Research Of Cistanche Polysaccharides Scavenging Free Radicals
Mar 06, 2022
For more information:ali.ma@wecistanche.com
Wang Guowei Zhao Fang
(School of Chemistry and Chemical Engineering, Shihezi University, Beisi Road, Shihezi City, Xinjiang 832000)
Keywords: Cistanche; Cistanche polysaccharide, polysaccharide from cistanche; free radical
The body continuously produces various reactive oxygen radicals (ROS) in the process of oxidative metabolism of life activities, which are intermediate products produced by the redox reaction of aerobic metabolism of the human body. Studies have found that excess free radicals will attack life macromolecular substances and various organelles, causing various damages to the body at the molecular, cellular, and tissue organ levels, accelerating the body’s aging process and inducing blood clots, diabetes, and atherosclerosis. , Tumors and other diseases [1]. As chemically synthesized antioxidants are toxic to the human body, the search for natural antioxidants with high efficiency and low toxicity that can scavenge oxygen free radicals has become a hot spot in the fields of medicine, food, and chemical engineering in recent years.
Cistanche [Cistanche deserticola (Y. C. Ma.)] mostly parasitizes on the roots of Tamarisk, Nitraria, and Haloxylon ammodendron. It is a rare Chinese medicine in my country and is known as "desert ginseng"[2]. Cistanche cistanche polysaccharide is one of the main biological functional active components of cistanche, but it has not been reported whether the cistanche polysaccharide has the effect of scavenging free radicals. In this paper, the method of spectral analysis was used to determine the scavenging effect of Cistanche polysaccharide on superoxide anion free radicals, hydroxyl free radicals, D PPH radicals, and singlet oxygen free radicals, so as to evaluate its antioxidant activity. Make full use of this natural resource to provide a scientific basis.
Click to Cistanche stem for free radical
This research studies Cistanche polysaccharide in vitro antioxidant activity. Taking ascorbic acid as a control, the Fenton-like reaction produces hydroxyl free radicals, the autooxidation of dopamine produces superoxide anion free radicals, the reaction of sodium hypochlorite and hydrogen peroxide to produce singlet oxygen, diphenyl bitter hydrazine radicals as the experimental model, using ultraviolet 2Visible spectrophotometry and chemiluminescence analysis method, for the first time to determine the scavenging effect of Cistanche polysaccharide on superoxide anion free radicals, hydroxyl free radicals, D PPH · free radicals (diphenyl hydrazino radicals) and singlet oxygen. The results showed that the polysaccharides of Cistanche had obvious scavenging effects on the above-mentioned free radicals, and the scavenging effects were positively correlated with the concentration of polysaccharides.
Apparatus and reagents: UV 22401 ultraviolet-visible spectrophotometer (Shimadzu Corporation, Japan); Lum at LB9507 ultra-sensitive tube luminometer (Berthold Technologies, Germany); R E252CS rotary evaporator (Shanghai Yarong Biochemical Co., Ltd.) Instrument Factory); HW 2SY electronic constant temperature water bath pot (Jintan Xinhang Instrument Factory); pH S23C acidity meter (Shanghai Precision Scientific Instrument Co., Ltd.); D ZF26020 vacuum drying oven (Shanghai Yiheng Technology Co., Ltd.).
Cistanche extract is purchased from Kuitun Medicinal Materials Corporation. Luminol (Luminol, Sigma, USA), Dopamine (China National Institute for the Control of Pharmaceutical and Biological Products), FeSO 4, ED TA, Vitamin C, H 2O 2, NaClO, Na2CO 3, NaHCO 3, etc. It is a domestic analytical reagent. The experimental water is ion-exchanged distilled water.
The extraction method of Cistanche polysaccharides: Take 30g of cistanche powder and place it in a 500mL Erlenmeyer flask, and soak in petroleum ether (60-90℃), ether and a volume fraction of 80% ethanol hot solution (30℃) for 6h, and the residue will be evaporated to dryness. . The filter residue was soaked in water at a solid-liquid ratio of 1:10 for 24 hours, and heated at 30°C to extract 100 min. After the extraction was completed, the supernatant was obtained by centrifugation. The supernatant was concentrated under reduced pressure and then precipitated by adding 3 times the volume of 95% ethanol, standing for 24 hours at 4 ℃, collecting the precipitate after centrifugation, and vacuum drying to obtain the crude Cistanche cistanche polysaccharide. Weigh 1. 00g of crude polysaccharide, dissolve it in water and dilute to a 50mL volumetric flask, and set aside for testing.
The test of cistanche polysaccharide on superoxide free radical (O) scavenging ability: the use of dopamine auto-oxidation to produce superoxide free radical test the cistanche polysaccharide on superoxide free radical scavenging ability [3]. Add 0.1mm olöL pH = 10.12 to the dry and graduated test tube in sequence, 3.0mL of carbonate buffer, 3.0mL of water, and 0.5mL of polysaccharide sample solution of different concentrations, and place it in a 30℃ water bath for preheating for 5 minutes. Add 1.0mL of 2.0mm olöL dopamine preheated in a water bath at 30°C, mix it immediately, keep the reaction at 30°C for 5 minutes, and immediately stop the reaction with 2 drops of 10molöL HCl. Use buffer as a reference, measure the absorbance at 221nm (Sample A), the blank group replaces the sample with 0.5 mL of water (A blank), and the control group replaces the sample with 0.5 mL of ascorbic acid. Each sample is tested in parallel 3 times and takes Its average value. O superoxide radical scavenging rate (%) = (A blank-A sample) öA blank × 100%.
Experiment on the Scavenging Ability of Cistanche Polysaccharide on ·OH Free Radical
The EDTA 2Fe (Ê) 2 methylene blue 2H 2O 2 system was used to test the ability of Cistanche polysaccharide to scavenge hydroxyl free radicals. The principle is that the Feton-like reaction between ED TA 2Fe(Ê) and hydrogen peroxide produces ·OH radicals, which reduces the characteristic absorption peak of methylene blue solution at 660nm. In a series of 10mL test tubes with stoppers, add 5mm oLöL FeSO 4 solution 1.0mL, 5mm oLöL EDTA solution 1.2mL, 0.03mol N aH 2 PO 4 2N a2H PO 4 (pH = 7.4) buffer solution 2 .0mL, 20mm olöL hydrogen peroxide solution 1.3mL, after shaking, add 0.2mm olöL methylene blue solution 0.7mL, and then add different concentrations of the test solution, vitamin C 1.0mL, and dilute with water to 10. 00mL, after mixing, incubate at 37°C for 30min, measure the absorbance of each solution at 660nm, and replace the sample solution with water in the blank group. Each sample was tested 3 times in parallel, and the average value was taken. The sample's OH radical scavenging rate (%) = [(A 2-A 1) ö(A 0-A 1) × 100%. In the formula: A 0 - absorbance value of the undamaged tube; A 1 - absorbance value of the damaged tube; A 2 - absorbance value after adding the sample.
Experiment on the scavenging ability of Cistanche polysaccharide on D PPH Free Radical
Take 0.1 mL of Cistanche deserticola polysaccharide solution and add it to 5.0 mL of D PPH · solution, mix quickly, and after standing for 10 min at room temperature, the absorbance change at 517 nm wavelength is measured [4]. Each sample was tested 3 times in parallel, and the average value was taken. The sample's clearance rate of D PPH · (%) = 1- [(A-B) öA 0] × 100%.
In the formula: A 0 - absorbance value of D PPH without sample addition (1.9mL D PPH + 0.1 mL 50% ethanol); A - absorbance value after the reaction between sample and D PPH; B - blank The absorbance value of the sample (sample 0.1mL + 1. 9mL 50% ethanol).
Cistanche polysaccharide scavenging singlet oxygen 1O 2 test
Singlet oxygen (1O 2) is produced by the chemical reaction between NaClO and H 2O 2 at 37°C [5]. The solution used for the determination of 1O 2 is 100L 2. 8mm olöL NaClO solution, 400L 5mm olöL NaOH solution, 300L 10mm olöL H 2O 2 solutions, 50L 1mm olöL Luminol solution. During the reaction, the reaction solution is added to the sample solution, and the chemiluminescence analyzer automatically records the reaction process at the same time. Take NaClO 2H 2O 2 2N NaOH 2 Luminol as a blank control. Start the reaction, and add the reaction solution from the reservoir of the chemiluminescence instrument to the sample tube according to the amount set by the program. Subsequently, the chemiluminescence intensity of the reaction mixture was recorded on the recording paper every 5s, and each sample was measured in parallel 3 times, and the average value was taken. The clearance rate is calculated according to the following formula: clearance rate (%) = (control luminous intensity-sample luminous intensity) ö control luminous intensity × 100% 3.
The scavenging effect of cistanche polysaccharides on superoxide free radicals
The chemical nature of superoxide free radicals is active, which can cause fatty acids on the biofilm to produce peroxides, which in turn destroys cells [6]. In this paper, the dopamine method was used to determine the scavenging ability of superoxide free radicals. See Figure 1 for the scavenging ability of Cistanche on superoxide free radicals. The results showed that: Cistanche polysaccharides can inhibit the superoxide free radicals produced by the auto-oxidation of dopamine, and within the measured concentration range (0-8m gömL), it increases with the increase of the concentration, but the scavenging ability is lower than that of ascorbic acid.
Scavenging effect of cistanche polysaccharide OH superoxide free radical
OH superoxide radical is one of the most active oxygen free radicals in active oxygen. It can react with almost any biological macromolecule in living cells, which can lead to lipid peroxidation of body tissues, protein polymerization, and depolymerization, nucleic acid fragmentation, etc. The biochemical process triggers tissue cell pathology, leading to the occurrence of various diseases and accelerating the body's aging [7]. Figure 2 shows the scavenging ability of cistanche polysaccharides on OH superoxide free radicals. The experimental results show that the polysaccharide of Cistanche has a good scavenging effect on ·OH free radicals, and it increases with the increase of the concentration.
The scavenging ability of Cistanche cistanche polysaccharide on D PPH free radical
D PPH radical (diphenyl-bitter hydrazino radical) is a stable free radical, which is purple in organic solvents and has a strong absorption near 517nm. After adding antioxidants, a part of free radicals will be eliminated and the absorption will be weakened, which can be used to evaluate the antioxidant activity of the substance [8]. The scavenging ability of Cistanche polysaccharides on D PPH free radicals is shown in Figure 3. The experimental results show that the scavenging ability of Cistanche deserticola polysaccharide on D PPH free radicals is positively correlated with its concentration, and the scavenging ability is greater than that of ascorbic acid.
The scavenging ability of cistanche polysaccharides on 1O 2 free radicals
Singlet oxygen is excited molecular oxygen with active chemical properties. Since unpaired electrons have a tendency to pair, free radicals show more active chemical properties [9]. Figure 4 shows the scavenging ability of Cistanche deserticola polysaccharide on 1O 2 free radicals. The experimental results show that the ability of Cistanche polysaccharides to remove singlet oxygen is lower than that of ascorbic acid in the range of 0-4.0m gömL. When the concentration is greater than 4.0m gömL, the removal ability of Cistanche cistanche polysaccharides is greater than that of ascorbic acid.
In conclusion: Through different reactive oxygen-free radical systems, the in vitro antioxidant activity of Cistanche polysaccharide was studied in detail. It can be seen from the experimental results that within the test concentration range, Cistanche polysaccharide extract has scavenging effects on superoxide free radicals O, OH superoxide free radicals, D PPH free radicals, and 1 O 2 free radicals. The effects are not the same. The results showed that Cistanche polysaccharide is an effective exogenous natural antioxidant, which is worthy of further research, development, and comprehensive utilization.
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