CN112778095A

CN112778095A - Extraction process of resveratrol in roselle leaves

Info

Publication number: CN112778095A
Application number: CN202110096029.4A
Authority: CN
Inventors: 杨路; 王台虎; 赵万洲; 郭永峰
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-01-25
Filing date: 2021-01-25
Publication date: 2021-05-11

Abstract

The invention discloses a process for extracting resveratrol from roselle leaves, which comprises the following steps: s1, raw material collection: selecting roselle leaves which naturally fall off in the production process of roselle and are artificially knocked off during planting, cleaning and airing the collected roselle leaves, and storing the collected roselle leaves in a dark place; s2, raw material pretreatment: taking roselle leaves, mechanically crushing, adding 20-22 times of ethanol, leaching for 3 days, taking out, filtering, carrying out rotary evaporation and concentration to obtain an extract, carrying out freeze drying, extracting for 3 times by using ethyl acetate, carrying out rotary evaporation and concentration again, carrying out freeze drying, and weighing to obtain the resveratrol leaf ethyl acetate extract. The method adopts the processes of ethanol extraction, multi-step chromatographic purification and the like, extracts the resveratrol from the roselle for the first time, has high extraction efficiency, high yield of the obtained product, is green and environment-friendly, and is easy to recover.

Description

Extraction process of resveratrol in roselle leaves

Technical Field

The invention relates to the technical field of extraction of components in plants, in particular to a process for extracting resveratrol from roselle leaves.

Background

Resveratrol (3, 4', 5-trihydroxystilbene; resveratrol) is an active polyphenolic compound and is also a natural antioxidant. Resveratrol has various pharmacological actions of resisting tumor, reducing blood fat, inhibiting platelet aggregation, regulating lipoprotein metabolism, protecting liver, resisting oxidation, inhibiting bacteria and the like on a human body, and is a natural substance with great development value.

Hibiscus sabdariffa Linn (scientific name) also called Hibiscus sabdariffaLinn, Hibiscus syriacus of chinquaiidae family annual herbaceous plant, native Africa, widely distributed in tropical and subtropical regions of the world. The roselle calyx is rich in organic acids, anthocyanidins, flavonoids, lignans and reducing saccharides, has the functions of inhibiting bacteria and promoting bile secretion, has the effects of preventing and treating cardiovascular diseases, hypertension, arteriosclerosis and the like, and has wide application and development prospects due to the nutritional and health-care values of roselle.

According to reports, resveratrol is widely existed in plants such as giant knotweed rhizome, grape, peanut, Maackia amurensis and the like, but in roselle, the separation of resveratrol is not reported. At present, the development and research on the roselle resources are concentrated in the deep processing of the roselle calyx, but the utilization of the roselle leaves is seriously insufficient, so that the waste of agricultural resources is caused. The research and development of the effective components in the roselle leaves have important significance for comprehensively utilizing the roselle plant resources and improving the economic value of the roselle.

Disclosure of Invention

The invention aims to solve the defect that agricultural resources are wasted due to serious insufficient utilization of the leaves of the roselle in the prior art, and provides an extraction process of resveratrol in the leaves of the roselle.

A process for extracting resveratrol from hibiscus sabdariffa leaves comprises the following steps:

s1, raw material collection: selecting roselle leaves which naturally fall off in the production process of roselle and are artificially knocked off during planting, cleaning and airing the collected roselle leaves, and storing the collected roselle leaves in a dark place;

s2, raw material pretreatment: taking roselle leaves, mechanically crushing, adding 20-22 times of ethanol, leaching for 3 days, taking out, filtering, carrying out rotary evaporation and concentration to obtain an extract, carrying out freeze drying, extracting for 3 times by using ethyl acetate, carrying out rotary evaporation and concentration again, carrying out freeze drying, and weighing to obtain an ethyl acetate extract of the resveratrol leaves;

s3, subjecting the ethyl acetate extract of resveratrol leaf to 300-mesh normal phase silica gel adsorption and column chromatography gradient elution, purifying by using a full-automatic rapid chromatography system, and confirming by liquid chromatography-mass spectrometry to obtain resveratrol.

Preferably, in S2, the raw material pretreatment: taking roselle leaves, mechanically crushing, adding 20 times of ethanol, leaching for 3 days, taking out, filtering, carrying out rotary evaporation and concentration to obtain an extract, carrying out freeze drying, extracting for 3 times by using ethyl acetate, carrying out rotary evaporation and concentration again, carrying out freeze drying, and weighing to obtain the resveratrol leaf ethyl acetate extract.

Preferably, in S3, the conditions of column chromatography gradient elution are as follows: removing most impurities with ethyl acetate/n-hexane as eluent, and further removing chlorophyll impurities with activated carbon and flash column chromatography.

Preferably, in S3, the conditions for purifying by the fully automatic flash chromatography system are as follows: dissolving with ethyl acetate, adding activated carbon to remove chlorophyll impurities, filtering, concentrating, mixing the concentrate with silica gel, and taking out half of the sample.

The invention adopts the processes of ethanol extraction, multi-step chromatographic purification and the like in the development and research of the roselle leaves, and the resveratrol is extracted from the roselle leaves for the first time after the confirmation of liquid chromatography-mass spectrometry. The process is characterized in that:

1. the process adopts ethanol as the extraction solvent because the resveratrol has good solubility in ethanol, and the ethanol belongs to a green solvent, has low toxicity and is easy to recycle;

2. the extraction process adopts a normal temperature percolation method, because the roselle leaves contain a large amount of chlorophyll, normal temperature or low temperature percolation can avoid the dissolution of chlorophyll impurities as much as possible, thereby simplifying the later purification process;

3. through small tests, a small amount of active carbon can well adsorb and eliminate the pigment, and a large amount or excessive amount of active carbon can cause the loss of resveratrol;

4. the process adopts a full-automatic rapid chromatography system to purify the resveratrol, the process is efficient, and the yield of the obtained product is high;

5. the LC-MS technology is applied to the identification of the resveratrol in the roselle leaves, the measurement is accurate, and the reliability of the result is ensured.

Drawings

FIG. 1 is a flow chart of the extraction process of the present invention;

FIG. 2 is a gradient chromatogram of a flash chromatography purification process;

FIG. 3 is a scanning diagram of a resveratrol standard solution in a parent ion negative ion mode;

FIG. 4 is a scanning diagram of the negative ion mode of the mother ion of the extract of the leaves of roselle.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples.

Examples

Referring to fig. 1, the extraction process of resveratrol in roselle leaves provided by the invention comprises the following steps:

s1, raw material collection: selecting natural falling in the production process of roselle and artificially knocking off leaves during the planting period; cleaning the collected roselle leaves, and storing the roselle leaves in a dark place;

s2, raw material pretreatment: taking 1 kg of roselle leaves, mechanically crushing, adding 20 liters of ethanol, and soaking for 3 days. After filtration, the mixture was concentrated by rotary evaporation to an extract state, and the weight of the extract was 90.3g by freeze-drying. The extract was extracted 3 times with 200 ml of ethyl acetate, concentrated by rotary evaporation, freeze-dried and weighed 80.4 g.

S3, primary purification: 80 g of the resveratrol leaf ethyl acetate extract is stirred with 65 g of 300 mesh normal phase silica gel, and is subjected to 400 g of silica gel column chromatography and ethyl acetate/n-hexane gradient elution (40/60 to 90/10), wherein ethyl acetate/n-hexane (70/30 to 80/20) fractions are combined and concentrated to 3.8 g. The chromatographic gradient is shown in table 1 below:

reagent (ml)	Ethyl acetate (%)	N-hexane (%)	Corresponding to the test tube number
				200	0	100	1-7
200	40	60	8-14
				200	50	50	15-21
200	60	40	22-28
				200	70	30	29-35
200	80	20	36-42
				200	90	10	43-49

TABLE 1

S4, secondary purification: 3.8 g of the concentrate are taken up in 25 g of silica gel, 100 g of silica gel are flash column chromatographed, eluting with an ethyl acetate/n-hexane gradient (50/50 to 60/40), the ethyl acetate/n-hexane (50/50 to 55/45) fractions being combined and concentrated to 1.1 g. The chromatographic gradient is shown in table 2 below:

reagent (ml)	Acetic acid B (%)	N-hexane (%)	Corresponding to the test tube number
				200	0	100	1-11
300	50	50	12-28
				200	55	45	29-40
200	58	42	41-52

TABLE 2

S5, purification by flash chromatography: dissolved in 30 ml of ethyl acetate, and then added with 500 mg of activated carbon to remove chlorophyll impurities, filtered and concentrated to 1.0 g. Mixing the concentrate with 4g of silica gel, taking out 2g of the mixture, performing dry loading, and purifying by a full-automatic rapid chromatographic system, wherein the separation method comprises the following conditions: performing column chromatography with 25 g normal phase, gradient eluting with ethyl acetate/n-hexane, detecting by TLC to obtain 29-32 tubes containing resveratrol, mixing, recovering, and weighing 21 mg. The chromatographic gradient is shown in figure 2;

s6, product identification: and confirming by liquid chromatography-mass spectrometry to obtain the resveratrol.

In the invention, the liquid extraction method comprises the following steps: sample 1.00 g was weighed, 10 ml of methanol was added, sonicated for 30 minutes, and soaked for 24 hours. And (3) taking 5 ml of extracting solution, adding PSA + activated carbon for purification, shaking for 2 minutes, centrifuging for 5 minutes, taking 1 ml of supernatant, adding methanol to fix the volume to 2 ml, and measuring by using UPLC-MS/MS after membrane filtration.

UPLC-MS/MS working conditions: direct mass spectral injection, ESI negative ion mode, instrument model AB4000 +.

Mass spectrum conditions: the parent ion (m/z) was 227.1 and the daughter ions (m/z) were 142.7 and 184.5.

And (3) detection results: resveratrol with a parent ion (m/z) of 227.1 was detected in the roselle leaf samples at a concentration of about 17mg/kg, as shown in fig. 3 and 4 below.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims

1. A process for extracting resveratrol from roselle leaves is characterized by comprising the following steps:

2. The process for extracting resveratrol from roselle leaves as claimed in claim 1, wherein in S2, raw material pretreatment: taking roselle leaves, mechanically crushing, adding 20 times of ethanol, leaching for 3 days, taking out, filtering, carrying out rotary evaporation and concentration to obtain an extract, carrying out freeze drying, extracting for 3 times by using ethyl acetate, carrying out rotary evaporation and concentration again, carrying out freeze drying, and weighing to obtain the resveratrol leaf ethyl acetate extract.

3. The process for extracting resveratrol from roselle leaves as claimed in claim 1, wherein in S3, the conditions of column chromatography gradient elution are as follows: removing most impurities with ethyl acetate/n-hexane as eluent, and further removing chlorophyll impurities with activated carbon and flash column chromatography.

4. The process for extracting resveratrol from roselle leaves as claimed in claim 1, wherein in S3, the conditions of purification by a full-automatic flash chromatography system are as follows: dissolving with ethyl acetate, adding activated carbon to remove chlorophyll impurities, filtering, concentrating, mixing the concentrate with silica gel, and taking out half of the sample.