| In this experiment, we used the branches of cultivated mulberry842asexperimental material, and then used alcohol-water as the extract solution to get thecrude extraction. The crude extraction was further purified by macroporous resin,Sephadex LH-20, and used semi-preparative reversed-phase high-performance liquidchromatography (RP-HPLC) to obtain a high purity monomer finally. The monomerwas identified as mulberroside A by high performance liquid chromatographic diodearray detection (HPLC-DAD) and its UV spectrum. The content of mulberroside A insamples was quantitative analyzed by drawing a liner equation for preparativemulberroside A. The liner equation was y=7535.64716+1508590x, and there was agood liner relationship within the scope of0.10-2.00μg, the correlation coefficientwas0.997. This paper systematically analyzed the change of mulberrroside A level inthe isolated mulberry branch bark after UV irradiation and salicylic acid treatment onthe basis of HPLC-DAD.Firstly, we study the toxicity of mulberroside A by animal experiment. Treatedthe normal ICR mice with mulberroside A by tail intravenous injection every day andstudied the effect of mice’s growth and development caused by mulberroside A. Theresults showed that after treated by mulberroside A (50mg/kg), in the treatment groupthe weight growth slowed down, the liver coefficient was higher, but statisticalanalysis showed that there were no significant differences compared to the normalgroup; the activity of SOD, GSH-PX rise in the serum in the treatment group whilethe activity of SOD, T-AOC, GSH-PX fell in the liver, but there were no significantdifferences compared to the normal group; there were significant rise of the totalprotein content in the serum and liver in the treatment group, statistical analysisshowed that there were significant differences compared to the normal group; but theywere normal fluctuation; observe the liver HE pathological section, the treatment ofmulberroside A lead to venous congestion and liver cell vacuolating. These resultsindicated that there weren’t significant effects on mice weight growth, livercoefficient and oxidative index in blood and liver, but minor damage to normal mice. Secondly, lots of mulberry branches in our silkworm districts were harvestedannual, so it had an important practical significance to study the active ingredients andits efficient use in wasted mulberry branch. Mulberroside A was oxyresveratroldiglycoside, an important activity ingredient and existed in mulberry branch and root.It had many biological activity and pharmacological effects. In order to study themethods of improving mulberroside A level in the isolated mulberry branches andensure the veracity of the experiment, we used the fresh branches of cultivatedmulberry842as experimental material, chose the same location in mulberry branch,treated with ultraviolet light and salicylic acid.Ultraviolet (which differ in wavelengths called ultraviolet C (UVC), ultraviolet B(UVB), ultraviolet A (UVA)) were used to treat the cut down fresh842mulberrytwigs. The results show that: when fresh mulberry branches were treated by UVC8minutes, UVB two minutes and UVA six minutes, the mulberroside A content werereaching maximum, respectively2.2times(0.72μg/mg),2.7times(0.61μg/mg),1.2times(0.48μg/mg) over the untreated. These results indicated that three kinds of UVirradiation can stimulate the mulberry cortex cell, induce the secretion ofmulberroside A in cortex cell. The best effect was treated by UVB irradiation whichwavelength was290-320nm.The new cut fresh842mulberry branches were respectively treated by soakingsalicylic acid with different time or concentration. When salicylic acid was60μg/mL,soaking time was80s, the content of mulberroside A was reaching maximum(0.64μg/mg) about2.1times over the untreated; when shortening the soaking time(20s) and increasing the concentration (80μg/mL), the content of mulberroside A wasup to2.0times(0.62μg/mg) over the untreated. These results indicated that salicylicacid can stimulate the mulberry cortex cell, induce the secretion of mulberroside A incortex cell.Additionally, the content of mulberroside A in different parts of a samemulberry’s branch brark were apparently different. The content of mulberroside A inthe top tender branch was low, only0.14μg/mg, and the lower parts were graduallyincreasing, reaching15times over the top. Harvesting period of mulberry branchesalso had a great impact on the effects of UV radiation and salicylic acid treatment, the longer the mulberry branch grew, the phloem cells’ sensitivity to their treatmentcondition became worse, which slowed down the synthesize of mulberroside A instimulated cells.In conclusion, this paper used HPLC-DAD to develop a whole method ofextraction, purification, identification and rapid analysis of mulberroside A, provideda useful detected way to develop mulberroside A from lots of wasted mulberrybranches. The animal toxicity experiment indicated that mulberroside A didn’t haveeffect on mice’s growth and development, and had nontoxicity to normal animal. UVirradiation and salicylic acid soaking can activate isolated mulberry cortex cell,promoted the secretion of mulberroside A. These experiment methods laid thefoundation of separation of mulberroside A and development of health produces orbiological medicine, which promoted making efficient and full use of mulberrybranches resource. |
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