Displaying publications 1 - 20 of 30 in total

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  1. Yusufzai SK, Khan MS, Sulaiman O, Osman H, Lamjin DN
    Chem Cent J, 2018 Dec 04;12(1):128.
    PMID: 30515636 DOI: 10.1186/s13065-018-0497-z
    Coumarins are the phytochemicals, which belong to the family of benzopyrone, that display interesting pharmacological properties. Several natural, synthetic and semisynthetic coumarin derivatives have been discovered in decades for their applicability as lead structures as drugs. Coumarin based conjugates have been described as potential AChE, BuChE, MAO and β-amyloid inhibitors. Therefore, the objective of this review is to focus on the construction of these pharmacologically important coumarin analogues with anti-Alzheimer's activities, highlight their docking studies and structure-activity relationships based on their substitution pattern with respect to the selected positions on the chromen ring by emphasising on the research reports conducted in between year 1968 to 2017.
  2. Yusufzai SK, Osman H, Khan MS, Abd Razik BM, Ezzat MO, Mohamad S, et al.
    Chem Cent J, 2018 Jun 12;12(1):69.
    PMID: 29896651 DOI: 10.1186/s13065-018-0435-0
    A series of novel 4-thiazolidinone inhibitors SKYa-SKYg, containing coumarin as a core structure were synthesized via facile and efficient method. The structures of the synthesized compounds were established by extensive spectroscopic studies (FT IR, 1D NMR, 2D NMR, LC-MS) and elemental analysis. All the synthesized hybrids were further evaluated for their potential as anti-tubercular agents against Mycobacterium tuberculosis H37Rv ATCC 25618, and anti-bacterial agents against Escherichia coli, Enterobacter aerogenes, Salmonella typhi, Streptococcus pneumoniae and Staphylococcus aureus. Interestingly, the hybrids displayed potent bioactivity. However, compounds SKYc, SKYd, and SKYe appeared to be more effective against the tested bacterial strains, among which compound SKYb showed the highest inhibition against all the bacterial strains ranging from 41 to 165 μg/mL, as compared to the standards, streptomycin, kanamycin and vancomycin. Moreover, derivative SKYa was found to be the strongest against M. tuberculosis (83 μg/mL). Additionally, the anti-dengue potential of the coumarin hybrids as two-component NS2B/NS3 DENV flavivirus serine protease inhibitors was calculated using computational molecular docking approach, with reference to the standards 4-hydroxypanduratin, panduratin and ethyl 3-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)propanoate with DS of - 3.379, - 3.189 and - 3.381, respectively. The docking results revealed that the synthesized hybrids exhibited potent anti-dengue activity among which compounds SKYf, SKYd, SKYc and SKYe were found to be the best ones with docking scores of - 4.014, - 3.964, - 3.905 and - 3.889. In summary, we discovered 4-thiazolidinone coumarin derivatives as a new scaffold that may eventually yield useful compounds in the treatment of bacterial and viral infections.
  3. Widyasti E, Shikata A, Hashim R, Sulaiman O, Sudesh K, Wahjono E, et al.
    Enzyme Microb Technol, 2018 Apr;111:21-28.
    PMID: 29421033 DOI: 10.1016/j.enzmictec.2017.12.009
    Oil palm trunk (OPT) is one of the most promising lignocellulosic bioresources. To develop effective biodegradation, thermophilic, anaerobic microorganisms were screened from bovine manure compost using fibrillated OPT (f-OPT) pretreated by wet disk milling as the substrate. One thermophilic, anaerobic bacterium, strain CL-2, whose 16S rDNA gene has 98.6% sequence identity with that of Caldicoprobacter faecale DSM 20678T, exhibited high degradation activity (32.7% reduction in total dry solids of f-OPT). Strain CL-2 did not use cellulose as a carbon source, but used hemicelluloses such as xylan, arabinoxylan, starch and pectin at 70 °C. Phylogenetic and morphologic analyses and the polysaccharide use suggest that CL-2 may be classified as a novel species of Caldicoprobacter, named Caldicoprobacter sp. CL-2. To characterize enzymatic activities of CL-2, extracellular enzymes were prepared from culture broth using beechwood xylan as the carbon source. The extracellular enzymes showed high xylanase activity, but low cellulase activity, suggesting that f-OPT degradation may depend on xylanase activity. To understand the xylanase system of CL-2, a major xylanase was cloned and characterized. The xylanase (CalXyn11A) had a modular structure consisting of a glycoside hydrolase (GH) family-11 domain and a family 36 carbohydrate-binding module. CalXyn11A did not show f-OPT degradation activity, but a strong synergistic effect was observed when CalXyn11A was added to the extracellular enzyme preparation. These results indicate that, rather than working alone, CalXyn11A has an important role in enhancing total lignocellulose degradation activity by cooperation with other GHs.
  4. Wan Omar A, Sulaiman O, Yusof S, Ismail G, Fatmah MS, Rahmah N, et al.
    Malays J Med Sci, 2001 Jul;8(2):19-24.
    PMID: 22893756
    We have recently reported that a dipstick colloidal dye immunoassay (DIA) that detect parasite antigens in human serum is sensitive and specific for the diagnosis of active infection of lymphatic filariasis. Rabbit polyclonal antibodies (RbBmCAg) labelled with a commercial dye, palanil navy blue was used to detect filarial antigenemia among Indonesian and Bangladeshi immigrant workers (N= 630) at oil palm estates at Hulu Trengganu District, Peninsular Malaysia. Microfilaremia with Brugia malayi were detected in 51 (8.10 %) individuals, of which 42 (6.67 %) were among the Indonesians and 9 (1.98 %) among the Bangladeshis. Microfilaremia with Wuchereria bancrofti were detected in 33 (5.24 %) individuals of which 15 (2.38 %) were among the Indonesians and 18 (2.86 %) among the Bangladeshis workers. The DIA detected 96 (15.24 %) antigenemic cases which comprise of all the microfilaremic cases and 15 (2.38 %) amicrofilaremic cases. The amicrofilaremic cases with filarial antigenemia consisted of 9 (1. 43 %) Indonesians and 6 (0.95%) Bangladeshis. We have used 6 ul of the RbBmCAg and diluted (1:10) patients' sera per dipstick which make the DIA reagent conservative. The DIA is a rapid test and can be read in approximate 2 hours.. Additionally, coloured dots developed in the DIA can be qualitatively assessed visually for intensity. The DIA does not require sophisticated equipment or radioactivity, and therefore suitable for field application.
  5. Tani N, Abdul Hamid ZA, Joseph N, Sulaiman O, Hashim R, Arai T, et al.
    Sci Rep, 2020 01 20;10(1):650.
    PMID: 31959766 DOI: 10.1038/s41598-019-57170-8
    Oil palm is an important crop for global vegetable oil production, and is widely grown in the humid tropical regions of Southeast Asia. Projected future climate change may well threaten palm oil production. However, oil palm plantations currently produce large amounts of unutilised biological waste. Oil palm stems - which comprise two-thirds of the waste - are especially relevant because they can contain high levels of non-structural carbohydrates (NSC) that can serve as feedstock for biorefineries. The NSC in stem are also considered a potent buffer to source-sink imbalances. In the present study, we monitored stem NSC levels and female reproductive growth. We then applied convergent cross mapping (CCM) to assess the causal relationship between the time-series. Mutual causal relationships between female reproductive growth and the stem NSC were detected, with the exception of a relationship between female reproductive organ growth and starch levels. The NSC levels were also influenced by long-term cumulative temperature, with the relationship showing a seven-month time lag. The dynamic between NSC levels and long-term cumulative rainfall showed a shorter time lag. The lower temperatures and higher cumulative rainfall observed from October to December identify this as a period with maximum stem NSC stocks.
  6. Sulaiman O, Hashim R, Wahab R, Ismail ZA, Samsi HW, Mohamed A
    Bioresour Technol, 2006 Dec;97(18):2466-9.
    PMID: 16524726
    Studies were carried out on heat treatment of bamboo species Gigantochloa scortechinii Gamble using palm oil. The samples were laminated and glued. The adhesion results showed that the delamination of glue line increased as the temperature and duration of oil heat treatment increased. Maximum load and shear strength of the glue line reduced as the heat treatment become more severe. It was found that the palm oil used as the heating medium penetrated in some parts of the cell wall as well as in the cell lumen of the bamboo.
  7. Sohni S, Hashim R, Nidaullah H, Lamaming J, Sulaiman O
    Int J Biol Macromol, 2019 Jul 01;132:1304-1317.
    PMID: 30922916 DOI: 10.1016/j.ijbiomac.2019.03.151
    The utilization of renewable and functional group enriched nano-lignin as bio-additve in fabricating composite has become the focus of attention worldwide. Herein, lignin nanoparticles in the form of hollow spheres with the diameter of the order of 138 ± 39 nm were directly prepared from agro-industrial waste (palm kernel shell) using recyclable tetrahydrofuran in an acidified aqueous system without any chemical modification steps. We then fabricated a new chitosan/nano-lignin composite material as highly efficient sorbent, as demonstrated by efficient removal (~83%) of methylene blue (MB) dye under natural pH conditions. The adsorption process obeyed pseudo-second-order kinetics and adequate fitting of the adsorption data using Langmuir model suggested a monolayer adsorption with a maximum adsorption capacity of 74.07 mg g-1. Moreover, thermodynamic study of the system revealed spontaneous and endothermic nature of the sorption process. Further studies revealed that chitosan composite with nano-lignin showed better performance in dye decontamination compared to native chitosan and chitosan/bulk lignin composite. This could essentially be attributed to synergistic effects of size particularity (nano-effect) and incorporated functionalities due to lignin nanoparticles. Recyclability study performed in four repeated adsorption/regeneration cycles revealed recyclable nature of as-prepared composite, whilst adsorption experiments using spiked real water samples indicated recoveries as high as 89%. Based on this study, as-prepared bio-nanocomposite may thus be considered as an efficient and reusable adsorptive platform for the decontamination of water supplies.
  8. Samson DO, Jafri MZM, Shukri A, Hashim R, Sulaiman O, Aziz MZA, et al.
    Radiat Environ Biophys, 2020 08;59(3):483-501.
    PMID: 32333105 DOI: 10.1007/s00411-020-00844-z
    For the first time, Rhizophora spp. (Rh. spp.) particleboard phantoms were developed using defatted soy flour (DSF) and soy protein isolate (SPI) modified by sodium hydroxide and itaconic acid polyamidoamine-epichlorohydrin (IA-PAE) adhesive. The microstructural characterization and X-ray diffraction patterns of the material revealed that the modified DSF and SPI adhesives became more compact and homogeneous when NaOH/IA-PAE was added, which prevented damage by moisture. It was confirmed that the composite is crystalline with (101), (002), and (004) orientations. Phantoms made of this material were scanned with X-ray computed tomography (CT) typically used for abdominal examinations with varying energies corresponding to 80, 120, and 135 kVp, to determine CT numbers, electron densities, and density distribution profiles. The radiation attenuation parameters were found to be not significantly different from those of water (XCOM) with p values [Formula: see text] 0.05 for DSF and SPI. The DSF- and SPI-based particleboard phantoms showed CT numbers close to those of water at the three X-ray CT energies. In addition, electron density and density distribution profiles of DSF-SPI-Rh. spp. particleboard phantoms with 15 wt% IA-PAE content were even closer to those of water and other commercial phantom materials at the three X-ray CT energies. It is concluded that DSF-SPI with NaOH/IA-PAE added can be used as a potential adhesive in Rh. spp. particleboard phantoms for radiation dosimetry.
  9. Rathi DN, Amir HG, Abed RM, Kosugi A, Arai T, Sulaiman O, et al.
    J Appl Microbiol, 2013 Feb;114(2):384-95.
    PMID: 23176757 DOI: 10.1111/jam.12083
    Halophilic micro-organisms have received much interest because of their potential biotechnological applications, among which is the capability of some strains to synthesize polyhydroxyalkanoates (PHA). Halomonas sp. SK5, which was isolated from hypersaline microbial mats, accumulated intracellular granules of poly(3-hydroxybutyrate) [P(3HB)] in modified accumulation medium supplemented with 10% (w/v) salinity and 3% (w/v) glucose.
  10. Rafatullah M, Sulaiman O, Hashim R, Ahmad A
    J Hazard Mater, 2009 Oct 30;170(2-3):969-77.
    PMID: 19520510 DOI: 10.1016/j.jhazmat.2009.05.066
    The present study proposed the use of meranti sawdust in the removal of Cu(II), Cr(III), Ni(II) and Pb(II) ions from synthetic aqueous solutions. Batch adsorption studies showed that meranti sawdust was able to adsorb Cu(II), Cr(III), Ni(II) and Pb(II) ions from aqueous solutions in the concentration range 1-200mg/L. The adsorption was favoured with maximum adsorption at pH 6, whereas the adsorption starts at pH 1 for all metal ions. The effects of contact time, initial concentration of metal ions, adsorbent dosage and temperature have been reported. The applicability of Langmuir, Freundlich, and Dubinin-Radushkevich (D-R) isotherm was tried for the system to completely understand the adsorption isotherm processes. The adsorption kinetics tested with pseudo-first-order and pseudo-second-order models yielded high R(2) values from 0.850 to 0.932 and from 0.991 to 0.999, respectively. The meranti sawdust was found to be cost effective and has good efficiency to remove these toxic metal ions from aqueous solution.
  11. Rafatullah M, Sulaiman O, Hashim R, Ahmad A
    J Hazard Mater, 2010 May 15;177(1-3):70-80.
    PMID: 20044207 DOI: 10.1016/j.jhazmat.2009.12.047
    In this article, the use of low-cost adsorbents for the removal of methylene blue (MB) from solution has been reviewed. Adsorption techniques are widely used to remove certain classes of pollutants from waters, especially those which are not easily biodegradable. The removal of MB, as a pollutant, from waste waters of textile, paper, printing and other industries has been addressed by the researchers. Currently, a combination of biological treatment and adsorption on activated carbon is becoming more common for removal of dyes from wastewater. Although commercial activated carbon is a preferred adsorbent for color removal, its widespread use is restricted due to its relatively high cost which led to the researches on alternative non-conventional and low-cost adsorbents. The purpose of this review article is to organize the scattered available information on various aspects on a wide range of potentially low-cost adsorbents for MB removal. These include agricultural wastes, industrial solid wastes, biomass, clays minerals and zeolites. Agricultural waste materials being highly efficient, low cost and renewable source of biomass can be exploited for MB remediation. It is evident from a literature survey of about 185 recently published papers that low-cost adsorbents have demonstrated outstanding removal capabilities for MB.
  12. Lamaming J, Hashim R, Leh CP, Sulaiman O, Sugimoto T, Nasir M
    Carbohydr Polym, 2015 Dec 10;134:534-40.
    PMID: 26428155 DOI: 10.1016/j.carbpol.2015.08.017
    In this study cellulose nanocrystals were isolated through acid hydrolysis process from parenchyma and vascular bundle of oil palm trunk (Elaeis guineensis). The morphological properties of obtained cellulose nanocrystals were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The microscopy images showed smoother and cleaner surface of parenchyma cellulose nanocrystals when compared to vascular bundle cellulose nanocrystals. The TEM image shows a higher length and diameter for parenchyma cellulose nanocrystals compared to vascular bundle cellulose nanocrystals. The Fourier transform infrared (FTIR) spectra showed changes in functional groups after acid hydrolysis due to removal of lignin, hemicelluloses and other impurities in both type of cellulose nanocrystals. Crystallinity index of cellulose nanocrystals was observed higher for vascular bundle as compared to parenchyma. Thermogravimetric analysis (TGA) was performed to study the thermal stability of cellulose nanocrystals and it was observed higher for parenchyma cellulose nanocrystals compared to vascular bundle.
  13. Lamaming J, Hashim R, Sulaiman O, Leh CP, Sugimoto T, Nordin NA
    Carbohydr Polym, 2015;127:202-8.
    PMID: 25965475 DOI: 10.1016/j.carbpol.2015.03.043
    In this study cellulose nanocrystals were isolated from oil palm trunk (Elaeis guineensis) using acid hydrolysis method. The morphology and size of the nanocrystals were characterized using scanning electron microscopy and transmission electron microscopy. The results showed that the nanocrystals isolated from raw oil palm trunk (OPT) fibers and hot water treated OPT fibers had an average diameter of 7.67 nm and 7.97 nm and length of 397.03 nm and 361.70 nm, respectively. Fourier Transform Infrared spectroscopy indicated that lignin and hemicellulose contents decreased. It seems that lignin was completely removed from the samples during chemical treatment. Thermogravimetric analysis demonstrated that cellulose nanocrystals after acid hydrolysis had higher thermal stability compared to the raw and hot water treated OPT fibers. The X-ray diffraction analysis increased crystallinity of the samples due to chemical treatment. The crystalline nature of the isolated nanocrystals from raw and hot water treated OPT ranged from 68 to 70%.
  14. Lamaming J, Hashim R, Leh CP, Sulaiman O
    Carbohydr Polym, 2017 Jan 20;156:409-416.
    PMID: 27842840 DOI: 10.1016/j.carbpol.2016.09.053
    Cellulose nanocrystals were isolated from oil palm trunk by total chlorine free method. The samples were either water pre-hydrolyzed or non-water pre-hydrolyzed, subjected to soda pulping, acidified and ozone bleached. Cellulose and cellulose nanocrystal (CNC) physical, chemical, thermal properties, and crystallinity index were investigated by composition analysis, scanning electron microscopy, transmission electron microscopy, fourier transform infrared, thermogravimetric analysis and X-ray diffraction. Water pre-hydrolysis reduced lignin (<0.5%) and increased holocellulose (99.6%) of ozone-bleached cellulose. Water pre-hydrolyzed cellulose exhibited surface fibrillation and peeling off after acid hydrolysis process compared to non-fibrillated of non-water pre-hydrolyzed cellulose. Water pre-hydrolysis improved final CNC crystallinity (up to 75%) compared to CNC without water pre-hydrolysis crystallinity (69%). Cellulose degradation was found to occur during ozone bleaching stage but CNC showed an increase in crystallinity after acid hydrolysis. Thus, oil palm trunk CNC can be potentially applied in pharmaceutical, food, medical and nanocomposites.
  15. Kunasundari B, Arai T, Sudesh K, Hashim R, Sulaiman O, Stalin NJ, et al.
    Appl Biochem Biotechnol, 2017 Sep;183(1):412-425.
    PMID: 28361245 DOI: 10.1007/s12010-017-2454-z
    The availability of fermentable sugars in high concentrations in the sap of felled oil palm trunks and the thermophilic nature of the recently isolated Bacillus coagulans strain 191 were exploited for lactic acid production under non-sterile conditions. Screening indicated that strain 191 was active toward most sugars including sucrose, which is a major component of sap. Strain 191 catalyzed a moderate conversion of sap sugars to lactic acid (53%) with a productivity of 1.56 g/L/h. Pretreatment of oil palm sap (OPS) using alkaline precipitation improved the sugar fermentability, providing a lactic acid yield of 92% and productivity of 2.64 g/L/h. To better characterize potential inhibitors in the sap, phenolic, organic, and mineral compounds were analyzed using non-treated sap and saps treated with activated charcoal and alkaline precipitation. Phthalic acid, 3,4-dimethoxybenzoic acid, aconitic acid, syringic acid, and ferulic acid were reduced in the sap after treatment. High concentrations of Mg, P, K, and Ca were also precipitated by the alkaline treatment. These results suggest that elimination of excess phenolic and mineral compounds in OPS can improve the fermentation yield. OPS, a non-food resource that is readily available in bulk quantities from plantation sites, is a promising source for lactic acid production.
  16. Kosugi A, Tanaka R, Magara K, Murata Y, Arai T, Sulaiman O, et al.
    J Biosci Bioeng, 2010 Sep;110(3):322-5.
    PMID: 20547348 DOI: 10.1016/j.jbiosc.2010.03.001
    Old oil palm trunks that had been felled for replanting were found to contain large quantities of high glucose content sap. Notably, the sap in the inner part of the trunk accounted for more than 80% of the whole trunk weight. The glucose concentration of the sap from the inner part was 85.2g/L and decreased towards the outer part. Other sugars found in relatively low concentrations were sucrose, fructose, galactose, xylose, and rhamnose. In addition, oil palm sap was found to be rich in various kinds of amino acids, organic acids, minerals and vitamins. Based on these findings, we fermented the sap to produce ethanol using the sake brewing yeast strain, Saccharomyces cerevisiae Kyokai no.7. Ethanol was produced from the sap without the addition of nutrients, at a comparable rate and yield to the reference fermentation on YPD medium with glucose as a carbon source. Likewise, we produced lactic acid, a promising material for bio-plastics, poly-lactate, from the sap using the homolactic acid bacterium Lactobacillus lactis ATCC19435. We confirmed that sugars contained in the sap were readily converted to lactic acid with almost the same efficiency as the reference fermentation on MSR medium with glucose as a substrate. These results indicate that oil palm trunks felled for replanting are a significant resource for the production of fuel ethanol and lactic acid in palm oil-producing countries such as Malaysia and Indonesia.
  17. Khan JF, Shah DM, Sivapakiam S, Mokhtar S, Subramaniam M, Raman K, et al.
    Transplantation, 2021 Dec 01;105(12):2507-2512.
    PMID: 34818304 DOI: 10.1097/TP.0000000000003591
  18. Ibrahim MN, Ngah WS, Norliyana MS, Daud WR, Rafatullah M, Sulaiman O, et al.
    J Hazard Mater, 2010 Oct 15;182(1-3):377-85.
    PMID: 20619537 DOI: 10.1016/j.jhazmat.2010.06.044
    The present study explores the ability of modified soda lignin (MSL) extracted from oil palm empty fruit bunches (EFB) in removing lead (II) ions from aqueous solutions. The effect of contact time, point zero charge (pH(pzc)) and pH of the solution, initial metal ion concentration and adsorbent dosage on the removal process were investigated. Furthermore, the MSL is characterized by SEM, XRF, FT-IR and surface area analysis. Equilibrium adsorption isotherms and kinetics were investigated. The experimental data were analyzed by the Langmuir, Freundlich and Temkin models of adsorption. The kinetic data obtained at different initial concentrations were analyzed using pseudo-first-order and pseudo-second-order models. The results provide strong evidence to support the hypothesis of adsorption mechanism.
  19. Hashim R, How LS, Kumar RN, Sulaiman O
    Bioresour Technol, 2005 Nov;96(16):1826-31.
    PMID: 16051090
    The flame retardancy of medium density fiberboard (MDF) made from mixture of rubberwood fibers and recycled old corrugated containers was studied. Aluminum trihydroxide (ATH) was used as a fire retardant additive and mixed with the fibers to manufacture experimental MDF panels using wet process. Phenol formaldehyde (PF) resin in liquid, 2% based on oven dry weight of fibers, was used along with 0%, 10%, 15% and 20% of ATH. The flame retardant test was done using the limiting oxygen index (LOI) test. The other properties investigated include internal bond strength, thickness swelling and water absorption. The results showed that ATH loading increased as the LOI of MDF increased. This demonstrated that ATH could improved the fire retardant property of MDF at sufficient loading. An increase in concentration of ATH showed an increase in the IB values of MDF made without resin. MDF panels made without resin showed a progressive increase in internal bond as the composition of recycled old corrugated containers fiber increased. Addition of resin improved internal bond strength and reduced thickness swelling, and water absorption. Thickness swelling of panel increased as the composition of recycled old corrugated containers fiber increased. Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDAX) showed that there is indication of ATH and resin filling the void space in between fibers.
  20. Ghalib RM, Hashim R, Sulaiman O, Mehdi SH, Anis Z, Rahman SZ, et al.
    Nat Prod Res, 2012;26(22):2155-8.
    PMID: 22181707 DOI: 10.1080/14786419.2011.633083
    The leaves of Cinnamomum iners (Reinw. ex Blume-Lauraceae) have been refluxed successively with chloroform and alcohol to get chloroform extract and alcoholic extract. Both the extracts have been assayed for cytotoxicity against human colorectal tumour cells. The chloroform extract exhibited significant cytotoxicity with IC(50) 31 µg mL(-1) (p  200 µg mL(-1). The chloroform extract has been further proceeded for chemical analysis by GC-TOFMS and 178 components were identified including acids, amines, amides, aldehydes, alcohols, esters, benzene derivatives, bicyclic compounds, terpenes, hydrocarbons, naphthalene derivatives, furan derivatives, azulenes, etc. Nine components representing 51.73% of the total chloroform extract were detected as major components. Caryophyllene (14.41%) and Eicosanoic acid ethyl ester (12.17%) are the most prominent components of the chloroform extract. β-Caryophyllene (14.41%) as most abundant compound supports potent cytotoxicity as shown by chloroform extract.
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