The Isolation And Pharmacology Of Main Active Constituents Of Ramulus Mori

There is overwhelming evidence to indicate that the inner circumstance of the organism can produce the free radicals all the time and free radicals cause oxidative damage to lipids, proteins, and nucleic acids. Free radicals may lie at the heart of the etiology or natural history of a number of diseases, including cancer, heart, vascular, diabetes and neurodegenerative diseases. At the same time, the ability of the protective system of the organism was wear off with the age increasing, the chance of the disease such as diabetes, cancer, heart disease, arteriosclerosis and cataract in corrective with the aging was increasing. So the mechanism of the relation with the free radical and the disease with aging has become the hotpot of research.In order to minimize the oxidative damage largely, the people begin to search for the antioxidant, synthetic antioxidants are known to have toxic and carcinogenic effects on human, Thus, with increasing consciousness of consumers with regard to safety of food additives and the higher manufacturing cost and lower efficiency of natural and safer sources of food antioxidant is created. Therefore, search for natural antioxidants, especially of plant origin, has notably increased in recent years.Sangzhi (Ramulus mori) is the branch of Morus alba L., family Moraceae. According to theories of traditional Chinese medicine, it is slightly bitter in taste, mild in nature, and attributive to the liver meridian. In traditional Chinese medicine,its pharmacological actions are to expel wind, dredge the meridians, and ease joint pain. In the clinic, it was used to cure the disease of joint of shoulder and arm, ache and anaesthesia of hand, rheumatism and paralysis. In modern pharmacology, it has the activities of lowering the blood sugar and press, anticancer, antibacterium, antivirus, anti-inflammation and easing pain. There is consanguineous correlation between the activity of pharmacology and the chemical composition. In our country, there is rich resource of Ramulus mori, the utilization of it was mostly limited to the primary exploitation. So it is important to find out the main medical composition and the pharmacological function of Ramulus mori for developing the depth exploitation of the natural preparation of Chinese traditional medicine. The present study focus on the antioxidant activity of Ramulus mori, analysis the active compound with the modern methods of measurement, separation, purification, and identification, carry out a series of pharmacological experiments with the isolated compound, prove up the mechanism of pharmacological function, and try to provide the scientific basis for the further exploitation of Ramulus mori. The main results as follows: 1. The experiment optimized the condition of the extraction and determination the flavonoid in Ramulus mori. The flavonoid content could be exactly and stably measured with ultraviolet spectrophotometer after it was treated with the process:it was sifted with 160 item, use the solvent of 80% ethanol, extracted with the ultrasonic for 20 minuates and the circumfluence for 30min at 80℃for three times.2. The present study optimized the condition of the determination of the polysaccharide in Ramulus mori with the orthogonal methods. The content of polysaccharide could be exactly and stably measured with spectrophotometer after it was treated with the following process:it was sifted with 160 item, extracted with the ultrasonic for 40 minuates and the extraction for 90 min at 100℃in water for twice. The present study isolated and refined the polysaccharide of Ramulus mori too, and got the conversion factor between the polysaccharide and glucose (4.96). There was rich polysaccharide in Ramulus mori and the content of polysaccharide were significantly different to the mulberry varieties. In the same variety, the order of the content was new Ramulus mori in spring> Ramulus mori in winter > one year old Ramulus mori in spring.3. The DNJ could not be determined directly because there was no chromogenic group in its structure, the present study marked it with 9-fluorenylmethyl chloroformate for detecting the content of DNJ. The optimum condition of DNJ derivation including reaction for 50 min at 30℃with pH7 potassium borate buffer was established with orthogonal design. There was rich DNJ in Ramulus mori, and the content significantly different according to the mulberry varieties. In the same variety, the order of the content was one year old Ramulus mori in spring> Ramulus mori in winter> new Ramulus mori in spring. The DNJ can be accumulated in Ramulus mori.4. The polysaccharide of Ramulus mori was purified with the combination of the protein rapid separated appearance and detection, the molecular weight was determined by HPLC, the monose composition of the polysaccharide in Ramulus mori was measured with ICS2500 ion chromatography and its structure was determined with infrared spectral. The results showed that the polysaccharide is a-configuration and mainly composed of five monoses:rhamnose, arabinose, galactose, glucose and xylose and the ratio of composition of isrhamnose:arabinose:galactose:glucose:xylose is 9.12:24.12:44.09:12.22:7.18.5. The preliminary research found that the polysaccharide showed very poor antioxidant activity, in the present study, different polysaccharide derivatives were prepared to give sulfated, phosphorylated, acetylated and benzoylated polysaccharide. The antioxidant activity of the polysaccharide and its derivatives in vitro was determined, including scavenging activity against DPPH, superoxide and hydroxyl radical, reducing power, and chelating ability. Among the five samples, the polysaccharide derivatives showed stronger antioxidant activity than the polysaccharide in Ramulus mori. The activities of the tested samples varied with pH and temperature. The antioxidant activity in the linoleate emulsion improved with increasing pH from 3 to 11, while it decreased with ascending storage temperature between 20 and 90℃.6. Based on the character of DPPH in HPLC, in the present study, the result of HPLC was used as direction to isolate the main antioxidant compound in the ethanol extract of Ramulus mori. The main antioxidant compound was identified as oxyresveratrol. This method is simple, rapid, accurate, sensitive and avoiding the repeating operation and decomposing of the active compounds.7. On-line HPLC method and liquid chromatography-electrospray ionization tandem mass spectrometry was used to separate and identify the main antioxidant compound in Ramulus Mori, and the result of the above method was used as a reference to isolate and identify the main antioxidant compound, which is oxyresveratrol. The content of oxyresveratrol in different mulberry varieties were also performed by high performance liquid chromatography. The method is simple, quick and sensitive, the result is satisfactory.8. To investigate the antioxidant effect of compounds isolated from Ramulus mori structure-activity relationship, the present study determined the antioxidant activities of rutin, morin, oxyresveratrol and cis-mulberroside A and establish the important role of 4 and 3'hydroxyl groups in oxyresevertrol for eliminating superoxide radical, hydroxyl radial and DPPH. The 3 hydroxyl group in morin was important for riding the superoxide radical and hydroxyl radial and the 3 glucose in rutin was important for its reducing power.9. The present study revealed that alloxan could induce the liver damage through the elevation of transaminases activities and MDA accompanied by significant reductions in GSH level and SOD and CAT activities. The treatment with ethanol extract of Ramulus mori can bring those changes to near normal level. The protective effect of it was further supported by the histopathological observation and the expression of pro-inflammatory cytokines. Considering the mechanism of alloxan-induced damage and the above result, we can conclude that the protective effect of EER primarily involves three mechanisms:(1) acts as a free radical scavenger, directly scavenges the free radical caused by alloxan, (2) acts as a metal chelator, inhibits radical generations by stabilizing transition metals, consequently reducing free radical damage, and (3) acts as a a-glucosidase inhibitor, through lowering the blood glucose concentration to inhibit the glycation of antioxidant enzyme, which can elevate the activity of antioxidant enzyme.10. Based on the character of DPPH, the model of liver injured was established. Considering that oxidative stress plays a role in most of clinical conditions, the findings of this experiment will be very useful for evaluating oxidative stress mechanisms and as an experimental model for testing the benefits of therapeutic substances on oxidative stress-related diseases.11. Rutin treatment could protected against the acute TAA-induced liver injury in mice was investigated, Form the results, we can predict the possible mechanisms of the prevention of the rutin from Ramulus Morus in four different ways:(A) stimulate the regeneration of the liver through promoting the synthesis of ribosomal RNA; (B) act as an antioxidant through scavenging and regulating the intracellular content of glutathione; (C)prevent hepatotoxic agents from entering hepatocytes throught stablizing cell brane and regulating the permeability; (D) inhibit the tansformation of stellate hepatocytes into myofibroblasts, which is responsible for the deposition of collagen fibres leading to cirrhosis. The key mechanism that ensures hepatoprotection appears to be free radical scavenging.12. Oxyresveratrol and its glucoside treatment could protect against the ethanol-induced liver injury in mice as demonstrated by increasing the activities of SOD, CAT, GPx and GST and the content of GSH as well as decreasing the content of MDA. The protective function was further supported by the histopathological observation and the expression of TNF-a. The result showed that the protective efficiency of oxyresveratrol was more pronounced than its glucoside. Considering their antioxidant activities in vitro and the mechanism of the metabolism of ethanol, their protective effect may mainly be related to the antioxidant properties.13. The present research isolated the active compounds from Ramulus mori through bioassay method and investigated their anti-inflammatory and anticoncept through acetic acid-induced writhing response, formalin-induced licking, carrageenan-induced air pouch, acetic acid-induced vascular permeability and the content of PGE2. cis-mulberroside A exhibited high anti-inflammatory and anticoncetipt activities. So cis-mulberroxide A may be the main active compound with the anti-inflammatory activity in Ramulus mori.