Isolation and antimicrobial activities of a novel discolornolide and other compounds from Monanthotaxis discolor

Characterisation of a novel discolornolide ( 1 ) isolated from a first time investigation of Monanthotaxis discolor is described. Other 6 known compounds, karatavin ( 2 ), N acetylanonaine ( 3 ), quercetin-3- O --arabinose ( 4 ), stigmasterol ( 5 ), a mixture of stigmasterol and  -sitosterol ( 6 ) and octahydro-5-isopropyl-3-methyl-2-methyleneazulene-8,8-diol ( 7 ) isolated are also reported. The structures were established by spectroscopic methods. Citotoxicities and antimicrobial activities of the compounds and crude extracts are also reported whereby compound 1 showed in vitro antifungal activity against Candida albicans and Aspergilus niger at concentrations of 0.13 and 0.17 mg/ml with zones of inhibitions of 7.0 and 5.5 mm respectively. The compound also showed cyitotoxic activity in the brine shrimp test with LC 50 of 5.88  g/ml. Also compound 4 exhibited antibacterial activity against E. coli and S. aureus . The compound also exhibited cytotoxic activities in the brine shrimp test with LC 50 of 24.73  g/mL. The crude extracts exhibited varying citotoxic and in


Chromatography and Spectroscopy experiments
Column chromatography was carried out using silica gel of 230-400 mesh (Merck) and Sephadex ® LH-20 (Pharmacia). Fractions were monitored by thin layer chromatography (TLC) (60 F 254 , Merck) developing with different solvent systems and spots visualized under UV light at 254 nm and 366 nm. Detection was done under UV and thereafter spraying with panisaldehyde reagent and heating at 105  C. Vacuum liquid chromatography (VLC) used for fractions and was carried out using normal phase silica gel of particles size 400 mesh ASTM (Merck) applying gradient elution. The vacuum was generated from a membrane pump.
The Melting Points were determined using electrothermal melting point apparatus and are uncorrected. The IR spectra were recorded on Perkin Elmer FT-IR spectrum 100 spectrophotometer fitted with UATR with absorptions given in wave number (cm -1 ). UV/Vis spectra were run on a scanning spectrophotometer Shmadzu UV-2101PC using chloroform or methanol of which maximum absorption ( max ) are given in nm. Mass spectra were recorded under electron impact (EI) at 70 eV. 1 H and 13 C NMR spectra were recorded on a Bruker 300 spectrophotometer operating at 300 MHz for 1 H NMR and 75 MHz for 13 C NMR. CDCl 3 or MeOH or DMSO were used as solvents. Chemical shift are given in  value relative to an internal standard TMS  = 0 ppm) for 1 H NMR or  value relative to the internal standard CDCl 3 for 13 C NMR. All 13 C spectra were obtained as 13 C decoupled spectra. The 1 H and 13 C NMR spectra data are summarized in table S1.

. Plant Material
The leaves, root and stem barks of Monathotaxis discolor were collected from Chimala Scarp Forest Reserve in Mbeya region, Tanzania. The plant material materials were identified in the field by a botanist and the identification was confirmed at the herbarium of the Department of Botany of the University of a Dar es Salaam where a voucher specimen was deposited under number FMM 3403 T3.

Extraction and isolation
The air-dried and pulverized plant material was soaked twice consecutively in petroleum ether, dichloromethane and methanol for 48 hrs twice at room temperature (25-30 o C). The extracts were concentrated using a rotary evaporator and fractionated by VLC over silica gel. Repeated column chromatography on silica gel and Sephadex® LH-20 (Pharmacia) yielded compounds 1 -7.

Brine shrimp lethal testing
The brine shrimp test (BST) was used to evaluate the citotoxicity of crude extracts and pure compounds. It was carried out using brine shrimps (Artemisia salina) larvae as test organisms using the standard method. (Meyer et al. 1982).

Anti-microbial Assays
Antimicrobial activity of the plant crude extracts, some VLC fractions and pure compounds were evaluated by the agar well diffusion method as describe by Meyer et al. (1982)  The crudes of dichloromethane, methanol, petroleum ether of root and stem bark of M.
discolor extracts exhibited higher antimicrobial activity (Table S4) showed activity against both bacteria and fungi (Table 5).

The minimum inhibitory concentrations (MIC)
The minimum inhibitory concentrations (MIC) were determined using the micro plate serial dilution method. A duplicated experiment was done for each extract/ pure compound against each organism. Nutrient broth was prepared and 1 mL of it was transferred into each of the test tubes (Perez et al.1990). For antifungal MIC, Malt Extract Broth (MEB) was prepared according to the manufacturer's instructions and dispensed in capped test tubes to about 10 mL in each tube. For the determination of antimycotic activity, all the fungal isolates, were first adjusted to the concentration of 10 6 CFU/mL of subcultured fungal colonies were then asceptically transferred into the broth and adjusted to a turbidity equivalent to 0.5 McFarland standard. The results for tested compound 1, 2 and 3 showed mild activity against tested fungi and bacteria (Table 6). Table S1. 1 H and 13 C NMR spectral data, and HMBC interactions for compound 1  Figure S1: The 1 H NMR spectra for discolornolide Figure S2: The 13 C NMR spectrum for discolornolide Figure S3. The DEPT-135 o spectrum for disclornolide Figure S4: The H/H COSY spectrum for discolornolide Figure S5: The HSQC Spectrum for discolornolide Figure S6. Important long-range C/H interactions as observed in the HMBC plot for compound         Figure S3. The DEPT-135 o spectrum for discolornolide   Figure S5: The HSQC Spectrum for discolornolide