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 (Figure
2). 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 (Figure
1). 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 file
1). 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 (Table
2). 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 (Table
3). 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.