INTRODUCTION
Plants have been classified as an essential source of medicinal agents for
centuries and a huge number of novel drug components have been isolated from
natural plant source and their extract used for in traditional medicine (Obeidat
et al., 2012).
Cassia alata is one of the most important species of the genus Cassia
which is rich in anthraquinones and polyphenols. The leaves of C. alata
have been qualitatively analyzed for the presence of primarily five pharmacologically
active anthraquinones: rhein, aloe-emodin, chrysophanol, emodin and physcion
as well as the flavonoid, kaempferol (El-Mahmood and Doughari,
2008; Moriyama et al., 2001). The flavonoid,
kaempferol has been reported to have anticancer properties (Fernand
et al., 2008).
Idu et al. (2007) observed that, preliminary
phytochemical analysis of Cassia alata showed the presence of phenols,
tannins, anthraquinones, saponins and flavonoids. Odunbaku
and Lusanya (2011) also corroborated this study; they further stated that,
the plant also had alkaloids and cardenolides.
Sharma et al. (2010) also reported in their
study that Preliminary phytochemical screening of alcoholic extract revealed
the presence of anthraquinone glycosides, Phenolic compounds; Saponin glycoside
and while aqueous extract showed presence of glycosides and Phenolic compounds,
Saponin glycoside.
These anthraquinone derivatives are well known to exhibit a variety of biological activities such as antimicrobial, antifungal, antitumor, antioxidant, cytotoxic and hypoglycaemic activities. Other chemicals contained in the plant include saponin which acts as a laxative and expels intestinal parasites. Rhein and chrysophanol are also known to be present in the roots in addition to two other quinone pigments.
Pieme et al. (2006) investigated the acute and
sub-acute toxicities of hydro-ethanolic extract of leaves of Cassia alata
on Swiss mice and Wistar albino rat. This study presents strong evidence of
the nontoxic effect of the hydroethanolic extract of Cassia alata. Cassia
alata showed some protective effect on hepatocytes and improved liver architecture.
These results showed that the use of the extract of Cassia alata is safe
and explained the extensive utilisation of the plant in traditional medicine.
In the light of this, Chukeatirote et al. (2007)
stated that since ancient time, plant and animal products have been used for
treatment of diseases and disorders and that, plants in particular, have been
used to treat infections due to its antimicrobial properties.
The effects of water, methanol and chloroform extracts on some pathogenic Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes, Pseudomonas aeruginosa and Proteus mirabilis showed that the plant parts can be used to treat infections caused by these bacteria. S. aureus, S. pyogenes and P. mirabilis were more susceptible while E. coli and P. aeruginosa were less sensitive.
Uddin et al. (2008) observed in their study
that methanol extract of Cassia tora had strong antioxidant properties
and that the results they obtained provided a support for the use of this plant
in traditional medicine. They also observed that the methanol and aqueous extracts
of Cassia tora showed significant antibacterial activities against few
gram positive negative bacterial strains.
Pieme et al. (2006) observed that, the ethanol-aqueous
extract of S. alata showed moderate antibacterial and antifungal activity.
Also, Sharma et al. (2010) stated that Ethanol
and aqueous extract of Cassia alata had antibacterial activity. Awal
et al. (2004) also observed that ethanol leave extract of Cassia
alata had antibacterial properties. They further stated that, ethanol extract
of Cassia alata has cytotoxic effect against Artemia and postulated
that C. alata can be used for the treatment of cancer cell line in humans.
Krishnan et al. (2010) observed that acetone
and Methanol extracts of Cassia spectabilis were very effective on Bacillus
subtilis.
Reezal et al. (2002), opined that C. alata
has been proven to be effective against C. albicans growth culture by
using the ethanol and aqueous barks extracts. They further stated that Miconazole
when compared to the Cassia alata barks aqueous and ethanol extract on
C. albicans growth, showed only a slight differences between them. 30
mg mL-1 Miconazole with 18 mm inhibition zone and 30 mg mL-1
of barks aqueous and ethanol extracts with inhibition of 16 mm and 14 mm. This
proved that this plant has potential to be exploited as a natural source of
antifungal remedy in the future. With an increase of discs concentration, the
extracts might produce at least the same or better effects than Miconazole.
Esimone et al. (2008) observed that there was
an excellent effect of the in vitro study of the antimicrobial potency
of herbal soap formulated with ethanol extract of Cassia alata. They
stated that the antimicrobial activity of the soap is predominantly against
Gram-positive and opportunistic yeast. The herbal soap formulated with Cassia
alata demonstrated high potency against common pathogens of the skin and
therefore a potential excipient in the production of antiseptic soaps. They
observed that the findings have high medical, industrial and economic significance
as extracts of Cassia alata could be harnessed in the formulation of
medicated soaps.
Oladunmoye et al. (2007) observed in their study
that ethanol extract of Cassia hirsuta inhibited the activities of some
microorganisms by altering their genome. They further stated that the extract
can be mutagenic and also possess antimicrobial activities against pathogenic
bacteria.
Abubacker et al. (2008) stated that aqueous
extract of Cassia alata can be used as potential antifungal agent. They
observed that the aqueous extract of Cassia alata had effect on Aspergillus
flavus, A. parasiticus, Fusarium oxysporum and Candida
albicans.
Cassia alata traditional usage varies greatly in different places and
countries. This plant is well known to treat external infections mostly the
skins. The leaves, however, shown to be the choice for treatment rather than
the flowers, barks and seeds. Throughout the tropics, the leaf juice is collected
and found to be useful in treating skin diseases. Whereas, an infusion of the
roots is used to treat rheumatism and as a strong laxative (Reezal
et al., 2002).
Plants have been classified as an essential source of medicinal agents for
centuries and a huge number of novel drug components have been isolated from
natural plant source and their extract used for in traditional medicine (Obeidat
et al., 2012).
Historically, plants have provided a good source of anti-infective agents with
the compounds which are highly effective instruments in the fight against microbial
infections (Idu et al., 2007).
Plants with different medicinal properties have been employed by traditional
medical practice for the treatment of different disease conditions. In eastern
Nigeria, some plants which have frothing or foaming ability have been employed
as soap for bathing and for treatment of skin and wound infections (Esimone
et al., 2008).
Cassia alata is known to have laxative properties. Elujoba
et al. (1989). Traditionally, tea are made from the leaves and taken
as a treatment for constipation and intestinal worms. The leaves are pounded
and rubbed on the skin to cure eczema and ringworm (Pieme
et al., 2006) and white-spot fungal skin infections (Palanichamy
and Nagarajan, 1990). The leaves contain a fungicide, chrysophanic acid
which is a common ingredient in soaps, shampoos and lotions (Ajose,
2007; Villasenor et al., 2002). The leaves
are boiled with water and simmered before applied over the infected area 2 times
a day due to wound healing properties. In treatment for ringworm, usually, the
leaves are crushed and made into paste. Then it is spread upon the affected
parts. For treatment of eczema, the infected part is washed repeatedly with
strong decoction of the leaves and flowers. In cases of bronchitis and asthma,
decoction of the leaves and flowers in herpetic constitutions administered repeatedly
taken during the day relieves dyspnoeal oppression and promotes expectoration.
The medicine also acts on the bowels slightly and increases the secretion of
urine.
Several traditional uses of Cassia alata have been discovered in several places. In Indo China and Philippines Island, the leaves are considered most effective against herpes and the wood in decoction is used as a mild purgative. In Guinea, the pounded fresh leaves are applied or rubbed on to all kind of skin affections. In the Gold Coast, the leaves are applied to dhobey itch, craw-craw and ringworm on the forehead or on the skin. This is one of the most effective amongst native medicine. The leaves are also boiled and drunk by women to hasten the delivery of children.
This study investigates the antibacterial activity of the aqueous and methanol extracts of sundried leaves of Cassia alata by testing the extracts against selected bacteria.
MATERIALS AND METHODS
Plant materials: Cassia alata leaves were collected from the premises of the Federal Government College in Warri, Delta State-Nigeria and authenticated at the Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos. The collected leaves were cleaned of unwanted foreign materials, cut up into small pieces and dried in sunlight for a week, ground and weighed. The dried material was coarsely milled, packed into a brown paper bag and stored at room temperature in the laboratory until used.
Chemicals and reagents: Sterile petri dishes, Mueller Hinton agar and Sabouraud Dextrose Agar (SDA) were purchased in Lagos. Cup borer, diameter 6 mm, Hot air oven, Cooling incubator C1-10S (REMI Instruments Ltd., Mumbai, India), Incubator (Astell Hearson, England), Uniscope SM801A Laboratory Water bath (Surgifield medicals, England), Vertical Heating pressure steam sterilizer LDZX-30FB (Labnet international Inc, Woodbridge, USA),Mettler P1210 balance (GallenHorup) was provided by the Department of Pharmaceutics and Pharmaceutical Technology, University of Lagos.
Extraction of plant materials
Methanol extraction: The leave material was soaked in a Winchester
bottle with methanol for 48 h (maceration). The extract was concentrated using
a Buchi V-801 rotary evaporator at 35°C.
Aqueous extraction: The leave material was boiled with water in a 2000 mL (2 L) of pyrex beaker at 60°C in a water bath for an hour twice, filtered and concentrated on a water bath at 70°C.
The coarsely milled leaves and stem bark of Cassia alata were extracted separately using water and methanol as solvents. About 140 g of the powdered sample was continuously extracted with a particular solvent by use of a Soxhlet extraction apparatus for 24 h. The extracts were filtered and concentrated to dryness under reduced pressure and controlled temperature (50-55°C) to obtain solvent-free semisolid extracts. The solvent-free semisolid extracts obtained were used for the antimicrobial studies.
Test microorganism and growth media: The microorganisms (Gram-positive bacteria: Bacillus subtilis, Staphylococcus aureus, Gram-negative bacteria: Escherichia coli, Pseudomonas aeruginosa) used for the study were obtained from the stocks of the Pharmaceutical Microbiology laboratory of the Department of Pharmaceutics and Pharmaceutical technology, Faculty of Pharmacy, University of Lagos.
Assay of organism: The bacterial strains were grown and maintained on Mueller Hinton agar at 37°C. All bacteria were first cultivated in their various diagnostic media and then subcultures into nutrient Agar plates. This was to remove the effects of inhibitory agents and indicators in the diagnostic media. All bacteria were sub cultured into various sterile nutrient broths in universal bottles and incubated overnight. Using sterile saline in a universal bottle, the culture suspension was calibrated to about 108 CFU mL-1 using sterile Pasteur pipette. All standardized assay organisms were kept for the assay.
Antibacterial activity evaluation: The antibacterial activity of aqueous and methanol extracts of the leaves of Cassia alata at concentrations of 50, 100, 150 and 200 mg mL-1 were determined using the cup plate method. A molten Mueller Hinton agar stabilized at 45°C was seeded with 0.1 mL of a 24 h broth culture of the test organism (B. subtilis, E. coli, P. aeruginosa and S. aureus) containing approximately 108 CFU mL-1 in a sterile petri dish and allowed to set. Wells of 6 mm diameter were created with a sterile cork borer and filled to about three-quarters full with solutions of the aqueous and methanol extracts of the leaves of Cassia alata. The plates were pre-incubated for 1 h at room temperature to allow for diffusion of the solutions and then incubated for 24 h. The zones of inhibition were measured (mean, n = 2). Streptomycin and propylene glycol were used as positive and negative controls, respectively.
Statistical analysis: The experiments were run in duplicate and the zones of inhibition were determined and recorded (mean, n = 2). The different effects of methanol and aqueous extract of Cassia alata extracts on the test organisms were analyzed using ANOVA.
Minimum inhibition concentration (MIC): The Agar dilution technique was used to determine the Minimum Inhibition Concentration (MIC) of the extract of Cassia alata against the test organisms. A stock concentration of 500 mg mL-1 of extract was prepared by dissolving 15 g of extract in 30 mL of propylene glycol. Ten working concentration were subsequently prepared from the stock.
Ten different concentration of the extract was used for this determination ranging from 0.625-320 mg mL-1. Sterile petri dishes containing varying volumes of extract and molten agar (total volume 20 mL in each petri dish) depending on the concentration of extract intended were inoculated with 0.2 mL of the test organisms previously diluted to contain approximately 105 CFU mL-1 for bacteria and 105 CFU mL-1 for fungi. A plate without an extract and another without a test organism were used as controls. The plates were incubated at 37°C for 24 h and observed for growth. The experiments were conducted in duplicate. The plate with the lowest concentration of the extract which showed no growth after incubation was taken and recorded as the MIC.
RESULTS AND DISCUSSION
Crude methanol and aqueous extract of leaves and barks from Cassia alata were tested for selected bacteria (Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa).
All the crude extracts inhibited the growth of S. aureus and B. subtilis,
though to varying degrees (Table 1, 2) as
indicated by the zones of inhibition. The extract did not show any activity
against E. coli and Pseudomonas which are gram negative bacteria
contrary to observations of El-Mahmood and Doughari, 2008).
The activity of the extract also varied between solvents with the aqueous extracts
demonstrating the highest activity against all the susceptible bacteria as revealed
on Table 2 similar to observations of Roy
et al. (2006) and the results showed that the extracts demonstrated
a concentration-dependent antibacterial activity with higher concentrations
of 150 and 200 mg mL-1 showing greater zones of inhibition than lower
concentrations of 50 and 100 mg mL-1. High MIC values are indication
of low activity while low MIC values are indication of high activity. In this
study, S. aureus and B. subtilis had low MIC values thus suggesting
higher activity against the corresponding organisms (Table 3).
| Table 3: |
Minimum inhibitory concentration (MIC) profile of Cassia
alata against test organisms (mg mL-1) |
 |
CONCLUSION
This study has shown that, crude extracts of C. alata possessed reasonable
activity against some bacteria and has high potential as antibacterial agent.
This finding is in line with Pieme et al. (2008)
and Krishnan et al. (2010). This finding provides
an insight into the usage of these plants in traditional medicine for the treatment
of common skin disorder since the common bacterial skin infections are usually
caused by Staphylococcus and Streptococcus species which are gram
positive organisms. This plant can be locally sourced since it grows well in
any Nigerian soil. However, the effect of this plant against a wider range of
bacteria and fungi and toxicological studies of the extracts is recommended.
ACKNOWLEDGMENTS
We are grateful to Mr. A.R. Usman of the Pharmaceutical Microbiology Laboratory, Faculty of Pharmacy, University of Lagos, for his technical assistance in carrying out this study and Mrs. Nancy Alalor for her Moral and financial support during this project.
