CN107628954A - A kind of method that polyphenol substance is isolated and purified in the jasmine tea from lavender - Google Patents
A kind of method that polyphenol substance is isolated and purified in the jasmine tea from lavender Download PDFInfo
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- CN107628954A CN107628954A CN201710951797.7A CN201710951797A CN107628954A CN 107628954 A CN107628954 A CN 107628954A CN 201710951797 A CN201710951797 A CN 201710951797A CN 107628954 A CN107628954 A CN 107628954A
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Abstract
The invention discloses a kind of method that polyphenol substance is isolated and purified in jasmine tea from lavender, specifically include sample pretreatment, polyphenol slightly carries, extract and separate and by the ethyl acetate phase after extract and separate through the purifying of macroreticular resin polyamide column, aqueous phase is through macroreticular resin ODS post purifying process.The present invention is using lavender jasmine tea as raw material, isolate and purify by column chromatography chromatogram method to obtain a variety of polyphenol compounds with researching value with the aqueous solutions of organic solvent of different quality concentration, simple to operate, easy realization, product purity provides technical support up to 99.5%, for the acquisition and activity research of natural polyphenol material.
Description
Technical field
The present invention relates to a kind of method for isolating and purifying polyphenol substance.
Background technology
Lavender, also known as perfume plant, Lysimachia foenum-graecum, are Labiatae lavender category perennial herb or fruticuli plant, former
Originate in Mediterranean Region, after planted extensively in Britain and Yugoslavia.China in early in the twentieth century from overseas introduction, it is existing oneself
The ground such as Xinjiang, Shaanxi, Jiangsu are widely applied plantation.Lavender jasmine tea, it is that a kind of fragrance is dense also known as " quiet perfume plant "
Strongly fragrant purple little Hua, for make tea can effectively dispelling fatigue and anxiety, have the effect of calmness, sleeping, beauty and headache of releiving.Research
Show, drink lavender jasmine tea and can alleviate anxiety symptom using Lavender smelling, and the former can preferably drop
Low blood pressure.
Lavender flower spike is not only to extract the very good material of essential oil, with the jasmine tea that it is made except taste is sweet, also
Effective fatigue-relieving and pressure, promotion sleep and other effects.There are some researches show have physiologically active containing polyphenol etc. in lavender
Phytochemistry element, this kind of plant polyphenol anti-mutagenesis, it is antitumor, suppress microorganism, slow down osteoporosis, reducing blood lipid and blood glucose etc.
Aspect has good action, and its multiple efficacies has also been widely accepted.
At present, China is for contained active matter Quality Research application in extraction lavandulae flos, on the one hand greatly both for essence
Oil component；On the other hand, the active material extracted is also mostly general flavone mixture therein.And in lavender jasmine tea
Contained plant polyphenol, the purifying of especially a large amount of phenolic acid class glycosides derivatives and activity research are rarely reported, present invention process
With wide market prospects.
The content of the invention
It is an object of the invention to provide a kind of method that polyphenol substance is isolated and purified in jasmine tea from lavender, specific steps
It is as follows：
(1) sample pretreatment：Lavender jasmine tea through ultracentrifugation beveller crushing after cross 60 mesh sieves, extract oil removing, dry in-
20 DEG C save backup；
(2) polyphenol slightly carries：Using acetone-water solution as solvent, according to solid-liquid ratio 1:20, extraction, filter, collect filtrate,
Rotary evaporation, with centrifugation after pH=2 water dissolving to without precipitation, obtains lavandulae flos Tea Polyphenols crude extract, in 4 DEG C of ice to paste
Case preserves stand-by；
(3) extract and separate：Above-mentioned polyphenolic extract is divided equally in centrifuge tube, three are extracted with the ethyl acetate of two volumes
It is secondary, centrifugation revolving, dissolved with PBS stand-by (ethyl acetate phase) after 4 DEG C of preservations；Remaining aqueous phase merges rotary evaporation, uses pH=2
Water dissolve and saved backup after 4 DEG C again；
(4) purify：Respectively by ethyl acetate phase through the purifying of macroreticular resin-polyamide column, aqueous phase through macroreticular resin-ODS posts
Purifying, produce separation polyphenol substance Rosmarinic acid and glucose-phenols acids.
Further, in the step (4), ethyl acetate phase is purified, from macroreticular resin NKA-2, successively with
The ethanol that water, mass fraction are respectively 10%, 30%, 45%, 60% and 75% is eluant, eluent, is entered using constant flow pump with 2BV/h
Row gradient elution, 10%-45% ethanol sections are collected, it is stand-by to merge concentration；
Above-mentioned ethyl acetate phase separate fraction concentrate wet method is splined on polyamide column, is with water, mass fraction successively
10%th, 20%, 30%, 40%, 55%, 70% and 85% ethanol, and the acetone that mass fraction is 50%, 70%, 90%
Eluted, elution flow rate 2BV/h；70% acetone eluant is collected, and 90% acetone eluant, as Rosmarinic acid evaporate
Point.
Further, in the step (4), aqueous phase is purified, from macroreticular resin NKA-2, successively with water, quality
The ethanol that fraction is 10%, 30%, 45%, 60%, 75% and 95% is eluant, eluent, and gradient is carried out with 2BV/h using constant flow pump
Elution；10%, 30% and 45% ethanol eluate is collected, merges concentration, is freezed after water-soluble stand-by in -20 DEG C of preservations；
The freeze-dried powder for weighing above-mentioned aqueous phase separation cut is dissolved in 95% water-methanol, after fully being adsorbed by ODS resins,
Respectively using 95% water-methanol, 15% methanol, 30% and 45% methanol as eluant, eluent, using constant flow pump with 2BV/h carry out gradient
Elution；Fractional Collections glucose-phenols acids cut.
Further, in the step (2), in the acetone-water solution, VAcetone:VWater=70:30.
Further, in the step (2), the Extracting temperature is 30 DEG C -60 DEG C, preferably 40 DEG C；Extraction time
0.5h-2h, preferably 1h.
The technological merit of the present invention is that present invention process is using lavender jasmine tea as raw material, with having for different quality concentration
The solvent aqueous solution isolates and purifies to obtain a variety of polyphenol compounds with researching value, operation letter by column chromatography chromatogram method
Single, easily realization, product purity provides technical support up to 99.5%, for the acquisition and activity research of natural polyphenol material.
Brief description of the drawings
Fig. 1 is the HPLC figures of the Rosmarinic acid cut of the embodiment of the present invention 2 identification；
Fig. 2 is one-level, second order mses figure and the counter structure formula of the Rosmarinic acid of the embodiment of the present invention 2；
Fig. 3 is the Rosmarinic acid of the embodiment of the present invention 21HNMR collection of illustrative plates；
Fig. 4-7 is the HPLC figures of 3 four kinds of glucose of the embodiment of the present invention-phenols acids cut identification；
Fig. 8-11 is one-level, second order mses figure and the counter structure formula of 3 four kinds of glucose-phenols acids of the embodiment of the present invention；
Figure 12-15 is 3 four kinds of glucose-phenols acids of the embodiment of the present invention1HNMR collection of illustrative plates.
Embodiment
Present invention is described further below in conjunction with highly preferred embodiment of the present invention.
Polyphenol substance is isolated and purified in the lavender jasmine tea of embodiment 1：
(1) sample pretreatment：Lavender jasmine tea crosses 60 mesh sieves after the crushing of ultracentrifugation beveller, and the part of sieving is with oil
Ether is solvent, 40~60 DEG C of boiling range, and oil removing is extracted through 8~10h with Soxhlet oil extraction plant, and it is overnight in ventilating kitchen dry, fill
Enter valve bag to save backup in -20 DEG C.
(2) polyphenol slightly carries：Use VAcetone:VWater=70:30 acetone-water solution, according to solid-liquid ratio 1:20, pass through magnetic force temperature control
Stirring instrument extracts 1h under the conditions of 40 DEG C, merges after being repeated twice and filters, gained supernatant is concentrated into cream by Rotary Evaporators
Body shape, with centrifugation after pH=2 water dissolving to without precipitation, lavandulae flos Tea Polyphenols crude extract is obtained, preserves and treats in 4 DEG C of refrigerators
With.
During said extracted, Extracting temperature is chosen as 30 DEG C -60 DEG C, extraction time 0.5h-2h.
(3) extract and separate：Above-mentioned polyphenolic extract is divided equally in centrifuge tube, with ethyl acetate according to volume ratio 1:2 (slightly carry
Liquid：Ethyl acetate) extract three times, upper strata is drawn after centrifuging 5min every time, all merging rotate complete, with 0.1mol/L pH=
7.4PBS dissolvings are stand-by (ethyl acetate phase) after 4 DEG C of preservations；Remaining aqueous phase all merges, by Rotary Evaporators concentrate with except
The ethyl acetate wherein remained is removed, is dissolved with pH=2 water and is saved backup (aqueous phase) after 4 DEG C again；Analyzed through HPLC sample introductions, second
Acetoacetic ester mutually mainly includes the Rosmarinic acid in lavender jasmine tea, and aqueous phase main component is glucose-tonka-bean acid esters and grape
The isomers two-by-two of sugar-ferulic acid ester.
The purifying of Rosmarinic acid in the ethyl acetate phase of embodiment 2：
(1) macroreticular resin just separates
Take 100cm3Macroreticular resin NKA-2 fills post, makes macroreticular resin sedimentation complete by 2-3BV water elutions, by lavender
Jasmine tea ethyl acetate phase concentrate is slowly added in macroreticular resin chromatographic column, macroreticular resin is fully adsorbed polyphenol.Respectively with
Water 200mL, 10% ethanol 400mL, 30% ethanol 400mL, 45% ethanol 550mL, 60% ethanol 450mL and 75% ethanol
360mL is eluant, eluent, carries out gradient elution with 2BV/h using constant flow pump, collects 10%-45% ethanol sections, 200mL water before discarding
With the rear ethanol of 450mL 60%, the ethanol of 360mL 75%, it is stand-by to merge concentration.
(2) purifying of Rosmarinic acid
Take 180cm3Polyamide fills post, and above-mentioned ethyl acetate phase separate fraction concentrate wet method is splined on into polyamides post, point
Yong not water 980mL, 10% ethanol 700mL, 20% ethanol 780mL, 30% ethanol 600mL, 40% ethanol 900mL, 55% ethanol
650mL, 70% ethanol 530mL, 85% ethanol 770mL and 50% acetone 900mL, 70% acetone 1650mL, 90% acetone
3200mL is eluted, elution flow rate 2BV/h.Collecting 70% acetone 650mL-1650mL, (1000mL flows out after 70% acetone
Liquid) and 90% acetone 3200mL eluents be Rosmarinic acid cut.
Glucose-phenols acids isolates and purifies in the aqueous phase of embodiment 3：
(1) macroreticular resin just separates
Take 300cm3Macroreticular resin NKA-2 fills post, makes macroreticular resin sedimentation complete by 2-3BV water elutions, by lavender
Jasmine tea aqueous phase concentrate is slowly added in macroreticular resin chromatographic column, macroreticular resin is fully adsorbed polyphenol substance.Respectively
With water 1680mL, 10% ethanol 1350mL, 30% ethanol 1150mL, 45% ethanol 1280mL, 60% ethanol 1100mL, 75% second
Alcohol 800mL and 95% ethanol 970mL is eluant, eluent, and gradient elution is carried out with 2BV/h using constant flow pump.Collect 10% ethanol
350-1350mL (1000mL effluxes after 10% ethanol), 30% ethanol 1150mL and 45% ethanol 1280mL eluents, merge
Concentration, is freezed stand-by in -20 DEG C of preservations after water-soluble.
(2) purifying of glucose-phenols acids
Take 210cm3Reverse phase silica gel filler (ODS, 50 μm, 120A) dress post, with 95% water-methanol after being settled completely with methanol
Environment in post is replaced as Initial Gradient, the freeze-dried powder 2.0g for weighing above-mentioned aqueous phase separation cut is dissolved in 95% water-methanol, leads to
Cross after ODS resins fully adsorb, respectively with 95% water-methanol 650mL, 15% methanol 620mL, 30% methanol 330mL and
45% methanol 190mL is eluant, eluent, and gradient elution is carried out with 2BV/h using constant flow pump.95% water 100mL-200mL is collected (to abandon
100mL95% water before going) efflux is 6 ' the o- tonka-bean acid esters cuts of-O- β-glucose-；Collect 95% water 200mL-650mL streams
It is 6 '-O- β-glucose-ferulic acid ester cuts to go out liquid；Collect 30% methanol 130mL-330mL and (discard 15% methanol and 30% first
130mL before alcohol) eluent is coumaric acid -2-O-6 '-beta-glucosidase cut；Collect 45% methanol 0-75mL and (discard 45% first
115mL after alcohol) eluent is forulic acid-O-6 '-beta-glucosidase cut.
Claims (7)
1. the method for polyphenol substance is isolated and purified in a kind of jasmine tea from lavender, it is characterised in that comprise the following steps that：
(1) sample pretreatment：Lavender jasmine tea crosses 60 mesh sieves after the crushing of ultracentrifugation beveller, extracts oil removing, dries in -20 DEG C
Save backup；
(2) polyphenol slightly carries：Using acetone-water solution as solvent, according to solid-liquid ratio 1:20, extraction, filter, collect filtrate, rotation
Paste is evaporated to, with centrifugation after pH=2 water dissolving to without precipitation, lavandulae flos Tea Polyphenols crude extract is obtained, is protected in 4 DEG C of refrigerators
Deposit stand-by；
(3) extract and separate：Above-mentioned polyphenolic extract is divided equally in centrifuge tube, with the ethyl acetate extractions of two volumes three times, from
The heart rotates, and is dissolved with PBS stand-by (ethyl acetate phase) after 4 DEG C of preservations；Remaining aqueous phase merges rotary evaporation, with pH=2 water
Again dissolve and saved backup after 4 DEG C；
(4) purify：Ethyl acetate phase is purified through macroreticular resin-polyamide column purifying, aqueous phase through macroreticular resin-ODS posts respectively,
Produce separation polyphenol substance；The polyphenol substance is Rosmarinic acid, glucose-phenols acids.
2. according to the method for claim 1, it is characterised in that in the step (4), ethyl acetate phase is purified,
From macroreticular resin NKA-2, successively using water, mass fraction be respectively 10%, 30%, 45%, 60% and 75% ethanol to wash
De- agent, gradient elution is carried out with 2BV/h using constant flow pump, 10%-45% ethanol sections is collected, it is stand-by to merge concentration；
Then, above-mentioned ethyl acetate phase separate fraction concentrate wet method is splined on polyamide column, is with water, mass fraction successively
10%th, 20%, 30%, 40%, 55%, 70% and 85% ethanol, and the acetone that mass fraction is 50%, 70%, 90%
Eluted, elution flow rate 2BV/h；70% acetone eluant is collected, and 90% acetone eluant, as Rosmarinic acid evaporate
Point.
3. according to the method for claim 1, it is characterised in that in the step (4), aqueous phase is purified, from big
Hole resin NKA-2, successively using water, mass fraction 10%, 30%, 45%, 60%, 75% and 95% ethanol as eluant, eluent,
Gradient elution is carried out with 2BV/h using constant flow pump；10%, 30% and 45% ethanol eluate is collected, merges concentration, after water-soluble
Freeze stand-by in -20 DEG C of preservations；
Then, the freeze-dried powder for weighing above-mentioned aqueous phase separation cut is dissolved in 95% water-methanol, after fully being adsorbed by ODS resins,
Respectively using 95% water-methanol, 15% methanol, 30% and 45% methanol as eluant, eluent, using constant flow pump with 2BV/h carry out gradient
Elution；Fractional Collections glucose-phenols acids cut.
4. according to the method for claim 3, it is characterised in that the glucose-phenols acids include：6 '-O- β-glucose-
O- tonka-bean acid esters, 6 '-O- β-glucose-ferulic acid ester, coumaric acid -2-O-6 '-beta-glucosidase, forulic acid-O-6 '-β-Portugal
Polyglycoside.
5. according to the method for claim 1, it is characterised in that in the step (2), in the acetone-water solution, VAcetone:
VWater=70:30.
6. according to the method for claim 1, it is characterised in that in the step (2), the Extracting temperature is 30 DEG C -60
DEG C, extraction time 0.5h-2h.
7. according to the method for claim 6, it is characterised in that the Extracting temperature is 40 DEG C, extraction time 1h.
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| CN201710951797.7A CN107628954B (en) | 2017-10-12 | 2017-10-12 | Method for separating and purifying polyphenol substances from lavender scented tea |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6444839B1 (en) * | 2000-04-28 | 2002-09-03 | Salad Cosmo Co., Ltd. | Method of producing rosemarinic acid |
| CN105017345A (en) * | 2015-04-13 | 2015-11-04 | 湖南中医药大学 | Method for simultaneously extracting four kinds of compounds from selfheal, and application of extracted compounds |
| CN106565489A (en) * | 2016-10-10 | 2017-04-19 | 广西来宾绿翔生物科技有限公司 | Process for extracting rosmarinic acid from perilla |
| CN106928173A (en) * | 2017-03-07 | 2017-07-07 | 中国科学院新疆理化技术研究所 | Phenolic acid compound in lavender and its production and use |
-
2017
- 2017-10-12 CN CN201710951797.7A patent/CN107628954B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6444839B1 (en) * | 2000-04-28 | 2002-09-03 | Salad Cosmo Co., Ltd. | Method of producing rosemarinic acid |
| CN105017345A (en) * | 2015-04-13 | 2015-11-04 | 湖南中医药大学 | Method for simultaneously extracting four kinds of compounds from selfheal, and application of extracted compounds |
| CN106565489A (en) * | 2016-10-10 | 2017-04-19 | 广西来宾绿翔生物科技有限公司 | Process for extracting rosmarinic acid from perilla |
| CN106928173A (en) * | 2017-03-07 | 2017-07-07 | 中国科学院新疆理化技术研究所 | Phenolic acid compound in lavender and its production and use |
Non-Patent Citations (3)
| Title |
|---|
| M. GABRIELA GALLEGO, ET AL.: "Antioxidant Properties of Three Aromatic Herbs (Rosemary, Thyme and Lavender) in Oil-in-Water Emulsions", 《JOURNAL OF THE AMERICAN OIL CHEMISTS" SOCIETY》 * |
| 张特 等: "薰衣草精油提取残渣中多酚的提取纯化工艺研究", 《化学与生物工程》 * |
| 李晨阳 等: "狭叶薰衣草药材中迷迭香酸的含量测定", 《西北药学杂志》 * |
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