Flavonoid Glycosides from Hosta longipes , Their Inhibition on NO Production , and Nerve Growth Factor Inductive Effects

Investigação fitoquímica das folhas da Hosta longipes identificou um novo flavonóide glicosídeo, o caempferol-3-O-β-D-glucopiranosil-(1→2)-[6’’’-O-acetil-β-D-glucopiranósido]7-O-β-D-glucopiranósido, e mais cinco derivados flavonóides conhecidos. As estruturas de dois compostos foram reveladas por vários métodos de RMN (H e C RMN, H-H COSY, HMQC HMBC) e hidrólise química. Dados de RMN de um deles são publicados pela primeira vez. As atividades biológicas de seis compostos revelaram que cinco inibiram fortemente a produção de óxido nítrico (NO), com valores de IC50 de 11,56-15,97 μm em células BV-2 estimuladas por lipopolissacarídeo (LPS), sem toxicidade celular. Dois compostos mostraram indução moderada da secreção no fator de crescimento do nervo (NGF) em linhagem de células C6 de glioma (124,70 ± 7,71% e 117,02 ± 3,60%, respectivamente).


Introduction
More than 8,000 flavonoids have been isolated from plant sources.Flavonoids have a variety of pharmacological effects that include anti-cancer, anti-microbial, anti-oxidant, and anti-inflammatory activities. 1,2Neuroprotective effects of flavonoids from Citrus species are reportedly associated with their anti-inflammatory action, the ability to traverse the blood-brain barrier, and multiple neuroprotective mechanisms. 3NO and NGF have important roles in neuropathological conditions.These roles include regulation of inflammatory response and recovery of tissue damage in brain injury. 4,5Regulation of NO production or/ and NGF secretion in microglia and astrocytes is a good target for the treatment of neurodegenerative disorders.
As part of our efforts to screen bioactive constituents of Korean medicinal plants with anti-neuroinflammatory activities, we found that the MeOH extract of the leaves of Hosta longipes (FR.et SAV.) MATSUMURA (Liliaceae) inhibited NO production in murine microglia BV-2 cells.H. longipes is an edible vegetable in Korea and has long been used as a traditional Korean medicine for treating cough, sputum, laryngopharyngitis, and burns. 6,7Previous phytochemical investigations from this source led to the isolation of cytotoxic steroidal saponins. 8,9Our earlier phytochemical investigation of H. longipes resulted in the isolation of steroidal constituents capable of inhibiting NO production. 10Our continuing research for active constituents in MeOH extracts led to the isolation of six flavonoid glycosides (1-6) (Figure 1), including one new compound (1).All six compounds were tested for their inhibitory effects on NO production in a LPS-activated murine microglial cells and their effects on NGF secretion from C6 glioma cells.

General procedures
Optical rotations were measured on a Jasco P-1020 polarimeter in MeOH (Jasco, Easton, MD).IR spectra were recorded on a Bruker IFS-66/S FT-IR spectrometer (Bruker, Karlsruhe, Germany).UV spectra were recorded using a Schimadzu UV-1601 UV-Visible spectrophotometer (Shimadzu, Tokyo, Japan).HR-FAB and ESI mass spectra were obtained on a JEOL JMS700 mass spectrometer (JEOL, Peabody, MA).NMR spectra, including COSY, HMQC, and HMBC experiments were recorded on a Varian UNITY INOVA 500 and 900 NMR spectrometer with chemical shifts given in ppm (Varian, Palo Alto, CA).Preparative high performance liquid chromatography (HPLC) was conducted using a Gilson 306 pump (Gilson, Middleton, WI) with Shodex refractive index detector (Shodex, New York, NY).Silica gel 60 and RP-C 18 silica gel (230-400 mesh, Merck, Darmstadt, Germany) were used for column chromatography.The packing material for molecular sieve column chromatography was Sephadex LH-20 (Pharmacia, Uppsala, Sweden).Spots were detected by thin layer chromatography (TLC) under UV light or by heating after spraying with 10% H 2 SO 4 in C 2 H 5 OH (v/v).

Plant materials
Leaves of H. longipes were collected in Taebaek City, Korea, in June 2010.The plant was identified by one of the authors (K.R. Lee).A voucher specimen (SKKU-NPL 1103) of the plant has been deposited at the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Alkaline hydrolysis of 1 and 2
A solution of compound 1 (2.0 mg) in 0.1 N KOH (3 mL) was stirred at room temperature for 24 h.The reaction mixture was neutralized with Dowex HCR W2 (H + form) and the resin was removed by filtration.A portion of the reaction product was partitioned between CHCl

Measurement of NO production and cell viability in LPSactivated BV-2 cells
BV-2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin.To measure NO production, BV-2 cells were dispensed in wells of a 96-well plate (3 × 10 4 cells/well).After 24 h, the cells were pretreated with compounds for 30 min and stimulated with 100 ng/mL LPS for 24 h.Nitrite, a soluble oxidation product of NO, was measured in the culture media using the Griess reaction.The supernatant was harvested and mixed with an equal volume of Griess reagent (1% sulphanilamide, 0.1% N-1-naphthylethylenediamine dihydrochloride in 5% phosphoric acid).After 10 min, the absorbance at 540 nm was measured using an Emax microplate reader (molecular devices).Sodium nitrite was used as a standard to calculate the nitrite concentration.Cell viability was measured using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay.N G -Monomethyl-L-arginine (L-NMMA), a well-known NO synthase inhibitor, was tested as a positive control.

NGF and cell viability assays
C6 glioma cells were used to measure NGF release into the medium. 11C6 cells were purchased from the Korean Cell Line Bank and maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator with 5% CO 2 .To measure NGF content in medium and cell viability, C6 cells were seeded into 24-well plates (1 × 10 5 cells/well).After 24 h, the cells were treated with DMEM containing 2% FBS and 1% penicillin-streptomycin with 20 μM of each sample for one day.Media supernatant was used for the NGF assay using an ELISA development kit (R&D Systems).Cell viability was assessed by the MTT assay.

Results and Discussion
Leaves of H. longipes (2.5 kg) were extracted with 80% MeOH and the extract was partitioned with n-hexane, CHCl 3 , EtOAc, and BuOH.The BuOH layer (24 g) was successively chromatographed over silica gel, Sephadex LH-20, and preparative HPLC to give one new flavonoid glycoside ( 1 16 NMR spectral data have not been reported.By extensive NMR studies ( 1 H and 13 C NMR, 1 H-1 H COSY, HMQC, and HMBC), full NMR data were assigned for the first time.
Compounds 1-6 were tested using an ELISA development kit for their influence on secretion of NGF from C6 glioma cells into the medium. 17As shown in Table 3, compounds 4 and 5 were moderate stimulants of NGF release (124.70 ± 7.71% and 117.02 ± 3.60%, respectively) without cell toxicity (97.49% and 89.21% survival, respectively) at a concentration of 20 mM.anti-neuroinflammatory activity by suppressing the release of NO in LPS-stimulated microglial cells and by inducing NGF secretion in C6 glioma cells.These results suggest that these compounds might be promising candidates for treatment of Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases.

Conclusion
are thankful to the Korea Basic Science Institute (KBSI) for the measurements of NMR and MS spectra.

From
the leaves of H. longipes a new flavonoid glycoside (1) and five known flavonoid derivatives (2-6) were isolated by chromatographic methods.The structure of compounds 1 and 2 was revealed by extensive NMR methods ( 1 H and 13 C NMR, 1 H-1 H COSY, HMQC and HMBC) and chemical hydrolysis.The NMR data of compound 2 are published for the first time.Compounds 4 and 5 exhibited significant

a C6 cells were treated with 20 μM of compounds 1 - 6 .
After 24 h, the content of NGF secretion in C6-conditioned media was measured by ELISA.The level of secreted NGF cells is expressed as percentage of the untreated control.The data shown represent the means ± SD of three independent experiments performed in triplicate; b cell viability after treatment with 20 μM of each compound was determined by MTT assay and is expressed in percentage (%).The results are averages of three independent experiments, and the data are expressed as mean ± SD; c 6-Shogaol as positive control.

Table 1 .
13 and13C NMR data for compounds 1 and 2 in CD 3 OD (d in ppm)

Table 2 .
Effects of compounds 1-6 and L-NMMA on LPS-induced NO production in BV-2 microglia cells IC 50 value of each compound was defined as the concentration (μM) that caused 50% inhibition of NO production in LPS-activated BV-2 cells; b cell viability after treatment with 20 μM of each compound was determined by MTT assay and is expressed in percentage (%).The results are averages of three independent experiments, and the data are expressed as mean ± SD; a c L-NMMA as positive control.

Table 3 .
Effects of compounds 1-6 on NGF secretion in C6 cells a