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Microsoft word - 7_vol.3_2_, 2012, ijcepr-232, 142-148
Inhibitory Effect of Varying Concentrations of Leaves’ Extracts of Centella
Asiatica (Gotu Kola) on Some Microorganisms of Medical Importance
Dora Imefon Udoh1,*, Naomi Udo Asamudo1, Danladi Ngyan Bala2 and Otung Enwongo1
1Department of Microbiology, Faculty of Science, University of Uyo
2Department of Pharmacognosy and Natural Medicine, Faculty of Pharmacy, University of Uyo, Uyo
*E-mail: [email protected]
The inhibitory concentration of Centella asiatica (Gotu kola) on some microorganisms of clinical importance was investigated
using standard microbiological methods. The phytochemical screening revealed the presence of Alkaloids, Saponins, Tannins,
Flavonnoids, Anthraquinones, Cardiac glycosides and Phlobatannins. The antimicrobial tests were carried out with ethanolic and
aqueous extracts using agar disc diffusion method. The bacterial species used were Staphylococcus aureus, Bacillus subtilis,
Escherichia coli, Proteus species, Shigella species, Salmonella typhi and Vibrio cholerae. The results obtained for ethanolic
extracts revealed that Staphylococcus aureus exhibited the largest zones of inhibition 23.0mm (35mg/ml) while Shigella and
Escherichia coli had the smallest of 15.0mm (15mg/ml). For the aqueous extracts Proteus species had the largest zone of
inhibition of 18.0mm (35mg/ml) while Vibrio cholerae had the least zones of inhibition of 12.1mm (15mg/ml). The higher
inhibitory effects of these ethanolic extracts may be due to some bioactive substances present in the extracts. The minimum
inhibitory concentrations ranged from 100mg/ml to 500mg/ml for both aqueous and ethanolic extracts. The results obtained
qualify Centella asiatica to be a medicinal plant that is recommended for use in the treatment of some diseases and infections.
Key words: Inhibitory effect, leaves extracts, bioactive components, Centella asiatica (Gotu kola), microorganisms.
2012 ijCEPr. All rights reserved
It would be of interest to know that most plants that we have around us contain one or more chemical substances
which are of therapeutic value. That is why in developing countries especially in rural areas, people often turn to
traditional medicine dealers when ill or infected with one particular disease or another.
Herbs had been used by all cultures throughout history to cure or prevent a disease in both animals and man . It
has become an integral part of the development of modern civilization. In Nigeria, a variety of herbal preparations
are used by traditional medicine practitioners for treatment of different kinds of diseases including microbial
The search for new antimicrobial agents led to the screening of bioactive compounds of plants. Medicinal use of
plants range from the plant roots, bark, stem, flowers, leaves, seeds and extracts from the whole plants . Some
plants whose antimicrobial properties have been demonstrated include some Euphoriaceae plants which the family
provides food [13,19].They varied in medicinal properties and are used in ethnobotany such as in the treatment of
ailments like respiratory infection, venereal diseases, toothache, rheumatism, cough, ulcer and wounds [1,2,7,12,16].
Others include Allium sativum, which is active against E. coli, Salmonella typhi, Staphylococcus aureus and some
There is some traditional usage of some plants such as Centella asiatica (Gotu kola) in Nigeria. Centella asiatica
belongs to the kingdom Plantae ,Family of Mackinlayaceae and Genus Centella .It is a small herbaceous annual
plant of subfamily Mackinlayoideae of the Apiaceae and is a native to India ,Sri Lanka, Northern Australia,
Indonesia, Iran, Malaysia, Papua New Guinea,and other parts of Asia[5,15,24]. It has small fan-shaped green leaves
with white or light purple to pink flowers. It bears small oval-shaped fruits. The stems are slender, creeping
stolons, green to reddish green in colour, interconnecting one plant to another. It has a long –stalked, green reinform
leaves with rounded apices which have smooth texture with palmate netted veins. It is mainly found in as a weed
growing in waste lands, agricultural lands and river basins. It grows abundantly during the rainy season. It contains
no caffeine and is not a stimulant [5,11,15]. The plant has been exploited for several hundred years by traditional
medicine dealers for the treatment of wounds, mental fatigue, skin conditions such as leprosy and psoriasis. It has
been also used for treatment of syphilis, hepatitis, stomach ulcers, epilepsy, diarrhea, fever and asthma [4,24].
Centella asiatica has been widely used in traditional medicine in Africa, India, China, Japan, Indonesia, Sri Lanka
and in South pacific for treatment of microbial infections . Centella asiatica is often called one of the miracles
elixirs of life because legend has it that an ancient Chinese herbalist lived for more than 200 years as a result of
using this herb . Researchers reported that an American and Europeans herbalist used Centella asiatica for disorders that cause connective tissue swelling such as Scleroderma, Ankylising Spondylitis (arthritis of the spine) rheumatoid arthritis, depression and to improve memory . Another reported that Centella asiatica has been used traditionally in lowering of high blood pressure, treating of various insufficiency (pooling of blood in the veins usually in the leg), boosting of memory and intelligence, easing anxiety and would healing  Despite all these alleged used, there is need to ascertain some inhibitory effects of various concentrations of this plant extract on some medically important microorganisms especially infectious bacteria. This verification of
its inhibitory effects on some of these bacteria of medical importance will help to establish a scientific rationale and
basis, thereby validating its use. Hence the reason that proffers this research.
MATERIALS AND METHODS
Centella asiatica plant part (leaves) was collected from Ikot Abasi Local Government Area of Akwa Ibom State.
The leaves were identified by a taxonomist in the Department of Pharmacognosy and Traditional Medicine,
University of Uyo, Uyo. The leaves were sun-dried for two weeks. The dried leaves were then grounded into fine
particles with mortar and pestle for homogenicity.
Extraction (Ethanolic Extract)
400 grams of pulverized Centella asiatica was weighed out in 300ml of 50% ethanol for 72 hours at room
temperature and then filtered using Whatman No. 1 filter paper. The filtrate was concentrated in water bath and
evaporated to dryness at 400C [2,22]. The dried sample was stored in the refrigerator at 40C for further analysis.
Table-1: Phytochemical Screening of both Aqueous and Ethanolic Leaf Extracts of Centella Asiatica Violent ring observed with Keller-Killani KEY:
+ = bioactive component in small amount
++ = bioactive component moderate amount.
+++ = bioactive component high amount
++ ++ = bioactive component much higher amoun
400 grams of pulverized Centella asiatica weighted out into 300ml of distilled water. The beaker was shaken every
30 minutes for 6 hours before leaving it undisturbed on a work bench for 48 hours. The mixture was then filtered
using Whatman No. 1 paper. The filtrate was kept in the refrigerator at 40C until used [2,22].
To test for alkaloids, 0.5g of the extracts were stirred with 5ml of 1% hydrochloric acid (HCl) on a steam bath and
then filtered. 1ml of the filtrate was treated with few drops of Dragnendroffs reagent and observed .
In Saponin test, two methods were employed, namely frothing test and sodium bicarbonate test. (a) Frothing test, about 0.5g of the plant’s extracts were shaken vigorously with distilled water in a test-tube and allowed for 15 minutes then observed . (b)) Sodium bicarbonate test, about 0.5g of the plant’s extracts was added with 5% sodium bicarbonate and Fehling’s solutions A and B were added and boiled. Then observation was made . Also to test for the presence of tannin, about 0.5g of the extracts were dissolved with 100ml of distilled water and filtered. The filtrated 5% Ferric Chloride reagent was added and observed. Hydrochloric acid test was used for detection of Phlobatannins. 0.5g of the extracts were dissolved in water and filtered, the filtrate was boiled with 1% hydrochloric acid and observed . Borntrager’s test was used in detecting the presence of anthraquinones whereby 0.5g of the extracts were shaken with 10ml benzene and filtered. To the filtrates, 10% ammonia solution was added to each tube and the mixture were shaken and observed . To test the presence of Flavonnoids was carried according to method described by . Whereby few pieces of magnesium metal were added to 5ml of each plant extract solution. The solution was obtained by dissolving the extract in concentrated hydrochloric acid. The presence of cardiac glycosides was confirmed using Liberman’s test, and Keller-Killani test [9,22,23]. Table-2: Zones of Inhibition (mm) Showing Inhibitory Effect of Varying Concentration of Centella asiatica Aqueous Leaves Extracts on the Test Organisms Concentration (mg/ml) and Zones of Inhibition (in mm)
Collection of Bacterial Test Isolates
Bacterial isolates used for this analysis were obtained from Microbiology Laboratory, University of Uyo, University
of Uyo Health Centre as well as University of Uyo Teaching Hospitals, all in Akwa Ibom State, Nigeria. The
organisms used were Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Proteus mirabilis, Shigella
species, Proteus mirabilis, Salmonella species and Vibrio cholerae.
Evaluation and Antimicrobial properties of the Plant’s Leaf Extracts
The ability of the plant’s leaf extract to inhibit growth of the bacterial isolates of medical importance was
determined using the disc diffusion method . Sterile filter paper discs of 6mm in diameter were soaked in equal
volumes of varying concentrations of the extracts and left for 2 hours undisturbed. The 0.2ml of 18 hours peptone
water culture of each of the test bacterial isolates was spread on the sterile Mueller Hinton Agar plates. The discs
about 6mm in diameter containing varying concentrations of these extracts were picked with sterile forceps and
placed at different areas on the surface of each plate were incubated at 370C for 24 hours. Control experiments
comprising of plates were also set up. Antimicrobial activity of extracts or the inhibitory effect of the extracts on the
test organisms was determined by measuring the zones of inhibition in Milliliter (mm) diameter of the respective
Measurement and Interpretation of Zones of Inhibition
The diameters of the zone of inhibition of the growth were measured by the use of scale ruler in milliliter (mm) clear
zones of inhibition indicated the susceptibility of the organism to the extracts while absence of such zones showed
resistance or no inhibitory effect of extracts on the test organism.
Table-3: Zones of Inhibition (mm) Showing Inhibitory Effect of Varying Concentration of Ethanolic Leaves Extracts of Centella asiatica on the Test Organisms Concentration (mg/ml) and Zones of Inhibition (in mm)
Susceptibility Test of the Organisms of Selected Commercial Antibiotics
A control experiment was set up to test the susceptibility of these test organisms on the commercial antibiotics.
Each test isolates were inoculated and spread on sterile Mueller-Hinton Agar plates and multi-discs of antimicrobial
drugs were placed on each plate using a sterile forceps and incubated also at 370C for 24 hours .
Table-4: Zones of Inhibitions of Gram-Positive Isolates to Commercial Antibiotics Used Test Organisms/Zones of Inhibitions (mm) Zones of Inhibition > 12mm = Sensitive Zones of inhibition < 12mm = Resistance Table-5: Zones of Inhibitions of Gram-Negative Isolates to Commercial Antibiotics Used Test Organisms/Zones of Inhibitions (mm) Table-6: Minimum Inhibitory Concentrations of Aqueous Leaves Extracts of Centella asiatica on the Test Test Organisms/Zones of Inhibitions (mm) Table-7: Minimum Inhibitory Concentrations of Ethanolic Leaves Extracts of Centella asiatica on the Test Test Organisms/Zones of Inhibitions (mm) Determination of Minimum Inhibitory Concentration of the Extracts of Each Isolates
The minimum inhibitory concentration (MIC) of the leaf extracts on each isolate was determined using tube dilution
method as described by . Different concentrations of the extract ranging 31.5mg/ml to 500mg/ml were prepared
by making a serial dilution with the 500mg/ml to the power of five for each of the extracts. The different
concentration was seeded on sterile Petri-dishes and then a molten nutrient agar was poured on these seeded Petri-
dishes and the swirled, allow to cool test. Organism was inoculated on these Petri-dishes with different
concentration of the extracts and controls for all the cultures were set up without the extracts. The plates were
incubated at 370C for 24 hours. The concentration without any visible growth was reported as the minimum
inhibitory concentration of the plant’s leaf extracts for that particular organism.
RESULTS AND DISCUSSION
The phytochemical screening of both Aqueous and Ethanol extract of Centella asiatica leaves carried out showed
the presence of some bioactive components in varying amounts. The components are alkaloids, Saponins, tannin,
Phlobatannins, Flavonnoids, anthraquinones and cardiac glycosides. This is shown on Table 1. The result of phytochemical analysis of Centella asiatica leaves showed that the leaves contain some bioactive components namely Saponins, tannins, Phlobatannins, anthraquinones, Flavonnoids, alkaloid and cardiac glycoside. These bioactive components when found on a plant, have some inhibitory effects on some microorganisms, some of which were reported in literature as antimicrobial constituents. For instance, Flavonnoids are known to be antimicrobial in nature . Tannins were also identified to have antimicrobial activities . The aqueous and ethanolic extracts obtained from Centella asiatica leaves of various concentrations were used for antimicrobial sensitivity assay to determine the inhibitory effects of these extracts on the test organisms. The results revealed that both in aqueous and ethanolic extracts, the inhibitory effects determined by the zone of inhibition (in mm) increases with increase in concentration of extracts. However the highest zones of inhibitions were greatly recorded with ethanolic extracts than that of aqueous extract. A zone of inhibition of 14.2mm was observed with Staphylococcus aureus at the aqueous concentration of 100mg/ml but as the concentration of the extracts increased to 200mg/ml, the inhibition effect of the extracts also increased with zones of inhibition of 15.1mm. Zones of inhibitions by other test organisms also increased with increased concentrations. Similarly in ethanolic extracts, inhibitory effect was also recorded for Staphylococcus aureus and Salmonella species with clear zones of inhibition of 16.2mm and 16.1mm respectively, with an extracts concentration of 100mg/ml but 18.8mm and 18.7mm when extracts concentration was 200mg/ml. However the highest zone of inhibitions was observed with Staphylococcus aureus with 23.0mm at the ethanolic leaves extracts concentration of 400mg/ml and 500mg/ml respectively. These are illustrated on Tables 2 and 3. Although the aqueous leaves extracts produced some inhibitory effects on the clinical isolates, the ethanolic leaves extracts were observed to produce high inhibitory effects. This confirmed the report of  who reported ethanol to be the best solvent for extraction over aqueous when working with plants of medicinal importance. It was discovered that in the commercial antibiotics which are commoly used as a routine drugs, S. aureus was sensitive to some antibiotics such as Floxapen, Erythromycin, Ciprofloxacin, Streptomycin and Gentamycin but resistant to Rifampicin and Ampiclox. Also sensitivity patterns for other organisms were also observed. However, the results showed that the highest zones of inhibitions when using the commercial antibiotic susceptibility discs to be 16.5mm from Staphylococcus aureus on Lincocin (LND) and 16.5mm was observed with Streptococcus pyogenes on Norfloxacin (NB) while 18.1mm were recorded for Salmonella species on Gentamycin (CN) and Proteus species on Septrin (SXT) respectively (Table 4 and 5). Thus, since higher zones of inhibitions from the leaves extracts of Centella asiatica on these organisms were observed on these isolates, the plants leaves could be used to cure infections from the tested isolates in the place of commonly used antibiotics which the isolates have started to develop resistance. Minimum inhibitory concentrations (MIC) of both the aqueous and ethanolic leaves extracts of Centella asiatica were determined for each test organism. In aqueous extracts Streptococcus pyogenes had the MIC of
250mg/ml while MIC for Staphylococcus aureus was 125mg/ml and 62.5mg/ml was observed Proteus mirabilis. In
ethanolic extracts, the lowest MIC of 62.5mg/ml was observed with Staphylococcus aureus and Salmonella species
respectively, while the highest MIC was observed with Shigella species with 500mg/ml (Table 6 and 7).
The results of antimicrobial activities of the extracts revealed that Centella asiatica extracts have high antimicrobial
activity with some zones of inhibition of >15mm from ethanolic leaves extracts against some clinical isolates while
some susceptibility patterns were also observed with aqueous extracts. Thus, both aqueous and ethanol could be
used as solvents to extract bioactive components from this plant. However, the strong inhibitory effects of these
extracts over commercial antibiotics and zone of inhibition greater than 12mm in diameter for the isolates are
noteworthy as some organisms used were noted to be resistant to commercial antibiotics, for instance S. aureus was
resistant to Ampiclox but highly sensitive to the extracts. The MIC of both aqueous and ethanolic extract of
Centella asiatica leaves showed the lowest concentration of 62.5mg/ml for some isolates, thus, small quantity of the
plants leaves could be used to treat infections from these organisms.
The findings in the study showed the inhibitory effects of both the aqueous and ethanolic leaves extracts of Centella
asiatica on some clinical organisms commonly involved in wound infection, urinary tract infection, gastrointestinal
tract infections, sore throat, respiratory tract infection and inflammations, thereby justifying its use for therapeutic
purposes. Therefore there is need to conserve this plant species for exploitation by pharmaceutical industries for the
production of new drugs.
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Correlation of the electrical and intrinsic optical signals in theN.L.V. Peixotoa,*, V.M. Fernandes de Limab, W. HankecaMicroelectronics Laboratory, University of SaÄo Paulo-USP, Esc. Politecnica, Avenue Professor Luciano Gualberto, 158, Trav.3,bDepartment of Neuroscience, FUNREI, SaÄo JoaÄo del Rey, MG, BrazilcInstitute of Zoophysiology, Hohenheim University, Stuttgart, GermanyReceive