CN112858513B - Stress method for improving resveratrol content in roselle - Google Patents
Stress method for improving resveratrol content in roselle Download PDFInfo
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Abstract
The invention discloses a stress method for improving the content of resveratrol in roselle, which comprises the following steps: s1: selecting healthy roselle, spraying and stressing by using a mixed solution containing 0.04-0.6% of acetic acid, 0.01-0.02% of ammonium molybdate, 0.01-0.02% of sodium selenate, 0.01-0.02% of tin dichloride and 0.02-0.03% of potassium iodide, and then carrying out drought stress for three days; s2: separating cacao trichomonad from tropical fruit, adding into culture solution, standing and fermenting at 28-30 deg.C for 6 days to obtain high-concentration jasmonic acid fermentation liquid, and spraying on Hibiscus sabdariffa to stress; s3: preparing 10-100 mu mol/L jasmonic acid solution, spraying the roselle under stress in sunny days, and after spraying, respectively carrying out random sampling at different time periods and then carrying out extraction detection. The content of resveratrol in roselle can be improved by 83 times to the maximum under the combined action of three stresses, and the high performance liquid chromatography and the ultra high performance liquid chromatography tandem mass spectrometry can be used for qualitative and quantitative determination of the resveratrol in the roselle.
Description
Technical Field
The invention relates to the technical field of biosynthesis, in particular to a stress method for improving the content of resveratrol in roselle.
Background
Resveratrol (Resveratrol), a polyphenol compound, was isolated by japanese from the root of a plant Resveratrol (Veratrum grandiflorum) in 1940 at the earliest, is a chemopreventive agent for tumors, and also for reducing platelet aggregation, preventing and treating atherosclerosis, cardiovascular and cerebrovascular diseases, and delaying aging. It has its stature in up to 72 plants, such as peanut, grape, giant knotweed, mulberry, pomegranate, cranberry, blueberry, and the like.
At present, the industrialized raw material of resveratrol is the root of giant knotweed. Giant knotweed rhizome is a drug which is legally not allowed to be used in foods, health products or cosmetics. The extraction of resveratrol with giant knotweed rhizome is accompanied by the difficult removal of emodin, which causes diarrhea.
The resveratrol content of natural sources in edible plants is very low, and it is found that Roselle, also known as Roselle, belladonna and Hibiscus, which are annual herbaceous plants or perennial shrubs of the genus Hibiscus of the family malvaceae, grows in tropical and subtropical regions and can be up to 2-2.5 m high, contains resveratrol in a Roselle plant, which is translated from the english name Roselle. Under the stress of jasmonic acid with certain concentration, the content of resveratrol in roselle is increased by several times to dozens of times, the industrial value of extracting resveratrol from roselle is greatly improved, and the medical and health care values are also improved. In addition, roselle contains flavone, protocatechuic acid, anthocyanin, isoflavone, abundant amino acids, vitamins, saccharides, organic acids and other chemical components, can reduce cholesterol and triglyceride, inhibit oxidation of low density lipoprotein, inhibit aggregation of blood platelet, reduce thrombosis, reduce atherosclerosis, and effectively prevent cardiovascular diseases.
Roselle protocatechuic acid PCA can accelerate the death of blood cancer cells; hibiscus sabdariffa extract (RS) is effective in inhibiting colon cancer carcinogen (AOM); roselle polyphenol (HPE) accelerates the death of gastric cancer cells; hibiscus sabdariffa anthocyanosides (HAC) accelerate the death of blood cancer cells. Roselle can also regulate blood pressure and improve sleep.
Jasmonic acid (also known as jasmonic acid) is a fragrant cyclopentane type compound, chemical name: trans-3-oxo-2- (cis-2-pentenyl) -cyclopenta-cetic acid, english name: jasmonic Acid has good application value in the fields of agriculture, industry and medicine, and particularly, in recent years, jasmonic Acid and esters thereof are found to play a plurality of special physiological roles on organs and cells of plants, thereby causing physiological growthGeneral attention of the chemist. Biologically synthesizing jasmonic acid, i.e. using a special bacterium, culturing for a certain time under the proper condition in a specific culture medium, then producing a certain amount of jasmonic acid in the culture solution, different strains, different culture media, different culture conditions, and the yield of jasmonic acid is from 1 mg.L -1 To 1080 mg. L -1 The difference is very large, the yield is mainly influenced by the strain, and the culture medium and the culture conditions are the second.
Disclosure of Invention
The invention aims to solve the defects of low resveratrol content and difficult extraction in roselle in the prior art, and provides a stress method for improving the resveratrol content in roselle.
A stress method for improving the content of resveratrol in roselle is characterized by comprising the following steps:
s1: selecting healthy roselle, spraying and stressing by using a mixed solution containing 0.04-0.6% of acetic acid, 0.01-0.02% of ammonium molybdate, 0.01-0.02% of sodium selenate, 0.01-0.02% of tin dichloride and 0.02-0.03% of potassium iodide, and then carrying out drought stress for three days;
s2: separating the cocoa-trichoderma from tropical fruits, and adding the cocoa-trichoderma into a culture solution, wherein the culture solution comprises the following raw materials in parts by weight: 25-75g/L of carbon source, 1-3g/L of corn steep liquor, 3-7g/L of sodium nitrate, 0.001-0.003g/L of manganese sulfate, 0.001-0.003g/L of copper sulfate, 0.01-0.03g/L of zinc sulfate, 2-4g/L of magnesium sulfate, 0.3-0.6g/L of ferrous sulfate, 0.3-0.5g/L of potassium chloride and 1-2g/L of potassium dihydrogen phosphate, standing and fermenting for 6 days at the temperature of 28-30 ℃ to obtain high-concentration jasmonic acid fermentation liquor, and spraying roselle with stress;
s3: preparing 10-100 mu mol/L jasmonic acid solution, spraying the roselle under stress in sunny days, and respectively carrying out random sampling at different time periods after spraying, and then carrying out extraction detection;
s4: weighing 1-2g of the roselle sample stressed in the steps S1, S2 and S3, adding 10-12mL of methanol, carrying out ultrasonic treatment for 30-35min, soaking for 24-26h, taking 5-6mL of extracting solution, adding PSA + activated carbon for purification, shaking for 2-3min, centrifuging for 5-6min, taking 1mL of supernatant, adding methanol to a constant volume of 2mL, and carrying out mass spectrometry after passing through a membrane.
Preferably, in the S1, robust roselle is selected, a mixed solution containing 0.05% of acetic acid, 0.01% of ammonium molybdate, 0.01% of sodium selenate, 0.01% of tin dichloride and 0.02% of potassium iodide is used for carrying out spraying stress, and then drought stress is carried out for three days.
Preferably, in S2, the tropical fruit is one or more of mango, longan, lychee, banana, pineapple and clausena lansium.
Preferably, in S4, the mass spectrometry conditions are: the parent ion was 227.1m/z, and the daughter ions were 142.7m/z and 184.5m/z.
After the jasmonic acid culture solution is diluted to a certain concentration, the jasmonic acid culture solution is sprayed on the surface of roselle to generate a stress effect, so that the content of resveratrol is increased. Before the jasmonic acid is sprayed, roselle is sprayed with a solution of 0.1% of trace elements and acetic acid, stress of the trace elements and the acetic acid is carried out, and then drought stress is carried out. Under the combined action of the three stresses, the content of resveratrol in roselle can be improved by 83 times to the maximum, and simultaneously, high Performance Liquid Chromatography (HPLC) and ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) are used for carrying out qualitative and quantitative determination on the resveratrol in the roselle.
Drawings
FIG. 1 is a process flow diagram of the stress process for increasing the resveratrol content in roselle according to the invention;
FIG. 2 is a scanning diagram of a methanol parent ion in positive ion mode under chromatographic detection;
FIG. 3 is a methanol parent ion scan in negative ion mode under chromatographic detection;
FIG. 4 is a scanning image of a parent ion of a resveratrol standard solution in positive ion mode under chromatographic detection;
FIG. 5 is a scanning image of a mother ion of a resveratrol standard solution in a negative ion mode under chromatographic detection;
FIG. 6 is a scanning image of the parent ion of Hibiscus sabdariffa extract in positive ion mode under chromatographic detection;
FIG. 7 is a scanning image of the anion mode of the mother ion of roselle extractive solution under the chromatographic detection.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Examples
Referring to fig. 1, the stress method for improving the resveratrol content in roselle provided by the invention comprises the following steps:
(1) The original creativity is that the roselle contains resveratrol for the first time, and the content of the leaves is higher than that of the flowers and the trunks.
(2) In October each year, relatively strong roselle is selected, sprayed with a solution containing 0.05% of acetic acid, 0.01% of ammonium molybdate, 0.01% of sodium selenate, 0.01% of tin dichloride and 0.02% of potassium iodide for stress, and then subjected to drought stress for three days.
(3) Preparing solutions with different jasmonic acid contents for spraying stress, spraying jasmonic acid solutions with concentrations of 10 mu mol/L, 20 mu mol/L, 40 mu mol/L, 60 mu mol/L and 100 mu mol/L (500 mL/plant) in a sunny day, then respectively carrying out random sampling on 4H, 8H,24H, 48H and 72H after spraying, and then carrying out extraction detection, wherein the result shows that the content of resveratrol is increased along with the increase of the concentration of jasmonic acid, and the stress effect is weakened after the concentration of the resveratrol is higher than 60 mu mol/L.
(4) The content of the new variety of roselle is between 100 and 160mg/kg, and after three stresses, the optimal condition can reach 4500mg/kg, thereby completely meeting the requirement of large-scale industrial production.
(5) The extraction method comprises the following steps: weighing 1.00g of sample, adding 10mL of methanol, carrying out ultrasonic treatment for 30min, and soaking for 24h. And (3) adding PSA + activated carbon into 5mL of extracting solution for purification, shaking for 2min, centrifuging for 5min, taking 1mL of supernatant, adding methanol to fix the volume to 2mL, and performing on a machine for determination after membrane filtration.
The operating conditions of the machine are as follows: the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry UPLC-MS/MS adopts direct sample injection by mass spectrometry, ESI negative ion mode and instrument model AB 4000+. Mass spectrum conditions: the parent ion was 227.1m/z, and the daughter ions were 142.7m/z and 184.5m/z.
The determination results of the resveratrol content in roselle under different stress modes and common stress action are shown in the following table:
the concentrations of the components of the medium and the culture conditions all affect the yield of jasmonic acid, and the types and concentrations of the carbon source (sucrose, lactose, starch, maltose, glucose), the types and concentrations of the nitrogen source (sodium nitrate, ammonium nitrate, corn steep liquor, yeast extract, beef jelly), the salts and the trace elements (manganese sulfate, magnesium sulfate, ferrous sulfate, potassium chloride and potassium dihydrogen phosphate) are determined by orthogonal experiments. The highest yield of jasmonic acid produced by a correct method can reach 1080 mg.L -1 After the culture solution is diluted to a certain concentration, the surface of roselle is sprayed to generate a stress effect, and the content of resveratrol is improved. Meanwhile, before the jasmonic acid is sprayed, roselle is sprayed with a solution of 0.1% of trace elements and acetic acid to stress the trace elements and the acetic acid, and then drought stress is carried out. Under the combined action of three stresses, the content of resveratrol in roselle can be improved by 83 times to the maximum. Performing qualitative and quantitative analysis on resveratrol in Hibiscus sabdariffa by High Performance Liquid Chromatography (HPLC) and ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), with the determination results shown in FIGS. 2-7.
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 (4)
1. A stress method for improving the content of resveratrol in roselle is characterized by comprising the following steps:
s1: selecting healthy roselle, spraying and stressing by using a mixed solution containing 0.04-0.6% of acetic acid, 0.01-0.02% of ammonium molybdate, 0.01-0.02% of sodium selenate, 0.01-0.02% of tin dichloride and 0.02-0.03% of potassium iodide, and then carrying out drought stress for three days;
s2: separating the cocoa-trichoderma from tropical fruits, and adding the cocoa-trichoderma into a culture solution, wherein the culture solution comprises the following raw materials in parts by weight: 25-75g/L of carbon source, 1-3g/L of corn steep liquor, 3-7g/L of sodium nitrate, 0.001-0.003g/L of manganese sulfate, 0.001-0.003g/L of copper sulfate, 0.01-0.03g/L of zinc sulfate, 2-4g/L of magnesium sulfate, 0.3-0.6g/L of ferrous sulfate, 0.3-0.5g/L of potassium chloride and 1-2g/L of potassium dihydrogen phosphate, standing and fermenting for 6 days at the temperature of 28-30 ℃ to obtain high-concentration jasmonic acid fermentation liquor, and spraying roselle with stress;
s3: preparing 10-100 mu mol/L jasmonic acid solution, spraying the roselle under stress in sunny days, and respectively carrying out random sampling at different time periods after spraying, and then carrying out extraction detection;
s4: weighing 1-2g of the roselle sample stressed in the steps S1, S2 and S3, adding 10-12mL of methanol, carrying out ultrasonic treatment for 30-35min, soaking for 24-26h, taking 5-6mL of extracting solution, adding PSA + activated carbon for purification, shaking for 2-3min, centrifuging for 5-6min, taking 1mL of supernatant, adding methanol to a constant volume of 2mL, and carrying out mass spectrometry after passing through a membrane.
2. The stress method for increasing the content of resveratrol in roselle as claimed in claim 1, wherein in S1, robust roselle is selected, sprayed with a mixed solution containing 0.05% of acetic acid, 0.01% of ammonium molybdate, 0.01% of sodium selenate, 0.01% of tin dichloride and 0.02% of potassium iodide for stress, and then subjected to drought stress for three days.
3. The stress method for increasing the content of resveratrol in roselle according to claim 1, wherein in S2, the tropical fruit is one or more of mango, longan, lychee, banana, pineapple and Chinese wampee.
4. The stress method for increasing the content of resveratrol in roselle according to claim 1, wherein in S4, the conditions of mass spectrometry are as follows: the parent ion was 227.1m/z, and the daughter ions were 142.7m/z and 184.5m/z.
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