Investigation of the Phytochemicals , Antioxidant , and Antimicrobial Activity of the Andrographis paniculata Leaf and Stem Extracts

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 IC50 value for all the A. paniculata extracts were ~0.05 μg/μL, and ~1 μg/μL, respectively.


Introduction
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 [1].Due to the toxicity and side effects of modern therapeutic drugs, people across the world are looking for the alternative medicines from natural sources and herbal plants are the most sought after [2].
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 [3].Among other medicinal plants, Andrographis paniculata is one of the most potential herbs for the treatment of various deadly diseases.Because A. paniculata extracts have antihypertensive effects and decreases plasma angiotensin converting enzyme (ACE) activity, and lipid peroxidation in kidneys [4].They also have antimicrobial, antioxidant and anti-inflammatory activities, antihyperglycemic and hypoglycemic activities [5].Moreover, A. paniculata extracts have hepatoprotective effects which help to cure damaged liver caused by agents with different hepatotoxic mechanisms [4].
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) [5].It has also been reported that the crude aqueous as well as the ethanolic extracts of A. paniculata do not show any antibacterial effect against gram-negative E. coli [6] [7].Inconsistency in in vitro antibacterial effects could be due to several factors, the variation of the constituents in the material tested being the prime suspect [4].The negative antibacterial results have been reported from Thailand by Leelarasamee et al. [7] [8] while the results reported from India [9] and Malaysia [6] have been positive.It is due to the place and timing of collection of the medicinal herbs, storage, and extraction conditions may affect the extracts constituents both qualitatively and quantitatively [4].Herein, in this study we have collected A. paniculata herbs from the plain areas of Bangladesh during the rainy season (from July to September).The A. paniculata leaf and stem extracts were prepared using both the polar solvents (i.e., water, and 70% ethanol), and nonpolar solvent (i.e., hexane).The phytochemical contents, antioxidant, and antibacterial activity of all the extracts were investigated in vitro.To perform the antibacterial activity of the extracts, both the gram-positive (i.e., B. subtillis), and gram-negative (i.e., E. coli, and S. typhi) bacterial strains were used.

Chemicals and Reagents
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-picryhydrazyl (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.

Plant Sample Collection
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.

Preparation of Extracts
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.

Determination of Total Phenol Content (TPC)
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 [10] [11].Briefly, different sample extracts (i.e., 100 μL) were taken into separate test tubes containing 500 μL Folin-Ciocalteu's reagent (10% w/v).The concentration of phenolic contents in the samples were derived from a standard curve of Chlorogenic acid (i.e., gallic acid) diluted with absolute methanol (0 -100 μg/mL).Folin-Ciocalteu's reagent (500 μL, 10% v/v) was then added to all the test tubes containing different concentrations of gallic acid.Finally, 400 μL sodium carbonate solution (7.5% w/v) was added to all the test tubes containing the A. paniculata extracts as well as the gallic acid.
The tubes were then incubated at room temperature for 60 minutes before measuring the absorbance at 765 nm using a UV-visible spectrophotometer (Optizen POP, Korea).The total phenol contents of the A. paniculata extracts prepared from both the polar and nonpolar solvent were expressed as μg CAE/mL.

Determination of Antioxidant Activity by DPPH Method
The antioxidant activity of A. paniculata extracts were determined according to the protocol reported by Manzocoo et al., with slight modification [12] [13].
Briefly, sample extract (200 μL) or different concentrations of standard (i.e., freshly prepared ascorbic acid) solution were taken into different test tubes.50 μL HCL (1.0 M) was added to all the test tubes containing the sample extracts and ascorbic acid.The final volume of all the test tubes were made up to 500 μL with absolute methanol.Methanolic solution of DPPH (500 μL, 0.004%) was then added to all the test tubes and incubated in the dark and cold environment for 30 minutes to complete the reaction.The absorbance of the solution was measured at 517 nm.
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 IC 50 (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.

Determination of the Phytochemicals
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) [14].

Preparation of Bacterial Starter Cultures and Test Plates
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.

Preparation of Sample Discs with Test Samples
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.

Determination of Antibacterial Activity by Disc Diffusion Method
Antibacterial activity of the A. paniculata extracts were investigated by disc dif-fusion method [15].Briefly, LB agar medium was used to grow bacteria.Dried and sterile filter paper discs (6 mm diameter) containing A. paniculata extracts of known concentrations were placed on nutrient agar medium uniformly seeded with the test microorganisms.The plates were then incubated at 37˚C for 24 hours for optimum growth of the microorganisms.The extracts having antibacterial property prevent bacterial growth in the media surrounding the discs and yield a clear, transparent area defined as 'zone of inhibition'.The antimicrobial potency of the extract samples were measured by their activity to prevent the growth of the microorganisms surrounding the discs which gives clear zone of agar or inhibition.After incubation, the antimicrobial activities of the extract samples were determined by measuring the diameter of the zone of inhibition in millimeter with a transparent scale.

Determination of MIC (Minimum Inhibitory Concentration) and IC50 (Inhibitory Concentration 50) Value of the A. paniculata Extracts
The MIC and IC 50 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.

Estimation of Total Phenol Content (TPC)
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 (Figure 1).The TPC of the A. paniculata extracts showed the following order: 70% ethanolic extract > aqueous ex- tract > hexane extract.The minimum amount of TPC in the hexane extract of leaf and stem is due to the use of nonpolar hexane as the solvent of extraction as because most of the phenolic compounds are soluble in polar solvents [16].
Therefore, the TPC was found more in the extracts obtained from the polar solvents (i.e., water, and 70% ethanol) than that of the nonpolar solvents as shown in Figure 1.

Antioxidant Activity Assay
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 (Figure 2).The antioxidant activity of a plant extract depends on the presence of the compounds with free hydroxyl groups such as flavonoids [17].The amount of flavonoids and phenols in the aqueous extracts of A. paniculata was found higher when compared to that of the ethanol and hexane extracts (Table 1), since water is the most polar solvent among the solvents used and flavonoids have free hydroxyl groups.Therefore, the highest antioxidant activity of the aqueous extract of stem was due to the presence of higher amount of free hydroxyl group containing flavonoids which can scavenge the free radicals more efficiently.Our data support the previously published reports that the antioxidant activity is present in the water soluble compounds [18] and high polarity solvents are the most efficient in the extraction of natural antioxidants [19] [20].
The IC 50 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 (Figure 3).The lowest IC 50 value was obtained from the aqueous extract of stem (i.e., 4.42 μg/mL) followed by ethanolic stem extract (i.e., 6.84 μg/mL).Hence, the aqueous extract of stem is the most efficient in scavenging the free radicals formed due to the oxidation of proteins, nucleic acids, and per oxidation of lipids.

Determination of the Phytochemicals
The qualitative phytochemical analysis data of both the polar and nonpolar Free radical scavenging activity of both the leaf and stem extracts of A. paniculata.Leaf and stem extracts were prepared using polar solvents (i.e., water, and 70% ethanol) and nonpolar (i.e., hexane) solvent.Three different concentrations of the plant extracts (i.e., 2.5 μg/mL, 5 μg/mL, and 10 μg/mL) were used and it showed that the free radical scavenging activity is proportional to the sample concentrations.Here, six different samples were taken: aqueous leaf extract (AL), aqueous stem extract (AS), ethanolic leaf extract (EL), ethanolic stem extract (ES), hexane leaf extract (HL), and hexane stem extract (HS).All the values are mean ± SD of three replications performed on three different times.solvent extracts of A. paniculata leaf and stem showed that most of the phytochemicals are present in the aqueous and ethanolic extracts (Table 1).However, all the tested phytochemicals are absent in the hexane extracts except the glycosides which is present in a very little amount.Both the aqueous stem and ethanolic stem extracts contain the maximum amount (+++) of flavonoids followed by the aqueous and ethanolic leaf extracts.The flavonoid is known to be responsible for the antioxidant activity [21] [22] and, therefore, the aqueous stem extract showed the highest antioxidant activity (Figure 2).The aqueous stem and ethanolic stem extracts also contained the maximum amount (+++) of saponin and glycosides.The maximum amount (+++) of tannin and phenol were found in the ethanolic stem extracts, and aqueous leaf extracts, respectively.

Antimicrobial Activity
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 (Table 2) in both the concentrations.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 (Table 2(a)).The aqueous stem extract also showed the maximum antibacterial activity against gram-negative E. coli and the zone of inhibition was 21 mm in diameter followed by the ethanolic stem extract which was 12 mm in diameter (Table 2(b)).However, the maximum antibacterial activity against S. typhi was performed by the ethanolic stem extract (8.15 mm) followed by the aqueous leaf, and stem extracts (Table 2   The antibacterial activity of the medicinal plant extracts depends on the presence of different concentrations of secondary metabolites such as tannin, saponin, and glycosides [23] [24] [25].Herein, the highest amount (+++) of tannin was present in the ethanolic stem extract followed by the aqueous stem extract (++) (Table 1).The aqueous stem as well as the ethanolic stem extract contained the maximum amount (+++) of saponin and glycoside.The antimicrobial activity of hexane extracts was found due to the presence of 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 [26] [27].The antibacterial activities of all the extracts at a same concentration against B. subtillis were stronger when compared to that of E. coli and S. typhi.Our results suggest that gram-negative bacteria are generally more resistance to A. paniculata extracts when compared to that of gram-positive bacteria.A possible explanation for these findings may lie in the significant differences in the outer layers of gram-negative and gram-positive bacteria.Gram negative bacteria possess an outer membrane and a unique periplasmic space which is not found in grampositive bacteria and, therefore, they showed different antimicrobial activity against the tested sample extracts [28].Stem extracts have higher antimicrobial activity than that of the leaf extracts.Thus, A. paniculata stem extracts prepared from the polar solvents (i.e., water, and ethanol)possess excellent antimicrobial property that can be used as a potential source of antibiotics.

Determination of MIC (Minimum Inhibitory Concentration) and IC50 (Inhibitory Concentration 50) Value
The absorbance of overnight grown bacterial culture was measured at different wavelengths ranging from 560 to 620 nm to determine the λ max for the bacterial culture and observed that λ max was 600.The minimum inhibitory concentration (MIC) value for all the extracts was ~0.05 μg/mL (Figure 4).Furthermore, the It is due to the absence of most of the phenolics except glycosides in hexane extract of A. paniculata leaf and stem (Table 1).
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.

Conclusions
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 IC 50 value.A. paniculata leaf

Salmonella typhi
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.

Figure 1 .
Figure 1.Total phenol content (TPC) of both the leaf and stem extracts of A. paniculata.Leaf and stem extracts were prepared using polar (i.e., water, and 70% ethanol) and nonpolar (i.e., hexane) solvents.Gray bar indicates the TPC of leaf extract and black bar indicates the TPC of stem extract.Data represent mean value ± SD (n = 3).

Figure 2 .
Figure 2. Free radical scavenging activity of both the leaf and stem extracts of A. paniculata.Leaf and stem extracts were prepared

Figure 3 .
Figure 3.Comparison of IC 50 value (μg/mL)) of A. paniculata leaf and stem extracts.After investigation of the free radical scavenging activity (%) of A. paniculata extracts in vitro, IC 50 values were calculated.
(c)).The highest antibacterial activity of the aqueous stem extracts of A. paniculata is because of the presence of tannin, saponin, and glycosides which are known as potential antimicrobial agents.The minimum antibacterial activity was performed against the gram-positive and gram-negative strains of bacteria by the hexane extracts of both the leaf and stem.It was due to the presence of very little amount of glycosides (+) and the absence of excellent antimicrobial agents such as tannin, and saponin.

Figure 4 .
Figure 4. Absorbance of overnight grown bacterial culture in the presence of different amounts of A. paniculata leaf and stem extracts.Six sample were taken under investigation: aqueous leaf (AL), aqueous stem (AS), ethanol leaf (EL), ethanol stem (ES),hexane leaf (HL), and hexane stem (HS).Figures4(a)-4(c) depict the growth inhibitory response for Bacillus subtilis, Escherichia coli, and Salmonella typhi, respectively.In all the cases, the bacterial culture showed the maximum absorbance at 600 nm in the absence of any extracts.However, the absorbance value decreased with the increased amounts of sample extracts.All the values are mean ± SD of three determinations.

Table 1 .
Phytochemical tests of A. paniculata leaf and stem extracts prepared using water, ethanol, and hexane.

Table 2 .
Antibacterial activity of the A. paniculata leaf and stem extracts to (a) B. subtilis (b) E. coli, and (c) S. typhi.[Two different concentrations (5 μg/mL, and 10 μg/mL) of different extracts were used for the antibacterial assay.Values represent mean ± SD of three different replications].