Antibacterial Activity of Cycas rumphii Miq. Leaves Extracts against Some Tropical Human Pathogenic Bacteria
Abdul Viqar Khan,
Qamar Uddin Ahmed,
Athar Ali Khan
Cycas rumphii is traditionally used to cure wide variety of ailments including malignant ulcers, wounds healing, sore throats and tuberculosis showing its great potential as an antimicrobial agent. Hence, aim of the present study was to explore antibacterial potential of the leaves of C. rumphii Miq. Dried leaves were separately macerated with methanol and double distilled water at room temperature to obtain methanol and aqueous (AQ) extracts. The methanol extract was further successively extracted with light petroleum ether (LPE), benzene (BZ), ethyl acetate (EtOAc) and methanol (MeOH) on hot water bath to get respective extracts. These extracts were evaluated for their antibacterial efficacy at six different concentrations (500 μg, 1, 2, 5, 10 and 15 mg mL-1) against seven G+ve and eleven Gve hospital isolated bacterial strains causing several tropical diseases using disc diffusion method. EtOAc and MeOH extracts showed maximum antibacterial activity against most of the bacteria taken into account. The most susceptible Gram positive bacteria was S. albus with inhibition zone of 22 mm while most susceptible Gram negative bacteria was S. boydii with 21 mm inhibition zone with EtOAc extract while the most resistant bacteria was gram negative S. typhi. Present findings are suggestive of antibacterial agents in the leaves of C. rumphii which can be used in future for formulation of broad spectrum herbal antibacterial products. Results of present study also support various traditional uses associated with this plant which were suggestive of possible antibacterial potential of this plant. Hence, EtOAc and MeOH extracts of the leaves of C. rumphii deserve further investigations.
July 26, 2011; Accepted: September 16, 2011;
Published: October 28, 2011
Since antiquity, plants have traditionally been used to cure a broad continuum
of human diseases (Abu-Rabia, 2005). Ayurveda, Unani
and Siddha are important Indian medical practices which have played a significant
role to congregate information on traditional medicinal plants in the course
of many centuries (Muthu et al., 2006). According
to survey conducted by ethnobotanists in India, it was found out that about
2500 different plant species are used to cure various disorders by folklore
practitioners in India (Shengji, 2001). During the past
few decades, various scientific attempts have been made to study medicinal plants
and their traditional uses in different parts of the world (Lev,
2006; Karim et al., 2011). Ethnomedical studies
on traditional medicinal plants have significantly helped pharmaceutical industries
around the world in finding out new therapeutic agents for the various fields
of tropical biomedicine (Cordell, 2000). Recently in
the past twenty years, many of the plant species have been documented pharmacologically
which are endowed in phytochemicals with marked activity on human pathogenic
bacterial strains (Cox, 1994; Khan
et al., 2010, 2011).
Inability of existing antibiotics to cure clinical infections efficaciously
due to resistant bacteria has urgently demanded the need for new antimicrobial
agents to overcome chronic bacterial infections (Sule et
al., 2011a). Alleviating the problem may require the use of new antimicrobials
to mimic the bodys natural defense against infection and plants are considered
potent candidate for this aim. Employment of antibiotic resistance inhibitors
of plant origin is one of the ways to reduce antibiotic resistance and adverse
effects on host associated with pathogenic microbes (Nostro
et al., 2000; Houghton, 2001).
Cycas rumphii Miq. (C. rumphii) (family: Cycadaceae; common name:
Queen Sago) is a native species of Indonesia that inhabits closed wood-lands
or forest near shorelines in S. Borneo, N.E. Java, Maluku Papua and Sulawesi
(Barceloux, 2008). C. rumphii grows up to 10 m
in height with the trunk diameter reaches up to 400 mm. The grey bark is characteristically
fissured into diamond and rectangular-shaped (Lindstrom
and Hill, 2007). Different parts (leaves, seeds and bracts) of C. rumphii
are traditionally used in India, Bangladesh and Indonesia to cure wide variety
of ailments including malignant ulcers, wounds healing, boils, itchy skin lesions,
nephritic pains, edematous swellings, dizziness, headaches, sore throats, flatulence,
vomiting of blood, gynecological disorders and tuberculosis (Jain,
1991; Barceloux, 2008). In India, male bracts are
also used as a narcotic, stimulant and aphrodisiac agents (Jain,
Phytochemical investigations of C. rumphii have revealed the presence
of different class of phytoconstituents including cycasin; β-glycosidase;
amentoflavone; podocarpusflavone A; 2, 3-dihydroamentoflavone; 2, 3-dihydrohinokiflavone;
isoginkacin and bilobetin (Rastogi and Mehrotra, 1991,
1993; Rastogi, 1995; Uddin
et al., 2004).
Literature has revealed many medicinal uses associated with this plant species particularly in the management of malignant ulcers, wounds healing, sore throats and tuberculosis showing its great potential as an antimicrobial agent. However, its antibacterial potential has never been evaluated scientifically. Hence, present study was an attempt to evaluate the possible antibacterial potential of the crude leaves extracts of C. rumphii Miq. against some tropical human pathogenic bacterial strains.
MATERIALS AND METHODS
Collection of plant material: Fresh leaves (5 kg) of C. rumphii were collected during winters (December 2009) from different localities of Aligarh district, U.P., India. Plant samples (Voucher specimen number: AV023, AV2080) were deposited in the Herbarium of Department of Botany, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, U.P., India.
Preparation of non-polar and polar extracts: Shade dried leaves of C.
rumphii (1.75 kg) were pulverized in an electric grinder and powder was
stored in a desiccator. The grinded material was used for the extraction process.
Five hundred grams plant powder was macerated with 95% methanol in a round bottom
flask at room temperature for about 24 h. Mother liquor was filtered out and
residual plant material was again macerated with 95% methanol for another 24
h. The entire process was repeated four times to ascertain the maximum yield
of methanol soluble compounds. The extract was evaporated to dryness at 35°C
under reduced pressure using rotary evaporator to get concentrated methanol
extract. The extract was freeze-dried to give a final yield of 55 g (11.0%)
methanol extract and kept in the chiller at -18°C till further use. Freeze-dried
methanol extract was reflexed with light petroleum ether (60-80°C) for 5
h on the hot water bath. Light petroleum ether soluble portion upon filtration
was evaporated under reduced pressure and freeze-dried to obtain light petroleum
ether extract (LPE) (10.0 g, 2.0%). Light petroleum ether insoluble fraction
of methanol extract obtained in above process was further reflexed with benzene
for 5 h, filtered and evaporated under reduced pressure and freeze-dried to
obtain benzene extract (BZ) (12.5 g, 2.5%). Benzene insoluble fraction was reflexed
with ethyl acetate for 5 h, filtered and evaporated under reduced pressure and
freeze-dried to obtain ethyl acetate extract (EtOAc) (8.0 g, 1.6%). Ethyl acetate
insoluble fraction was further reflexed with methanol for 5 h, filtered and
evaporated under reduced pressure and freeze-dried to obtain methanol extract
(MeOH) (13.0 g, 2.6%) while methanol insoluble residue was discarded. During
the entire successive extraction, each step was repeated five times to ensure
maximum yield of every desirable extract.
Five hundred gram plant powder macerated with double distilled water and left
for 72 h at room temperature under dark. The flask was then reflexed over hot
water bath for 10 h and the mother liquor was filtered. The distilled water
was again added reflexed and filtered and the entire process was repeated for
four times. The filtrate, thus obtained, was evaporated to complete dryness
under reduced pressure and freeze-dried to obtain aqueous extract (AQ) (40.0
g, 8.0%) (Harborne, 1998).
Microorganisms: Leaves extracts were tested for possible antibacterial activity through disc diffusion method against eighteen (18) human pathogenic bacteria viz., Staphylococcus aureus, Staphylococcus aureus ATCC 25953, Staphylococcus albus, Streptococcus haemolyticus Group A, Streptococcus haemolyticus Group B, Streptococcus faecalis, Bacillus subtilis, Escherichia coli, Edwardsiella tarda, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi, Shigella boydii, Shigella dysenteriae, Shigella flexneri and Plesiomonas shigelloides. All bacterial strains taken into consideration were obtained from the bacterial stock, Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh, U.P., India. The bacterial cultures were maintained at 4°C on nutrient agar.
Bacterial susceptibility test: Disc diffusion method (Bauer
et al., 1966; Colle and Marr, 1989; Vaghasiya
et al., 2007) was used to determine susceptibility of plant extracts.
Standardized inoculum (1-2x107 CFU mL-1 0.5 McFarland
standard) was introduced on the surface of the plates containing Mueller Hinton
agar (NCCLS, 2004) which was spread evenly with a glass
spreader. Paper disc (6 mm) were impregnated with 500 μg mL-1,
1, 2, 5, 10 and 15 mg mL-1 plant extracts. The discs were dried in
an oven at 37°C and placed on the agar surface with the help of a sterile
forceps, aseptically. Finally the discs were pressed with forceps to make complete
contact with the surface of the medium. Inoculated petridishes were incubated
at 37°C overnight and the inhibition zones were recorded. The experiments
were repeated thrice and the mean of the triplicate of the results is summarized
in Table 1. Standard sensitivity discs of chloramphenicol
(10 μg/disc) (Span Diagnostics Limited, Surat, India) were used to test
the sensitivity profile against the reference bacteria. Each experiment was
done in triplicate and the mean values are presented. The diameters of the inhibition
zones were measured in mm with respect to evaluate antimicrobial activity of
all extracts in vitro.
|| Yields of non-polar and polar freeze dried extracts of the
leaves of C. rumphii
Five hundred gram pulverized leaves of C. rumphii produced = 40.0 g (8.0%) freeze-dried AQ extract. Five hundred gram pulverized leaves of C. rumphii yielded = 55.0 g (11.0%) freeze-dried methanol extract. Methanol extract upon successive extraction with non-polar and polar organic solvents at room temperature subsequently yielded LPE = 10.0 g (2.0%), BZ = 12.0 g (2.5%), EtOAc = 8.0 g (1.6%) and MeOH =13.0 g (2.6%) freeze-dried extracts (Table 1).
In the present study, the investigation of antibacterial activity of the aforementioned extracts of the leaves of C. rumphii against seven Gram-positive and eleven Gram-negative bacteria was studied using disc diffusion method. The data pertaining to the antimicrobial potential of non-polar (LPE and BZ) and polar (EtOAc, MeOH and AQ) extracts of the leaves of C. rumphii are presented in Table 2. The results revealed variability in the inhibitory concentrations of each extract for given bacteria. All extracts presented antimicrobial activity to at least four of the tested microorganisms. None of the extracts except EtOAc extract of the leaves of C. rumphii were active against S. typhi while all the extracts were active against S. aureus, S. haemolyticus (group B) and K. pneumoniae; the LPE extract was inactive against S. albus, S. faecalis, B. subtilis, E. coli, S. typhi and S. boydii. BZ extract was found inactive against S. aureus (ATCC 25953), S. haemolyticus (group A), P. vulgaris, P. aeruginosa, S. typhi, S. dysenteriae and S. flexneri, however, EtOAc extract did not show any antibacterial activity against E. tarda, P. vulgaris and S. typhi. AQ extract showed no inhibitory activity against E. coli, P. mirabilis, P. aeruginosa, S. typhi and S. dysenteriae. The most susceptible Gram-positive bacteria was S. albus, inhibition zone = 22 mm (EtOAc extract) and Gram-negative bacteria was S. boydii, inhibition zone = 21 mm (EtOAc extract), respectively. The most resistant bacteria was Gram negative S. typhi strain with almost no antibacterial activity against all extracts except EtOAc extract which displayed 11 mm inhibition zone at 15 mg mL-1 (Table 2).
Moreover, the EtOAc and MeOH extracts presented the highest activity, i.e., they were able to inhibit 15 (83.33%) types of microorganisms of interest. Furthermore, they also had the highest activity rate against bacteria. On the other hand, AQ extract showed inhibitory activity against 12 (66.66%) types of microorganisms and non-polar extracts (LPE and BZ) revealed antimicrobial activity against 11 (61.11%) microorganisms of interest, respectively. The antibacterial activity was more prominent on the Gram-positive bacteria than the Gram-negative bacteria. Gram-positive bacteria were the most susceptible to growth inhibition by EtOAc and MeOH extracts. The results showed that all the extracts have variable degrees of antibacterial activity and that the inhibition of bacterial growth was dose dependent as inhibitory action of the extracts was found to increase with the increase of concentration against all bacterial strains.
All extracts displayed similar antibacterial activity compared to positive
control (Chloramphenicol) though at higher concentration. All extracts up to
1 mg mL-1 did not exert any antibacterial activity against all bacterial
|| Antibacterial activity of Cycas rumphii Miq. leaves
extracts against some tropical human pathogenic bacteria
|Gram positive bacteria: 1. Staphylococcus aureus 2.
Staphylococcus aureus ATCC 25953 3. Staphylococcus albus 4.
Streptococcus haemolyticus, Group-A 5 and Streptococcus haemolyticus,
Group-B 6. Streptococcus faecalis 7. Bacillus subtilis.
Gram negative bacteria: 1. Escherichia coli 2. Edwardsiella tarda
3. Klebsiella pneumoniae 4. Proteus mirabilis 5. Proteus
vulgaris 6. Pseudomonas aeruginosa 7. Salmonella typhi
8. Shigella boydii 9. Shigella dysenteriae 10. Shigella
flexneri 11. Plesiomonas shigelloides. Values are the mean of
replication of three experiments; -: no inhibition. Zone of inhibition in
mm LPE: Light petroleum ether extract, BZ: Benzene extract, EtOAc: Ethyl
acetate extract, MeOH: Methanol extract, AQ: Aqueous extract
Highest antibacterial activity was observed at 15 mg mL-1 by all
extracts against most of the bacterial strains. EtOAc extract exhibited the
highest antibacterial activity against most of the bacterial strains in comparison
to other extracts of the leaves of C. rumphii.
Plants are imperative source of potentially important compounds for the development
of new therapeutic agents. Natural products from medicinal plants as secondary
metabolites (minor phytoconstituents) have been associated with the conferment
of antibacterial activities by many studies (Rios and Recio,
2005; Tamokou et al., 2008; Sule
et al., 2011b). The presence of some of such biological active substances
in a substantial amount in the explored part of C. rumphii may have bestowed
the strong and significant antibacterial activity on the leaves extracts. In
this regard, higher concentration of these phytochemicals might have been responsible
for a higher degree of inhibition on the bacterial strains.
Formerly, in many studies of similar kinds, polar extracts i.e., acetone, ethanol
and methanol have been used as extractants, however this orchestrated strategy
could not reveal the furthermost sensitivity in yielding strong antimicrobial
compounds that might pave the ways to synthesize new antimicrobial drugs (Cowan,
1999; Ncube et al., 2008). For this reason,
in the present study, five different solvents based on their different degrees
of polarity as determined by the functional group present, i.e., light petroleum
ether, benzene, ethyl acetate, methanol and water were used and taken into account
to obtain maximum active compounds in the extracts. It is quite noteworthy to
observe that a relationship was detected between the extract solubility and
antibacterial activity of different fractions. This suggests that in sequential
extraction, maximum biological active compounds were solubilized according to
their degree of solubility in polar solvents as EtOAc and MeOH extracts (fractions)
displayed the highest antibacterial activity followed by AQ extract, BZ and
LPE fractions. Parekh et al. (2005) and Sule
et al. (2011a) accounted that methanol is a better solvent for consistent
extraction of antimicrobials from medicinal plants. Our results also comply
with the same study as polar fractions (EtOAc and MeOH) obtained from methanol
extract revealed potent antibacterial activity in comparison to non-polar solvents
(LPE and BZ). These results further confirm that significant antibacterial compounds
are polar in nature as evidenced by the higher degrees of antibacterial activity
of EtOAc and MeOH fractions from the methanol extract of the leaves of C.
All the extracts exhibited strong antibacterial activity against Gram-positive
bacteria in comparison to Gram-negative bacteria. The susceptibility of Gram-positive
bacteria towards various plant extracts than those of Gram-negative bacteria
has already been reported previously (Bele et al.,
2009; Vaghasiya and Chanda, 2010). This has been
attributed to the fact that the Gram-negative bacterial cell wall outer membrane
appears to act as a permeability barrier to many substances including amphipathic
(cationic, neutral and anionic) broad spectrum antibiotics (Lewis,
2001). The activity of C. rumphii leaves extracts against both
Gram-positive and gram-negative bacteria might indicate the presence of broad
spectrum antimicrobial compounds. Biological active substances in the form of
pure compounds and standardized plant extracts endow infinite prospects for
the development of new drugs owing to bountiful unparalleled availability of
chemical miscellany (Sati et al., 2011; Cos
et al., 2006; Chanda et al., 2011;
Sule et al., 2011b). These results explicitly
suggest that the bioactive compounds present in polar extracts have good potential
for development of novel antibacterial herbal products.
Results obtained from our study further confirm the traditional use of C.
rumphii in different ailments associated with pathogenic bacteria. Non-polar
and polar extracts revealed varying degrees of antibacterial activity. However,
polar extracts i.e., EtOAc and MeOH extracts demonstrated most pronounced antibacterial
activities against bacterial strains taken into consideration. Hence, EtOAc
and MeOH extracts of the leaves of C. rumphii deserve further investigations
to develop new antibiotics that may help in combating several bacterial diseases
in tropical countries. This is the first ever report on the antibacterial potential
of the leaves of C. rumphii.
Thanks are due to Department of Science and Technology SERC Division, New Delhi for financial support to the author Dr. Abdul Viqar Khan.
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