Extraction and Transformative Reaction of Triterpenoid and Comparison of their Biological Activity

Objective: Fusion of heterocyclic ring system of the pentacyclic triterpenoids at position 2 and 3 of the steroidal nucleus has been found to afford chemically useful compounds. Method/Analysis: Lupeol (I) was used as the starting material to carry out a transformative reaction to introducing oxadiazole moiety to ring A of the triterpenoid which was identified as lupan (2,3-C) 1′,2′,5′-oxadiazole (VI). The structures of the compounds were established based on spectroscopic (UV, IR, NMR) analysis. Finding: The derivative of the pentacyclic triterpenoid obtained has been selected for their antibacterial, fungicidal and Phytotoxicity at different concentration with respect to parent compound and found oxadiazole derivative almost at all concentration give better result compare to parent compound. Despite their widespread existence in the vicinity and their direct and indirect defensive activities, systematic study regarding their biocidal activity has not been attempted sufficiently. Since limited chemical work has so far been attempted on pentacyclic triterpene skeleton so there is a possibility of ample opportunity to study the effect of number of reagents on them. Novelty/Importance: These investigations not only enrich the triterpene chemistry but at the same time may yield a number of very useful pharmacologically important derivatives.


Introduction
Throughout history, humankind has always been interested in naturally occurring compounds from prebiotic, microbial, plants and animals sources. Various extracts of different parts of plants have been widely used in folk medicines and perfumes as well as in food flavor and preservatives and are more commonly utilized in chronic diseases like cancer, diabetes and asthma 1,2 . Plant secondary metabolite such as terpenoids is believed to be synthesized primarily as a chemical defense against harmful insects, bacteria and fungi and vertebrate herbivores 3 . Recently this group of compounds attracting attention from the pharmaceutical industry because of its considerable range of medicinal properties such as antibacterial, antifungal, anticancer and anti HIV activities 4,5 . The role of certain compounds is to act as chemical messengers, such as sex-attractants in insects, terrestrial and marine animals and humans. A number of derivatives of them have also been prepared either to establish the structure of the compound or to study the reactivity of such compounds towards some specific oxidizing and reducing agents [6][7][8] .The author interested in the biocidal activity of these plant products include understanding their mode of action, determining Structure Activity Relationship (SAR) and making suitable derivatives with improved biocidal activity.

Isolation of the Compound from the Barks of Xanthoxylum budrunga
The powdered plant materials were collected from the Sukna belt of Darjeeling foothills (barks of Xanthoxylum budrunga) and extracted with toluene using soxhlet apparatus for 36 hours. The solvents were then removed under reduced pressure and a sticky brown residue was obtained. This residue of the compounds present in the bark of the plant was than purified by column chromatography 9 using silica gel (60-120) mesh and suitable proportions of petroleum ether and ethyl acetate mixture as the eluent.

Elucidation of the Structures
All the melting points were determined by open capillary method and are uncorrected. The NMR spectra were recorded in CDCl 3 solutions at ambient temperature on a Bruker Avance 300 MHz-FT NMR spectrometer using 5mm BBO probe. The chemical shift δ are given in ppm related to Tetra Methyl Silane (TMS) as internal standard. The coupling constant (J) are reported in Hz. The IR spectra were recorded in Shimadzu FT-IR spectrophotometer in KBr discs.

Characterization of the Isolated Compounds
Different fractions of isolated compounds were mixed and crystallised by chloroform and methanol mixture which gave white crystals, m.p. 210− 212 o C; [α] D = +30.4 (conc. 0.58 in CHCl 3 ). Its IR spectrum exhibited hydroxyl at max 3610, 1020 cm -1 and exomethylene at max 3070, 1640, 887 cm -1 absorption and was identified as lupeol (I) 10 from other spectral data (NMR, Mass) and by comparison with authentic sample of lupeol (I) (Figure 1).

Hydrogenation of Lupeol: Preparation of Lupanol(II)
Lupeol (I) dissolved in a mixture of ethyl acetate and acetic acid (100 ml each) was shaken in an atmospheric hydrogen in presence of PtO 2 catalyst. The solid obtained after crystallization by using a mixture of chloroform and methanol had m.p.

Jone's Oxidation of Lupanol(II): Preparation of Lupanone(III)
To a solution of lupanol(II) in pure acetone, Jone's reagent was added until complete the reaction. The

Preparation of Lupan[2,3-C] 1 / ,2 / ,5 / -oxadiazole (VI)
Lupan [2, 3-C] 1 / ,2 / ,5 / -oxadiazole(VI) was prepared by cyclization of 2, 3-dioximino lupine (V) in dry DMF under microwave irradiation (100W, 100 o C) for 10 minutes. The compound obtained from the reaction was purified by repeated crystallization from chloroform-methanol mixture to obtain a compound (VI), m.p.249-50 o C, analyzed for C 30 11 . Interestingly in the NMR spectrum different methyl group signals got separated which may be due to the presence of heterocyclic ring attached with ring 12 . It showed the presence of six tertiary methyl of which four of them have been shifted downfield about 0.5 ppm in comparison to that oflupanone (III). A pair of one-proton doublets each at 2.1 (J=16 Hz) ppm and 3.15 (J= 16 Hz) ppm may be due to the germinal coupling of the C-1 proton which are adjacent to the furazan ring. Thus from spectral analysis the structure of the compound has been established as lupan[2,3-C]1 / ,2 / ,5 / -oxadiazole(VI) (Figure 7).

Assay of Antibacterial Activity by the Disc Diffusion Method
One ml of 48 h old culture of the test bacterium was taken in a Petri dish of 90 mm diameter. Then 20 ml of sterile Nutrient Agar (NA) medium was poured in the Petri dish and shaken carefully to mix the bacterial suspension with the medium. The Petri dishes were allowed to cool. Filter paper discs of 5 mm diameter containing desired concentration of the test samples were placed on the surface of the solidified media and incubated at 37 o C in an incubator for 48 h. Diameter of the inhibition zones were measured. In control sets, no chemicals were used in the filter paper discs but sterile distilled water was used to soak the filter papers.

Assay of Antifungal Activity
Fungi were grown on Potato Dextrose Agar (PDA) medium at 28±1 o C for mycelial growth. The fungicidal activities were determined using agar cup bioassay and spore germination bioassay. The purified eluents (10ml) were placed on two spots 3 cm apart on a clean grease free slide and the solvent was allowed to keep for some time to evaporate. One drop (0.02 ml) of spore suspension (10spores/ml) prepared from 15 days old culture of the test fungi was added on the same place where the purified eluents were placed and subsequently evaporated. Various compounds of five different concentrations were prepared (500 ppm, 400 ppm, 300ppm, 200 ppm, 100 ppm). The studies were performed at 28±1 o C for 24 h under humid conditions in Petri plates. Finally after proper incubation period, one drop of a cotton blue-lacto phenol mixture was added to each spot to fix the germinated spores.
The number of spores germinated was compared with that of germinated spores of control (where no chemicals were used). Moist chamber was used for germination of spores. The number of germinated spores was calculated on the basis of an average of 300 spores per treatment.

Phytotoxicity
In order to show phytotoxic activities different concentrations of compounds were prepared and applied to check germination of both root and shoot of the germinating seeds. Phytotoxicities of the parent compound and its derivative were determined on the healthy seeds of wheat (Sonalika variety), rice (IR-20, Jaya variety) and pea purchased from the local market. These healthy seeds were dipped in acetone-water suspensions of the compounds Vol 12 (5) | February 2019 | www.indjst.org Mohammed Golam Rasul of different concentration(500 ppm, 250 ppm, 100 ppm) and incubated for 1, 4 and 8 hours. The treated seeds of wheat, rice and pea were allowed to germinate on a mat of moist filter paper. The roots and shoots of germinated seeds were kept in a covered Petri plates. Each experiment was based on 80 seeds of each varieties or plants. After five days the germinated seeds (treated with compounds) were counted. Treated experimental sets were compared with that of control sets where no compounds were used to treat the seeds. Each experiment was repeated in triplicate. All apparatus and materials used were sterilized where necessary.
It was evident that significant inhibition of growth was observed in the case of E. coli and Lactobacillus by all the concentrations ofoxadiazole derivative (VI)with respect to lupeol(I)tested in disc diffusion method. For oxadiazole derivative (VI) maximum inhibition was observed at 500 ppm when tested on E. coli; the inhibition zone was 2.9 cm, but at 250 ppm and 100 ppm concentration the diameter of inhibition zones were 2.5 cm and 2.6 cm respectively. Thus there is a gradual increase in the zone of inhibition with the increase in concentration of the compounds. Similar results were also observed in case of the parent compound (I) at 500 ppm, 250 ppm and 100 ppm concentrations the diameters of inhibition zones were 2.7 cm, 2.5 cm and 2.4 cm respectively.
Although the inhibition was found relatively less with B. subtilis, S. aureus in comparison to above two microorganisms but the trends of activity were found identical.
The growth of inhibition zone with Pseudomonas was not prominent for both (VI) and (I).
The parent compound (I) and the respective oxadiazole derivative (VI) showed inhibition of spore
In case of rice, the parent compound lupeol(I) at 100 ppm concentration showed 0.59 cm root germination and 0.24 cm shoot germination; whereas 0.68 cm root and 0.30 cm shoot germination was observed in the case of control set. Higher concentrations of the compounds gradually reduce the germination of root as well as shoot.
In case of wheat, the parent compound (I) at 100 ppm concentration showed 2.52 cm root germination and 0.77 cm shoot germination, in comparison to 2.62 cm root and 1.1 cm shoot germination experienced in control set. The rate of germinations (root and shoot) was found to be reduced at higher concentrations of the compounds.
In case of pea, the parent compound (I) at 100 ppm concentration showed 1.65 cm root germination and 0.74 cm shoot germination, in comparison to 2.6 cm root and 1.16 cm shoot germination observed in control set. The rates of germinations (root and shoot) were found to be reduced at higher concentrations of the compounds.
Similar results were observed when the experiments were carried out with the respective oxadiazole derivative (VI). In case of rice, at 100 ppm concentration showed 0.60 cm root germination and 0.25 cm shoot germination, whereas 0.68 cm root and 0.30 cm shoot germination was experienced in control set. At 500 ppm concentration slightly reduced the germination of both root and shoot. It showed 0.52 cm root germination and 0.10 cm shoot germination. In case of wheat, the oxadiazole derivative (VI) at 100 ppm concentration showed 2.5 cm root germination and 0.91 cm shoot germination; whereas 2.62 cm root and 1.1 cm shoot germination was observed in control set. At 500 ppm concentration slightly reduced the germination of both root and shoot. It showed 1.18 cm root germination and 0.43 cm shoot germination.
In case of pea, the oxadiazole derivative (VI) at 100 ppm concentration showed 0.65 cm root germination and 0.52 cm shoot germination; whereas 1.63 cm root and 1.16 cm shoot germination was observed in control set. The 500 ppm concentration slightly reduced the germination of both root and shoot. It showed 0.30 cm root germination and 0.22 cm shoot germination.

Conclusion
The derivative showed better activity than parent compound. The introduction of oxadiazole moiety to ring A of the pentacyclic triterpenoid enhanced the activity of the compound obtained from natural sources. The present study will be extremely helpful to enrich the present knowledge about the structure activity relationship for this type of triterpenoid skeleton and encouraged the author towards a biological potential on a library of pentacyclic triterpenoids and their derivatives.

Acknowledgement
The author expresses deep sense of gratitude to Supervisor Prof. Pranab Ghosh, Department of Chemistry, University of North Bengal and Authorities of Raiganj University for providing Laboratory facilities and support.