| Mulberry is a perennial woody plant belonging to Moraceae, Rosales, and also has important medicinal properties. It was first record as traditional medicine in “Shen Nong Ben Cao Jing” in the eastern Han Dynasty, two thousand years ago. The parts of tree, such as foliage, fruit, branch, and bark, contain special material and functional ingredients. They have long been used as medicine with their unique medicinal value. In addition, mulberry leaf, branch, and fruit can be used to produce health food and products.Alkaloid is a kind of alkaine organic compounds containing nitrogen, which exists in the organism. Most of them are isolated from the botany and usually have strong and special pharmacological and physiological properties. The active alkaloids in mulberry leaf are polyhydroxylated piperidine alkaloids including 1-deoxynojirimycin and its derivatives. Their structures are similar to monosaccharide, also known as N-containing sugars. Recent studies on alkaloids focus on their anti-diabetic properties. Different mulberry resources have different alkaloid contents, and led to their differences in medicinal value. In the present study, we investigated the contents of alkaloid in different mulberry resources in order to provide the basic data for mulberry resource screening for further application. The main results are as follows:1. Establishment the detection method of the total alkaloids and DNJ extracted from mulberry leaves The contents of total alkaloids in mulberry leaves were determined by Reinecke salt colorimetry: the total alkaloids have been extracted from leaves are concentrated and re-dissolved. The solution(2m L) was mixed with 3m L of 2% Reinecke salt. After incubation at ice bath for 2 hour, the solution was filtered and the filter paper was then flushed by ice bothed ethyl acetate until the paper invariant. The paper was dried and put in 50 m L centrifuge tube, then dissolved with 70% of acetone. The absorbance was measured at 523 nm against a 70% of acetone blank. 4-hydroxypiperidine was chosen as a standard. Using a six point standard curve(0.002-0.02mol/L), the total alkaloids contents in mulberry leaves based on the molar mass of DNJ were determined. The precision, repeatability, and stability of this method were validated. The mulberry leaves extract contains impurities such as pigment, flavonoids, protein, et al. They should be removed before measure. Active carbon was used to remove impurity and decoloring. The ratio of carbon to material of 1:2(g/g) and 1 hour oscillation time were then determined after several tests. The contents of 1-deoxynojirimycin(DNJ) in the mulberry leaves were measured by ultra performance liquid chromatography(UPLC) coupled with UV detector. A CORTECSTM C18 column was used and the column temperature was kept at 28℃. The mobile phase was acetonitrile-0.5% acetic acid solution(28:72,V/V) with a flow rate of 0.6 m L/min. The UV detection wavelength was 254 nm. The tests for precision, stability, reproducibility and recovery rate showed that the method was stable and reliable, and could be used as an effective method for the determination of DNJ contents.2. Extraction of total alkaloids from mulberry leaves Ultrasound-assisted extraction of total alkaloids from mulberry leaves was studied. The effects of six factors on the yield of total alkaloids were investigated by the single factor experiment. The results showed that optimum conditions were extraction solvent of 50% ethanol-0.025 mol/L HCl, a solid/liquid ratio of 1:20(g/ml), extraction temperature of 40℃, extraction time of 10 min, and number of repeated extractions were 2. Effects of such factors on the total alkaloids extraction ratio were studied and optimized by conducting orthogonal test. The results showed that there have no significant influence factors. Finally, the best experimental condition were 30% ethanol-0.025 mol/L HCl, 1:20(g/ml), 40℃, 10 min and 2, respectively. Under this condition, the yield of total alkaloids was 2.1176 mg/g in leaves of Husang 32.3. Analysis the content of total alkaloids and DNJ in mulberry leaves Under the optimized extraction conditions, the total alkaloids and DNJ contents of mulberry leaves had been analyzed. The results showed that 52 mulberry resources have difference contents of total alkaloids and DNJ. The black mulberry from Xinjiang is rich in total alkaloids and DNJ compared to other resources. The degree of maturity of the mulberry leaves significantly affected the alkaloid content. The younger leaves contained higher alkaloid concentration. Comparison of the content of alkaloid from two varieties of mulberry leaves in different periods, we found that the total alkaloid of leaves gradually increased from April、May、July、September to October. The highest content of DNJ is in September, and then decreased in October. In contrast, the DNJ content of Gui 14 is higher in May rather than that in September. The contents of alkaloid in YUN somatoplasm were related to leaf type. The cleft leaf and the entire-cleft leaf contain higher total alkaloids, and the cleft and the entire leaf contain higher DNJ. |
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