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Method for extracting antioxidant from myricaria laxiflora and application thereof

A technology of thinning flowers and cypress branches and antioxidants, which is applied in the directions of antidote, medical preparations containing active ingredients, and pharmaceutical formulas.

Inactive Publication Date: 2015-01-07
CHINA THREE GORGES UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no relevant report on the application of anti-oxidation by obtaining alcohol from the branch of Cypress laxatives, especially for the application of anti-oxidative protection of nerve cells from oxidative damage

Method used

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  • Method for extracting antioxidant from myricaria laxiflora and application thereof
  • Method for extracting antioxidant from myricaria laxiflora and application thereof
  • Method for extracting antioxidant from myricaria laxiflora and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Separation and extraction steps of antioxidants:

[0018] Dry and pulverize the Shuiba branch in a drying oven at 50°C; (2) Weigh 100g of the pulverized powder and put it into a 1000mL Soxhlet extractor, add 500mL of 95% ethanol according to the ratio of material to liquid at 1:5, and heat at 80°C Reflux at normal pressure for 2 hours, repeat the operation 3 times until the solution in the extractor is almost colorless; (3) combine the extracts for 3 times and filter, concentrate under reduced pressure and low temperature to obtain 95% ethanol extract extract; (4) extract the extract Soak with an appropriate amount of 4% HCL to obtain acid water; (5) acid water with saturated NaCO 3 Adjust the pH to 7; (6) extract the solution in step 5 with chloroform, and concentrate under reduced pressure at low temperature to obtain the chloroform extract, (7) the chloroform extract is subjected to chloroform-methanol gradient elution silica gel column chromatography to collect chlo...

Embodiment 2

[0025] Cytotoxicity evaluation: Dilute the SH-SY5Y cells in the logarithmic growth phase and inoculate them in 96-well plates. When the nerve cells grow to 80% confluence, add concentrations of 100ug / mL, 50ug / mL, 25ug / mL, 12.5 ug / mL, 6.25ug / mL of this antioxidant and propyl gallate, the dosage is 100uL / well. After culturing for 24 hours, discard the culture medium, add 100 μL medium containing 0.5 mg / mL MTT to continue culturing for 4 hours, discard the supernatant, add 150 μL DMSO to each well, shake the 96-well plate gently to fully dissolve the formazan particles, and use whole The automatic enzyme-linked immunosorbent assay instrument measures the absorbance (OD) value at a wavelength of 492nm, and calculates the cell survival rate (survival rate = OD value of the treatment group / OD value of the blank group × 100%). Table 1 shows the absorbance of the antioxidant and PG (OD) value. When the concentration of antioxidants is 0.5-12.5ug / mL and the dosage is 100uL, the toxici...

Embodiment 3

[0029] h 2 o 2 Oxidative damage model: when the nerve cells grow to 80% confluence, discard the old culture medium and add different concentrations of 0, 400, 600, 800, 1000 μmol / L H 2 o 2 solution (made by diluting the basal medium), the blank control group only added the basal medium, and after continuing to culture for 8 hours, the absorbance value was measured by the MTT method, and the cell survival rate was calculated. select H 2 o 2 Concentration 800μmol / L, 50%-60% dead cells for subsequent experiments.

[0030] Evaluation of antioxidant capacity: SH-SY5Y cells were pre-administered when they grew to 80% confluence, and PG with concentration gradients of 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, and 1.0625 μg / mL were added (Propyl gallate) and this antioxidant (prepared from basal medium), each concentration of 5 replicate wells. After culturing for 24 h, the culture medium was discarded, and the model group was added with H at a concentration of 800 μmol / L ...

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Abstract

The invention relates to an extraction method of an antioxidant and an application in protection of nerve cells against oxidation by utilizing the antioxidant, belonging to the technical field of biological medicines. The extraction method disclosed by the invention comprises the following steps of: taking myricaria laxiflora as a raw material, taking ethanol as an extraction solvent, performing Soxhlet extraction, performing cold leaching on a crude extraction solution with HCl, adjusting the pH with saturated NaCO3, extracting with chloroform, performing chloroform-methanol gradient elution by silica gel column chromatography, performing Sephadex LH-20 gel column methanol elution, and further performing crystallization and re-crystallization to obtain the antioxidant. The antioxidant is identified as 3, 4-dimethoxy-5-methylparaben by 1H-NMR and 13C-NMR, the molecular formula is C10H12O5, and the anti-oxidation property and the free radical scavenging activity are stronger. The concentration of the antioxidant is 0.5-12.5mu g / mL, when the dosage is 100mu L, the toxicity of the natural antioxidant against SH-SY5Y cells is less than 3.4%, the relative survival rate against human neuroblastoma cells SH-SY5Y after oxidative damages by hydrogen peroxide is 50-80%, and the antioxidant has a better anti-oxidation protection effect in comparison with PG (propyl gallate), can be used as a natural non-toxic antioxidant and can be applied to the fields of foods, medicines, cosmetics and the like.

Description

technical field [0001] The present invention relates to an antioxidant extraction method and the application of the antioxidant to protect nerve cells from oxidation, in particular to an antioxidant extraction method using S. Antioxidative protection of damaged human neuroblastoma cells SH-SY5Y. Background technique [0002] Antioxidants have various functions and are widely used in food, medicine, cosmetics and other fields. Antioxidants can be divided into two categories: synthetic antioxidants and natural antioxidants. Synthetic antioxidants such as BHT (dibutyl hydroxytoluene), BHA (butyl hydroxyanisole), PG (propyl gallate), etc. Synthetic antioxidants are potentially harmful to health, while natural antioxidants have strong antioxidant and free radical scavenging capabilities, and are more popular because of their natural non-toxicity. [0003] Myricaria laxiflora, belonging to the genus Myricaria of the Tamaricaceae family, has the effects of dispelling wind and re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/235A61P39/06C07C67/48
Inventor 刘士平田伟薛艳红曾文孔玉珊仇敏
Owner CHINA THREE GORGES UNIV
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