INTRODUCTION:

Zanthoxylum armatum DC. (syn. Z.alatum Roxb.) of Rutaceace family is an important medicinal plant which is commonly known as Indian prickly ash, Toothache tree (English) Tejphal(Hindi) Tejowati (Sanskrit) Timur (Nepal).It is widely distributed in India from Kashmir to Bhutan at altitudes up to 2,500 m, also occurs throughout North east India. It is also found throughout most of china, Taiwan. Nepal, Phillipines, Malasiya, Pakistan, Japan at altitudes of 1,300-1,500 m. valleys and thickets in the mountains, wasteland and the under storey of mixed forest are customary locations of the species.^1-11

Z.armatum DC is extensively used in Indian system of medicine as a carmanative, stomachic and anthelminthic and in treatment of Toothache. The fruit, seeds are employed as an aromatic tonic in fever, dyspepsia and expelling round worms. Phytochemical investigations on bark have afforded many non volatile constituents such as lignans, alkaloids, hydroxy-enoic acids as well as flavanoids. The following accounts on zanthoxylum essential oils and consists of mostly Terpenes, Geraniol, cineol, citronellal, limonene, β-myrecene, 1,8 cineol, Methyl cinnamate, also dipentene was found besides several minor constituents^12-16. The present research work on zanthoxylum armatum describes the isolation and structural elucidation of a new compound cinnamoyl amide from Dichloromethane extract of bark and In-vitro microbial assay of crude extract.

Chemical constituents

Various phytochemical constituents like alkaloids, sterols, phenolics, lignans, coumarins, terpenoids, flavanoids and their glycosides and benzenoids, fatty acids, amino acids have been isolated from this plant. A volatile oil consisting mainly linalool¹⁷monoterpetriol-3,7-dimethyl 1-octane 3,6,7-triol,trans cinnamic acid, nevadensin umbelliferone, β-sitosterol and its glucoside¹⁸3,5,dihydroxy7,8,4 trimethoxy flavones and tambulin¹⁹, 3-methoxy-11-hydroxy-6,8-dimethylcarboxylatebiphenyl,3,5,6,7-tetrahydroxy, 3¹,4¹-dimethoxyflavone-5-β-d-xylopyranoside²⁰, armatamide, lignans, asarinin and fargesin, α and β-amyrins lupeol, β-sitosterol β-d-glucoside²¹ have been previously reported from the plant. Anthelminthic²², antiproliferative²³ antifungal²⁴ and anti-insecticidal²⁵ activities have also been studied with different parts of the plant.

MATERIAL AND METHOD:

General:

Melting points were recorded on a perfit melting point apparatus. IR spectra recorded on a Bruker FTIR spectrophotometer (KBr discs). NMR spectra were obtained on Bruker Advance 300 and 500 spectrometers (300 MHz for 1H and 125 MHz for 13C, CDCl₃ as solvent (TMS as internal standard).

Plant material:

The plant Zanthoxylum armatum DC was collected from Ananthagiri hills, Andhra pradesh in the month of Dec-2011 and identified from plant identification laboratory (Dept. of Botany, Andhra University, Visakhapatnam) a voucher specimen (AUPH – 1287) has been deposited in the laboratory for future records.

Extraction and Isolation:

The stem bark of Z.armatum (2.5 Kg) then rinsed, air dried and pulverized into powder. The material was then macerated using organic solvents and extracted exhaustively 5 times following sequential extraction with nonpolar solvent (Hexane) and with solvents of (Dichloromethane, Ethyl acetate, Methanol, Ethanol, Water) increasing polarity. The extract was filtered and the filtrate was concentrated using rotary evaporator at low temperature and high pressure to remove the solvent under reduced pressure which yielded a dark brown, greenish semi solid mass. The Dichloromethane extract (30gm) was fractionated initially with n-Hexane, and then chromatographed over silica gel (600 gm) column. The column was eluted with increasing polarity using different solvents.

Structure Elucidation:

Spectral Data:

The compound showed characteristic IR absorption bands at IR (KBR) Vmax 3446.39, 2967.99, 2901.59, 2850.21, 1497.97 cm^-1. In the 1H NMR spectrum characteristic signals for a methylene dioxy group as a sharp singlet H (S δ 5.906), H (q δ 3.028, j = 4.6), H (q δ 3.857, j=3.6), H (M δ 4.222 j=2.0), H (d δ 4.692 j=4.0), H (m δ 6.792 j=1.2) (H- Aromatic) H (s δ 6.8333) (H-Aromatic), were assigned. The c13 NMR also corroborated the proposed structure. Diagnostic signals were readily observed and assigned as δ148.00 (C12)(C=0), δ 147.12(C19,C1), δ 135.25(C20,C2), δ 119.31 (C17,C5) (CH=CH),δ 108.16(C22,C4), δ 106.51 (C21,C3), δ 101.7(C18,C6)(O-CH-O), δ 85.78 (C24,C8), δ 77.49 (C15), δ 77.17(C16), δ 76.85(C11), δ 71.71(C10), δ 54.39 (C9), (CH₂CH₂NH). The identity of the compound was confirmed by its comparison with reported data of compound²⁶. On the basis of above spectral data, the compound was identified and the structure was established as N-(3’, 4’- Methylene dioxy phenyl ethyl) –3, 4-Methylene dioxy cinnamoyl amide.

Structure:

Invitro Microbial Assay:

The anti-bacterial activity²⁷ was determined against Gram –ve strains ( Serratia sp, Pseudomonas sp) and Gram +ve bacteria (Bacillus aureus, Staphylococus aureus) obtained from culture collection of bacteriological laboratory, Dept. of Botany, Faculty of Science, Acharya Nagarjuna University, Guntur using Whatman filter paper no-0.1, 1 cm diameter, disc diffusion assay method. Five replicates were performed with two concentrations (200µg/ML and 400µg/ML). Discs were soaked in the test compound for 30 seconds, evaporated, then over load on the surface of the nutrient agar media cultured with the tested bacterium. All the plates were incubated at 30C for 48 hrs. Ampicillin and Amoxicillin were used as reference compounds.

Anti-Bacterial Screening:

Invitro screening experiments for antibacterial activity^27-29 of Dichloromethane Bark extract was subjected to biological testing. To substantiate the antibacterial results, we screened against two Gram positive and two Gram negative bacteria using reference standard antibiotics. The minimum inhibitory concentrations³⁰ (MIC, µgm/ml) are determined using standard dilution method. The MIC value is summarized in table 1.

Antimicrobial activity of DCM extract:

(Dry DMSO as solvent)

Table.1:

Test Organism DCM Extract Ampicillin Amoxicillin

Gram +ve Strain

B. cereus 11^a 10^a No effect^a 19^b

S. aureusNo effect^a 8^aNoeffect^a 6^b

Gram –ve Strain

Serratia. sp No effect^a 11^a 13^a No effect^b

Pseudomonas sp No effect^a 11^a 13^a

No effect^b

a- Values show the zone of inhibition in mm: concentration of samples was 200µg/ML.

b- Values show the zone of inhibition in mm: concentration of samples was 400µg/ML.

c- Data are the mean of five measurements with neglected standard errors.

d- Reference antibiotics were carried out at 200µg/ML only.

RESULTS AND DISCUSSION:

From the data obtained, it is clear that Dichloromethane bark extract possess higher activity against Gram +ve strain particularly Bacillus cereus on the contrary Gram –ve strains are not affected at tested concentrations as shown in table 1. Our results are in good agreement with antibacterial activity and are comparative with standard reference antibiotics.

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Received on 06.04.2012 Modified on 12.06.2012

Asian J. Research Chem. 5(6): June, 2012; Page 730-732