Separation And Purification, Chemical Structure Characterization And Biological Activity Of Flavonoid Glycosides In Lotus Leaves

Owning to the increasing serious question about the environmental pollution and toxicity caused by those chemical synthetic compounds, the demand for natural products of plant was paid more attention. The bioactive components of natural plant has been become one of important objects researched by people. The leaves of Nelumbo nuclfera Gaertn (family Nymphaceae) which is named Lotus leaf is a kind of plant proclaimed by national Ministry of Health, could be used in food and medicine field, distributed mainly in India, China, Japan and Egypt, inexpensive and received easily. The leaves of Nelumbo nucifera are bitter and aromatic, which are reported to resolve summer heat syndrome, eliminate stasis to activate blood circulation and stop bleeding, exhibit antihyperlipidemic, antiobesity and antioxidant effects, and has been also used as an effective drugs for hematemesis, epistaxis and hematuria in clinical intervention. The results of previous work showed that all biological activities and physiological functions of Lotus leaf are closely related to these two functional components including alkaloids and flavonoids present in the leaves. At present many studies mainly focus on alkaloids, only few investigations on flavonoids of Lotus leaf have been done, which bring negative effects of utilities and exploitation of active components in Lotus leaf.The flavonoids, the main functional components in Lotus leaf, have been researched in system in this paper, which will be expected to open up a new way for the development and deep processing of Lotus leaf, provide a useful guideline for investigation of new drugs, and enhance the commercial value of Lotus leaf in an all-round way. The main investigations and experimental results in this paper were listed as following, respectively:1. The process conditions for flavonoids from Lotus leaf extracted by alcohol solution were optimized by orthogonal experiment. The results showed that the optimum process conditions for flavonoids from Lotus leaf extracted by 60% ethanol: extracting temperature being 75℃, extracting time being 2.0 h, proption of solvent and material being 40:1. Under the condition the total flavonoids content of Lotus leaf was 6.63%.2. The adsorption characteristics of flavonoids from Lotus leaf on AB-8 and H103 macroporous resin were investigated by static adsorption experiments in order to choose one resin suitable for the preliminary separation and purification of those flavonoids. It was found that AB-8 resin had excellent adsorption and desorption capacity to the flavonoids from Lotus leaf. The dynamic adsorption characteristics of the flavonoids from Lotus leaf on AB-8 resin were also studied. 36.73%. of flavonoids could be obtained in the purified sample by AB-8 macroporous resin.3. Using high performance liquid chromatography (HPLC) method to analyze the flavonoids preliminarily cleaned up by AB-8 macroporous resin, the optimum HPLC condition which could efficiently separated and determined these flavonoids was obtained: immobile phase was reversed phase Agilent ODS C18 column (250 mm×4.6 mm, I.D., 5μm), mobile phase A was acetonitrile, mobile phase B was Ultra-pure water/formic acid (99.9/0.1; v/v), sample volume was 10μL, flow rate was 0.8 ml/min, temperature of column was 30℃, wavelength of detection was 254 nm, the linear gradient elution system was: 0～20min, 10% A; 10～20min, 10～20% A; 20～35 min, 20～30% A。4 The preparative separation and purification of flavonoid glycosides from Lotus leaf were studied by high-speed counter-current chromatography (HSCCC). Four flavonoid glycosides, namely Hyperoside, Isoquercitrin, Astragalin and Quercetin-3-O-sambubioside, were isolated and their chemical structures were identified by IR, ESI-MS, 1D NMR and 2D NMR, and Quercetin-3-O-sambubioside was obtained from Nelumbo nucifera for the first time. The optimum HSCCC condition which could efficiently separated and determined these four flavonoid glycosides of lotus leaves was obtained: (1) A total of 9.1 mg of Hyperoside, 4.6 mg of Isoquercitrinand 3.0 mg of Astragalin from 80 mg of the flavonoids preliminarily cleaned up by AB-8 macroporous resin with the purity of 97.5 %, 95.8% and 98.3% determined by HPLC, respectively in one-step HSCCC separation by using a two-phase-solvent system composed of n-hexane-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v), fixing revolution speed, separation temperature , flow rate and detection wavelength to be 800rpm/min, 30℃, 1.8m.L/min and 254 nm, respectively. (2) A total of 5.0 mg of Quercetin-3-O-sambubioside with the purity of 98.6% determined by HPLC were obtained from 100 mg of the flavonoids preliminarily cleaned up by AB-8 macroporous resin in one-step HSCCC separation with a two-phase-solvent system composed of ethyl acetate-n-butanol-water (4:1:5, v/v), regulating revolution speed, separation temperature , flow rate and detection wavelength to be 800rpm/min, 30℃, 1.8m.L/min and 254 nm, respectively .5. The interaction between bovine serum albumin (BSA) and flavonoids and Astragalin from Lotus leaf in physicological condition (pH=7.4) was studied by fluorescence spectroscopy and ultraviolet absorption spectroscopy. The results demonstrated that flavonoids from Lotus leaf and astragalin could bind to BSA and quenched the intrinsic fluorescence of BSA through static quenching mechanism. The quenching rate constants of biomoleculer, the binding constants and the number of binding sites between flavonoids from Lotus leaf, astragalin and BSA were Kq = 5.133×10¹³ L·mol^-1·S-land 4.31×10¹³ L·mol^-1·S^-1, K_d = 12.97×10⁵ L·mol^-1 and 2.009×10⁵ L·mol^-1, n = 1.07 and 0.943 at 298 K , respectively. When temperature goes to 308K, these parameters between flavonoids from Lotus leaf, astragalin and BSA turns to Kq = 3.982×10¹³ L·mol-1·S^-1 and 3.72×10¹³ L·mol^-1·S^-1, K_d = 9.12×10⁵ L·mol^-1 and 0.927×10⁵ L·mol-1, n = 1.06 and 0.893, respectively. The interaction between BSA and flavonoids from Lotus leaf was driven mainly by hydrophobic interaction force and hydrogen bonds, but hydrogen bonds and van der Waals forces played a major role in the interaction between BSA and astragalin.6. The interaction between flavonoids and Astragalin from lotus leaf and deoxyribonucleic acid (DNA) in Tris-HCl buffer (pH=7.4) was investigated by the application of fluorescence spectroscopy and ultraviolet absorption spectroscopy, the effects of ionic strength and anion quencher KI on the fluorescence intensity of the system of DNA - flavonoids and DNA-Astragalin were explored, the competitive binding to DNA between flvonoids and Astragalin from lotus leaf and Neutral Red(NR) dye were also studied at the same time. The results demonstrated that flavonoids from Lotus leaf and astragalin could bind to DNA and the formed complex quenched their corresponding intrinsic fluorescence of flavonoids from Lotus leaf and astragalin through static quenching mechanism. The binding constants (K_d) of the reaction of flavonoids and Astragalin from Lotus leaf and DNA were computed to be 8.204×10⁴ L·mol^-1 and 3.412×10⁴ L·mol^-1, and the number of binding sites (n) were counted to be 1.013 and 1.007 between the two flavonoids and DNA at 298 K, respectively. When temperature goes to 308K, these parameters between flavonoids from Lotus leaf, astragalin and BSA turns to K_d = 6.855×10⁴ L·mol^-1 and 1.762×10⁴ L·mol^-1, n=1.025 and 0.962, respectively. When bound to DNA, flavonoids from Lotus leaf and astragalin showed hypochromic effect in the absorption spectra. The results showed that flavonoids from Lotus leaf could combine with DNA in the mode of intercalation, but Astragalin could combine with DNA in the mode of interaction and groove bindings.7. The anti-oxidative effects of flavonoids and flavonoids glycosides from Lotus leaf were measured and evaluated by using DPPH free radical scavenging and NaS₂O₃-I₂ methods, respectively. The results showed all the total flavonoids from Lotus leaves and the four flavonoids glycosides purified by HSCCC had strong capacity for scavenging DPPH free radical, and could effectively inhibit lard's oxidation.