| Lotus leaves, dry leaves of Nelumbo nucifera Gaertn., were cultured in both north and south of China. It was reported in traditional Chinese medicine that lotus leaves had the efficacies of losing weight, reducing fat and decreasing hypertension, etc. In modern research, scientists found that lotus leaves had the activities of stop bleeding, decreasing blood pressure, reducing weight and resisting arrhythmia and so on. The compositions in it include alkaloids, flavonoids, organics acid and tannins, etc. Alkaloids are known of the main bioactive ingredients in it. We investigated the best methods of extracting the alkaloids from lotus leaves and the best experimental conditions by orthogonality method. On the basis of the above research, identification of alkaloids was established by using nuciferine as reference substance with thin-layer chromatography (TLC). The content of nuciferine in lotus leaf was determined by RP-HPLC. The result will give a guideline for studying the pharmacological characteristic of the alkaloids.Objective: To investigate the best technology of extracting alkaloids in lotus leaves; to determine the content of nuciferine by RP-HPLC and establish identification method with nuciferine as reference substance by TLC. Methods: (1)Identification: Lotus leaf was identified with nuciferine as reference substance by TLC, the sample solution and standard solution were applied separately to the same thin layer plate of silica gel GF254, which was developed and dried. The spots were observed under ultraviolet light at 254 nm. (2)Extraction: We investigated the best method of extracting the alkaloids from lotus leaves and the best experimental conditions with orthogonality optimized method. Ultraviolet spectrophotometer was used and nuciferine was adopted as control in the determination. (3)Determination of the nuciferine content: ①Extraction: We extract nuciferine with HCl-CH3OH (2:98) from lotus leaves by supersonic wave-extraction. ②Determined condition: Choose appropriate column, different formulation and proportion of mobile phase and adjust optimal flow rate and wavelength in order to separate nuciferine well. ③Preparation of regress equation: Prepare a series of the reference solution, determine peak area, then the regress equation was obtained with the content of nuciferine as abscissa, the relevant peak area (×10000) as ordinate.④Lowest detection limitation: dilute the reference solution to the value of S/N was more than 3. The relevant concentration was the lowest detection limitation. ⑤Stability: Prepare the sample solution from lotus leaves to determine the relevant peak areas at 0, 1, 2, 4, 6, 8, 10 and 12 hour, respectively. ⑥Precision: Take the same test solution, inject it into the apparatus for six times, determine the peak area of nuciferine according to the same regress equation with the same apparatus in the same day, record the chromatograms, then calculate the precision. ⑦Reproducibility: Repeat preparing the sample solution of lotus leaves five times in the same way, respectively. ⑧Recovery: Take five shares of lotus leaves, and then add same volume of reference solution of nuciferine to it and prepared the mixture with the above method, inject them into the apparatus and determine the content of nuciferine of every sample. ⑨The content of nuciferine in lotus leaves was analyzed under above-mentioned conditions.Results: (1) Identification: The result showed that the spots of the test solution were in the same position as those of the reference solution. (2) Extraction: Extraction of the total alkaloids was performed by ion-exchange resin, the best conditions were decocting ground lotus leaves with 0.1% HCl solution at the ratio of 20:1 at 90℃. The leaves were extracted twice for duration of 2h each time. The content of total alkaloids was 2.12%. (3) Determination of the nuciferine content: ①Method of supersonic wave-extraction with acidic solvent HCl-CH3OH (2:98) was simple, quick and stable. ②The HPLC system was carried out on Hypersil-C18 (...
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