Chemical Constituents from the Leaves of Annona reticulata and Their Inhibitory Effects on NO Production

In the present study, the chemical investigation of the leaves of Annona reticulata has resulted in the identification of nine compounds, including annonaretin A, (1), a new triterpenoid. The purified compounds were subjected to the examination of their effects on NO inhibition in LPS-activated mouse peritoneal macrophages and most of them exhibited significant NO inhibition, with IC50 values in the range of 48.6 ± 1.2 and 99.8 ± 0.4 μM.


Introduction
The Annona genus (Annonaceae) consists of about 119 species, most of which are shrubs and trees widely distributed in the tropical and subtropical regions, including the Southeast Asia countries such as Malaysia, Indonesia, Thailand, Cambodia, Laos, and Vietnam. In Indian folk medicine, various species of Annona have been used as vermifuges, anti-inflammatory agents, in wound healing, as antimalarial agents and in the treatment of diarrhoea and dysentery [1]. The bark of the plant Annona reticulata L. is a powerful astringent and given as tonic. The plant has been used as an anti-inflammatory agent in wound healing, anti-anxiety, anti-stress, anti-mutagenic, and spasmolytic agent. Leaf and stem extract shows inotropic, positive chronotropic and spasmolytic activities [2]. The plant is reported to contain acetogenins mainly cis-and trans-isomurisolenin [3], annoreticuin, bullatacin, squamosine, rolliniastatin [4], reticullacinone, rolliniastatin-2, molvizarin [5], 14-hydroxy-25-deoxy-rollinicin [6]. Reticulatacin and kaurane diterpenes were also identified from the bark of the plant [7]. Other terpenes such as spathenelol, muurolene, copaene and eudesmol were reported in the previous literature [8]. In our continued program aimed at the identification of anti-inflammatory drug leads from natural sources, the chemical composition of the leaves of A. reticulata was investigated to search for the bioactive constituents by assays of inhibitory effects on NO inhibition in LPS-activated mouse peritoneal macrophages. In the present study, we wished to report the characterization of nine compounds, including the structural determination of new compound 1, as well as their NO inhibitory effects.

Structural Elucidation of New Compound 1
The purified colorless powder 1 was visualized by spraying with 1% (w/v) Ce(SO 4 ) 2 in 10% (v/v) aqueous H 2 SO 4 followed by heating at 120 °C and displayed purplish black spots on TLC plate. It also displayed a positive response in the Lieberman-Burchard test. These results suggested that compound 1 possessed a basic triterpenoid skeleton [15]. The molecular formula of 1 was established as C 33 H 56 O 3 by the pseudomolecular [M+Na] + ion peak at m/z 523.4122 in HR-ESI-MS analysis and was further supported by its 13 3 -19), and 1.60 (3H, s, CH 3 -30); and two doublets at δ 0.80 (3H, d, J = 9.5 Hz, CH 3 -32) and 0.93 (3H, d, J = 6.0 Hz, CH 3 -33), respectively. In addition, two high field cyclopropyl proton doublets at δ 0.

The Inhibitory Effects of Isolated Compounds on NO Production
The isolated compounds 1-9 were subjected into the examination of their effects on LPS-induced iNOS-dependent NO production in RAW 264.7 cells. Cells cultured with 1-9 at different concentrations used in the presence of 100 ng/mL LPS for 24 h (Table 1). Some cell toxicity was observed in cells treated with compounds 1, 2, 4, and 9, whereas other compounds had no influence on cell viability. NO production was significantly decreased by the treatment with 2, 3, and 5-8 in a dosedependent manner, with IC 50 values in the range of 48.6 ± 1.2 and 99.8 ± 0.4 μM ( Table 1). The inhibitory effects of 1, 4 and 9 were less obvious. 2, 3, and 5-8 did not exhibit significant cytotoxicity in the concentration range of 12.5-100 μM (Table 1), thus the NO inhibiting effects were probably not due to cytotoxicity. Among the tested compounds, 2, and 5-8 belonged to the ent-kaurane diterpenoids noted for the occurrence in the Annona species [16,17]. In the previous literature, kaurenoic acid (2) was reported to inhibit NO production, prostaglandin E2 release, cyclooxygenase-2, and inducible nitric oxide synthase expression in LPS-induced RAW264.7 macrophages [18]. It also exhibited anti-inflammatory [19,20], cytotoxic [21,22], antiplasmodial [21], antimicrobial [16], hypoglycaemic [23], vasorelaxant [24,25], and antispasmodic [25] bioactivities. 16α-Hydro-19-al-ent-kauran-17-oic acid (5) displayed the most significant inhibitory effect on NO production with the lowest IC 50 value of 48.6 ± 1.2 μM. It was also reported from the stems of A. squamosa to exhibit anti-platelet aggregation activity [26]. In addition, taraxerol (3) could downregulate the expression of proinflammatory mediators in macrophages by interfering with the activation of TAK1 and Akt, thus preventing NF-κB activation [27]. In our experimental results, 2, 3, and 5 displayed NO inhibitory effects similar to these anti-inflammatory reports.

General Procedures
Melting point was determined using an Electrothermal IA-9200 melting point measuring apparatus without correction. The UV spectrum was recorded on an Agilent UV-VIS recording spectrophotometer. The IR spectra (KBr) were obtained, on a Hitachi 270-30 type spectrometer. Optical rotations were measured with a Jasco DIP-1000 KUY polarimeter. The electrospray ionization (ESI) mass spectra were determined using an Agilent 1200 LC-MSD Trap spectrometer, and the HR-ESI-MS was completed with the aid of a Bruker APEX II mass spectrometer. 1 H-and 13 C-NMR, COSY, NOESY, HMQC, and HMBC spectra were recorded on a Bruker Avance-500 NMR spectrometer, using tetramethylsilane (TMS) as the internal standard. Standard pulse sequences and parameters were used for the NMR experiments and all chemical shifts were reported in parts per million (ppm, ). Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, E. Merck, Darmstadt, Germany).

Plant Materials
The leaves of Annona reticulata L. (Annonaceae) were collected from Tiengiang, Vietnam, during October 2010 and the plant materials were identified and authenticated by Dr. Tran Huy Thai, Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology. A voucher specimen (Viet-TSWu-20101015) was deposited at the Herbarium of the Vinh University.

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, 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 [28]. 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). 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. Statistical comparisons were made by Student's t-test. 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.

Conclusion
In summary, nine compounds were characterized from the leaves of A. reticulata L. and their inhibitory activity on NO production was examined. The results provide a potential explanation for the use of the leaves of A. reticulata as a herbal medicine in the treatment of inflammatory diseases, and they may be potentially useful in developing new anti-inflammatory agents.