Study On The Anti-hypoxia Effect Of Garlic Total Saponins And Its Mechanisms

Hypoxia is a very pervasive pathological process in many cases such as breathing, blood and circulation system diseases, and some special environments including high altitude, deep sea and aerospace. Hypoxia is very harmful even life-threatening to our human beings, it can result in diseases such as acute high altitude reaction or mountain sickness because of high altitude hypoxia, or make existing diseases more serious. It is very necessary to take active measures for the prevention and treatment of hypoxia. Therefore, the research and development of more effective and economic anti-hypoxia-products is very important.Garlic is planted worldwide, and it has been used as common foods and for the treatment of many diseases since ancient times. Many important effects have been gradually confirmed by experimental and clinical effects, including antibiosis, eliminate inflammation, lower hypertension, lower serum cholesterol, anti-rheumatism, a stimulation of immune function and antitumor, et al. Garlic is known for their production of steroid saponins, as well as organosulfur compounds. Now Garlic preparations in the market are mainly some crude products and small amounts of organosulfur compounds preparations. More potential garlic preparations are needed to be researched and developed for the health benefits of garlic.In this paper, the anti-hypoxia effects of garlic total saponins (GSP) extracted from garlic powder were discovered for the first time. GSP had obvious anti-hypoxia effects: It prolonged the survival time of the mice in the airtight bottles, of the Sodium nitrite poisoning mice and of the decapitated mice. And it also improved the total antioxidation capacity in the cerebrum, myocardium and liver of mice in an altitude chamber, increased the activity of CAT (catalase) in the cerebrum or SOD in the liver of mice, and decreased the MDA content in the liver and carbonylated proteins in the cerebrum. These results suggested that GSP had the effects of anti-hypoxia, as well as anti-oxidation on the mice, which may be the important mechanism of the anti-hypoxia effects.Oxidative stress and its the responses seem to be central to many of the key biological questions. An excessive load of reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated under severe hypoxic conditions can induce oxidative stress, which will affect the structure and function of proteins, nucleic acids and lipids, and result in cell injury and dysfunction.PC12 cells were used for the further investigation of the anti-hypoxia effects and its mechanisms.It showed that GSP had obvious anti-hypoxia effects: A certain dosage of GSP improved cell viability of hypoxic differentiated PC12 (dPC12) determined by MTT reduction assay, decreased the LDH leakage, and made cells keep more neuronal-like characteristic shape. Furthermore, the production of MDA and 8-OH-dG were significantly attenuated by pretreatment with GSP before exposing to hypoxia. The activity of antioxidase SOD and especially CAT in dPC12 treated with GSP were significantly higher than those of the hypoxia control. Meanwhile, GSP upregulated the expression of CAT, SOD2 and tuj1, and decreased the nuclear translocation of p65 in hypoxic cells. These suggested that GSP can protect dPC12 cells against hypoxia via antioxidant effects, which may be related to the upregulation of the activity and expression of SOD2, especially those of CAT, and the decrease of the nuclear translocation of p65 through redox-sensitive signalling pathways mediated by ROS and RNS.In this paper, the extraction technology and the determination methods were set up for the preparation of GSP. (1)The extraction technology: The crude total saponins was isolated by alcohol-extracting from dried Garlic powder. After being defated by ligarine, the remaining aqueous solution was extracted by using acetoacetate and n-butanol in turn, subsequently the n-butanol extracts was purified with D101 macroporous resin Collumn. The alcohol-extracting parameters were selected by using orthogonal design. The optimum alcohol-extracting method was to soak 1Kg garlic powder in 3 L 70% alcohol for 72 h. GSP with higher purity can be extracted with this technology, being comfirmed by assaying and TLC analysis.(2) The determination method: Diosgenin being used as the reference substance, the vanillin-glacial acetic acid-perchloric acid colorimetry was applied to determine the content of saponins at 532 nm. The determination method had good precision and repeatability, and the average recovery of control compound was 97.8 %( RSD=1.98%). It can be used for evaluating the extraction quality of total saponins.These results suggested that GSP is the important anti-hypoxia component of garlic. The anti-oxidation effects of GSP, which may be related to the upregulation of the activity and expression of SOD2 and CAT, and the decrease of the nuclear translocation of p65, may be the important mechanisms of the anti-hypoxia.This paper provided a certain experimental data for the development and utilization of the anti-hypoxia and anti-oxidation effects of garlic.