Study On Analysis And Pharmacokinetics Of Alkaloids From Nelumbo Nucifera Leaves

Lotus leaves, leaves of the plant which belong to Nelumbo of Nymphaeaceae, produced in most places of China. Lotus leaf is a kind of natural plant resource used in both food and medicine field. The alkaloids are the main active compounds in lotus leaves. Pharmacological studies show that alkaloid compounds have effects on lipid-lowering, anti-virus, anti-bacterial, sedation and anti-spasm. Current researches mostly focus on extraction and purification, quantitative determination, the pharmacological functions of total alkaloids or nuciferine. However, few other researches have been reported. Therefore, a series of investigation on lotus leaves were performed in the paper, including the isolation of total alkaloids, identification of single alkaloid; quantitative determination of main alkaloid compounds and comparison of their contents in the samples collected from different regions; pharmacokinetic studies in rat plasma and brain, thereby providing scientific evidences for the further development of this plant. The main researches and results are indicated as follows:1. Extraction and purification of alkaloids in lotus leaves The Nelumbo nucifera leaves were collected from Hunan province in China. Four major factors of extraction of total alkaloids, concentration of HCl, the ratio of material to liquid, extraction time and times, were optimized by the orthogonal experiment. Furthermore, the effects of four kinds of cation exchange resins XSI-004, XSI-008, D001, and D113 were compared. As a result, the herb was extracted 3 times with 0.2% HCl water solution as solid-liquid ratio of 1:30,3 h for each extraction. After purified with D001 column, the alkaloid fraction was obtained with yield of 10.9 g/kg crude material. Further, by RP-C18 silica gel column chromatography and preparative high performance liquid chromatography, two alkaloid compounds were obtained with purities higher than 97% determined by HPLC. The structures of the isolated alkaloids were identified by ESI-MS,1H-NMR and I3C-NMR. Compared spectroscopic data and some related references, they are confirmed to 2-hydroxy-l-methoxyaporphine (HMA) and N-nornuciferine (N-NF), respectively.2. The contents of alkaloids in Nelumbo nucifera leaves collected from different regions and time periods A HPLC-UV method was developed for simultaneous determination of three alkaloids HMA, N-NF and nuciferine (NF) in Nelumbo nucifera leaves. Meanwhile, their contents from six regions of China and different time periods were assayed and compared. The samples were extracted in 80% methanol containing 0.2% HCl with ultrasound-assisted extraction. The chromatographic separation was achieved on an Agilent Extend-C18 column, graduate eluting with acetonitrile and water containing 0.1% triethylamine. The results showed that the analytical method achieved excellent linearity, precision, reproducibility and extraction recovery, which was suitable for analysis of the alkaloids in lotus leaves. The sum of the three alkaloids were greatly different among the samples collected from different regions, being in the range of 0.11%～ 1.15%, with the highest contents in the samples from Zhejiang (1.15%), followed by those from Guizhou (1.02%) and Hunan (0.98%). NF showed higher level in the same place of origin. The contents of the alkaloids were also significantly different in the samples collected in Beijing from different time periods, with the highest contents in the June. With the increasing of growing period, both NF and N-NF contents were gradually decreased. However, the content of HMA was gradually increased in the periods from June to October. Maybe a biotransformation exists among the alkaloid compounds.3. Pharmacokinetics of alkaloids in Nelumbo nucifera leaves in rat plasma P.o. (50 mg/kg) and i.v. (10 mg/kg) administration were given to SD rats respectively. The plasma samples were extracted by ethyl acetate and analyzed by UPLC. The chromatographic separation was achieved on a Thermo Syncronis C18 column, eluting with a gradient mobile phase of acetonitrile and water both containing 0.05% triethylamine at a flow rate of 0.4 mL/min. The detection wavelength was set at 270 nm. Pharmacokinetic parameters and absolute bioavailability were estimated by the noncompartmental model of WinNonlin 6.2. The results showed that the calibration curves of NF and N-NF were both linear over the ranges of 10-200 ng/mL and 200-5000 ng/mL. The intra-day and inter-day precision and the accuracy meet the analytical requirements for biological samples. NF and N-NF had similar concentration-time profiles and parameters. Specifically, absorption and elimination rates of NF were faster than those of N-NF. N-NF had a better bioavailability of 79.91% than 58.13% of NF.4. Pharmacokinetics of alkaloids in Nelumbo nucifera leaves in rat brain The pharmacokinetics of unbound alkaloid compounds in brain of rat was studied by brain microdialysis. The probe was perfused with artificial cerebrospinal fluid containing 5 mM p-cyclodextrin at a flow rate of 2 μL/min. Dialysate samples from lateral ventricle were continuously collected in awake and freely-moving rat after an intravenous dose of total alkaloid extract at 20 mg/kg. The concentration of NF and N-NF in samples were measured by UPLC and calculated with recovery of probe. The results demonstrated that NF and N-NF both showed higher concentration of free drug in rat brain, and had similar concentration-time profiles. They were easily transported across the blood-brain barrier. It is guessed that NF and N-NF may be potential compounds to demonstrate neural activity.