Medicinal plants for the past two decades have been of keen interest as potential
major source of new drugs mainly due to presence of various bioactive secondary
metabolites such as alkaloids, phenolics compounds, terpenoids and essential
oils. Hence, natural products in particular medicinal plants remain as a potential
source of new antimicrobial agents since microbes has been found increasingly
resistant to clinically used antimicrobial drugs (Cowan,
1999). Medicinal plants become a good choice as these natural products have
ordinary less side effects, are costless and effective against broad spectrum
of antibiotic resistant microbes (Motamedi et al.,
The genus Cassia is well known due to their excellent medicinal values.
It has been dispersed broadly in the tropical countries such as, India, Thailand,
Malaysia, Indonesia and certain region in Australia (Chukeatirote
et al., 2007). It consists of 600 species, for example Cassia
spectabilis, Cassia alata and Cassia tora and owing to their
beautiful flowers; these plants are used as ornamental plant (Pivatto
et al., 2005).
They are reported to have laxative, purgative, antimicrobial, antipyretic,
anti-inflammatory agent and antiviral properties (Silva
et al., 2005; Viegas et al., 2007).
In Brazil, the crushed leaves tea of Cassia species is used to treat
throat inflammation and diarrhea (Viegas et al.,
2007). Malaysia is endowed naturally with diverge flora and among the promising
crops that should receive more attention is C. spectabilis (sin Senna
spectabilis) (DC) Irwin et Barn. It is traditionally known in Malaysia as
The leaves and pods of C. fistula, C. spectabilis and C. podocarpa
possess laxative and antimicrobial activities (Ayo et
al., 2007). However the in depth antimicrobial properties of C. spectabilis
plant has not been well characterized except for the antimicrobial study reported
by Sangetha et al. (2008) and (Chukeatirote
et al., 2007). With this in view, the present study was undertaken
to characterize the in vitro antimicrobial properties of various crude
extracts of the C. spectabilis leaf against bacteria and yeast.
MATERIALS AND METHODS
Plant material: The fresh leaves of C. spectabilis were collected from Penang, Malaysia in 2009. The plant material was examined and washed to remove dirt before oven dried at 40°C for 3 days and ground into powder form.
Chemical and reagents: Sabouraud 4% Dextrose Agar (SDA), Sabouraud 2%
Dextrose Broth (SDB), Mueller-Hinton Agar (MHA), ethyl acetate (EtOAc), ethanol
96% (EtOH), methanol (MeOH), n-hexane, acetone, dichloromethane (DCM) and sulphuric
acid (H2SO4) were purchased from Merck (Darmstadt, Germany),
while Mueller-Hinton broth (MHB) from Becton, Dickson and Company (Le Pont de
Claix, France). Barium chloride was obtained from BDH laboratory Supplies (Poole,
England). All other chemicals and reagents used are of analytical grade.
Extraction of plant material: Briefly, 10 g of the dried powdered plant materials were extracted separately in 50 mL of acetone, n-hexane, DCM, EtOAc and MeOH for 4 days. Then, the whole extracts were decanted and filtered using Whatman No. 1 filter paper. This process was repeated three times for each solvent. The filtrates obtained were concentrated under vacuum with rotary evaporator (BUCHI R-110, USA) at 40°C to obtain the crude extracts. The extracts were subsequently freeze dried.
Antimicrobial activity evaluation
Test microorganism and growth media: All bacteria (Bacilus subtilis,
Staphylococcus aureus, Escherichia coli, Salmonella typhi and
Pseudomonas aeroginosa) and yeast (Candida albicans) were obtained
from the laboratory stock culture. The bacteria were cultured on MHA slants
while C. albicans was cultured on SDA slants at 37°C for 18 h.
Minimum Inhibition Concentration (MIC): The Minimum Inhibitory Concentration
(MIC) was performed based on a microdilution method in 96 multi-well microtiter
plates according to Valgas et al. (2007). The
crude extracts were dissolved in 50% acetone, respectively. The use of acetone
as a solvent was necessary for the miscibility of the extracts. The stock concentrations
of the samples were 10 mg mL-1. Then the stock concentration was
serially diluted two-fold by using MHB as diluents. There after, each well was
inoculated with 5 μL of suspension containing 108 cfu mL-1
(equivalents to McFarland 0.5) of the culture and incubated at 37°C overnight.
To account for the inhibitory effect of the solvent, a negative control was
included for all pathogens. This was achieved by preparing the control acetone
(25%) sample with broth in place of test plant material. The commercial drugs,
amoxillin and miconazole nitrate serve as positive controls for bacteria and
yeast respectively. Microorganisms growth was detected by addition of 40 μL
of 0.2 mg mL-1 of p-iodonitrotetrazolium violet (INT) dissolved in
water into each of the microplate wells (Eloff, 1998).
The covered microplates were incubated further for 30 min at 37°C. The MIC
was recorded as the lowest concentration of the extract that inhibited the microorganisms
growth after 24 h.
Determination of Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal
Concentration (MFC): The MBC and MFC were determined for each of the extracts
by sub-culturing the media from each well showing no visible growth onto MHA
for bacteria and SDA plates for yeast. The plates were incubated at 37°C
until growth was seen in the control plates. The MBC/MFC was defined as the
consequent concentrations required killing 99.9% of the cells (Scorzoni
et al., 2007).
Determination of Total Activity (TA): The total activity of a plant
is the quantity of material extracted from one gram of dried plant material
divided by the minimum inhibitory concentration value using the formula following
formula described by Eloff (2004).
The units mL g-1 indicate the amount to which the active extracts,
fractions or compounds in one gram of plant material can be diluted and still
inhibit the growth of the test organism.
RESULTS AND DISCUSSION
The purpose of this study was to determine the antimicrobial activity of various
organic solvent extracts of C. spectabilis leaf. Organic solvents of
different polarities namely n-hexane, DCM, EtOAc, acetone and MeOH were used
in this study. The degree of extraction of chemical constituents from C.
spectabillis leaf depends on the solubility of the solvents used. In general,
studies have shown that n-hexane extracts wax, lipids, fat soluble oils and
ester; DCM extracts terpenoids; EtOAc and acetone are used for ester extraction
and MeOH for extraction of alkaloids (Samy and Gopalakrishnakone,
The solvent extraction yield decreased in the following order: MeOH>DCM>acetone>EtOAc>n-hexane
(Table 1). MeOH resulted in a higher yield when compared to
other solvents which was in agreement with reported by Masoko
and Eloff (2005). They reported earlier higher yield and recovered more
chemical compounds when MeOH was used as extractant for leaves of Combretum
||Percentage yield of various organic extracts of Cassia
||Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal
Concentration (MBC)/Minimum Fungicidal Concentration (MFC) activity of various
organic extracts of Cassia spectabilis leaves
|NA: Not active at concentration 5 mg mL-1; ND:
In the present study, the MeOH, n-hexane, DCM, EtOAc and acetone extracts
were re-dissolved in acetone for further use in bioassay. The highest acetone
concentration used was 25% and was found not to be inhibiting the growth of
any of the tested bacteria and yeast. Acetone appears to be good solvent for
use in bioassays and most pathogens are found to be resistant to acetone even
at concentration of 51% as postulated by Eloff et al.
Susceptibility of the microorganisms was determined quantitatively using 96
wells plate method. Acetone and MeOH extracts of C. spectabilis leaf
showed a potentially good antimicrobial activity. The MIC values of acetone
extract range from 0.625 to 1.25 mg mL-1 for the tested bacteria
and C. albicans whereas the corresponding MIC values of MeOH extract
range from 1.25 to 2.5 mg mL-1 (Table 2). The MIC
values of these extracts were 10 to 80 times less potent than standard antimicrobial
drugs, amoxilin and miconazole nitrate. Acetone and methanol crude extracts
showed MBC or MFC values in range of 1.25 to 5 mg mL-1 which was
10 to 80 times less potent than standard antimicrobial drugs used. The variation
between plant extracts and standard antimicrobial drugs may due to the mixtures
compound in the plant extracts compared to pure compound in standard (Gatsing
et al., 2010).
The acetone extract was most active against Bacillus subtilis with MIC
of 0.625 mg mL-1 which was only 10 times less potent than the standard
antimicrobial drug, amoxilin (62.5 μg mL-1). The DCM and EtOAc
extracts of C. spectabilis leaf were less active against most of the
tested pathogens including C. albicans with MIC value 5 mg mL-1
or more. On the other hand n-hexane extract was not active (MIC>5 mg mL-1)
against the tested microorganisms. Antimicrobial substances are considered as
bacteriostatic agents when the ratio MBC/MIC>4 and bactericidal agents when
the ratio MBC/MIC≤4 (Gatsing et al., 2009).
In present study, MeOH and acetone showed the ratio MBC/MIC≤4, suggesting
that these extracts may be classified as bactericidal agent. Whereas for DCM,
EtOAc and n-hexane the MBC/MIC was not determined.
Earlier Chukeatirote et al. (2007) showed both
ethanol and water crude extracts of various parts (leaf, flower, stem and pod)
of Senna spectabilis (synonym of C. spectabilis) had no activity
against C. albicans at 75 mg mL-1 concentration using the
agar disk diffusion method. Only crude water extracts of S. spectabilis
showed inhibitory effect on B. cereus growth with a MIC of 30 mg mL-1
while it was inactive against E. coli. In contrast, using disk diffusion
technique and broth dilution method, Sangetha et al.
(2008) reported that C. spectabilis leaf extracts has favorable antifungal
activity against C. albicans with MIC of 6.25 mg mL-1. Present
study showed that C. spectabilis methanol and acetone crude extracts
was active against C. albicans or other microbes with a smaller MIC values.
Though ethanol (polarity index: 5.2; viscosity: 1.2) and water (polarity index:
9; viscosity: 0.89) have high polarity but they are highly viscous than methanol
(polarity index: 5.1; viscosity: 0.6). Methanol with low viscosity has low density
and high diffusivity and can easily able to diffuse into the pores of the plant
materials (Hemwimol et al., 2006).
||Total Activity (TA) of various organic extracts of Cassia
|ND: Not determined
Methanol was found to be quantitatively the best extractant, extracting a greater
quantity of plant material than any of the other solvents used. This observation
suggest that the bioactive(s) responsible for tested microorganism could be
to high polarity properties.
The effectiveness of the plant materials against microorganisms not only determined
based on the MIC values but also based on the TA of the plant. TA in mL g-1
is a measurement of potency of the plant to inhibit the microbial growth. These
values indicated the volume that can be added in dried plant material and still
kill the microorganisms (Eloff, 2004). Based on average
TA, MeOH has the highest value (84.73 mL g-1) followed by acetone
(62.23 mL g-1) and these extracts were considered the best to work
with for detailed antimicrobial evaluation (Table 3).
A good antimicrobial activity of methanol and acetone C. spectabilis leaf extracts was observed when compared to those leaf extracted with low polarity solvents. This study supports the use of C. spectabilis leaf as medicinal plant by traditional healers. However further work is warranted to fractionize, isolate and characterize the bioactive(s) responsible for the antimicrobial activity.
This project was funded by Short Term Grant from University Sains Malaysia. NK is supported by the USM Fellowship Scheme from the Institute for Postgraduate Studies (IPS) of Universiti Sains Malaysia.