Chemical Constituents from Andrographis echioides and Their Anti-Inflammatory Activity

Phytochemical investigation of the whole plants of Andrographis echioides afforded two new 2′-oxygenated flavonoids (1) and (2), two new phenyl glycosides (3) and (4), along with 37 known structures. The structures of new compounds were elucidated by spectral analysis and chemical transformation studies. Among the isolated compounds, (1–2) and (6–19) were subjected into the examination for their iNOS inhibitory bioactivity. The structure-activity relationships of the flavonoids for their inhibition of NO production were also discussed.


Introduction
Andrographis (Acanthaceae) is a genus of about 40 species, various members of which have a reputation in indigenous medicine. In traditional Indian medicine, several Andrographis species have been used in the treatment of dyspepsia, influenza, malaria and respiratory infections, and as astringent and antidote for poisonous stings of some insects [1,2]. More than 20 species of Andrographis have been reported to occur in India. The phytochemistry of this genus has been investigated quite well in view of its importance in Indian traditional medicine and reported to contain several flavonoids [3,4] and labdane diterpenoids [5][6][7][8][9][10]. A. echioides, an annual herb occurring in South India, is listed in the Indian Materia Medica used as a remedy for fevers. However, information on the chemical composition and bioactivity of this species is very rare. There is only report of flavonoids as major components from the extracts of A. echioides in the previous literature [11][12][13][14]. As part of our program to study the bioactive constituents from Andrographis species [15,16], we have investigated the whole plant of A. echioides and four new compounds (1)(2)(3)(4) were characterized. Herein, we wish to report on the structure elucidations of compounds 1-5 and the effects of flavonoids on NO inhibition in LPS-activated mouse peritoneal macrophages.

Anti-Inflammatory Activity
Inflammation is related to morbidity and mortality of many diseases and is recognized as part of the complex biological response of vascular tissues to harmful stimuli. It is the host response to infection or injury, which involves the recruitment of leukocytes and the release of inflammatory mediators, including nitric oxide (NO). NO is the metabolic by-product of the conversion of L-arginine to L-citrulline by a class of enzymes termed NO synthases (NOS). Numerous cytokines can induce the transcription of inducible NO synthase (iNOS) in leukocytes, fibroblasts, and other cell types, accounting for enhanced levels of NO. In the experimental model of acute inflammation, inhibition of iNOS can have a dose-dependent protective effect, suggesting that NO promotes edema and vascular permeability. NO also has a detrimental effect in chronic models of arthritis, whereas protection is seen with iNOS inhibitors. The iNOS inhibiting potentials of 1-2 and 6-19 were evaluated by examining their effects on LPS-induced iNOS-dependent NO production in RAW 264.7 cells determined by MTT assays. Cells cultured with 1-2 and 6-19 at different concentrations except 18 (at 42 μM) used in the presence of 100 ng/mL LPS for 24 h did not change cell viability thus the NO inhibiting effects may not due to the cytotoxicity (Table 3). In the examined concentration ranges (5.25-74 μM), NO production decreased in the presence of 1-2 and 6-19 in a dose-dependent manner (Table 3). Flavonoids are widely distributed in the higher plants capable of modulating the activity of enzymes and affect the behavior of many cell systems, including NO inhibitory activity. The structure-activity relationships of 3',4'-oxygenated flavones were discussed by Matsuda [53] and Kim et al. [54]. In 1999, Kim et al. [54] examined the naturally occurred flavonoids for NO production inhibitory activity in LPS-activated RAW 264.7 cells and the following structural requirements were afforded: (a) the strongly active flavonoids possessed the C2-C3 double bond and 5,7-dihydroxyl groups; (b) the 8-methoxyl group and 4'-or 3',4'-vicinal substitutions favorably affected inhibitory activity; (c) the 2',4'-(meta)-hydroxyl substitutions abolished the inhibitory activity; (d) the 3-hydroxyl moiety reduced the activity; (e) flavonoid glycosides were not active regardless of the types of aglycones. Andrographis species are noted for profuse production of 2'-oxygenated flavones and in the present study, the bioactive data of the examined flavonoids using RAW 264.7 cells were in agreement with the previous report by Kim et al., and the additional structural requirements of flavonoids for NO production inhibitory activity were suggested as follows: (1) the glycosidic moiety reduced the activity, like 9 and 14; (2) the 2'-hydroxyl group did not cause significant effects on NO inhibitory activity; (3) methylation of 5-hydroxyl group enhanced the activity, like 13 and 14 ( Table 4). The structure-activity relationships of flavonoids for NO production inhibitory activity resulted from our study clarified the insufficiency in the previous report.

General
The UV spectra were obtained with Hitachi UV-3210 spectrophotometer. The IR spectra were measured with a Shimadzu FTIR Prestige-21 spectrometer. Optical rotations were recorded with a Jasco DIP-370 digital polarimeter in a 0.5 dm cell. The ESIMS and HRESIMS were taken on a Bruker Daltonics APEX II 30e spectrometer. The FABMS and HRFABMS were taken on a Jeol JMS-700 spectrometer. The ESIMS (negative ESI) data were measured using a Thermo TSQ Quantum Ultra LC/MS/MS spectrometer. The 1 H and 13 C NMR spectrums were measured by Bruker Avance 300, 400 and AV-500 NMR spectrometers with TMS as the internal reference, and chemical shifts are expressed in δ (ppm). The CD spectrum was recorded in a Jasco J-720 spectrometer. Sephadex LH-20, silica gel (70-230 and 230-400 mesh; Merck, Darmstadt, Germany) and reversed-phase silica gel (RP-18; particle size 20-40 μm; Silicycle) were used for column chromatography, and silica gel 60 F 254 (Merck, Darmstadt, Germany) and RP-18 F 254S (Merck, Darmstadt, Germany) were used for TLC. HPLC was performed on a Shimadzu LC-10AT VP (Tokyo, Japan) system equipped with a Shimadzu SPD-M20A diode array detector at 250 nm, a Purospher STAR RP-8e column (5 μm, 250 × 4.6 mm) and Cosmosil 5C 18

Plant Materials
The whole plant of A. echioides Nees was collected from Tirupati, Andhra Pradesh, India in May 1998. The plant was authenticated by Professor C. S. Kuoh, Department of Life Science, National Cheng Kung University, Taiwan. The voucher specimens (DG-199) have been deposited in the herbarium of the Department of Botany, Sri Venkateswara University, Tirupati, India; and Department of Chemistry, National Cheng Kung University, Tainan, Taiwan, respectively.

Determination of Aldose Configuration
Compounds 1-5 (each 0.5 mg) were hydrolyzed with 0.5M HCl (0.4 mL) in a screw-capped vial at 60 °C for 1 h. The reaction mixture was neutralized with Amberlite IRA400 and filtered. The filtrates were dried in vacuo, then dissolved in 0.1 mL of pyridine containing L-cysteine methyl ester (0.5 mg), and reacted at 60 °C for 1 h. To those mixtures were added a solution of O-tolylisothiocyanate in pyridine (5 mg/1 mL) at room temperature for 1 h. Those reaction mixtures were directly analyzed by HPLC (Cosmosil 5C 18 ARII (250 × 4.6 mm i.d. Nacalai Tesque Inc., Tokyo, Japan); 20% CH 3 CN in 50 mM acetate; flow rate 0.8 mL/min; detection, 250 nm). D-glucose (t R 40.5 min) was identified as the sugar moieties of 1-5 based on comparisons with authentic samples of D-glucose (t R 40.5 min).

Cell Viability
Cells (2 × 10 5 ) were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with samples in the presence of 100 ng/mL LPS for 24 h. After that, the cells were washed twice with DPBS and incubated with 100 μL of 0.5 mg/mL MTT for 2 h at 37 °C testing for cell viability. The medium was then discarded and 100 μL dimethyl sulfoxide (DMSO) was added. After 30-min incubation, absorbance at 570 nm was read using a microplate reader (Molecular Devices, Orleans Drive, Sunnyvale, CA, USA).

Measurement of Nitric Oxide/Nitrite
NO production was indirectly assessed by measuring the nitrite levels in the cultured media and serum determined by a colorimetric method based on the Griess reaction [55]. The cells were incubated with a test sample in the presence of LPS (100 ng/mL) at 37 °C for 24 h. Then, cells were dispensed into 96-well plates, and 100 μL of each supernatant was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride, and 5% phosphoric acid) and incubated at room temperature for 10 min, the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices, Orleans Drive, Sunnyvale, CA, USA). By using sodium nitrite to generate a standard curve, the concentration of nitrite was measured form absorbance at 540 nm.

Statistical Analysis
Experimental results were presented as the mean ± standard deviation (SD) of three parallel measurements. IC 50 values were estimated using a non-linear regression algorithm (SigmaPlot 8.0; SPSS Inc. Chicago, IL, USA). Statistical significance is expressed as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Conclusions
In the previous literature, there are four Andrographis species containing diterpenoids such as andrographolide, including A. paniculata, A. affinis, A. lineata, and A. wightiana. In our investigation, the major constituents of the titled plant were flavonoids rather than the crystalline bitter principle analogous to diterpenoids. In the evaluation of NO inhibition activity, compounds 10 and 14 were the most effective and the IC 50 values were 37.6 ± 1.2 μM and 39.1 ± 1.3 μM, respectively. These results suggested that the Andrographis species are valuable sources for the discovery of natural anti-inflammatory lead drugs.