CN110407906A - The method of triterpenoid is separated and prepared from hippophae rhamnoides - Google Patents
The method of triterpenoid is separated and prepared from hippophae rhamnoides Download PDFInfo
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Abstract
The present invention provides a kind of chromatography preparation method of compound of formula, including rhamnoides extract is made in sea-buckthorn, carries out medicinal extract and silica gel to fill column after mixing sample, and the mixed liquor of the petroleum ether and ethyl acetate that are mixed using petroleum ether and different volumes ratio carries out rushing column as mobile phase;Fraction obtained by step (1) is successively subjected to liquid-phase chromatographic analysis, normal-phase chromatography preparation, liquid-phase chromatographic analysis and reverse-phase chromatography preparation, finally carries out liquid-phase chromatographic analysis purity.The present invention extracts from hippophae rhamnoides prepare compound for the first time, with alpha-glucosidase inhibitory effect, can apply in preparing hypoglycemic Related product.
Description
Technical field
The present invention relates to the extraction fields of active principle in sea-buckthorn, more particularly to the separation from hippophae rhamnoides and prepare
The method of triterpenoid.
Background technique
Sea-buckthorn (Classification system: Hippophae rhamnoides Linn.) is a kind of Elaeangnaceae, Hippophne fallen leaves property
Shrub, characteristic are drought-enduring, anti-blown sands, can be survived on wetland with saline-alkaline, therefore be widely used in water and soil conservation.Western China
Northern a large amount of plantation sea-buckthorns, are used for desert afforestation.
Sea buckthorn fruit is full of nutrition, according to surveying and determination in its fruit containing multivitamin, fatty acid, microelement, sub- oil element,
The various amino acid of Hippophate flavone, superoxides isoreactivity substance and needed by human body.Wherein Vitamin C content is high, and every 100 grams
In fruit juice, Vitamin C content can reach 825~1100 milligrams, is 2~3 times of Kiwi berry, is known as the laudatory title of the king of vitamin C.
Now it is widely used in the production of sea buckthorn juice, Seabuckthorn fruit plasmogen liquid, in process of production, needs to remove the peel, the seabuckthorn fruit peel removed
It is often abandoned as waste material, or is produced into dry powder as feed, price is very cheap.
It has been reported in seabuckthorn fruit peel now containing ursolic acid and oleanolic acid the two triterpenic acids, there is liver protection shield
Liver reduces blood glucose and anti-skin oxidative and other effects, is expected to detect more active principles in sea-buckthorn, conducive to sea-buckthorn
Scientific development utilizes.
Summary of the invention
The purpose of the present invention is to provide the preparation method of compound shown in Formulas I,
The following steps are included:
(1) by silicagel column on rhamnoides extract, the petroleum ether and ethyl acetate mixed with petroleum ether and different volumes ratio
Mixed liquor be that mobile phase is eluted, wherein petroleum ether and ethyl acetate mixed proportion are (9~1): (1~9), collect triterpene
Compound eluent;
(2) eluent obtained by step (1) is successively subjected to the analysis of liquid phase normal-phase chromatography, normal-phase chromatography preparation, reverse phase liquid
Chromatography and reverse-phase chromatography preparation, finally carry out liquid-phase chromatographic analysis purity, obtain the compound of formula I.
Further, step (2) includes the following contents:
Fraction obtained by step (1) is subjected to positive liquid-phase chromatographic analysis, chromatographic condition includes:
Chromatographic column: hydrophilic HILIC chromatographic column;Preferably specification is 4.6mm*250mm, 5 μm, is more selected as
XAmide4.6mm*250mm, 5 μm of chromatographic columns;
Mobile phase: A is ethyl alcohol, and B is n-hexane;Isocratic condition: 2%~4%A, 98%~96%B;
Preferably, further include at least one of the following conditions:
Detection wavelength: 203 ± 10nm;Preferably 203 ± 5nm;
Column temperature: 30 ± 5 DEG C;Preferably 30 ± 2 DEG C;
Flow velocity: 1 ± 0.5mL/min;Preferably 1 ± 0.2mL/min;
Sample volume: 10 ± 5 μ L;Preferably 10 ± 2 μ L;
Further, the normal-phase chromatography preparation condition includes:
Chromatographic column: HILIC chromatographic column;It is preferred that specification be 20mm*250mm, 5 μm;More preferably XAmide20mm*250mm,
5 μm of chromatographic columns;
Mobile phase: A is ethyl alcohol, and B is n-hexane;Isocratic condition: 2%~4%A, 98%~96%B;
Preferably, further include following preparing at least one of chromatographic condition:
Detection wavelength: 203 ± 10nm;Preferably 203 ± 5nm
Column temperature: 30 ± 5 DEG C;Preferably 30 ± 2 DEG C
Flow velocity: 19 ± 2mL/min;Preferably 19 ± 1mL/min;
Sample volume: 1 ± 0.5mL;Preferably 1 ± 0.2mL;
Further, reversed-phase liquid chromatography analysis is carried out, the chromatographic condition of reversed-phase liquid chromatography analysis includes:
Chromatographic column: C18 chromatographic column, preferably specification are 4.6mm*250mm, 5 μm, more preferably Kromasil C18 column
(4.6mm × 250mm, 5 μm);
Mobile phase: A is water, and B is acetonitrile, isocratic condition: 20%~30%A, 80%~70% B;
Preferably, further include at least one of following chromatographiccondition:
Detection wavelength: 203 ± 10nm;
Column temperature: 30 ± 5 DEG C;
Flow velocity: 1 ± 0.5mL/min;
Further, reversed-phase preparative chromatography condition includes:
Chromatographic column: C18 chromatographic column, preferably specification be 21.2mm*250mm, 5 μm, 100~5~C18 of Kromasil (21.2
×250mm,5μm);
Column temperature: 30 DEG C;
Mobile phase: A is water, and B is acetonitrile, isocratic condition: the A of 20%-30%, 80%~70%B;
Preferably, further include at least one of following chromatography preparation condition:
Detection wavelength: 203 ± 10nm;
Column temperature: 30 ± 5 DEG C;
Flow velocity: 21 ± 2mL/min.
By the solution concentration after preparation, then the liquid phase analysis of the same terms is carried out, judges the purity of obtained component;If purity
Greater than 98%, target compound is obtained;If purity is less than 98%, then carries out chromatography preparation, it is preferable that liquid-phase chromatographic analysis packet
Include following chromatographic condition:
Chromatographic column: C18 chromatographic column, preferably specification be 4.6mm × 250mm, 5 μm;
Mobile phase: A is water, and B is acetonitrile, isocratic condition: 20%~30%A, 80%~70% B.
In above scheme of the invention, column temperature, Detection wavelength, sample volume, flow velocity etc. can select in Typical ranges.Wherein
Selection can be adjusted by conventional means in aforementioned scope of disclosure in Detection wavelength.When finding best detection wavelength,
The modes such as the matching used all band scanning of ultraviolet spectrophotometry, HPLC can be used to carry out, then cooperate HPLC detector
Detection effect (such as avoids solvent from interfering), and suitable Detection wavelength is found using routine techniques.One specific implementation of the present invention
In mode, Detection wavelength is selected from 201~205nm, such as in 203nm.
Further, step (1) successively uses petroleum ether and ethyl acetate mixed proportion for 9:1,8:2,7:3,6:4,
5:5,4:6,3:7,2:8,1:9.
Preferably, petroleum ether is determined by thin layer chromatography in step (1) elution process: when ethyl acetate is 7:3
Fraction be the highest fraction of triterpenoid content.
Further, after normal-phase chromatography preparation, collection retention time is the corresponding component of 14~-19min, is carried out in next step
Analysis.
Further, rhamnoides extract described in step (1) extracts to obtain using ethyl alcohol to sea-buckthorn;Further, it adopts
It is that 95% ethyl alcohol extracts with volumetric concentration;Further;By sea-buckthorn pomace and ethyl alcohol according to solid-liquid ratio be 1:(10~
30) kg/L is blended at 70~80 DEG C and extracts, and extracts 1.5~2.5h every time, extracts 3~4 times, and combined extract removes solvent and obtains
To medicinal extract.
The invention has the following advantages:
The present invention is prepared from hippophae rhamnoides for the first time by liquid-phase chromatographic analysis and positive, reverse-phase chromatography preparation method
The compound out, the compound have the activity suppression effect of alpha-glucosidase, can be used for preparing hypoglycemic Related product.
Detailed description of the invention
Fig. 1 is positive liquid-phase chromatographic analysis map;
Fig. 2 is that normal-phase chromatography prepares map;
Fig. 3 is that reversed-phase liquid chromatography analyzes map;
Fig. 4 is that reverse-phase chromatography prepares map;
Fig. 5 is the liquid-phase chromatographic analysis map after preparation.
The acarbose suppression curve of Fig. 6 various concentration;
The suppression curve of the inhibitor of Fig. 7 various concentration.
Specific embodiment
Below by specific embodiment, the present invention will be further described.As described below is only the embodiment of the present invention,
Be not intended to limit the scope of the invention, it is all using equivalent transformation made by present specification, or directly or
Other related technical areas are used in indirectly, are included within the scope of the present invention.
In following embodiment, Obtusol is compound of formula I of the present invention
Embodiment 1
1. preparing medicinal extract
Sea-buckthorn pomace 5kg is taken, 95% ethyl alcohol extracts in traditional Chinese medicine extracting machine, each 2h, solid-liquid ratio 1:10kg/L,
It extracts 3 times altogether, merges after No. 3 extracting solutions are concentrated under reduced pressure and obtain medicinal extract about 500g.
2. prepare compound
Key instrument and reagent
Instrument: 1260 series of high efficiency liquid chromatograph of Agilent is equipped with G1311C quaternary gradient pump, G1329B automatic sampling
Device, G1316A column oven, G1315D detector.
Reagent: analysis and preparation scale acetonitrile are all from scape chemical industry Co., Ltd, Yunnan Xinlan, and chromatographic grade water is baby Kazakhstan
Breathe out pure water.
2.1 carry out medicinal extract and silica gel after mixing sample, are rushed with the petroleum ether of petroleum ether and different proportion and ethyl acetate
Column, wherein petroleum ether and ethyl acetate ratio are respectively 9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:8,1:9, by silica gel
Pin deck plate determines petroleum ether: fraction position in ethyl acetate 7:3 is the higher position of triterpene acid content, by 4 in 7:3 fraction~
13 parts (referring to that every 500mL collects one bottle, take the 4th bottle~the 13rd bottle of fraction) carry out normal-phase chromatography analysis and preparation.
2.2 liquid phase chromatography analytical methods are established
4~13 parts in 7:3 fraction are analyzed on XAmide (4.6 × 250mm, 5 μm) analytical column, chromatographic condition
As follows: mobile phase: A is ethyl alcohol, and B is n-hexane;Isocratic condition: 0~45min, 4%A, 96%B;Flow velocity: 1mL/min;
Column temperature: 30 DEG C;Detection wavelength: 203nm, sample volume are 10 μ L.
The preparation of 2.3 normal-phase chromatographies
4~13 parts in 7:3 fraction are prepared into progress normal-phase chromatography system on column in XAmide (20mm × 250mm, 5 μm)
It is standby.Sample volume is 1mL when preparation, and flow velocity: 19mL/min, other conditions are identical as XAmide analytical column analysis condition.Component is received
Integrate: 3~3.8min is F~1;3.9~4.6min is F~2;6.2~8min is F~3;8.5~10min is F~4;10~
14min is F~5;14~19min is F~6;20~23min is F~7;23~25min is F~8;25~30min is F~9;
30~35min is F~10;35~37min is F~11.
The preparation of 2.4 reverse-phase chromatographies
By F~6 (the 6th fraction that 4~13 parts obtain after prepared by normal-phase chromatography in 7:3 fraction) In
After being analyzed on Kromasil100~5~C18 (4.6 × 250mm, 5 μm) analytical column, in 100~5~C18 of Kromasil
(21.2 × 250mm, 5 μm) prepares progress reverse-phase chromatography preparation on column, later with same liquid-phase condition in Kromasil 100
It is analyzed on~5~C18 (4.6 × 250mm, 5 μm) analytical column.
Chromatographiccondition are as follows: mobile phase: A: water, B: acetonitrile;Isocratic condition: 0~40min, 20%A, 80%B;
Analyze flow velocity: 1mL/min;Detection wavelength: 203nm;Column temperature: 30 DEG C.This analysis condition is also compound characterization quantitative detection
Liquid phase chromatogram condition.
Chromatography preparation condition are as follows: mobile phase: A: water, B: acetonitrile;Isocratic condition: 0~40min, 20%A, 80%B;
Flow velocity: 21mL/min;Detection wavelength: 203nm;Column temperature: 30 DEG C;
Liquid-phase chromatographic analysis is carried out to the component of preparation, as a result sees that Fig. 5, chromatography peak purity are 96.8%.Change to acquisition
It closes object and carries out NMR identification:
The NMR information of the compound is as follows: Obtusol compound, molecular formula: C30H50O2, white powder,1H NMR
(600MHz, MeOD) δ 5.14 (1H, t, J=3.6Hz, H~12), 3.56,3.03 (2H, (a) d, J=11.0Hz;(b) d, J=
11.0Hz, H~27), 3.16 (1H, dd, J=11.4,4.8Hz, H~3), 1.95 (1H, m, H~18), 1.94 (2H, m, H~
16), 1.91 (2H, m, H~11), 1.62 (2H, m, H~21), 1.61 (1H, m, H~19), 1.57 (2H, m, H~22), 1.55
(2H, m, H~1), 1.41 (2H, m, H~7), 1.39 (2H, m, H~6), 1.32 (1H, m, H~9), 1.25 (2H, m, H~2),
1.23 (2H, m, H~15), 1.12 (3H, s, H~28), 1.03 (3H, s, H~26), 1.0 (1H, m, H~20), 0.98 (3H, s,
H~23), 0.97 (3H, s, H~24), 0.93 (3H, d, J=6.6Hz, H~30), 0.81 (3H, d, J=6.6Hz, H~29),
0.78 (3H, s, H~25), 0.76 (2H, dd, J=12.0,1.2Hz, H~5)
13C NMR (151MHz, MeOD) δ 140.21 (s, C~13), 126.32 (s, C~12), 79.68 (s, C~3),
70.20 (s, C~27), 56.68 (s, C~18), 55.64 (s, C~5), 49.57 (s, C~9), 49.00,43.18 (s, C~
17), 41.29 (s, C~8), 40.86 (s, C~19), 40.74 (s, C~4), 40.14 (s, C~1), 39.86 (s, C~14),
39.17 (s, C~20), 37.99 (s, C~10), 36.61 (s, C~22), 34.04 (s, C~7), 31.82 (s, C~15),
28.73 (s, C~2), 27.92 (s, C~23), 27.11 (s, C~21), 24.45 (s, C~11), 24.13 (s, C~30),
23.86 (s, C~16), 21.78 (s, C~29), 19.46 (s, C~6), 17.93 (s, C~28), 17.34 (s, C~24),
16.37 (s, C~25), 16.22 (s, C~26)
Determine the compound molecule formula: C30H50O2
Molecular weight: 442.3811
Structural formula is
Experiment 1: the alpha-glucosidase Inhibition test of compound of the present invention
One, instrument and reagent
NaH2PO4·2H2O (Tianjin great Mao chemical reagent factory);
Na2HPO4·12H2O (Tianjin great Mao chemical reagent factory);
Anhydrous Na2CO3(Tianjin Hedong District red rock chemical reagent work);
Dehydrated alcohol (Tianjin Kai Tong chemical reagent Co., Ltd);
Yeast α-glucosidase (sigma company, the U.S., 100UN);
PNPG (Aladdin reagent (Shanghai) Co., Ltd.);
Acarbose (Shanghai Yuan Ye Science and Technology Ltd.);
Microplate reader (BioTek company, model: EPOCH2);
Constant temperature oscillator (THOMMO SHAKER, model: BE~9010).
Two, experimental method
1, the preparation of reagent
(1) configuration of solution A: NaH2PO4·2H2O weighs 15.603g, is settled to 500mL, 4 DEG C, brown bottle saves, standby
With.
(2) configuration of second liquid: Na2HPO4·12H2O weighs 35.822g, is settled to 500mL, 4 DEG C, brown bottle saves, standby
With.
(3) configuration of 0.1M phosphate buffer: measuring solution A 51mL, second liquid 49mL, and 100mL water is added, and mixes, obtains pH value
6.8 phosphate buffer, 4 DEG C, brown bottle saves, spare.
(4) 100U/mL proenzyme liquid yeast α-glucosidase: is diluted to 20U/mL's with phosphate buffer (pH 6.8)
Enzyme solutions, freezing is spare, is diluted to 1U/mL with phosphate buffer (pH 6.8) using preceding, spare.
(5) substrate pNPG is prepared: precision weighs 0.3766g pNPG, is added in suitable sodium phosphate buffer and dissolves, then
It is settled to 50mL, is configured to 25mmol/L mother liquor, is configured to 0.5mmol/L with sodium phosphate buffer using preceding, it is spare.
(6) acarbose inhibitor is prepared: precision weighs 13.9mg acarbose, is settled to 1mL with DMSO, is configured to
13.9mg/mL spare.
(7) Na of 0.1mol/L2CO3It prepares: weighing 1.06g Na2CO3In beaker, appropriate distilled water dissolution is added, and
Constant volume is saved at 4 DEG C to 100mL, spare.
2, the preparation of inhibitor
Precision weighs 2.5mg Obtusol into centrifuge tube, use 1mL dehydrated alcohol dissolve after as mother liquor, need using
When be diluted to concentration appropriate again to get the inhibitor of serial various concentration Obtusol.
3, activity suppression effect experiment of the Obtusol to alpha-glucosidase
Principle: p-nitrophenyl-alpha-D-glucose glycosides (pNPG) can produce p-nitrophenol through alpha-glucosaccharase enzyme hydrolysis,
It is absorbed in 405nm in specificity, therefore the activity of alpha-glucosidase can be detected by detecting the production quantity of p-nitrophenol.
Experiment is divided into blank group, control group, sample blank group and sample sets, preparation method: each reactant presses 1 middle dosage of table
It is loaded in 96 orifice plates, every group 3 parallel, and inhibitor, ethyl alcohol, buffer and enzyme solutions are uniformly mixed, and shakes in constant temperature
37 DEG C of heat preservation 10min in device are swung, after, it takes out, 50 μ L 0.5mmol/L pNPG solution is added, mix well, in 37 DEG C of water
Bath reaction 20min, after the Na of 50 μ L 0.1mol/L is added2CO3Solution stopped reaction is (blank group, right to get each test group
According to group, sample blank group and sample sets).
Have most at 405nm since PNPG can hydrolyze generation glucose and PNP, PNP under the action of alpha-glucosidase
It is big to absorb, measure its absorbance using microplate reader, according to formula can calculate each sample alpha-glucosidase inhibiting rate and
50 value of IC.
Formula:Wherein, Ac is blank group light absorption value, ABFor control
Group light absorption value, As are sample sets light absorption value, ASBFor sample blank group light absorption value.
Each reactant of table 1 addition metering and sequence (unit: μ L)
The inhibiting rate of the inhibitor of acarbose various concentration and various concentration Obtusol is shown in Table 2, table 3, respectively to two
Group data draw matched curve, and as shown in Figure 1 and Figure 2, using concentration as abscissa, inhibiting rate is ordinate, the Ah Ka of various concentration
Wave Glyco inhabiting curve is shown in Fig. 6, and the suppression curve of the inhibitor of various concentration Obtusol is shown in Fig. 7.Song is obtained by the curve being fitted again
Line equation:
The acarbose suppression curve equation of various concentration are as follows: y=0.3639x+0.0767, R2=0.9926.
The suppression curve equation of the inhibitor of various concentration Obtusol are as follows: y=19.107x+0.0546, R2=0.9963.
Acarbose and Obtusol can be acquired respectively to alpha-glucosidase 50% by above-mentioned two curvilinear equation
Concentration when inhibiting rate, acquiring acarbose is 1.1632mg/mL to 50 value of 503nhibiting concentration IC of alpha-glucosidase,
Obtusol is 0.0233mg/mL to 50 value of 503nhibiting concentration IC of alpha-glucosidase.
The inhibiting rate (n=3) of 2 acarbose various concentration of table
Concentration (mg/mL) | Inhibiting rate (%) | RSD |
0.07 | 9.82 | 14.32% |
0.139 | 13.22 | 18.68% |
0.278 | 19.11 | 8.53% |
0.556 | 25.79 | 7.79% |
1.112 | 48.82 | 2.72% |
IC50 value is 1.1632mg/mL
The inhibiting rate (n=3) of the inhibitor of 3 various concentration Obtusol of table
Concentration (mg/mL) | Inhibiting rate (%) | RSD |
0.05 | 100.54% | 1.67% |
0.025 | 55.29% | 7.99% |
0.0125 | 27.59% | 12.82% |
0.00625 | 15.01% | 7.23% |
0.003125 | 13.97% | 5.81% |
IC50 value is 0.0233mg/mL
As it can be seen that compound of the present invention has the activity suppression effect of significant alpha-glucosidase, and its IC50Far
Lower than acarbose.
Claims (10)
1. a kind of preparation method of compound shown in Formulas I, which comprises the following steps:
(1) by silicagel column on rhamnoides extract, with the mixed of the petroleum ether and ethyl acetate of petroleum ether and the mixing of different volumes ratio
Closing liquid is that mobile phase is eluted, and wherein petroleum ether and ethyl acetate mixed proportion are (9~1): (1~9), collects eluent;
(2) eluent obtained by step (1) is successively subjected to liquid phase positive liquid-phase chromatographic analysis, normal-phase chromatography preparation, reverse phase liquid
Chromatography and reverse-phase chromatography preparation, finally carry out liquid-phase chromatographic analysis purity, obtain the compound of formula I.
2. method according to claim 1, which is characterized in that step (2) positive liquid-phase chromatographic analysis condition includes:
Chromatographic column: hydrophilic HILIC chromatographic column;Preferably specification be 4.6mm*250mm, 5 μm;
Mobile phase: A is ethyl alcohol, and B is n-hexane;Isocratic condition: 2%~4% A, 98%~96% B;
Further, further include at least one of following chromatographiccondition:
Detection wavelength: 203 ± 10nm;
Column temperature: 30 ± 5 DEG C;
Flow velocity: 1 ± 0.5mL/min;
Sample volume: 10 ± 5 μ L.
3. method according to claim 2, which is characterized in that the normal-phase chromatography preparation condition includes:
Chromatographic column: HILIC chromatographic column;Preferably specification be 20mm*250mm, 5 μm;
Mobile phase: A is ethyl alcohol, and B is n-hexane;Isocratic condition: 2%~4%A, 98%~96%B;
Further, further include at least one of following chromatography preparation condition:
Detection wavelength: 203 ± 10nm;
Column temperature: 30 ± 5 DEG C;
Flow velocity: 19 ± 2mL/min;
Sample volume: 1 ± 0.5mL.
4. method according to claim 3, which is characterized in that reversed-phase liquid chromatography analysis includes following chromatographic condition:
Chromatographic column: C18 chromatographic column, preferably specification be 4.6mm*250mm, 5 μm;
Mobile phase: A is water, and B is acetonitrile, isocratic condition: 20%~30%A, 80%~70% B;
Further, further include at least one of following chromatographiccondition:
Detection wavelength: 203 ± 10nm;
Column temperature: 30 ± 5 DEG C;
Flow velocity: 1 ± 0.5mL/min.
5. method according to claim 4, which is characterized in that reversed-phase preparative chromatography condition includes:
Chromatographic column: C18 chromatographic column, preferably specification be 21.2mm*250mm, 5 μm;
Mobile phase: A is water, and B is acetonitrile, isocratic condition: 20%~30%A, 80%~70% B;
Further, further include following preparing at least one of chromatographic condition:
Detection wavelength: 203 ± 10nm;
Column temperature: 30 ± 5 DEG C;
Flow velocity: 21 ± 2mL/min.
6. method according to claim 5, which is characterized in that the solution concentration after preparation is carried out liquid phase analysis, if purity
Greater than 98%, target compound is obtained;If purity is less than 98%, then carries out chromatography preparation, liquid-phase chromatographic analysis includes following
Chromatographic condition:
Chromatographic column: C18 chromatographic column, preferably specification be 4.6mm × 250mm, 5 μm;
Mobile phase: A is water, and B is acetonitrile, isocratic condition: 20%~30%A, 80%~70% B.
7. method according to claims 1 to 6, which is characterized in that the petroleum ether and ethyl acetate that step (1) successively uses are mixed
Composition and division in a proportion example is 9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:8,1:9.
8. method according to claim 7, which is characterized in that determined in step (1) elution process by thin-layer chromatographic analysis
Petroleum ether: fraction when ethyl acetate is 7:3 is the highest fraction of triterpenoid content.
9. method according to claim 8, which is characterized in that after the preparation of step (2) normal-phase chromatography, collecting retention time is 14
The corresponding component of~19min carries out next step analysis.
10. method according to claims 1 to 6, which is characterized in that rhamnoides extract described in step (1) is using ethyl alcohol to sand
Spine extracts to obtain;Further, volumetric concentration is used to extract for 95% ethyl alcohol;Further;By sea-buckthorn pomace
According to solid-liquid ratio it is 1:(10~30 with ethyl alcohol) kg/L is blended at 70~80 DEG C and extracts, and extracts 1.5~2.5h every time, extract 3
~4 times, combined extract removes solvent and obtains medicinal extract.
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