 ' position hydroxyl of parent nucleus; δ 4.39 (Glc-H-6a) and 4.59 (Glc-H-6b) and δ 166.24 (C=O); δ 7.09 (H-2 " "; 6 " ") relevant to δ 166.24 (C=O); it is Nutgalls acyl group that prompting tetra-substituted phenyl is directly connected with fat carbonyl; meanwhile, fat carbonyl is connected with glucose 6. δ 3.86 (4 " "-O-CH3) relevant to δ 139.87 (GA-4 " "), prompting methoxyl group is connected to 4 of Nutgalls acyl group.
In conjunction with all nuclear magnetic spectrograms and analysis, target compound is accredited as two hydrogen trans-resveratrols-4 '-O-[6 " '-O-(4 " "-methyl Nutgalls acyl group)]-β-D-glucopyranoside. Looking into Scifinder database, this compound is novel substance, and molecular weight is 558, and purity can reach more than 98wt%, and chemical structural formula is:
In a word, the target compound that the extracting method through the embodiment of the present invention obtains all has identical structure after structure is identified, and this kind of compound belongs to Late Cambrian, and follow-up study has certain directive significance.
Experimental example 1
The embodiment of the present invention 1 is extracted the target compound obtained carry out testing as follows: MTS method detection compound is on the impact of RAW264.7 cell-proliferation activity. Cell being inoculated in 96 orifice plates, establishes blank group, test group (10 μ g/mL, 30 μ g/mL and 100 μ g/mL, tri-different concns groups) respectively, often group establishes three multiple holes.Every hole adds MTS solution 20 μ L, hatches 1-2 hour, utilizes microplate reader to survey its light absorption value when wavelength 490nm. Find that test group (three groups) and control group absorbancy are without significant difference, illustrate that compound can not affect the proliferation activity of RAW264.7 cell, can find out that the present invention extracts the target compound obtained to body without any side effect from this experiment;
And then undertaken testing as follows by the target compound of the embodiment of the present invention 1: utilize the LPS that concentration is 1ug/mL to induce RAW264.7 cell to set up inflammatory model. establish blank group respectively, LPS induction group, LPS and test group (totally three groups, compound concentration is respectively 10 μ g/mL, 30 μ g/mL and 100 μ g/mL), test group is compared with LPS induction group, pass through RT-qPCR, the method detections such as Westernblot, find that test group all can significantly suppress IL-1 β in RAW264.7 cell, TNF-α, transcribing and expression level of the relevant inflammatory factors such as iNOS, can find that the target compound of the present invention has inflammation-inhibiting thus reaches the effect suppressing the malignant tumours such as cancer further by this experiment, and effect is remarkable.
Also identical result is presented by being undertaken testing by the target compound that the embodiment of the present invention 2 extraction obtains.
Wherein, laboratory apparatus used in this experiment mainly contains: the inverted biologic microscope of Shanghai unity instrument manufacturing company limited, the analytical balance that Japan's Shimadzu is produced, the low-temperature centrifugation machine that eppendorf company of Germany produces, electrophoresis apparatus, gel imaging instrument, vertical slab electrophoresis groove and the horizontal electrophoresis chamber that Beijing Jun Yi company produces, the ultrapure water system that ELGA company of Britain produces, the horizontal shaker etc. that 61 instrument factories produce; Experiment reagent used mainly contains: LPS lipopolysaccharides, MTS, H-DMEM substratum, Trizol reagent, primer, Virahol, chloroform, tetrabromophenol sulfonphthalein, ethidium bromide, foetal calf serum etc.
Although illustrate and describing the present invention with specific embodiment, but it will be appreciated that can make when not deviating from the spirit and scope of the present invention many other change and amendment. Therefore, it means that comprise all such changes and modifications belonging in the scope of the invention in the following claims.
Claims (10)
1. a Bibenzyl compound, it is characterised in that, the chemical structural formula of this Bibenzyl compound is:
2. the extracting method of Bibenzyl compound according to claim 1, it is characterised in that, comprise the steps:
(A) by oil tea leaf through overheated extraction, concentrated obtain concentrated solution, cross after D101 macroporous resin adsorption completes, successively with distilled water, 20-30wt% ethanol, 75-80wt% ethanolic soln wash-out;
(B) elutriant adopting 75-80wt% ethanolic soln wash-out to obtain is carried out silica gel column chromatography, moving phase adopts methylene chloride-methanol, every 400-500mL is that unit collects elutriant, the elutriant of the 10th unit is carried out continue separation and Extraction and obtains polyvoltine compound extracting solution;
(C) polyvoltine compound extracting solution being carried out silica gel column chromatography, moving phase adopts methylene chloride-methanol, and every 40-50mL is that a unit collects elutriant, carries out continuing separation and Extraction to the elutriant of the 3rd unit, to obtain final product.
3. the extracting method of Bibenzyl compound according to claim 2, it is characterised in that, in described step (A), the temperature that oil tea leaf heat is extracted controls at 70-80 DEG C.
4. the extracting method of Bibenzyl compound according to claim 3, it is characterised in that, in described step (A), oil tea leaf is first pulverized into the powdery of more than 60 orders by heat before extracting;Preferably, heat extracts the ethanol adopting more than 50wt% is that solvent extracts.
5. the extracting method of Bibenzyl compound according to claim 2, it is characterized in that, in described step (B), after the elutriant that 75-80wt% ethanolic soln wash-out obtains first carries out concentrate drying, it is dissolved in methylene chloride-methanol, then carries out silica gel column chromatography;
Preferably, silica gel order Task-size Controlling used in silica gel column chromatography process is at 200-300 order.
6. the extracting method of the Bibenzyl compound in oil tea leaf extract according to claim 2, it is characterized in that, in described step (B), after the elutriant of the 10th unit is carried out liquid chromatographic detection, adopt methanol-water solution to carry out wash-out according to detected result and obtain polyvoltine compound extracting solution.
7. the extracting method of Bibenzyl compound according to claim 6, it is characterised in that, the mass percent concentration of described methanol-water solution is 60-65%.
8. the extracting method of Bibenzyl compound according to claim 2, it is characterized in that, in described step (C), after the elutriant of the 3rd unit utilizes semi-preparative liquid chromatography to detect, adopt acetonitrile-aqueous solution to carry out wash-out separation according to detected result and obtain target compound.
9. the extracting method of Bibenzyl compound according to claim 8, it is characterised in that, the mass percent concentration of described acetonitrile-aqueous solution is 25-30%;
Preferably, in described step (C), silica gel order Task-size Controlling used in silica gel column chromatography process is at 200-300 order.
10. the extracting method of Bibenzyl compound according to the arbitrary item of claim 2-9, it is characterised in that, in described step (C), in the extract of last gained, the content of Bibenzyl compound is at more than 95wt%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610129936.3A CN105669790B (en) | 2016-03-08 | 2016-03-08 | A kind of Bibenzyl compound and extracting method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610129936.3A CN105669790B (en) | 2016-03-08 | 2016-03-08 | A kind of Bibenzyl compound and extracting method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105669790A true CN105669790A (en) | 2016-06-15 |
| CN105669790B CN105669790B (en) | 2016-09-21 |
Family
ID=56307195
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610129936.3A Active CN105669790B (en) | 2016-03-08 | 2016-03-08 | A kind of Bibenzyl compound and extracting method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105669790B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107066788A (en) * | 2016-12-07 | 2017-08-18 | 辽宁科技大学 | The optimization method of object in a kind of complicated thing with preparation chromatographic isolation |
| CN112587536A (en) * | 2020-06-11 | 2021-04-02 | 广东盛普生命科技有限公司 | Application of resveratrol-4 '-O- (6' -galloyl) glucopyranoside in preparing anti-coronavirus medicine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104147026A (en) * | 2014-08-20 | 2014-11-19 | 安树君 | New application of resveratrol derivative |
| CN104496762A (en) * | 2015-01-12 | 2015-04-08 | 中南林业科技大学 | Method for separating two bibenzyl compounds from ordinary oil tea camellia |
-
2016
- 2016-03-08 CN CN201610129936.3A patent/CN105669790B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104147026A (en) * | 2014-08-20 | 2014-11-19 | 安树君 | New application of resveratrol derivative |
| CN104496762A (en) * | 2015-01-12 | 2015-04-08 | 中南林业科技大学 | Method for separating two bibenzyl compounds from ordinary oil tea camellia |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107066788A (en) * | 2016-12-07 | 2017-08-18 | 辽宁科技大学 | The optimization method of object in a kind of complicated thing with preparation chromatographic isolation |
| CN107066788B (en) * | 2016-12-07 | 2020-11-24 | 辽宁科技大学 | Optimization method for separating target object in complex impurities by using preparative chromatography |
| CN112587536A (en) * | 2020-06-11 | 2021-04-02 | 广东盛普生命科技有限公司 | Application of resveratrol-4 '-O- (6' -galloyl) glucopyranoside in preparing anti-coronavirus medicine |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105669790B (en) | 2016-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102617672B (en) | Camellia nitidissima flavonoid glycoside, and preparation method and application thereof | |
| CN104922196B (en) | The preparation of small pagodatree flower general flavone extract and quality determining method | |
| CN103884811B (en) | A kind of biological chromatography compares screening system and application thereof | |
| CN105111255A (en) | Extraction and separation method for echinacoside and verbascoside in cistanche | |
| CN102617673B (en) | Method for separating and purifying naringin and neohesperidin from white skin layer of citrus grandis | |
| CN101824067A (en) | Barrigenol-type triterpenoid saponins compound, preparation method and application thereof | |
| CN104262445B (en) | Camellia nitidissima saponin A, and preparation method and antitumor application thereof | |
| CN103113433B (en) | A kind of method extracting Oleuropein from Syringa pubescens | |
| CN105669790A (en) | Bibenzil compounds and extraction method thereof | |
| CN102757476B (en) | Preparation method and application of squid ink peptidoglycan | |
| CN106083788A (en) | A kind of quinoid chalcone carbon glycosides dimer compound with anti-tumor activity and anti-inflammatory activity and preparation method thereof | |
| CN105601600B (en) | A kind of coumarin kind compound and extracting method thereof | |
| Yang et al. | An effective method based on medium‐pressure liquid chromatography and recycling high‐speed counter‐current chromatography for enrichment and separation of three minor components with similar polarity from Dracocephalum tanguticum | |
| CN105601693B (en) | Ginseng saponin F1Preparation and its antitumor action | |
| CN109134583A (en) | The method of triterpene compound is extracted from schefflera leaf using ionic liquid | |
| CN105535154B (en) | Hall crabapple flower extract and reactive compound, preparation method and application | |
| CN101810696A (en) | Total triterpenoids of apples and method for preparing same | |
| CN106188198A (en) | A kind of method of cAMP in high efficiency extraction Fructus Jujubae | |
| CN103951723B (en) | A kind of Camellia nitidissima Chi flavonoid glycoside and its production and use | |
| CN109912582A (en) | The method of mangiferin is extracted from mango leaf | |
| CN109776515A (en) | The method of mangiferin is extracted from myrica rubra leaf | |
| CN105085453B (en) | A kind of utilization high-speed countercurrent chromatography method that separation prepares oligomeric stilbene compound from Chinese small iris | |
| CN109265494A (en) | The method of Kaempferol glucoside compounds is extracted from the camellia of Yunnan | |
| CN105541939A (en) | Ellagic acid compound and extraction method thereof | |
| CN105924484B (en) | The method of quercetin derivative is extracted from Camellia Leaves |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |