Andrographis paniculata (Kalmegh) has been considered as a medicinal shrub and used as a medicinal plant in the remote areas of Bangladesh. A. paniculata leaf and stem extracts were prepared using the polar ( i.e., water, and 70% ethanol) and nonpolar ( i.e., hexane) solvents. The phytochemical contents, total phenol contents (TPC), antioxidant activity, and antibacterial activity of all the extracts of A. paniculata leaf and stem were investigated. Both the gram-positive ( i.e., Bacillus subtillis) and gram-negative ( i.e., E. coli, and Salmonella typhi) strains of bacteria were used for the antibacterial activity assay of the sample extracts. The ethanolic stem extracts contained the maximum amount of TPC when compared to that of the leaf extracts. However, the aqueous stem extracts had the highest free radical scavenging activity in vitro. The extracts prepared from A. paniculata stem showed better antibacterial activity against all the strains of bacteria ( i.e., E. coli, S. typhi, and B. subtillis) when compared to that of the leaf extracts. More specifically, the aqueous stem extract showed superior antibacterial effect against E. coli, and B. subtillis, and the zones of inhibition were 21 mm, and 29 mm in diameter, respectively. On the other hand, the ethanolic stem extract showed the maximum antibacterial activity against S. typhi and the zone of inhibition was 8.15 mm. The minimum inhibitory concentration (MIC) value and IC 50 value for all the A. paniculata extracts were ~0.05 μg/μL, and ~1 μg/μL, respectively.
Free radicals and other reactive oxygen species are being constantly produced in the human body and they are known to be responsible for various deadly diseases such as cancer, aging, atherosclerosis, immunodeficiency, and infections. On the other hand, synthetic drugs bring about various side effects such as gastrointestinal disturbances, hypoglycemia, and liver dysfunction [
Medicinal plants are an important source of valuable therapeutic agents, both in modern and in traditional medicine. As powders, extracts, decoctions or infusions, plants are being used in the traditional systems of medicine in many parts of the world, especially in rural communities, for the control, management, and/or treatment of a variety of human and animal ailments [
However, when crude powder of A. paniculata was suspended in the water, it did not show any antibacterial activity against Salmonella, Shigella, E. coli, Streptococci, and Staphylococcus aureus in vitro even at a very high concentration (25 mg/mL) [
Absolute ethanol, methanol, hexane, and Folin-Ciocalteu reagent were purchased from Merck, Germany. Gallic acid was purchased from Ashland Inc. USA. Ascorbic acid was bought from VEGA, China. 1-1-diphenyl-2-picryhy- drazyl (DPPH) was collected from Sigma-Aldrich, USA. Agar powder was purchased from Titan Biotech Ltd., India. Peptone, yeast extract, sodium chloride and sodium carbonate were collected from UNI-CHEM, China.
Escherichia coli DH5α, Bacillus subtilis RBW, and Salmonella typhi were obtained from the department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh.
A. paniculata shrubs were collected from the surroundings of the department of Biotechnology & Genetic Engineering of Jahangirnagar University, Savar, Dhaka 1342, Bangladesh. Leaves and stems were first separated out and washed thoroughly with tap water followed by distilled water wash to remove any dirt, and dried naturally. The dried parts (i.e., leaves, and stem) of A. paniculata were ground separately into fine powder.
The powder (5 gm) of both the leaves and stems were extracted separately with 100 mL of both the polar (i.e., 70% ethanol, and distilled water) and nonpolar (i.e., hexane) solvents for 72 hours at 37˚C with gentle shaking at 120 rpm and filtered using Whatman No. 1 filter paper. The solvents were then removed completely to get the dried extracts. Finally, 1% of the extract sample was prepared using 0.9% NaCl solution.
The total phenolic contents (TPCs) of the extracts prepared from both the polar (i.e., water and ethanol) and nonpolar (i.e., hexane) solvents were determined by Folin-Ciocalteu’s reagent method [
The antioxidant activity of A. paniculata extracts were determined according to the protocol reported by Manzocoo et al., with slight modification [
The percentage (%) of free radical scavenging activity is calculated from (Ab ? As)/Ab × 100.
Here, Ab is the absorbance of the blank, and As is the absorbance of the standard or extract sample. Ascorbic acid was used as a positive control. Percentage (%) of free radical scavenging activity was plotted against the concentration of the plant extracts and the value of IC50 (i.e., the concentration of the plant extract required to inhibit the formation of free radicals by 50%) was calculated from the regression line obtained. Tests were carried out in triplicate and the average value was taken.
The presence of phytochemicals in A. paniculata leaf and stem extracts prepared using different solvents were determined qualitatively. Standard procedures were followed to determine the presence of flavonoids (alkaline reagent test), tanins (ferric chloride test), saponins (foam test), phenols (ferric chloride test), and glycosides (KellarKillani’s test) [
In a laminar air cabinet, Luria-Bertini (LB) broth was taken into three different test tubes and each test tube was inoculated with different microorganisms (i.e., E. coli, S. typhi, and B. subtilis) and incubated overnight at 37˚C upon gentle shaking to prepare fresh bacterial cultures. The bacterial cultures (100 μL) were then transferred to different agar plates and spreaded uniformly with a sterile spreader to prepare test plates for antimicrobial test.
Sterile metrical filter paper discs (Oxoid, UK) were taken in a blank petri plate. Then the discs were soaked with different concentrations (i.e., 5 μg/μL, and 10 μg/ μL) of the test sample extracts and wait for several minutes to dry them properly.
Antibacterial activity of the A. paniculata extracts were investigated by disc diffusion method [
The MIC and IC50 value of both the polar (i.e., water, and ethanol) and nonpolar (i.e., hexane) solvent extracts of A. paniculata leaf and stem were determined through serial dilution method. Briefly, different concentrations of the extracts (i.e., 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.0, and 1.5 μg/μL) were added to the bacterial cultures and the final volume was made up to 1.0 mL before incubating over night at 37˚C upon gentle shaking. The absorbance of all the tubes was taken at 600 nm using a UV-visible Spectrophotometer (Optizen POP, Korea). The experiment was performed multiple times and the data expressed as a mean of several replications (n = 3) and standard deviations.
The total phenol content (TPC) of the A. paniculata extracts was determined by Folin Ciocalteu method and the results were expressed in μg CAE/mL. The highest amount of TPC was found in the ethanolic extract of stem followed by its aqueous extract. On the other hand, the lowest amount of TPC was found in the hexane extract of both the leaf and stem (
The free radical scavenging activity of A. paniculata extracts was carried out by DPPH assay. The highest free radical scavenging activity was found to be
performed by the aqueous extract of stem followed by its ethanolic extract. On the other hand, the stem extract prepared by using hexane as the solvent showed the lowest antioxidant activity (
The IC50 value (i.e., the amount of extracts required to scavenge the formation of free radicals by 50%) of A. paniculata extracts prepared from polar solvents were calculated from the liner regression curve (
The qualitative phytochemical analysis data of both the polar and nonpolar
Test | Aqueous Leaf | Aqueous Stem | Ethanol Leaf | Ethanol Stem | Hexane Leaf | Hexane Stem |
---|---|---|---|---|---|---|
Tannin | + | ++ | + | +++ | ||
Flavonoid | ++ | +++ | ++ | +++ | ||
Saponin | + | +++ | + | +++ | ||
Phenol | +++ | ++ | ++ | + | ||
Glycoside | ++ | +++ | ++ | +++ | + | + |
solvent extracts of A. paniculata leaf and stem showed that most of the phytochemicals are present in the aqueous and ethanolic extracts (
All the A. paniculata extracts were investigated for their antimicrobial activity assay against both the gram-positive (i.e., B. subtilis) and gram-negative (i.e., E. coli, and S. typhi) bacteria by simple agar diffusion method. Different concentration of the sample extracts (i.e., 5 μg/μL, and 10 μg/μL) were used for the antimicrobial tests.
The extracts showed antimicrobial activity against all the microorganisms (
Among all the A. paniculata extracts, the aqueous stem extracts showed the maximum antibacterial activity against gram-positive B. subtillis and diameter of the zone of inhibition was 29 mm followed by the ethanolic stem extract which was 23 mm in diameter (
Concentration (μg/μL) | Diameter of zone of inhibiton (mm) | |||||
---|---|---|---|---|---|---|
Bacillus subtilis | ||||||
Aqueous leaf (AL) | Aqueous stem (AS) | Ethanol leaf (EL) | Ethanol stem (ES) | Hexane leaf (HL) | Hexane stem (HS) | |
5 | 9.02 ± 0.04 | 18.12 ± 0.12 | 15 ± 0.12 | 12.05 ± 0.08 | 15.1 ± 0.15 | 7.97 ± 0.03 |
10 | 11.15 ± 0.20 | 29.12 ± 0.19 | 21.1 ± 0.12 | 23.12 ± 0.12 | 15.9 ± 0.12 | 10.01 ± 0.04 |
Concentration (μg/μL) | Diameter of zone of inhibiton (mm) | |||||
---|---|---|---|---|---|---|
Eschericia coli | ||||||
Aqueous leaf (AL) | Aqueous stem (AS) | Ethanol leaf (EL) | Ethanol stem (ES) | Hexane leaf (HL) | Hexane stem (HS) | |
5 | 8.02 ± 0.08 | 13.97 ± 0.10 | 10.12 ± 0.39 | 9.02 ± 0.04 | 7.95 ± 0.11 | 7.05 ± 0.08 |
10 | 9.25 ± 0.32 | 21.02 ± 0.04 | 10.95 ± 0.08 | 12 ± 0.12 | 8.77 ± 0.04 | 7.97 ± 0.10 |
Concentration (μg/μL) | Diameter of zone of inhibiton (mm) | |||||
---|---|---|---|---|---|---|
Salmonella typhi | ||||||
Aqueous leaf (AL) | Aqueous stem (AS) | Ethanol leaf (EL) | Ethanol stem (ES) | Hexane leaf (HL) | Hexane stem (HS) | |
5 | 6.42 ± 0.06 | 6.9 ± 0.08 | 6.45 ± 0.12 | 7.37 ± 0.08 | 6.4 ± 0.14 | 6.37 ± 0.05 |
10 | 7.27 ± 0.05 | 7.15 ± 0.04 | 6.77 ± 0.04 | 8.15 ± 0.12 | 6.85 ± 0.05 | 6.77 ± 0.09 |
The antibacterial activity of the medicinal plant extracts depends on the presence of different concentrations of secondary metabolites such as tannin, saponin, and glycosides [
Our data support the previously published reports that the gram positive bacteria are more sensitive to plant extracts when compared to that of the gram negative bacteria. Therefore, the A. paniculata extracts were tasted against a variety of gram-positive and gram-negative bacterial strains [
The absorbance of overnight grown bacterial culture was measured at different wavelengths ranging from 560 to 620 nm to determine the λmax for the bacterial culture and observed that λmax was 600. The minimum inhibitory concentration (MIC) value for all the extracts was ~0.05 μg/mL (
inhibitory concentration 50 (IC50) value for all the extracts was ~1 μg/mL. The IC50 value explains the bacteriostatic as well as the bactericidal activity of the A. paniculata extracts that is required to inhibit the bacterial growth and multiplication by 50%. It is well known that the absorbance of bacterial culture increases with the increased concentration of bacteria. Since the extracts have both the bacteriostatic and bactericidal activity, the absorbance of bacterial culture reduced as the concentration of the extracts were increased. The IC50 values of HS and HL extracts were more among all other extracts and, therefore, it requires more amount of the extract to inhibit the growth of bacteria by 50% (i.e., bacteriostatic effect) or to kill the 50% of the existing bacteria (i.e., bactericidal effect). It is due to the absence of most of the phenolics except glycosides in hexane extract of A. paniculata leaf and stem (
In the disc diffusion method, we showed the antimicrobial activity of the extracts in terms of their zone of inhibition which explains the ability of the extracts to inhibit the growth of bacteria up to a certain distance around the disc of placement.
We have evaluated the phytochemical contents, antioxidant efficiency, and antimicrobial potentiality of A. paniculata extracts in terms of their zone of inhibition (i.e., diameter in millimeter), MIC value, and IC50 value. A. paniculata leaf and stem extracts were prepared using different solvents such as water and ethanol as the polar solvents and hexane as the nonpolar solvent. The ethanolic stem extract showed the highest total phenol content and the aqueous stem extract showed the maximum free radical scavenging activity.
The ethanolic, aqueous and hexane extracts of A. paniculata leaf and stem showed significant TPC, antioxidant, and antimicrobial activity which supports the traditional use of plants in various diseases. In this study, we found that Kalomegh (A. paniculata) stem extract has the highest TPC and free radical scavenging activity. Stem extract also showed the maximum zone of inhibition (29.12 ± 0.19 mm) against gram positive microbial strain (i.e., B. subtillis). So the A. paniculata can be further screened against various disease causing pathogens and can be a potential source of chemical and biologically important drug candidates.
This research was supported by Jahangirnagar University Research Grants 2014- 15, Savar, Dhaka 1342, Bangladesh, and University Grants Commission Research Grants 2015-16, Govt. of Bangladesh.
The authors declare no conflict of interest whatsoever.
Polash, S.A., Saha, T., Hossain, M.S. and Sarker, S.R. (2017) Investigation of the Phytochemicals, Antioxidant, and Antimicrobial Activity of the Andrographis paniculata Leaf and Stem Extracts. Advances in Bioscience and Biotechnology, 8, 149-162. https://doi.org/10.4236/abb.2017.85012