En el presente estudio se evaluó la actividad antimicrobiana in vitro, incluyendo la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM), de diferentes extractos obtenidos de hojas de Mesua nagassarium en 13 microorganismos patógenos. El extracto metanólico y sus fracciones solubles en éter de petróleo y tetracloruro de carbono, mostraron la mayor actividad antimicrobiana. La fracción de compuestos solubles en tetracloruro de carbono mostró la zona de inhibición máxima de 24.33 mm en Bacillus megaterium con valores de CIM y and CBM de 7.81 μg/ml y 250 μg/ml, respectivamente. Como agente antimicrobiano estándar se utilizó ciprofloxacina (30 μg/disco). En el bioensayo de mortalidad de Brine shrimp el extracto metanólico y su fracción soluble en tetracloruro de carbono mostraron importante citotoxicidad con CL50 de 2.99 y 1.74 μg/ml, respectivamente, comparadas con el sulfato de vincristina (CL50 0.543 μg/ml).In the present study the in vitro antimicrobial activity, along with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), of different extracts of leaves of Mesua nagassarium were evaluated against 13 pathogenic microorganisms. The methanol extract and its pet-ether and carbon tetrachloride soluble fractions showed the highest antimicrobial activity. The carbon tetrachloride soluble fraction showed the maximum inhibition zone of 24.33 mm against Bacillus megaterium with MIC and MBC values of 7.81 μg/ml and 250 μg/ml, respectively. Ciprofloxacin (30 μg/disc) was used as standard antimicrobial agent. In the Brine shrimp lethality bioassay, the crude methanol extract and its carbon tetrachloride soluble fraction showed significant cytotoxicity with LC50 of 2.99 and 1.74 μg/ml, respectively as compared vincristine sulphate (LC50 value 0.543 μg/ml

SIKDE, Md. Al Amin

English

Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas

  
© 2011 The Authors 
© 2011 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 10 (1): 83 - 87 
BLACPMA ISSN 0717 7917 
 
Comunicación | Communication 
83 
 
 
Preliminary Antimicrobial Activity and Cytotoxicity of Leaf Extracts of  
Mesua nagassarium (Burm.f.) 
 
 [Actividad antimicrobiana preliminar y citotoxicidad de extractos de hojas de Mesua nagassarium (Burm.f.)] 
 
 
Md. Al Amin SIKDER a, Mohammad A. KAISARb, Md. Masud PARVEZa, A.K.M. Nawshad HOSSIANa, Farzana AKHTERa and 
Mohammad A. RASHIDb,* 
 
 
a Department of Pharmacy,  State University of Bangladesh, Dhaka-1205, Bangladesh 
    b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh 
Contactos | Contacts: E-mail address: Mohammad A Rashid rashidma@univdhaka.edu 
 
 
 
Abstract  
 
In the present study the in vitro antimicrobial activity, along with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), 
of different extracts of leaves of Mesua nagassarium were evaluated against 13 pathogenic microorganisms. The methanol extract and its pet-ether and carbon 
tetrachloride soluble fractions showed the highest antimicrobial activity.  The carbon tetrachloride soluble fraction showed the maximum inhibition zone of  
24.33 mm against Bacillus megaterium with MIC and MBC values of 7.81 µg/ml and 250 µg/ml, respectively. Ciprofloxacin (30 μg/disc) was used as 
standard antimicrobial agent. In the Brine shrimp lethality bioassay, the crude methanol extract and its carbon tetrachloride soluble fraction showed 
significant cytotoxicity with LC50 of 2.99 and 1.74 μg/ml, respectively as compared vincristine sulphate (LC50 value 0.543 μg/ml). 
 
Keywords:  Mesua nagassarium, extracts, antibacterial activity, cytotoxocity.. 
 
Resumen 
 
En el presente estudio se evaluó la actividad antimicrobiana in vitro, incluyendo la concentración inhibitoria mínima (CIM) y la concentración bactericida 
mínima (CBM), de diferentes extractos obtenidos de hojas de Mesua nagassarium en 13 microorganismos patógenos. El extracto metanólico y sus fracciones 
solubles en éter de petróleo y tetracloruro de carbono, mostraron la mayor actividad antimicrobiana. La fracción de compuestos solubles en tetracloruro de 
carbono mostró la zona de inhibición máxima de 24.33 mm en Bacillus megaterium con valores de  CIM y and CBM de 7.81 µg/ml y 250 µg/ml, 
respectivamente. Como agente antimicrobiano estándar se utilizó ciprofloxacina (30 μg/disco). En el bioensayo de mortalidad de Brine shrimp el extracto 
metanólico y su fracción soluble en tetracloruro de carbono mostraron importante citotoxicidad con CL50 de 2.99 y 1.74 μg/ml, respectivamente, comparadas 
con el sulfato de vincristina (CL50  0.543 μg/ml). 
 
Palabras Clave: Mesua nagassarium, extractos, actividad antibacteriana, citotoxicidad.  
 
 
 
 
  
Recibido | Received: September 23, 2010. 
Aceptado en versión corregida | Accepted in revised form: December 15, 2010. 
Publicado en línea | Published online: January 30, 2011. 
Este artículo puede ser citado como  / This article must be cited as:  Md. Al Amin SIKDER, Mohammad A. KAISAR, Md. Masud PARVEZ, A.K.M. Nawshad HOSSIAN, 
Farzana AKHTER & Mohammad A. RASHID 2011. Preliminary Antimicrobial Activity and Cytotoxicity of Leaf Extracts of Mesua nagassarium (Burm.f.).  Bol Latinoam Caribe 
Plant Med Aromat 10(1): 83 – 87.  
Sikder et al Antimicrobial activity and cytotoxicity of Mesua nagassarium  
 
Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/84 
 
INTRODUCTION 
Mesua nagassarium (Bengali - nagesar, nageswar) is a 
medium-sized or fairly large evergreen tree up to 36 m 
tall. A mixture of pounded kernels and seed oil 
isolated from this plant is used for poultice as wounds. 
The seed-oil is used for treating itch and other skin 
eruptions, dandruff and rheumatism (Orwa et al., 
2009). The flowers are known to be useful  for the 
treatment of severe colds, bleeding haemorrhoids, 
dysentery with mucus, excessive thirst, excessive 
perspiration, cough and digestion, rheumatism and 
iron induced lipid peroxidation (Yadav et al ., 2010; 
Konwarh et al., 2010). Phenolic extract of seed oil of 
M. nagassarium revealed potent antiasthmatic effect 
(Bhide, 1977). As a part of our ongoing program to 
investigate the unexplored bioactivity of traditionally 
used medicinal plant of Bangladesh we studied the 
antimicrobial and cytotoxicity of leaves of M. 
nagassarium were evaluated.  
 
MATERIALS AND METHODS 
Plant Material  
The leaves of Mesua nagassarium were collected from 
Dhaka in March 2010. A voucher specimen for this 
plant has been maintained in Bangladesh National 
Herbarium, Dhaka, Bangladesh (Accession no. 
DACB- 35158). The powdered plant sample (600 gm) 
was soaked in 2.5 L of methanol for 7 days and then 
filtered through a cotton plug followed by Whatman 
filter paper number 1. The extract was concentrated 
with a rotary evaporator at low temperature (40-45 ºC) 
and reduced pressure. The concentrated methanolic 
extract (MES) was partitioned by modified Kupchan 
method (Van Wagenen et al., 1993) and subsequent 
evaporation of solvents yielded pet-ether (PESF), 
carbon tetrachloride (CTCSF), dichloromethane 
(DCMSF) and aqueous (AQSF) soluble fractions 
which were used for the experimental processes. 
 
Antimicrobial screening 
The disc diffusion method (Bauer et al., 1966, Rahman 
and Rashid, 2008)  was used to test antimicrobial 
activity of the extractives against thirteen bacteria 
(Table-1), collected as pure cultures from the Institute 
of Nutrition and Food Sciences (INFS), University of 
Dhaka, Bangladesh. Solutions of known concentration 
(400 μg/ml) of the test samples were made by 
dissolving measured amount of the samples in 
calculated volume of solvents. Dried and sterilized 
filter paper discs (6 mm diameter) were then 
impregnated with known amounts of the test 
substances using micropipette and the residual 
solvents were completely evaporated. Discs containing 
the test materials (400 μg/disc according to disc 
diffusion method (Bauer et al., 1966, Rahman and 
Rashid, 2008) were placed on to nutrient agar medium 
uniformly seeded with the test microorganisms. 
Standard disc of Ciprofloxacin (30 μg/disc) and blank 
discs (impregnated with solvents followed by 
evaporation) were used as positive and negative 
control, respectively. These plates were then kept at 
low temperature (4º C) for 24 hours to allow 
maximum diffusion of the test materials and 
Ciprofloxacin. The plates were finally incubated at 37º 
C for 24 hours to allow maximum growth of the 
organisms. The test material having antimicrobial 
activity inhibited the growth of the microorganisms 
and a clear, distinct zone of inhibition was visualized 
surrounding the discs. The antimicrobial activity of the 
test agents was determined by measuring the diameter 
of zone of inhibition expressed in mm. The experiment 
was carried out in triplicate and the mean value was 
taken. 
 
Minimum inhibitory concentration (MIC) and 
minimum bactericidal concentration (MBC) 
determination  
Minimum inhibitory concentrations are important to 
monitor the activity of new antimicrobial agents 
(Jennifer, 2001) and is generally regarded as the most 
basic laboratory measurement of the activity of an 
antimicrobial agent against an organism (Turnidge et 
al., 2003). The MIC was determined for MES, PESF 
and CTCSF extractives by serial tube dilution 
technique (Jennifer, 2001). Thirteen test tubes were 
taken, ten of which were marked as 1, 2, 3, 4, 5, 6, 7, 
8, 9, 10 and the rest three were assigned as TM (only 
medium), TMC (Medium +extractive solution) and TMI 
(medium + inoculum). In all the test tubes containing 
the calculated amount of broth media, the sample from 
the mother solution was added with serial dilution 
which gave different sample solution having 
concentration ranged from 1000 µg/ml to 0 µg/ml. 
Lastly the inoculum was added to the test tubes, 
shaken using rotamixer and incubated at 37 
o
C for 24 
hours. The control test tube TM, containing the medium 
only was used to ascertain the sterility of the medium. 
After 24 hours the test tubes were checked for the 
microbial growth and the clear test tubes compared to 
McFarland turbidity standards were marked for the 
MIC determination. Serial dilution of the liquid 
medium and the sample is inoculated with inoculum 
whose turbidity was compared with 0.5 Mcfarland 
standards. The lowest concentration (highest dilution) 
Sikder et al Antimicrobial activity and cytotoxicity of Mesua nagassarium  
 
Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/85 
 
of antibiotic preventing appearance of turbidity is 
considered to be the minimal inhibitory concentration 
(MIC). At this dilution the antibiotic is bacteriostatic. 
The minimum bactericidal concentration (MBC) was 
determined by sub-culturing the broth media into fresh 
agar media from each tube in which no growth was 
visible. The growth of one colony indicated a 99.8% 
fall in viable count (Brumitt et al., 1984). 
 
Brine shrimp lethality bioassay 
Brine shrimp lethality bioassay (Meyer et al., 1982, 
McLaughlin et al., 1998)
  
technique was applied for 
the determination of general toxic property of the plant 
extractives. DMSO solutions of the samples were 
applied against Artemia salina in a 1-day in vivo assay. 
For the experiment, 4 mg of each of the methanol 
extract as well as pet-ether, carbon tetrachloride, 
chloroform and aqueous soluble fractions of methanol  
extract were dissolved in DMSO and solutions of 
varying concentrations (400, 200, 100, 50, 25, 12.50, 
6.25, 3.125, 1.563, 0.781 μg/ml) were obtained by 
serial dilution technique using DMSO. Vincristine 
sulphate (10 µg/ml, 5 µg/ml, 2.5µg/ml, 1.25 µg/ml, 
0.625 µg/ml, 0.3125 µg/ml, 0.15625 µg/ml, 0.078125 
µg/ml, 0.0390 µg/ml) was used as positive control.  
 
RESULTS AND DISCUSSION 
The crude methanolic extract of leaves of M. 
nagassarium  (MES) and its pet-ether (PESF), carbon 
tetrachloride (CTCSF), dichloromethane (DCMSF) 
and aqueous (AQSF) soluble partitionates were 
subjected to antimicrobial screening at 400 g/disc. 
Among the extractives, MES, PESF and CTCSF 
exhibited very strong antimicrobial activity (Table 1). 
The carbon tetrachloride soluble fraction (CTCSF) 
revealed the highest inhibition against microbial 
growth having zone of inhibition ranged from 19.33 
mm to 24.33 mm. The maximum zone of inhibition 
produced by CTCSF was found to be 24.33 mm 
against B. megaterium. This partitionate also showed 
significant antimicrobial growth having zone of 
inhibition 22.67 mm against S. paratyphi (Table 1). 
The methanol extract also demonstrated significant 
inhibition of microbial growth having zone of 
inhibition ranging from 19.3 mm to 23.0 mm. This 
extract exerted highest inhibitory activity against B.  
megaterium (having zone of inhibition of 23.0 mm). 
Relatively less polar compounds revealed better 
antimicrobial activity as the pet-ether and carbon 
tetrachloride soluble partitionates showed significant 
antimicrobial activity than the aqueous and chloroform 
soluble partitionates of methanol extract. 
As MES, PESF, CTCSF showed better 
inhibitory activity against the microorganisms, the 
minimum inhibitory concentration (MIC) required to 
inhibit the growth of organisms were measured in this 
study. The MIC value of methanol extract (where no 
bacterial growth was observed in broth media) was 
found to be 7.81 µg/ml and the MBC value (where no 
bacterial growth was observed in agar media) was 
found to be 250 µg/ml against B. megaterium (Table 
2). No microbial growth was observed in the test tubes 
TM (containing medium only) and TMC (medium + test 
sample, no inoculum) indicating that the medium and 
the test sample were not contaminated by 
microorganism and the total investigation was 
performed properly in the sterile condition. Microbial 
growth was observed in the test tube TMI (medium + 
inoculums) revealing the fact that there was no 
problem with the subcultured microorganisms. 
In case of brine shrimp lethality bioassay, the 
lethality of the methanol extract (MES) and its pet-
ether (PESF), carbon tetrachloride (CTCSF), 
dichloromethane (DCMSF) and aqueous (AQSF) 
soluble fractions were evaluated against A. salina. 
(Table 3) shows the results of the brine shrimp 
lethality testing after 24 hours of exposure to the 
samples and the positive control, vincristine sulphate 
(VS). The carbon tetrachloride soluble fraction 
(CTCSF) showed potent cytotoxic activity having 
LC50 of 1.74 μg/ml as compared to 0.453 μg/ml for 
vincristine sulphate. 
 
 
 
 
 
 
 
 
 
Table 1: In vitro antibacterial activity of the extracts from M.  nagassarium leaves 
Sikder et al Antimicrobial activity and cytotoxicity of Mesua nagassarium  
 
Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/86 
 
Test microorganisms 
Diameter of zone of inhibition (mm) 
MES PESF CTCSF DCMSF AQSE Ciprofloxacin 
Gram positive bacteria 
Bacillus cereus 21.3±0.57 21.3±2.08 22.33±2.08 10.7±1.15 10.3±2.08 40.33±3.78 
B. megaterium 23.0±1.0 20.0±1.73 24.33±1.53 12.0±1.73 8.67±0.58 44.66±1.52 
B. subtilis 20.3±1.52 21.0±1.73 22.67±1.15 12.3±2.08 10.3±0.58 40.66±2.08 
Staphylococcus aureus 20.3±1.52 20.7±1.15 22.0±1.0 10.3±0.57 9.67±0.58 43.0±1.73 
Sarcina lutea 21.0±1.0 21.3±3.51 21.67±2.08 11.0±1.0 11.3±1.53 41.0±2.0 
Gram negative bacteria 
Escherichia coli 20.0±1.73 20.3±0.58 21.33±1.15 10.0±1.0 9.67±0.57 44.67±1.53 
Pseudomonus  
aeruginosa 21.3±1.52 20.0±0 20.0±2.0 10.3±0.57 11.0±1.73 44.0±2.65 
Salmonella paratyphi  21.7±2.08 22.3±2.08 22.67±2.52 9.67±0.57 10.7±2.08 44.33±2.08 
S. typhi 19.3±2.08 19.3±1.15 19.33±3.06 9.67±0.57 12.3±2.08 43.66±1.52 
Shigella boydii 22.7±2.51 20.7±3.07 22.0±2.65 9.67±0.57 11.7±1.53 44.33±2.08 
S. dysenteriae 20.7±1.15 20.7±1.53 19.67±2.08 10.0±2.0 10.7±3.06 41.33±1.52 
Vibrio mimicus 21.3±1.15 22.3±2.31 23.0±1.0 11.0±1.73 11.7±2.08 43.33±2.88 
V. parahemolyticus 21.7±2.08 22.7±2.52 22.67±2.31 11.0±1.0 12.0±2.0 44.0±1.73 
 
Values are expressed as mean ± S.D. (n=3) 
MES= Methanol extract, PESF= Pet-ether soluble fraction, CTCSF= Carbon tetrachloride soluble fraction,  
DCMSF= dichloromethane soluble fraction, AQSF= aqueous soluble fraction. 
 
Table 2: Determination of MIC and MBC (µg/ml) of the extracts from M.  nagassarium leaves 
 
  Determination of MIC and MBC (µg/ml)  
    MES PESF CTCSF Ciprofloxacin 
Test microorganisms MIC MBC MIC MBC MIC MBC MIC MBC 
Gram positive bacteria     
          
Bacillus megaterium   7.81 250 15.62 250 7.81 250 1.25 2.5 
Bacillus subtilis 31.25 500 15.62 250 62.5 500 0.62 1.25 
Staphylococcus aureus 31.25 500 15.62 500 7.81 250 0.31 0.62 
Sarcina lutea 15.62 500 15.62 500 7.81 250 1.25 5.0 
Gram negative bacteria     
          
Escherichia coli 62.5 1000 31.25 500 31.25 250 1.25 2.5 
Pseudomonas 
aeruginosa 31.25 500 62.5 500 15.62 500 0.62 2.5 
Salmonella typhi 62.5 1000 31.25 500 31.25 500 0.31 1.25 
Shigella dysenteriae 31.25 500 31.25 250 62.5 500 0.31 2.5 
Vibrio 
parahemolyticus 31.25 1000 15.62 500 15.62 500 0.62 2.5 
 
MIC = Minimum inhibitory concentration, MBC= Minimum bactericidal concentration. 
Table 3:  Cytotoxic activity of different partitionates of M. nagassarium. 
Sikder et al Antimicrobial activity and cytotoxicity of Mesua nagassarium  
 
Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/87 
 
Sample   
Cytotoxic activity  
(LC50 mg/ml) 
       
VS            0.453 ±  0.002 
MES            2.99   ±  0.188 
PESF            4.83   ±  0.025 
CTCSF            1.74   ±  0.11 
DCMSF          12.64   ±  1.03 
AQSF             7.86   ±  0.09 
 
The average values of three calculations are presented as mean ± S.D. (standard deviation); VS = Vincristine sulphate; MES = 
Methanolic extract; PESF = Pet-ether soluble fraction; CTCSF = Carbon tetrachloride soluble fraction; DCMSF= 
dichloromethane soluble fraction; AQSF = Aqueous soluble fraction of the methanolic extract of M. nagassarium 
 
 
 
CONCLUSIONS 
The results of in vitro microbial screening of M. 
nagassarium indicated that the methanol extract and 
its pet-ether (PESF) and carbon tetrachloride (CTCSF) 
soluble partitionates have   strong antimicrobial 
activity and the isolation of active compounds is 
underway.  
 
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Preliminary Antimicrobial Activity and Cytotoxicity of Leaf Extracts of Mesua nagassarium (Burm.f.).

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