CN109265494A - The method of Kaempferol glucoside compounds is extracted from the camellia of Yunnan - Google Patents

The method of Kaempferol glucoside compounds is extracted from the camellia of Yunnan Download PDF

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CN109265494A
CN109265494A CN201811459485.5A CN201811459485A CN109265494A CN 109265494 A CN109265494 A CN 109265494A CN 201811459485 A CN201811459485 A CN 201811459485A CN 109265494 A CN109265494 A CN 109265494A
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mobile phase
silica gel
fraction section
eluent
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CN109265494B (en
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王蔚婕
钟文惠
曹清明
易英建
陈嘉琳
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Central South University of Forestry and Technology
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
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    • C07H17/07—Benzo[b]pyran-4-ones

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Abstract

The method that the present invention provides a kind of to extract Kaempferol glucoside compounds from the camellia of Yunnan.The described method includes: Yunnan camellia tea cake is carried out ultrasonic extraction with ethanol water, extracting solution is obtained；Extracting solution is evaporated under reduced pressure to no alcohol and obtains concentrate；Macroreticular resin chromatographic column is added in concentrate and carries out gradient elution, eluent is collected and obtains thick fraction section, thick fraction section is concentrated, thick fraction section concentrate is obtained；Silica gel is added in thick fraction section concentrate, drying obtains sample to be processed after mixing evenly；Dry method loading row gradient elution into silica gel column chromatography collects eluent and obtains quasi- fraction section；Dry crude separation object is concentrated under reduced pressure；Use C18 chromatographic column, methyl alcohol-formic acid water solution system as mobile phase, sample introduction carries out isocratic elution after crude separation object is dissolved with solvent, collects the fraction section containing target compound, concentrated freeze-dried to obtain target compound.Method provided by the present application, isolates target compound from the camellia of Yunnan for the first time, and DNA purity is high.

Description

The method of Kaempferol glucoside compounds is extracted from the camellia of Yunnan
Technical field
The present invention relates to compounds to extract field, and Kaempferol glucose is extracted from the camellia of Yunnan in particular to one kind The method of glycosides compound.
Background technique
Yunnan camellia (Camellia reticulata Lindl.) also known as Yunnan tea or Tengchong safflower oil tea, Theaceae (Theaceae) Camellia (Camellia L.) perennial evergreen arbor is Chinese endemic species, national second level focused protection plant Object.Source area of the China as oil tea has more than 2300 years cultivation histories, and Yunnan Province is the big province of oil tea in China, has rich Rich oil tea resource.
Oil tea is mostly used to extract oil, and in general, the content of the fatty acid such as oleic acid, linoleic acid is evaluation oil tea product in tea seed The important indicator of matter.Yunnan camellia is compared with common oil tea, and oil content is high, oil body is bright, and unsaturated fatty acid content is up to 83.5%.Yunnan camellia bioactive substance rich in, such as flavones, organic acid, saponin(e, triterpene compound, these changes Closing object has certain nutritional health function, can reduce cholesterol, pre- preventing tumor, enhancing immunity of organisms, have biology well Utility value.
Kaempferol and its derivative are a kind of important compounds in oil tea, and there is anticancer, treatment diabetes and sclerotin to dredge The functions such as pine and protection damaging cells.
The prior art is not comprehensive for the analysis of active material contained in the camellia of Yunnan, and many effective components have to be separated mention It takes.By the effective active matter in the camellia of Yunnan, separation and Extraction is come out from complicated and diversified ingredient, obtains the higher product of purity, The good application of Yunnan camellia is played an important role.
In view of this, the present invention is specifically proposed.
Summary of the invention
The method that the purpose of the present invention is to provide a kind of to extract Kaempferol glucoside compounds from the camellia of Yunnan, institute State method and extract target compound from the camellia of Yunnan for the first time, and quickly and effectively, purity it is very high.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A method of it extracting Kaempferol glucoside compounds from the camellia of Yunnan, the described method comprises the following steps:
A. Yunnan camellia tea cake is subjected to ultrasonic extraction with ethanol water, obtains extracting solution；The extracting solution is depressurized and is steamed It evaporates to no alcohol and obtains concentrate；
B. macroreticular resin chromatographic column is added in the concentrate, successively uses water, 20% ethyl alcohol, 50% ethyl alcohol, 80% ethyl alcohol Gradient elution is carried out, is detected through chromatography, the eluent containing Objective extraction object is collected and obtains thick fraction section, the thick fraction section is dense Contracting, obtains thick fraction section concentrate；
C. silica gel is added in the thick fraction section concentrate, drying obtains sample to be processed after mixing evenly；It will be described Sample to be processed, into silica gel column chromatography, carries out gradient elution with dichloro methane-methanol system, through color using dry method loading Spectrum detection collects the eluent containing Objective extraction object and obtains quasi- fraction section；It is concentrated under reduced pressure, is dried to obtain crude separation object；
D. it uses C18 chromatographic column, use methyl alcohol-formic acid water solution system as mobile phase, by the crude separation object solvent Sample introduction after dissolution carries out isocratic elution, collects the fraction section containing target compound, concentrated freeze-dried to obtain target compound, institute State the structural formula of target compound are as follows:
It, can be effective by extraction-concentration-macroporous resin column chromatography-silica gel column chromatography-C18 chromatographic column preparation method By target compound, separation and Extraction is come out from Yunnan camellia numerous ingredient.
Preferably, in the step B, the column volume of the macroreticular resin chromatographic column is 2L；Every 500mL collects primary elution Liquid and sequentially label, the thick fraction Duan You A44-45 eluent merge to obtain.
It limits column volume and collects the opportunity of eluent, be to realize accurate separation and Extraction.It should be noted that working as column When volume, collection unit volume change, corresponding change can be occurred by collecting label.
Preferably, the model D101 of the macroreticular resin in the macroreticular resin chromatographic column, partial size 60-16 mesh, specific surface Product >=550m2/ g, adsorbance (phenol/butt) >=20mg/g is contrasted；The macroreticular resin is using preceding needing with ethanol elution to color It is limpid, then no alcohol taste is eluted to pure water.
It with the purpose that ethanol solution is eluted sufficiently is handled macroreticular resin, guarantees separating effect.
It is further preferred that in the step C, the production method of the silica gel column chromatography are as follows: take silica gel and methylene chloride Solution mixing, ultrasonic treatment is slowly fitted into chromatographic column after removing bubble removing, and carries out pressurized treatments to pillar with pressure pump, is obtained The silica gel column chromatography.
It is further preferred that in the step C, the dichloro methane-methanol system are as follows: mobile phase A is methylene chloride, Mobile phase B is methanol；The proportion of the gradient elution are as follows: the volume ratio of the mobile phase A and the Mobile phase B is followed successively by 7:1, 5:1,3:1,1:1,1:0, each gradient be eluted to it is colourless until.
Mobile phase and its ratio, gradient be based on the polarity of substance included in elution object, disengaging time, point It is determined from parameters such as degree.Suitable mobile phase and its ratio, gradient, can fast and effeciently separate target compound Out.
It is further preferred that silica gel described in every 25-35g corresponds to thick fraction section concentration described in 100mL in the step C Liquid；The temperature of the drying is 45-55 DEG C.
Appropriate mixes quadrat method, drying temperature, can effectively ensure the effect of silica gel column chromatography.
It is further preferred that the column volume of the silica gel column chromatography is 1.2L in the step C；Every 200mL collects primary Eluent and sequentially label, the quasi- fraction Duan You C4 eluent obtain.
Preferably, in the step D, the specification of the C18 chromatographic column are as follows: Venusil MP C18, size 10mm × 250mm, packing material size are 5 microns.
It is further preferred that the methyl alcohol-formic acid water solution system are as follows: mobile phase C is methanol, and mobile phase D is volume fraction 0.1% aqueous formic acid；The proportion of the isocratic elution are as follows: the volume ratio of the mobile phase C and mobile phase D is 25: 75。
Optionally, the volume fraction of the ethanol water is 50-60%；The method of the extraction are as follows: solid-liquid ratio 1kg The Yunnan camellia tea cake corresponds to ethanol water described in 3-4L, and ultrasound extraction 2-4 times, is obtained by filtration institute under the conditions of 30-40 DEG C State extracting solution.
Appropriate extraction solvent and leach extraction method, it is ensured that extract yield.
Compared with prior art, the invention has the benefit that
(1) Objective extraction object has been isolated from the camellia of Yunnan for the first time；
(2) extracting method provided by the present application is simple, effective, is suitable for promoting and applying.
(3) target compound DNA purity is high.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described.
Fig. 1 is the HPLC spectrogram that embodiment 1 prepares crude separation object；
Fig. 2 is the purity testing figure for the target compound that embodiment 1 is prepared；
Fig. 3 is the mass spectrogram for the target compound that the application is prepared；
Fig. 4 is the H spectrogram for the target compound that the application is prepared；
Fig. 5 is the C spectrogram for the target compound that the application is prepared.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is The conventional products that can be obtained by commercially available purchase.
Embodiment 1
The tea fruit of Yunnan camellia is dried, decladding, is dried after sloughing grease with cold-press and suitably crushing is to get to Yunnan camellia Tea cake.The ethanol water for being 50% with volume fraction ultrasound extraction 3 times, each 6h, solid-liquid ratio 1:4 under the conditions of 40 DEG C (m/v), it is then filtered, merges, obtain extracting solution.Extracting solution is concentrated under reduced pressure into no alcohol to screw out, obtains concentrate.
Grease is sloughed in decladding, is to reduce the interference in separation process to guarantee to extract yield to the greatest extent.Properly Material processing also help the loss for avoiding target compound.
Choose the model D101 type of macroreticular resin: partial size 60-16 mesh, specific surface area >=550m2/ g, adsorbance is contrasted (phenol/butt) >=20mg/g.It is fitted into after 2L macroreticular resin is mixed with suitable water in the chromatographic column that column volume is 2L, with 5L's 100% ethanol decolorization is limpid to color, then with pure water rinsing until entirely without alcohol taste.Concentrate 2L is taken, it is slowly added Into macroreticular resin chromatographic column, colourless, eluent use is then successively eluted to water, 20% ethyl alcohol, 50% ethyl alcohol, 80% ethyl alcohol Clean vial is collected, every bottle of 500mL, successively labeled as A1, A2, A3 ..., by the efficient liquid of the eluent marked Analysis of hplc instrument is detected, and is collected the A44-45 eluent containing target compound, thick fraction section is obtained, by thick fraction Section concentration, obtains thick fraction section concentrate.
Take 200-300 mesh silica gel and dichloromethane solution to mix, ultrasonic treatment go after bubble removing to be slowly packed into column volume be In the chromatographic column of 1.2L, and pressurized treatments are carried out to pillar with pressure pump, obtains silica gel column chromatography；It is dense in the thick fraction section of 200mL 60g silica gel is added in contracting liquid, 50 DEG C of drying obtain sample to be processed after mixing evenly；Extremely using dry method loading by sample to be processed In silica gel column chromatography, gradient elution, dichloro methane-methanol system are carried out with dichloro methane-methanol system are as follows: flowing Phase A is methylene chloride, and Mobile phase B is methanol；The proportion of the gradient elution are as follows: the body of the mobile phase A and the Mobile phase B Product ratio be followed successively by 7:1,5:1,3:1,1:1,1:0, each gradient be eluted to it is colourless until.In elution process, eluent is used Clean vial is collected, every bottle of 200mL, by reception sequence number C1, C2, C3 ... respectively, to the eluent of collection into Row efficient liquid phase chromatographic analysis, merges eluent according to testing result, and C4 eluent is taken to obtain quasi- fraction section；Quasi- fraction section is subtracted Pressure is concentrated and is dried up with nitrogen evaporator, and crude separation object is obtained after freeze-drying.
Crude separation object -0.1% aqueous formic acid of methanol of volume ratio 25:75 is dissolved, concentration 20mg/mL；It uses LC3000 type high performance liquid chromatograph, C18 chromatographic column use methyl alcohol-formic acid water solution system to carry out isocratic elution as mobile phase, The fraction section containing target compound is collected, it is concentrated freeze-dried to obtain target compound.Wherein, the specification of C18 chromatographic column are as follows: Venusil MP C18, size 10mm × 250mm, packing material size is 5 microns；Methyl alcohol-formic acid water solution system are as follows: mobile phase C For methanol, mobile phase D is the aqueous formic acid of volume fraction 0.1%；The proportion of isocratic elution are as follows: the mobile phase C with it is described The volume ratio of mobile phase D is 25:75.
The HPLC spectrogram that is prepared using Venusil MP C18 chromatographic column to crude separation object is as shown in Figure 1, wherein X II Number peak corresponds to target compound.
Embodiment 2
The tea fruit of Yunnan camellia is dried, decladding, is dried after sloughing grease with cold-press and suitably crushing is to get to Yunnan camellia Tea cake.The ethanol water for being 60% with volume fraction ultrasound extraction 4 times, each 6h, solid-liquid ratio 1:3 under the conditions of 30 DEG C (m/v), it is then filtered, merges, obtain extracting solution.Extracting solution is concentrated under reduced pressure into no alcohol to screw out, obtains concentrate.
Choose the model D101 type of macroreticular resin: partial size 60-16 mesh, specific surface area >=550m2/ g, adsorbance is contrasted (phenol/butt) >=20mg/g.It is fitted into after 2L macroreticular resin is mixed with suitable water in the chromatographic column that column volume is 2L, with 4L's 100% ethanol decolorization is limpid to color, then with pure water rinsing until entirely without alcohol taste.Concentrate 2L is taken, it is slowly added Into macroreticular resin chromatographic column, colourless, eluent use is then successively eluted to water, 20% ethyl alcohol, 50% ethyl alcohol, 80% ethyl alcohol Clean vial is collected, every bottle of 500mL, successively labeled as A1, A2, A3 ..., by the efficient liquid of the eluent marked Analysis of hplc instrument is detected, and is collected the A44-45 eluent containing target compound, is obtained thick fraction section.
Take 200-300 mesh silica gel and dichloromethane solution to mix, ultrasonic treatment go after bubble removing to be slowly packed into column volume be In the chromatographic column of 1.2L, and pressurized treatments are carried out to pillar with pressure pump, obtains silica gel column chromatography；It is dense in the thick fraction section of 200mL 50g silica gel is added in contracting liquid, 45 DEG C of drying obtain sample to be processed after mixing evenly；Extremely using dry method loading by sample to be processed In silica gel column chromatography, gradient elution, dichloro methane-methanol system are carried out with dichloro methane-methanol system are as follows: flowing Phase A is methylene chloride, and Mobile phase B is methanol；The proportion of the gradient elution are as follows: the body of the mobile phase A and the Mobile phase B Product ratio be followed successively by 7:1,5:1,3:1,1:1,1:0, each gradient be eluted to it is colourless until.In elution process, eluent is used Clean vial is collected, every bottle of 200mL, by reception sequence number C1, C2, C3 ... respectively, to the eluent of collection into Row efficient liquid phase chromatographic analysis, merges eluent according to testing result, and C4 eluent is taken to obtain quasi- fraction section；Quasi- fraction section is subtracted Pressure is concentrated and is dried up with nitrogen evaporator, and crude separation object is obtained after freeze-drying.
Crude separation object -0.1% aqueous formic acid of methanol of volume ratio 25:75 is dissolved, concentration 20mg/mL；It uses LC3000 type high performance liquid chromatograph, C18 chromatographic column use methyl alcohol-formic acid water solution system to carry out isocratic elution as mobile phase, The fraction section containing target compound is collected, it is concentrated freeze-dried to obtain target compound.Wherein, the specification of C18 chromatographic column are as follows: Venusil MP C18, size 10mm × 250mm, packing material size is 5 microns；Methyl alcohol-formic acid water solution system are as follows: mobile phase C For methanol, mobile phase D is the aqueous formic acid of volume fraction 0.1%；The proportion of isocratic elution are as follows: the mobile phase C with it is described The volume ratio of mobile phase D is 25:75.
Embodiment 3
The tea fruit of Yunnan camellia is dried, decladding, is dried after sloughing grease with cold-press and suitably crushing is to get to Yunnan camellia Tea cake.The ethanol water for being 55% with volume fraction ultrasound extraction 2 times, each 6h, solid-liquid ratio 1:3.5 under the conditions of 35 DEG C (m/v), it is then filtered, merges, obtain extracting solution.Extracting solution is concentrated under reduced pressure into no alcohol to screw out, obtains concentrate.
Choose the model D101 type of macroreticular resin: partial size 60-16 mesh, specific surface area >=550m2/ g, adsorbance is contrasted (phenol/butt) >=20mg/g.It is fitted into after 2L macroreticular resin is mixed with suitable water in the chromatographic column that column volume is 2L, uses 4-5L 100% ethanol decolorization it is limpid to color, then with pure water rinsing until entirely without alcohol taste.Concentrate 2L is taken, it is slowly added Enter into macroreticular resin chromatographic column, is then successively eluted to colourless, eluent with water, 20% ethyl alcohol, 50% ethyl alcohol, 80% ethyl alcohol Be collected with clean vial, every bottle of 500mL, successively labeled as A1, A2, A3 ..., by eluent mark use efficiently Chromatographic analyzer of liquid phase is detected, and is collected the A44-45 eluent containing target compound, is obtained thick fraction section.
Take 200-300 mesh silica gel and dichloromethane solution to mix, ultrasonic treatment go after bubble removing to be slowly packed into column volume be In the chromatographic column of 1.2L, and pressurized treatments are carried out to pillar with pressure pump, obtains silica gel column chromatography；It is dense in the thick fraction section of 200mL 70g silica gel is added in contracting liquid, 55 DEG C of drying obtain sample to be processed after mixing evenly；Extremely using dry method loading by sample to be processed In silica gel column chromatography, gradient elution, dichloro methane-methanol system are carried out with dichloro methane-methanol system are as follows: flowing Phase A is methylene chloride, and Mobile phase B is methanol；The proportion of the gradient elution are as follows: the body of the mobile phase A and the Mobile phase B Product ratio be followed successively by 7:1,5:1,3:1,1:1,1:0, each gradient be eluted to it is colourless until.In elution process, eluent is used Clean vial is collected, every bottle of 200mL, by reception sequence number C1, C2, C3 ... respectively, to the eluent of collection into Row efficient liquid phase chromatographic analysis, merges eluent according to testing result, and C4 eluent is taken to obtain quasi- fraction section；Quasi- fraction section is subtracted Pressure is concentrated and is dried up with nitrogen evaporator, and crude separation object is obtained after freeze-drying.
Crude separation object -0.1% aqueous formic acid of methanol of volume ratio 25:75 is dissolved, concentration 20mg/mL；It uses LC3000 type high performance liquid chromatograph, C18 chromatographic column use methyl alcohol-formic acid water solution system to carry out isocratic elution as mobile phase, The fraction section containing target compound is collected, it is concentrated freeze-dried to obtain target compound.Wherein, the specification of C18 chromatographic column are as follows: Venusil MP C18, size 10mm × 250mm, packing material size is 5 microns；Methyl alcohol-formic acid water solution system are as follows: mobile phase C For methanol, mobile phase D is the aqueous formic acid of volume fraction 0.1%；The proportion of isocratic elution are as follows: the mobile phase C with it is described The volume ratio of mobile phase D is 25:75.
The purity of the obtained compound monomer of embodiment 1-3 is measured, determination condition are as follows: high using LC3000 type Effect liquid phase chromatogram instrument, Venusil MP C18 (4.6mm × 250mm, 5 μm) chromatographic column, mobile phase A are methanol, and Mobile phase B is 0.1% formic acid water；Coutroi velocity 1mL/min, detector UV205nm, 2 μ L of sample volume carry out isocratic elution, elution proportion are as follows: The volume ratio of mobile phase A and Mobile phase B is 25:75.It is 97.8% that wherein embodiment 1, which obtains purity, extracts yield 22.6mg/ kg.Purity testing figure is as shown in Figure 2.The Objective extraction object purity and yield that embodiment 2-3 is obtained are all larger than equal to aforementioned value.
LC-MS measurement and magnetic resonance detection are carried out to the compound monomer that embodiment 1-3 is obtained.
As shown in figure 3, m/z951.2856 [M+H]+.Speculate that its molecular formula is C43H50O24。
Fig. 4 (1H-NMR in), δ 6.20 (H, s) and δ 6.37 (H, s) meet the feature of meta position hydrogen on phenyl ring, infer its point Belong to Kaempferol A ring H-6 and H-8.δ 8.01 (2H, d, J=8.5Hz) and δ 6.89 (2H, d, J=8.5Hz), pass through integral area It includes 2 protons that two groups of signals, which can be obtained, should belong to symmetrical structure, and chemical shift is split point and coupling constant meets phenyl ring The feature of upper ortho-hydrogens, the result are consistent with Kaempferol B ring structure, it is inferred that its belong to Kaempferol B ring H-2', 6' with H-3', 5'.δ 5.47 (H, d, J=7.3Hz) is glucosyl group anomeric proton signal, from coupling constant (J=7.3Hz > 7.0Hz) It may infer that the glucose is beta comfiguration；δ 4.59 (H, s), 4.67 (H, s) are rhamnopyranosyl anomeric proton signal；δ2.15(3H, S), 2.11 (3H, s), 2.04 (3H, s), 1.95 (3H, s), 1.94 (3H, s) are acetyl protons signal；δ 0.78 (3H, d, J= 6.2Hz), 1.12 (3H, d, J=6.2Hz) are rhamnopyranosyl methyl proton signal；4.0-3.0 sections of multiple groups signals of δ are that glycosyl is other Proton signal.
Fig. 5 (13C-NMR in), δ 103.13,75.85,76.58,71.97,78.00,67.11 is one group of glucosyl group letter Number, δ 99.92,70.91,75.33,73.76,67.50,17.62 and δ 99.92,70.91,75.33,73.76,67.50, 17.62 be two groups of group rhamnopyranosyl signals, δ 172.16,171.81,171.76,171.70,171.47 and δ 20.94, 20.74,20.72,20.67,20.52 be five groups of acetyl group carbon signals.Remaining signal is identical with Kaempferol characteristic signal.
The peak of H spectrum and C spectrum is belonged to:
1H-NMR:8.01 (2H, d, J=8.5Hz, H-2 ', 6 '), 6.89 (2H, d, J=8.5Hz, H-3 ', 5 '), 6.37 (H, s, H-8), 6.20 (H, s, H-6), 5.47 (H, d, J=7.3Hz, H-1 "), 4.59 (H, s, H-1 ' ") 4.67 (H, s, H- 1””)、2.15(3H,s,AcO-3””)、2.11(3H,s,AcO-2”’)、2.04(3H,s,AcO-4””)、1.95(3H,s,AcO- 4 " '), 1.94 (3H, s, AcO-2 " "), 1.12 (3H, d, J=6.2Hz, H-6 " "), 0.78 (3H, d, J=6.2Hz, H-6 " ').
13C-NMR:158.82 (C-2), 134.85 (C-3), 179.40 (C-4), 163.15 (C-5), 98.90 (C-6), 165.94(C-7)、94.74(C-8)、158.50(C-9)、105.67(C-10)、122.78(C-1’)、132.20(C-2’,6’)、 116.18(C-3’,5’)、161.49(C-6’)、103.13(C-1”)、75.85(C-2”)、76.58(C-3”)、71.97(C- 4”)、78.00(C-5”)、67.11(C-6”)、99.92(C-1”’)、70.91(C-2”’)、75.33(C-3”’)、73.76(C- 4”’)、67.50(C-5”’)、17.62(C-6”’)、99.98(C-1””)、70.00(C-2””)、71.07(C-3””)、72.55 (C-4””)、68.35(C-5””)、17.19(C-6””)、171.76,20.52(AcO-C-2”’)、172.16,20.67(AcO-C- 4””)、171.70,20.72(AcO-C-2””)、171.81,20.94(AcO-C-3””)、171.47,20.74(AcO-C-4””)。
Comprehensive all nuclear magnetic spectrograms and mass spectral results, compound identification are Kaempferol -3-O- [2 " ", 3 " ", 4 " "-triacetyls Base-α-L- rhamnose-(1 → 3) -2 " ', 4 " '-diacetyl-α-L- rhamnose (1 → 6)]-β-D-Glucose glycosides, structural formula Are as follows:
The method provided by the present application that Kaempferol glucoside compounds are extracted from the camellia of Yunnan, for the first time from the camellia of Yunnan Target compound is separated and extracted, method simple practical is suitable for scale application, and product purity is high, for Yunnan camellia Deep development, which utilizes, positive effect.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims Including belonging to all such changes and modifications in the scope of the invention.

Claims (10)

1. a kind of method for extracting Kaempferol glucoside compounds from the camellia of Yunnan, which is characterized in that the method includes Following steps:
A. Yunnan camellia tea cake is subjected to ultrasonic extraction with ethanol water, obtains extracting solution；By the extracting solution be evaporated under reduced pressure to No alcohol obtains concentrate；
B. macroreticular resin chromatographic column is added in the concentrate, is successively carried out with water, 20% ethyl alcohol, 50% ethyl alcohol, 80% ethyl alcohol Gradient elution is detected through chromatography, is collected the eluent containing Objective extraction object and is obtained thick fraction section, and the thick fraction section is concentrated, Obtain thick fraction section concentrate；
C. silica gel is added in the thick fraction section concentrate, drying obtains sample to be processed after mixing evenly；By described wait locate Managing sample uses dry method loading into silica gel column chromatography, carries out gradient elution with dichloro methane-methanol system, examines through chromatography It surveys, collects the eluent containing Objective extraction object and obtain quasi- fraction section；It is concentrated under reduced pressure, is dried to obtain crude separation object；
D. it uses C18 chromatographic column, use methyl alcohol-formic acid water solution system as mobile phase, the crude separation object is dissolved with solvent Sample introduction afterwards carries out isocratic elution, collects the fraction section containing target compound, concentrated freeze-dried to obtain target compound, the mesh Mark the structural formula of compound are as follows:
2. the method according to claim 1, wherein in the step B, the cylinder of the macroreticular resin chromatographic column Product is 2L；Every 500mL collects an eluent and sequentially label, the thick fraction Duan You A44-45 eluent merge It arrives.
3. according to the method described in claim 2, it is characterized in that, the model of the macroreticular resin in the macroreticular resin chromatographic column For D101, partial size 60-16 mesh, specific surface area >=550m2/ g, adsorbance (phenol/butt) >=20mg/g is contrasted；The macroreticular resin It needs limpid to color with ethanol elution before use, then is eluted to no alcohol taste with pure water.
4. the method according to claim 1, wherein in the step C, the production method of the silica gel column chromatography Are as follows: take silica gel and dichloromethane solution to mix, ultrasonic treatment is slowly fitted into chromatographic column after removing bubble removing, and with pressure pump to column Son carries out pressurized treatments, obtains the silica gel column chromatography.
5. according to the method described in claim 4, it is characterized in that, in the step C, the dichloro methane-methanol body System are as follows: mobile phase A is methylene chloride, and Mobile phase B is methanol；The proportion of the gradient elution are as follows: the mobile phase A and the stream The volume ratio of dynamic phase B is followed successively by 7:1,5:1,3:1,1:1,1:0, each gradient be eluted to it is colourless until.
6. according to the method described in claim 5, it is characterized in that, silica gel described in every 25-35g is corresponding in the step C Thick fraction section concentrate described in 100mL；The temperature of the drying is 45-55 DEG C.
7. according to the method described in claim 6, it is characterized in that, in the step C, the column volume of the silica gel column chromatography is 1.2L；Every 200mL collects an eluent and sequentially label, the quasi- fraction Duan You C4 eluent obtain.
8. the method according to claim 1, wherein in the step D, the specification of the C18 chromatographic column are as follows: Venusil MP C18, size 10mm × 250mm, packing material size is 5 microns.
9. according to the method described in claim 8, it is characterized in that, the methyl alcohol-formic acid water solution system are as follows: mobile phase C is Methanol, mobile phase D are the aqueous formic acid of volume fraction 0.1%；The proportion of the isocratic elution are as follows: the mobile phase C and institute The volume ratio for stating mobile phase D is 25:75.
10. -9 described in any item methods according to claim 1, which is characterized in that the volume fraction of the ethanol water is 50-60%；The method of the extraction are as follows: solid-liquid ratio is that Yunnan camellia tea cake described in 1kg corresponds to ethanol water described in 3-4L, Ultrasound extraction 2-4 times, is obtained by filtration the extracting solution under the conditions of 30-40 DEG C.
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