Antibacterial, antifungal and antioxidant activities of the ethanol extract of the stem bark of Clausena heptaphylla
- Md Fakruddin1Email author,
- Khanjada Shahnewaj Bin Mannan†2,
- Reaz Mohammad Mazumdar†3 and
- Hafsa Afroz†4
© Fakruddin et al.; licensee BioMed Central Ltd. 2012
Received: 14 May 2012
Accepted: 23 November 2012
Published: 27 November 2012
There is wide spread interest in drugs derived from plants as green medicine is believed to be safe and dependable, compared with costly synthetic drugs that have adverse effects.
We have attempted to evaluate the antioxidant, In vitro thrombolytic, antibacterial, antifungal and cytotoxic effects of Clausena heptaphylla (Rutaceae) stem bark extract ethanol extract.
Ethanolic stem bark extract of Clausena heptaphylla (CHET) contains flavonoids, alkaloids, saponins and steroids but it lacks tannins, anthraquinones and resins. Phenol content of the extract was 13.42 mg/g and flavonoid content was 68.9 mg/g. CHET exhibited significant DPPH free radical scavenging activity with IC50 value of 3.11 μg/ml. Reducing power of CHET was also moderately stronger. In the cytotoxicity assay, LC50 and Chi-square value of the ethanolic extract against brine shrimp nauplii were 144.1461 μg/ml and 0.8533 demonstrating potent cytotoxic effect of the extract. In vitro thrombolytic activity of CHET is significant with 45.38% clot lysis capability compared to that of Streptokinase (65.78%). In antibacterial screening, moderate zone of inhibition (6.5-9.0 mm in diameter) was observed against gram-positive Bacillus subtilis ATCC 11774, Bacillus cereus ATCC 10876, Staphylococcus aureus ATCC 25923, Bacillus polymyxa ATCC 842 and Bacillus megaterium ATCC 13578 and less promising zone of inhibition (3.0-4.5 mm in diameter) against gram-negative Salmonella typhi ATCC 65154, Shigella flexneri ATCC 12022, Proteus vulgaris ATCC 13315 and Escherichia coli ATCC 25922. Shigella sonnei ATCC 8992 did not show any sensitivity. The MIC values against these bacteria were ranged from 2,000 to 3,500 μg/ml. The extract showed significant zone of inhibition against Rhizopus oryzae DSM 2200, Aspergillus niger DSM 737 and Aspergillus ochraceus DSM 824 in antifungal assay.
Further advanced research is necessary to isolate and characterize the chemical components responsible for the therapeutic properties of the plant.
KeywordsAntioxidant Antibacterial Antifungal Cytotoxic Clausena heptaphylla
Medicinal plants are an important therapeutic aid for various ailments. Most of the people in rural and urban areas of the world depended on the medicinal plants for the treatment of infectious diseases.Today, there is wide spread interest in drugs derived from plants. Medicinal plant extracts offer considerable potential for the development of new agents effective against infections currently difficult to treat. As natural products have been elaborated within living systems, they are often perceived as showing more ‘drug likeness’ and biological friendliness than totally synthetic molecules, making them good candidates for further drug development.
Clausena heptaphylla (Roxb.) Wight and Arn. (Bengali name: Panbilash, Karanphul, Pomkaphur) is a small bushy shrub that is distributed throughout Bangladesh, India and other parts of south East Asia. Clausena species are known to be useful in paralysis, ulcerated nose, headache, muscular pain and malarial fever. They are also reputed as diuretic, astringent, insecticide, tonic and vermifuge. The leaves of the plants possess antimicrobial properties[8, 9]. A new carbazole alkaloid, named clausenal, was isolated from the leaves of C. heptaphylla and the alkaloid was found to be active against both Gram-positive and Gram-negative bacteria and fungi.
We have attempted to evaluate the antioxidant, In vitro thrombolytic, antibacterial, antifungal and cytotoxic effects of C. heptaphylla stem bark ethanolic extract in this study.
All animal experiments were carried out on shrimp nauplii, for which ethical approval is not required. All human blood samples were obtained from consenting, healthy individuals. There are no ethical review bodies in Bangladesh, so it was not possible for us to obtain approval for this procedure.
Media and chemicals
DPPH (2,2-diphenyl-1-picrylhydrazyl), TCA (trichloroacetic acid) and ferric chloride were obtained from Sigma Chemical Co. USA; Ascorbic acid was from SD Fine Chem. Ltd. India, ammonium molybdate from Merck, Germany. Mueller-Hinton broth and agar media (Hi media, India), final PH 7.3±0.2 (at 25ºC), was used for MIC determination and antibacterial. On the other hand, potato dextrose agar media (Hi media, India), final PH (at 25ºC) 5.6±0.2 and artificial seawater (3.8% NaCl solution) were used for the determination of antifungal and cytotoxic effect.
Collection of plant material
The stem bark of C. heptaphylla was collected from Jhalokathi, Bangladesh in june 2011 and was identified. The plant was identified by Sarder Nasir Uddin (Taxonomist, Bangladesh National Herbarium, Ministry of Environment and Forest, Dhaka, Bangladesh). A voucher specimen is preserved in Bangladesh National Herbarium with the accession No. DACD- 52876.
Extraction of plant material
The fresh stem barks of C. heptaphylla were washed with water immediately after collection. Then chopped into small pieces, air dried at room temperature for about 10 days and pulverized into powder (1 kg) which was macerated in 6 L pure ethanol for 7 days at room temperature (23±5)ºC. After 7 days, extract was filtered off through cotton plug and finally with a Whatman No. 1 filter paper. Then extract was concentrated under reduced pressure below 50ºC through rotatory vacuum evaporator (RE200 Sterling, UK). The concentrated extract (45 gm blackish-green, 4.5% w/w yield) was stored at 4ºC.
To identify the chemical constituents of plant extract standard procedures were followed. Freshly prepared crude extracts of C. heptaphylla were qualitatively tested for the presence of chemical constituents using the following reagents and chemicals, flavonoids with the use of Mg and HCl; tannins with ferric chloride and potassium dichromate solutions and saponins with ability to produce stable foam and steroids with Libermann Burchard reagent, reducing sugars with Benedict’s reagent and observed color change in respective.
Determination of total phenolic content
Folin-Ciocalteu method was used to determine the total phenolic content; Folin-Ciocalteu oxidized the extract whereas sodium carbonate neutralized it. Blue color formed and the absorbance was measured at 760 nm after 60 min by using gallic acid (GA) as standard. Total Phenolic content was expressed as mg GA equivalent/gm of extract.
Determination of total flavonoid content
Method described by Meda et al. was followed to determine the flavonoid content where quercetin was used as standard. 1 mg of plant extract in methanol was mixed with 1 ml of aluminium trichloride in Ethanol (20 mg/ml) and a drop of acetic acid was added. Then diluted up to 25 ml with ethanol and measured the absorbance at 415 nm after 40 min. The absorption of blank samples and standard quercetin solution (0.5 mg/ml) in methanol was measured under the same conditions.
DPPH radical scavenging activity
The antioxidant activity of C. heptaphylla ethanolic stem bark extract and the standard antioxidant ascorbic acid was assessed on the basis of the radical scavenging effect of the stable 2,2- diphenyl-1-picrylhydrazyl (DPPH) free radical activity according to the method described by Brand-Williams et al.. C. heptaphylla ethanolic extract with different concentrations (10, 50, 100, 200, 400, 600, 800 and 1000 μg/ml) were prepared in ethanol. Ascorbic acid was used as standard in 1- 100 μg/ml solution. The scavenging activity against DPPH was calculated using the following equation: Scavenging activity (%) = [(A-B)/A] x100, Where A was the absorbance of control (DPPH solution without the sample), B was the absorbance of DPPH solution in the presence of the sample (extract/ascorbic acid). Then, % scavenging activity was plotted against log concentration and from the graph IC50 (Inhibition concentration 50) value was calculated by linear regression analysis.
The reducing power of C. heptaphylla extract was determined according to the method of Oyaizu. Different concentration of C. heptaphylla extract in 1ml of distilled water was mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6 (2.5 ml, 1%) then the mixture was incubated at 50°C for 20 min. Trichloroacetic acid (10%) slightly added (2.5 ml)to the mixture and centrifuged at 3,000 rpm for 10min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%) then taken the absorbance at 700 nm. The reference standard was Ascorbic acid and the Blank solution contained Phosphate buffer.
Brine shrimp lethality bioassay
Cytotoxic activity of ethanolic stem bark extract was determined by Brine-Shrimp Lethality assay as described by Meyer et al.. Simple zoological organism (Artemia) was used as a convenient monitor for the screening. The eggs of the brine shrimp were hatched in artificial seawater (3.8% NaCl solution) for 48 hours to mature shrimp called nauplii. Then 30 mg of C. heptaphylla stem bark extract were separately dissolved in 3 ml of DMSO, and from these 1000, 500, 250, 125 and 62.5 μg/ml were prepared by serial dilution. Each concentration was tested in triplicate, giving a total of 15 test tubes for each sample. A control containing 5 ml of DMSO solvent was used for each solvent. The final volume of the solution in each test-tube was made up to 5 ml with seawater immediately after adding shrimp larvae. The test tubes were maintained under illumination. After 24 hours have elapsed, Survivors were counted with the aid of a 3x magnifying glass. The LC50 values were calculated from Probit Chart using computer software “BioStat-2007”.
In vitro thrombolytic activity
In vitro antibacterial screening was carried out by disc diffusion method against 6 gram-positive (Bacillus subtilis, Staphylococcus aureus, Bacillus cereus, Bacillus polymyxa, Bacillus megaterium, Enterococcus faecalis) and 8 gram-negative bacteria (Salmonella typhi, Klebsiella sp., Shigella flexneri, Shigella sonnei, Proteus sp., E. coli, Vibrio cholerae, Pseudomonas aeruginosa). The bacterial suspension turbidity adjusted to McFarland standard number 0.5, in Mueller Hinton Broth (Himedia, India). With a sterile cotton swab bacterial culture was streaked on previously prepared Mueller Hinton agar plate (Himedia, India). Dried and sterilized paper discs were treated separately with desired concentration of previously prepared ethanolic solution of the root extract using a micropipette dried in air under aseptic condition and placed at equidistance in a circle on the seeded plate. The concentrations of root extract used were 2 mg/disc and 3 mg/disc. These plates were kept for 4-6 hours at low temperature and the test materials diffuse from disc to the surrounding medium by this time. The plates were then incubated at 37°C for 18 hours. The diameter of zone of inhibition produced by root extract was then compared with standard antibiotic Kanamycin (30 μg/disc). Each sample was used in triplicate for the determination of antibacterial activity. Blank disc impregnated with solvent ethanol followed by drying off was used as negative control.
Determination of MIC
Minimum inhibitory concentrations (MIC) of crude extract of the C. heptaphylla were performed by macrodilution method. The crude extract was dissolved in 30% dimethyl sulfoxide (DMSO) to obtain 10% (w/v) solution. For MIC test of the selected bacteria, the extract was first diluted in sterilized Mueller-Hinton broth to the highest concentration of 10,000 μg/ml and then dilution were performed at concentration of 5000 μg/ml, 4000 μg/ml, 3500 μg/ml, 3000 μ/ml, 2500 μ/ml, 2000 μg/ml, 1500 μg/ml, 1000 μg/ml, 750 μg/ml, 500 μg/ml and 250 μg/ml in screw caped tube containing broth medium. Bacterial suspensions of the test organism were prepared in sterilized Mueller-Hinton broth. Then 1 ml of the dilution was added to each sterilized screw capped tube containing 1 ml of compound suitably diluted in the sterilized broth medium to give final volume of 2 ml. Culture medium without samples and others without microorganisms were used in the tests as control. Tubes were incubated at 370C for 20-24 hours and growth was indicated by turbidity.
where, I=Percentage of Inhibition, C=Diameter of the fungal colony in control, T=Diameter of the fungal colony in treatment.
All the in vitro experimental results were given as mean± SEM of three parallel measurements and data were evaluated by using student’s t-test. P values <0.001 were regarded as significant. Results were processed by Excel (2007) and BioStat[23, 24].
Result of phytochemical screening of ethanol stem bark extract of C. heptaphylla
Total phenol and flavonoid content
The total phenol and total flavonoid contents of Clausena heptaphylla of ethanol extract were expressed in Gallic acid and Quercetin equivalents respectively. Total phenolic content was 13.42 mg/g Gallic acid equivalent and total flavonoid content was 68.9 mg/g Quercetin equivalent.
Different studies suggest that different types of polyphenolic compounds such as flavonoids, phenolic acids which are found in plants have multiple biological effects, including antioxidant activity.
DPPH free radical scavenging activity
Antioxidant activity of C. heptaphylla extract was measured by DPPH free radical scavenging method and their scavenging activity was compared with the standard antioxidant ascorbic acid. Both ascorbic acid and extract showed a dose dependent activity. However, extract showed very strong DPPH free radical scavenging effect in regard to ascorbic acid. Among seven different concentrations used in the study (10 to 1000 μg/ml) 1000 μg/ml showed the highest scavenging activity 98.64% whereas ascorbic acid of the same concentration showed 99.65% which are very close to each other (Figure1). Percent (%) scavenging activity was plotted against log concentration and from the graph IC50 (Inhibition concentration 50) value was calculated by linear regression analysis. IC50 value of ascorbic acid and extract was found 5.15 μg/ml and 3.11 μg/ml, respectively.
The reducing power of a compound is related to its electron transfer ability and may therefore; serve as an indicator of its potential antioxidant activity. By using the potassium ferricyanide reduction method the reductive capabilities of the plant extracts was identified in comparison with ascorbic acid. The reducing power of the extracts was moderately strong while increasing dose it shows little increment. At 100 μg/ml concentration, reducing power of CHET was 0.73 whereas that of ascorbic acid was 0.85. But, with increasing concentration (100-200 μg/ml), reducing power of CHET increased from 0.73 to o.89 whereas that of ascorbic acid increased from 0.85 to 1.54.
Brine shrimp lethality bioassay
In Vitro clot lysis assay
Statistical representation of the effective clot lysis percentage by herbal preparations, positive thrombolytic control (Streptokinase) and negative control (sterile distilled water) were done by paired t-test analysis; clot lysis % is represented as mean ± S.D. and p of herbal preparation (C. heptaphylla) was < 0.05; which was considered as significant.
CHET showed antibacterial activity as measured by zone of inhibition against all the bacteria tested except Shigella sonnei. CHET showed better antibacterial activity against gram-positive bacteria than gram-negative. Highest activity was demonstrated against Enterococcus faecalis and lowest against Vibrio cholerae. Again better antibacterial activity was demonstrated with 4 mg/disc concentration than 2 mg/disc concentration.
Determination of MIC
Minimum Inhibitory Concentration of CHET and Doxycycline
Source ID (ATCC)
Doxycycline hydrochloride (μg/ml)
In-vitro antifungal activity of CHET and fluconazole
CHET (4 mg)
CHET (6 mg)
Fluconazole (100 μg)
Plants produce a huge variety of secondary compounds as natural protection against microbial and insect attack. Some of these compounds are toxic to animals, but others may not be toxic. Indeed, many of these compounds have been used in the form of whole plants or plant extracts for food or medical applications in human because plants are the natural reservoir of many antimicrobial, anticancer agents, analgesics, anti-diarrheal, antifungal as well as various therapeutic activities. Acceptance of medicines from such plant origin as an alternative form of healthcare is increasing because they are serving as promising sources of novel antibiotic prototypes. Some of the phytochemical compounds e.g. glycoside, saponin, tannin, flavonoids, terpenoid, alkaloids, have variously been reported to have antimicrobial activity.
Ethanolic stem bark extract of Clausena heptaphylla (CHET) contains flavonoids, alkaloids, saponins and steroids but it lacks tannins, anthraquinones & resins. Phenol content of the extract is 13.42 mg/g and flavonoid content is 68.9 mg/g.
Free radicals from oxidative stress are involved in many disorders like atherosclerosis, angina pectoris, neurodegenerative diseases and cancer. Antioxidants due to their scavenging activity are useful for the management of those diseases. The quantitative determination of antioxidants explored that high quantity of scavenging substances are found to be in C. heptaphylla which plays the key role in showing free radical scavenging activity of this plant. CHET exhibited significant DPPH free radical scavenging activity with IC50 value of 3.11 μg/ml. Reducing power of CHET is also moderately strong.
Brine shrimp lethality is a general bioassay which is indicative of cytotoxicity, antibacterial activities, pesticidal effects and various pharmacologic actions. In this research, six different concentrations (25, 31.25, 62.5, 125, 250, 500 μg/ml) of C. heptaphylla extract were used to determine its cytotoxicity by brine shrimp lethality bioassay (Figure2). The extract showed lethality in a dose dependent manner. LC50 value of C. heptaphylla ethanol extract was found 144.1461 μg/ml at confidence limit 95% with chi-square value of 0.8533 (Figure1). The LC50 value found in this study to be very significant suggesting the ethanol extract of C. heptaphylla has high potentiality to kill cancer cells as well as pests. This significant lethality of the crude plant extract (as LC50 value less than 100 ppm or μg/ml) to brine shrimp is indicative of the presence of potent cytotoxic compounds which warrants further investigation.
In vitro thrombolytic activity of CHET is significant with 45.38% clot lysis capability compared to that of Streptokinase (65.78%) it can be considered for compound isolation in order to detect future anti-tumour compounds.
In antibacterial screening, moderate zone of inhibition (6.5-9.0 mm in diameter) were observed against gram-positive Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Bacillus polymyxa and Bacillus megaterium and less promising zone of inhibition (3.0-4.5 mm in diameter) against gram-negative Salmonella typhi, Shigella flexneri,, Proteus sp. and Escherichia coli. Shigella sonnei did not show any sensitivity. Stem bark extract showed significant zone of inhibition against Rhizopus spp., Aspergillus niger and Aspergillus ochraceus in antifungal assay.
CHET showed zone of inhibition to almost all the strains (at dose 2 mg and 4 mg/disc) except Shigella soneii. Crude extract at the concentration of 2mg/disc showed 5.5, 6.0, 5.9, 6.2 and 6.4 mm zone of inhibition diameter against Gram-positive Bacillus subtilis, Staphylococcus aureus, Bacillus cereus, Bacillus polymyxa and Bacillus megaterium, respectively and 3.1, 2.5, 1.8, 3.6 mm diameter against Gram-negative Salmonella typhi, Shigella flexneri, Proteus sp. and E. coli. On the other hand, standard antibiotic Kanamycin (30 μg/disc) showed significant antibacterial activity against all tested gram-positive and Gram-negative bacteria. Results implicated that the Gram-positive bacteria were more sensitive to the extract than the gram-negative bacteria. Bacillus megaterium, Enterococcus faecalis, Staphylococcus aureusand Klebsiella spp. were the most susceptible bacteria in this study (see Additional file1). In a previous study, ethanolic stem bark extract of Terminalia arjuna showed 8-12 mm zone of inhibition diameter at 2 mg/disc concentration and 12-16 mm zone diameter at 4 mg/disc concentration against multi-antibiotic resistant Vibrio cholerae. CHET has almost similar activity as Terminalia arjuna in this study. The present study justifies the claimed uses of C. heptaphylla in the traditional system of medicine to treat various infectious diseases caused by the microbes. The obtained results may provide a support to use of the plant in traditional medicine. Based on this, further chemical and pharmacological investigations to isolate and identify minor chemical constituents in C. heptaphylla and to screen other potential bioactivities may be recommended.
The MIC values against these bacteria were ranged from 2,000 to 3,500 μg/ml. The lowest MIC (2000 μg) was recorded against Bacillus subtilis and the highest MIC (3500 μg) recorded against Salmonella typhi and Shigella sonnei.C. heptaphylla showed a significant degree of anti-fungal activity (Table2). The maximum anti-mycotic activity 43.66% was shown against A. ochraceus. Plant natural compounds are important source of mycotoxic compounds and they may provide a renewable source of useful fungicides that can be utilized in antimycotics drugs against infection of A. ochraceus. The effect of extract against A. niger was also higher implying that this plant can be utilized as anti-mycotics drugs against infection of A. niger in patients with pulmonary tuberculosis. Moderate anti-mycotic effect was found against Aspergillus ustus at the concentration of 4 and 6 mg/ml. Fluconazole was used as standard antifungal agent to compare the potentials of extract (Table3). There are, however, alarming reports of opportunistic fungal infections which describe that the resistance of the organisms increased due to indiscriminate use of commercial anti-microbial drugs commonly used for the treatment of infectious disease. This situation forced the researchers to search for new anti-microbial substance from various sources including medicinal plant. Our research findings revealed that medicinal plant C. heptaphylla can play a vital role in combating fungal resistance.
This study delineates that C. heptaphylla extract possesses some potentials in free-radical scavenging activity and cytotoxic effect but it has low antimicrobial (antibacterial and antifungal) activity. Since, crude ethanol extract of C. heptaphylla showed antibacterial and cytotoxic effect, it can be assumed that different active secondary metabolites were present in this extract. However, further studies are necessary to elucidate the mechanism lying with these effects. This study may serve as a foot step regarding the biological and pharmacological activities of stem bark extract of C. heptaphylla.
The authors acknowledge Abhijit Chowdhury and Md. Nur Hossain, Scientific Officer, IFST, BCSIR, Dhaka for reviewing the manuscript critically.
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