Research of the effect of Cistanche On Free Radicals

For more information:Ali.ma@wecistanche.com

Second Cloud Forest An Old Zhang Hongjing

At present, it is believed that the decline in the immune function of the machine is related to aging. Based on the study that Cistanche can enhance the humoral and cellular immune functions of mice and fight against the aging model caused by d-galactose, we further studied the effect of the drug on mice. Inhalation of ozone <. 3) Induces strong antagonistic effects of aging and aging caused by strong free radicals in the body. The chemiluminescence expression rate of mice in the drug group increased, SOD Increased vitality "The content of lipid peroxides in plasma, kneeling and thigh tissues and MAOB vitality of corridor tissues decreased; there was a dose-effect relationship between different dose groups. The anti-aging effect of mice against subacute aging is consistent with the study of Cistanche's enhancement of immune function in mice. Cistanche can enhance immune function, improve the body's anti-stress ability, and it is appropriate to receive or indirectly eliminate the damage of free radicals to the body. , Inhibit the vitality of 1WMAO-B. So as to achieve the effect of delaying aging. This experiment provides the experimental basis for the application of Cistanche in anti-aging and treating certain senile diseases.

Click to cistanche benefits and side effects for free radicals

1 Material and methods

1.1 Material

1.1.1 Drug and reagents: Cistanche deserticola Y. C. Ma was decocted in water 3 times, the decoctions were combined, concentrated to an appropriate volume, and a certain amount of ethanol was added. After standing for 24 hours, it was filtered. The filtrate was evaporated to no alcohol. Prepare with steaming water before using 0.25%~0.5% concentration. ml contains crude drugs about lg). Luminol (luminol), ®National E Merck company products Huang Howling Oxidation 88 (HO), Hypochromatic Howling (HX), standard tetra ethoxy propane, all are Sigma The company's product, salt, benzalkonium, Changzhou Guangming Biochemical Research Institute; cyclohexane, a product from Shanghai Reagent No. 1; Thiobarbituric acid, a product from Shanghai Reagent No. 2, Luzhan Superoxide Dismutase, Shanghai Chinese Academy of Sciences Products of the Institute of Biochemical Products.

Cistanche

1.1. 2 Instrument Beckman LS-98OO33 liquid scintillation counting only, Shimadzu RF-540 fluorescence spectrophotometer, 752 ultraviolet gratings spectrophotometer-G1-20A high-speed refrigerated centrifuge, CPS-1A ultrasonic pulverizer.

1.1.3 Animal ICR mice, 2 months old, weighing 20±2g, provided by Jiangsu Experimental Animal Center.

1.2 method

The mice were divided into control (do not enter the Q environment), model, and medication group, each with 10 mice, fast water eating without restriction. The medication group was divided into two dose groups, Volume 2, Issue 7, 1994, Chinese Journal of Chinese Materia Medica Cistanche extract100 and 200 mg/kg (equivalent to crude drugs 20 and 4Qg/kg) were fed respectively. The control group and the model group were given an equal volume of normal saline every day. The administration group and the model group were given O, environment (0.9 X 10-^Oaft degree), continuous 2. Day. The test of each finger strain was carried out on the 21st day of the experiment.

1. 2.1 Endurance test Put the mice in each of the above groups into a water depth of 40cm and 12±l. In the container of Water®L, start timing until it sinks to the bottom and does not move, which is the swimming time for the mice.

   
   

1.2.2 Warm-proof test The mouse is set to 15 first. Swim in the water for 2 minutes, and then enter a 15P freezer to observe the complete freezing time.

1.2.3 Atmospheric hypoxia tolerance test Put the small gluten into a 250ml wide-mouth bottle filled with sodium-lime, and record the survival time of the mice in each group after sealing.

1.2.4 Determination of brain type B monoamine oxidase (MACXB) According to the literature oral method, take the whole brain of mice, add an appropriate amount of pre-cooled 0.2mol/L phosphate buffer (PH7.4), homogenate, and ultrasound (24kC) Crush 3 times, a total of two times, with an interval of 30sr1000r/min, 0C for 30min, take the supernatant at 17000r/min, 4C for 30min, discard the supernatant and take the precipitate, and use an appropriate amount. .2mo(/L PH7.4) The acid is buffered and resuspended to make a crude fermentation solution. Take an appropriate amount of crude fermentation solution, add 16mmol/L benzamidine 0.3ml, use the above phosphate buffer to make up 3mL, and use phosphoric acid drum 3ml of buffer solution is used as a blank tube. All test tubes are placed at 37. Shake in a water bath for 3 hours, take it out, add 6% peroxy acid to terminate the reaction, then add 3m [cyclohexane, shake to mix, centrifuge at 1000r/min for 1 Omin, and take it up The absorbance of the clear solution is measured at 242nm, and the blank tube is to be kept warm before adding Chinese amine. The protein turbidity is determined by the Folin-phenol method, with calf serum albumin as the standard protein. One enzyme activity unit and one enzyme activity unit are defined as 37P produces the amount of enzyme with a change in absorbance of 0.01/3h.'

1. 2. 5 Serum superoxide dismutase (SOD) activity determination (5 mu I, mouse serum 10M, added to the luminescent mixture (mixed by 0, 1mmol/L luminol and 0.1mmol/L hypoxanthine) In 980", keep it in a 37C incubator for 20 minutes. After taking it out, add Huang Guanyin oxidase to start the reaction. In the Beckman LS~98OO liquid flash medium, the number of luminescence at the end of 1 min is calculated based on the degree of single-photon detection. Com is used as the unit. The luminescence inhibition rate is calculated with the inferior serum group as a blank. In addition, the standard superoxide dismutation concentration (abscissa) and luminescence inhibition rate (coordinate) is used as the standard protein content determination as before. 1. 2.6 Determination of Lipid Peroxide (LPO) in Plasma, Brain, and Liver ④ Take 50 cans of mouse plasma, homogenize liver and brain (use 0.2mol/L phosphate buffer pH7.4, in a homogenizer Made in 10% concentration) every 50 ancestors, add l/24mol/L sulfuric acid 4ml, 1% Phosphoric acid 0.5ml, mixed and placed for 5min, centrifuged at 1000r/mm for 10min, and aspirated the supernatant. Add 2ml of l/24mol/L sulfuric acid and 0.3ml of 10% calcium phosphate to the precipitate, mix well, centrifuge at 1000r/min, and add 1ml of resisted cowardly water to the precipitate, mix well to form a suspension, add broken barbital Acid reagent 1ml, at 95. After cooling for 14 in an oil bath, add 5ml n-butanol, mix well *100Or/min centrifugation 10mtn, take the n-butanol phase, and measure the fluorescence intensity on the RF-54OS spectrophotometer (excitation wavelength 515mn, emission length 553nm), In addition, use standard tetra ethoxy propane and relative fluorescence intensity as a standard curve.

   
   

2. 

2 results

2.1 The impact on the endurance test of mice injured by free radicals

The swimming time of mice in the model group was significantly shorter than that of the control group, while the swimming time of the administration group lasted longer, and the high-dose group was similar to the control group (Table 1).

Table 1： The effect of Cistanche on swimming endurance test of free tomb injury mouse model

2.2 Impact on the ability to keep warm

Mice in the model group freeze quickly at low temperatures, and the time is shorter than that of the control group, while the freezing time of the two-dose groups of Cistanche is not only longer than the model group but also longer than the control group, indicating that Cistanche not only has the effect of anti-free radical damage but also has obvious effects. Increase the ability to keep warm from the cold (Table 2).

Table 2： The effect of Cistanche on the ability of Senhan in mice model of free good injury

2.3 Impact on the ability to withstand hypoxia at atmospheric pressure

After administration of Cistanche, mice survived under normal pressure and hypoxia. Cistanche’s effect on the ability of free-kind injury mouse model Senhan was significantly longer than that of the model group and also higher than that of the control group, indicating that Cistanche can increase the mice’s tolerance to hypoxia. Force (Table 3)

Table 3： Cistanche's mouse model of free radical damage

2.4 Impact on the vitality of MAO-B

After inhalation of O, the brain tissue MAO-B activity of the model group was significantly higher than that of the counterpart group, and the increase of MAO-B activity of the administration group was significantly inhibited

Table 4： The effect of Cistanche on the activity of 1SB type monoamine oxide vine (MAO~B) in the free tomb injury mouse model

2. 5 Impact on SOD vitality

The chemiluminescence inhibition rate of serum in the model group was significantly lower than that of the other groups, and the SOD activity was significantly decreased. The SOD activity of the drug group was significantly increased, and the high-dose group was higher than that of the control group (see below table 5）

Table 5： Model serum superoxide dismutase (SOD) activity is the loudest2.6 Effect on the content of lipid peroxide (ZLPO)

2.6 The lipid peroxide (LFO) content in the plasma, liver and brain tissue of the model group was significantly increased, and the LPO content was significantly decreased after administration. The large dose and dose group had reduced the LPO in the brain tissue to the level of the control group (Table 6).

Table 6： The effect of meat Axun on plasma, liver, and lipid peroxide (LPO) levels in a mouse model of free radical damage

3 Discussion

Aging is a positive phenomenon in the process of life. The traditional medicine of the motherland has accumulated rich experience and medicine sources in anti-aging years. In recent years, domestic scholars have focused on finding effective anti-aging drugs from traditional Chinese medicine. At present, the free radical theory of body aging is increasingly being looked at. Although free radicals are generated in the body under normal conditions, the tissue cells are protected due to the continuous removal of SOD and other anti-oxygen toxicity enzyme systems. Oa is a strong oxidant. , Can start the free radical reaction in the body. Excessive inhalation of oxygen causes the generation of free radicals in the body, which exceeds the scavenging ability of the body's anti-tolerance stele system, causing damage to nucleic acids, proteins, amino acids, carbohydrates, and lipid compounds, thereby damaging the formation and function of cells, and leading to aging ⑶. This experiment shows that after inhaling oxygen7, the mice's physical strength, ability to keep warm, and tolerance to hypoxia are all reduced, similar to the performance of the physiological function decline of old animals. It shows that the young mice can cause an aging model after receiving d stimulation. Cistanche has significantly improved the changes in the above indicators.

Superoxide free radicals exist in the body for a short time and are mainly eliminated by SOD. Generally, the level of or is reflected by measuring the activity of SOD. Various antioxidants in the body of naturally aging mice and free radical-damaged mice are reduced, The activity of sod decreases, thereby reducing the ability to scavenge free radicals, leading to an increase in the content of lipid peroxides. The activity of type B monoamine oxidase (MA0-B) in the brain increases with age. MAOB is the main control of the aging biological clock in the central hypothalamus Clockwork is closely related to aging. ⑴. The activity of MA0-B in the brains of mice in this experimental model group is increased, showing biochemical changes consistent with those of aging animals.