Study On The Antioxidant Activities Of Phenylethanoid Glycosides From Cistanche Deserticola

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

LI Li et al ( Central Laboratory of Changchun Normal University, Changchun, Jilin 130032)

Abstract [ Objective ] The research aimed to study systematically the antioxidant activities of phenylethanoid glycosides from Cistanche deserticola. [Method ] Two model systems, the free radical 2,2-diphenyl-l -picrylhydrazyl ( DPPH ' ) assay and the photo chemiluminescence (PCL) assay, were used to measure the antioxidant activity of the C. deserticola extract, acteoside, isoacteoside, and echinacoside. [ Result] The results showed that the total phenolic contents (TPCs) in echinacoside, acteoside, isoacteoside, and 80% ethanol extract of C. deserticola were 753.95, 659.94, 356.14, and 14.73 mg gallic acid /g sample. The C. deserticola extract showed strong antiox记ant activity in the PCL experiment, followed by echinacoside, acteoside, and isoacteoside. However, echinacoside exhibited strong antioxidant activity in DPPH experiment, followed by acteoside, isoacteoside, and the C. deserticola extract.

[ Conclusion ] This research could provide references for the pharmacology and biological activity study of phenylethanoid glycosides from C. deserticola.

Key words Cistanche deserticola ； Acteoside ； Isoacteoside ； Echinacoside ； Antioxidant activity ； DPPH ； PCL

Cistanche (Cistanche deserticolaY.C. Ma.) is a plant of the Cistanche genus (Oro-banehaeeeae) of the Cistanche genus (Cistanche). The dried fleshy stem of Cistanche with scaly leaves. A large number of studies have found that the main active ingredients of Cistanche plants are phenethyl alcohol glycoside compounds. . Phenylethanol glycoside compound belongs to a kind of polyphenol compound, which is widely distributed in many kinds of plants. Pharmacological test studies have shown that these compounds have a wide range of pharmacological and biological activities, such as anti-hepatotoxin, anti-inflammatory, analgesic, and antioxidant activities. Among them, the most striking thing is that the phenoxyethanol glycoside compound has very good antioxidant activity. The author mainly conducted a systematic analysis of the antioxidant activity of the phenethyl alcohol glycoside compounds in Cistanche, using two anti-oxidant in vitro evaluation methods, namely Photoehemilumineseence (PCL) and 2,2-diphenyl-1-pieryl-hydroxyl (DPPH') Two different systems were used to measure the antioxidant activity of the 80% ethanol extract of Cistanche, ergosteroside, isoergoside, and echinacoside.

1 Material and methods

1.1 Samples and reagents

Cistanche (Cistanche deserticola) medicinal materials were purchased from Beijing Tongrentang Pharmacy. Ergosteroside, isoergoside, echinacoside, Folin. Ciocalteu reagent, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH.), etc. were purchased from SigmaChemicalCo. Company (St. Louis, Mo). The solvent used for PCL analysis was provided by Analytik Jena AG (Berlin, Germany). Other reagents are all analytical reagents (produced by Beijing Chemical Plant). The water is ultrapure water (18.2 nPa).

1.2 Sample extraction

Weigh accurately 2.0005g of Cistanche sample powder, use 50rnl of 80% ethanol for ultrasonic extraction at room temperature for 0.5h, extract twice, filter the sample solution, rotary evaporate to dryness (<40oC), use 100ml of distilled water Dissolve, degrease twice with 100ml n-hexane, discard the n-hexane solution, extract the water part with 100 ml of water-saturated n-butanol 3 times, combine the n-butanol extracts, rotary evaporate to (<40oC), and use 4ml for the residue The volume of absolute ethanol is fixed to obtain a sample of Cistanche deserticola.

1.3 Determination of the total polyphenol content of the sample

The total phenol content of the sample is determined using the F0-lin-Ciocaheu method, and the result is expressed as milligrams of gallic acid per gram of extract. Take a sample solution with a certain concentration of 0.2rnI (to make the absorbance fall on the standard curve), add 1ml of Folin-Ciocaheu reagent with a concentration of 0.5mol/L, and then add 0.8ml of a sodium carbonate solution with a concentration of 7.5% to mix well, Placed at room temperature for 0.5h, and then measured the absorbance at 765nm (ultraviolet-visible spectrophotometer: DU800, Beckman Couher, Inc. USA). Use gallic acid solution (the mass concentration range is 0.02～0.20me,/m1) to make the standard curve, and all the tests are repeated twice.

1.4 Antioxidant activity in vitro analysis

1.4.1 PCL method determination.

The test used Photochem@(Berlin, Ger-many) ultra-fast antioxidant and free radical automatic analyzer. The principle is to generate free radicals by photochemical method and use the chemiluminescence method to detect free radicals. In the experiment, a photosensitizer with a photochemical excitation effect was used to excite the reaction molecules, so that under the action of the ultraviolet lamp, the oxidation reaction occurred 1000 times faster than normal conditions, and the free radicals were quickly generated." Choose the PCL-ACW-Kit method Measure the antioxidant activity of the water-soluble components of the psyllium sub-samples. Pipette 1.0ml reaction buffer (including the concentration of 0.1moL/L SodiumCarbonate, pH value of 10.5 and concentration of 0.1mmol/LNA, one EDTA, 1.5ml ACW- diluent (water), concentration 1mmol/LLumino1) acts as a photosensitizer to excite the reaction molecules, so that free radicals are quickly generated, and at the same time as a photochemiluminescent substance. Both the sample and the standard are 10 "l. By measuring different concentrations (0.5, 1.0, 2.0 and 3.0 nmol/L) of L. The standard curve equation for ascorbic acid is Y = 24.640Ox + 2.9311, R² = 0.9969. It is necessary to dilute the sample or standard solution in the test so that the lag time interval falls within the linear range of the standard curve. Photochem generates the standard curve and tests the antioxidant activity of the sample automatically, and the antioxidant activity of the sample is equivalent to L． Calculated by the number of nanomoles of ascorbic acid.

1.4.2 DPPH method determination.

Diphenylpicrylhydrazino radical [DPPH ·] is a stable nitrogen-centered radical with maximum absorption at a wavelength of 515nm. [DPPH ·] methanol solution is purple, and its concentration has a linear relationship with absorbance." After adding antioxidants to [DPPH ·] methanol solution, antioxidants can combine with [DPPH ·] free radicals or replace them to make [DPPH ·] ] The number of free radicals decreases, and the color of the solution becomes lighter, which is manifested by the continuous decrease in the absorbance at the wavelength of 515 nm until it reaches stability." In the test, ethanol was used instead of methanol. Accurately weigh [DPPH ·] and configure the concentration to 0. [DPPH ·] ethanol solution of 0.040 76mg/ml, the standard curve of [DPPH ·] is obtained: Y: 18.032x-0.0016, R = 0.9993. The method for measuring the residual rate of free radicals refers to the literature [16]. Using ethanol as the solvent, prepare different concentrations of sample and standard solution, take 0.1ml sample or standard solution respectively, and add 3.9ml [DPPH·] standard solution with a mass concentration of 2.5mg/L (currently used now) ), replace the sample solution with ethanol as a blank. Shake the mixed solution, use a cuvette to measure its absorbance at different times at a wavelength of 515nm, and convert it to the mass concentration of [DPPH·] according to the standard curve to calculate the residual rate of [DPPH·]. According to the residual rate of [DPPH ·] and the corresponding amount of sample added, a curve for removing [DPPH -] free radicals from the sample can be drawn. According to the relationship curve between the mass concentration of [DPPH ·] and the absorbance, the absorbance measured after the addition of the free radical scavenger can be converted into the mass concentration of [DPPH ·] to calculate the [DPPH ·] after the addition of the free radical scavenger. The calculation formula for the residual rate is as follows: [DPPH ·]REM=[DPPH·]/[DPPH·]r=0×100% where [DPPH·] is the mass concentration of DPPH· at a certain moment in the free radical scavenging process, [DPPH·]:. Is the original mass concentration of DPPH·. According to the number of antioxidants added and the residual rate of [DPPH ·], the relationship curve between the two is made, and the regression equation can be obtained through linear regression analysis. According to the regression equation, the antioxidant dose when the residual rate of [DPPH ·] is 50% can be calculated. Half inhibition. According to the [DPPH·] free radical scavenging curve of different concentrations of samples or standards, calculate the amount EC of the sample when the original mass concentration of [DPPH·] is reduced to 50% (steady-state). The smaller the EC, the free radical scavenging ability The stronger". Test 3 times in parallel.

cistanche

2 Results and analysis

2.1 Determination of total polyphenol content of the sample In the determination of total polyphenol content, the total polyphenol content of echinacoside is the highest, which is 753.95 mg gallic acid/g. Followed by ergosteroid glycosides, isoergosteroid glycosides, and crude extracts of Cistanche, which were 468.3, 356.14, and 14.73 mg gallic acid/g, respectively.

2.2 Determination of sample antioxidant activity

2.2.1 PCL- ACW method determination results. In the PCL-ACW antioxidant test, the antioxidant capacity of 1txg Cistanche medicinal material is equivalent to the antioxidant capacity of 15.68nmol L-ascorbic acid; 1 g echinacoside is equivalent to the antioxidant capacity of 14.03nmol ascorbic acid Activity: 1txg ergosteroside is equivalent to 11.44nmolL an antioxidant capacity of ascorbic acid; 1Ixg isomergosteroside is equivalent to the antioxidant activity of 8.24nmol ascorbic acid.

2.2.2 DPPH method measurement results. It can be seen from Table 1 that it is cleared. The order of the ability of free radicals is ergosteroside, echinacoside, isoergoside and the crude extract of Cistanche, among which ergo side and echinacoside have basically the same antioxidant capacity in the DPPH test. It can be seen from this. Because different in vitro antioxidant test systems are used to measure samples, the mechanism of antioxidant effects is different, and the results may be inconsistent. This has also been verified in the test. Therefore, it is necessary to select other antioxidant analysis systems for further verification.

echinacosie

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