Comparison of antioxidant role of methanol, acetone and water extracts of Andrographis paniculata Nees

1 Department of Pharmacy, Faculty of Science, Noakhali Science and Technology University, Noakhali-3814, Bangladesh. 2 Department of Chemistry, Faculty of Science, University of Chittagong, Chittagong-4331, Bangladesh. 3 Pratyasha Health Biomedical Research Center, Dhaka-1230, Bangladesh. 4 Department of Pharmacy, School of Science and Engineering, Southeast University, Dhaka-1213, Bangladesh. 5 Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh. 6 Department of Pharmacy, Faculty of Health Sciences, Atish Dipankar University of Science and Technology, Dhaka1230, Bangladesh.


INTRODUCTION
The use of herbal medicines has rapidly increased in the last few decades and is expanding swiftly across the world day by day (Ekor, 2014;Welz et al., 2018;Ali et al., 2019). Around 75-80% of individuals of developing countries and 25% of individuals of the developed countries are using traditional herbal medicines for their primary source of treatment (Bodeker et al., 2005;Bandaranayake, 2006;Mukherji, 2008;Hussain et al., 2018). With the introduction of many more herbal products in the market to suffice the surging demand for herbal medicines, public health issues surrounding their safety have become a major concern for the community. Albeit, some herbal medicines have been known for their potential biological activity, and are widely used, the vast majority of them yet remain untested, and their uses are poorly monitored as well. This scenario is also prevalent in Bangladesh, where herbal medicines play a pivotal role in the treatment of different diseases. Top pharmaceutical companies are now opening their herbal plants to appease the burgeoning exigency of herbal medicines in the country. Nevertheless, there are no specific parameters up to date based on which we can control the quality of these therapeutic plants. MDA is the end product of lipid peroxidation. Increased level of MDA generates oxidative stress. Antioxidants work against oxidative stress by converting superoxide-free radicals into hydrogen peroxide and oxygen molecules (Das et al., 2020). This study was conducted to evaluate the antioxidant potential of Andrographis paniculata. However, In Bangladesh, A. paniculata commonly referred to as ' Kalmegh' in Bangla, has markedly been pervasively consumed for ages as one of the most vital medicinal plants.
A. paniculata (Family: Acanthaceae) is plentifully abundant in Southeast Asia where it engenders from seed on all kinds of soil (Purohit et al., 2006) and has the ubiquitous presence of bitter andrographolide. Since, it has a wide spectrum of pharmacological action (Singha et al., 2003;Mamatha 2011), it is termed "king of bitters". With the virtue of the combined effect of the arabinogalactan proteins and andrographolides, this plant has also been reported to exhibit antimicrobial action (Roy et al., 2010). A. paniculata was propounded by Charaka Samitha for the treatment of jaundice (Sharma, 1983) and has been used against sluggish liver in colic dysentery as well as dyspepsia (Handa and Sharma, 1990;Sheeja et al., 2006;Tatiya et al., 2012). In addition, it turns out to have beneficial effects on hepatitis and dysentery (Singha et al., 2007). The new leaf juice obtained from the plant is an established household remedy in treating colic pain, diminished appetite, irregular stools (Madav et al. 1995, Sheeja et al., 2006Nugroho et al., 2012). The most opulent and sought-after constituent andrographolide isolated by Valdiani et al. (2014) carries certain other activities mainly liver protection (Nagalekshmi et al., 2011), anticancer activity (Menon and Bhat, 2010), anti-diabetic activity (Nugroho et al., 2012) and antimalarial activity (Dua et al., 2004).
Studies in terms of seeking bioactivities such as the antioxidant potential of myriad plants including A. Paniculata have always been deemed with utmost priority since the effectiveness can deviate concerning geographic ancestry, age of the plant, part of the plant used as well as the use of the solvents for extraction. Recently, an increase of immense interest for plantderived natural antioxidants has been substantially on the rise due to their promising possibility to be used as an effective therapeutic drug, nutraceuticals, food, cosmetics because they have multifacetedness when it comes to activity and serves enormous opportunity in turning the tide of imbalance in the body.
According to Hamdard Laboratories (WAQF), in Bangladesh, the annual demand for Kalmegh (A. paniculata) is 1,000 tons (Hasan 2003). Bangladesh satiates this needs mostly by importing from foreign countries (e.g., India, China, etc.) and the rest is accrued from wild sources and cultivated in the country, especially in some parts of the Rajshahi division. But, still now, no such research has been done to determine the quality of Kalmegh cultivated in the country particularly. Since the antioxidant properties of any products can be implicated in the retardation of numerous diseases like cancer, coronary heart diseases, bronchitis, asthma, ulcers, scabies, insomnia, senile debility, diabetes, dysentery, etc., for the first time, this study was designed to determine the antioxidant properties of Kalmegh samples available in Bangladesh. Therefore, the main objective of this study is to assess the antioxidant activity of Kalmegh and to perform a comparative study among these samples.

Plant materials collection
A. paniculata (Kalmegh) samples (stem along with leaves) were garnered from Owshudi Gram, Natore, Bangladesh, during September 2018. Having properly identified by an expert botanist, a voucher specimen (DACB Accession Number: 35939) of the plant sample was then deposited to Bangladesh National Herbarium, Mirpur, Dhaka. After collection, cleansing and chopping of the sample into small pieces, in the next step dried samples were grained into powder with the help of a cyclone grinder machine. The powdered samples were eventually used for different experiments.

Preparation and extraction of extracts
The powdered samples of A. paniculata were then subjected to extract preparation. For extract preparation, powdered samples were extracted with solvents using a Soxhlet apparatus. The extraction solvents used for the extraction were methanol, acetone, and water. Then the extracted solvents were evaporated and concentrated using a rotary evaporator. Finally, it was desiccated in a vacuum drier and stored for further analysis (WHO, 2011).

DPPH radical scavenging activity
The stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was assessed concurring with the modified method explicated (Chang et al., 2001;Gupta et al., 2004;Uddin et al., 2020). Concisely, 2 ml volume of 0.1 mM DPPH solution having purple color and another 2 ml of plant sample of varying concentrations (5,10,20,40,60,80,100,250 and 500 µg/m) were first brought together and stirred vigorously for 15 s. To get a uniform mixture followed by keeping in the dark at room temperature for a time period of half an hour. After the specified interval, a UV/Visible spectrophotometer was used to read absorbance against a condign blank at 517 nm (Model 205, Germany). The antioxidant viability was estimated from the ability of the extract to eradicate the purple color that appeared in DPPH solution and the percentage of DPPH radical-scavenging activity was computed as: Where A 0 is the absorbance of the control solution; A is the absorbance of the DPPH solution containing plant extract. IC 50 values for the plant extract and standard were obtained by analysis of the respective percentage scavenging of DPPH radical.

Determination of reducing power assay
It was performed with faint modifications, based on the method expounded by Oyaizu (1986). First of all, a mixture of 0.75 ml of plant extract at varying concentrations with 1% potassium hexacyanoferrate (0.75 ml) and 0.2 M potassium dihydrogen phosphate (0.75 ml, pH 6.6) was conducted with subsequent incubation in a water bath at 50°C for 20 min. The mixture was then subjected to centrifugation for 10 min at 800 rpm after the addition of trichloroacetic acid (10%). After that, the obtained upper layer was blended with 0.1% ferric chloride (w/v, 0.1 ml) and distilled water (1.5 ml) for ten minutes. The optical density (OD) against a blank was ascertained at 700 nm. Increased OD value of the mixture of reaction indicates increased reducing power.

Total antioxidant capacity determination
The total antioxidant capacity was ascertained following the phosphomolybdenum assay method (Prieto et al., 1999) which operates via the reduction of Mo (VI) to Mo (V) by extracting and subsequently forming a green phosphate-Mo (V) complex in an acidic state. The extract was permitted to amalgamate with 3.0 ml of reagent solution (0.6 M H 2 SO 4 , 28 mM Na 3 PO 4 , 4 mM ammonium molybdate), and afterward, the reaction mixture was incubated at 95 0 C for 90 min. Upon cooling at room temperature, a The antioxidant activity was elicited as the number of ascorbic acids UV-Visible spectrophotometer was used to measure the solution's absorbance against an opposite blank at a wavelength of 695 nm. tantamount in gram.

ABTS radical scavenging activity test
The antioxidant capacity was appraised by Fan et al. (2009), with petty alterations. In succinct, ABTS radical cation was synthesized by reacting with 2.45 mM potassium persulfate solution of 7 mM ABTS and leaving the reaction blend to stay at room temperature in the dark for a period of 16 h. At the time of use, the ABTS solution was then diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm. Application of 1 ml of each sample with varying concentrations (5 to 250 μg/ml) to 1 ml of ABTS solution was conducted, afterward. At the subsequent stage, the reaction admixture was held to stand for another 6 min at room temperature, and momentarily the absorbance was reported at 734 nm. The ABTS scavenging effect was calculated based on the following formula: Where A o = Absorbance of control and A s = Absorbance of sample

Nitric oxide (NO) scavenging activity
Nitric oxide scavenging activity was measured spectrophotometrically (Marcocci et al., 1994;Balakrishnan et al., 2009). In this experiment, a reaction mixture of 5 ml, comprising SNP or sodium nitroprusside (5 mM) dissolved in phosphate buffer (pH 7.3), with the presence or absence of various concentration of plant samples was subjected to incubation for 180 min at 25°C in the existence of a light source ( visible and polychromatic). Thus the generation of nitric oxide radical (NO . ) took place that subsequently combined with oxygen to form nitrite ion which was estimated at every 30 min by amalgamating incubation mixture (1 ml) with Griess reagent of the same volume. Absorbance measured for the chromophore produced due to the reaction was then noted at 546 nm. Estimation of nitrite ion formed with or without adding extracts was performed based on the standard sodium nitrate curve obtained by using a known concentration of the solution. The nitric oxide (NO) radical scavenging activity was expressed as the inhibition percentage (I%) and calculated as: Where A blank is the absorbance of the control reaction, and A sample is the absorbance of the test compound. The IC 50 value was calculated from the plot of inhibition (%) against extract concentration. Ascorbic acid was used as a standard for this study.

Determination of total phenolic content
The total phenolic content of the extracts was gauged in compliance with the modified Folin-Ciocaltu method (Wolfe et al., 2003). The absorbance was noticed at 765 nm with a spectrophotometer (Specord 205, Germany). The standard curve was arranged using 0, 0.25, 0.50, 0.75 and 100 mg/ml solutions of gallic acid in methanol: water (50:50, v/v). Total phenolic content was finally stated as mg of gallic acid equivalent per gram using the equation obtained from a standard gallic acid calibration curve y =6.9104x -0.0937, R 2 = 0.9972.

Determination of total flavonoids
The aluminum chloride colorimetric method was used for flavonoids  Values are expressed as mean ± SD (n=3) and same column with a different letter are significantly different (P < 0.05).
determination (Chang et al., 2002). In this prescribed assay, a quantity of 0.6 ml of fractions and 2% aluminium chloride (6 ml) were blended with each other. Following mixing, incubation of the solution at room temperature was carried out for 1 h and the absorbance of the reaction mixture was then reported at 415 nm wavelength. The calibration curve was attained in the same way using Quercetin solutions instead of fractions at concentrations 0, 0.25, 0.50, 0.75, 100 mg per ml in methanol. Total flavonoids content was expressed as mg of Quercetin equivalent per gram using the equation acquired from a standard Quercetin calibration curve y = 4.7385x + 0.0355; R 2 = 0.9993.

Statistical analysis
All data presented in the study as mean ± SD and were statistically analyzed by SPSS software package (version 20) and MS Excel for windows version 2010.

RESULTS AND DISCUSSION
In this study, crude extracts of A. paniculata have been investigated for methanol, acetone, and water extracts. All extracts were tested for antioxidant potential evaluation. Antioxidant potential was assessed by the DPPH scavenging activity, reducing power assay, total antioxidant capacity, ABTS radical scavenging activity, nitric oxide (NO) scavenging activity, amount of total phenolic and flavonoid content determination.

DPPH scavenging activity of different solvent extracts
A constant free radical DPPH is decolorized in the existence of antioxidants (Hasanuzzaman et al., 2013) and has an unpaired electron which exhibits absorbance of 515 to 517 nm providing a clear dark purple color. Decolorization of DPPH occurs as it accepts electron generated by antioxidant compound, and is quantitatively measured from the changes in absorbance (Subhan et al., 2008). All extractives showed substantial DPPH free radical scavenging activity suggested by IC 50 values (Table 1 and Figure 1). The IC 50 value implies the magnitude of scavenging potential. Scavenging activity of hot methanol, acetone, and water extract of A. paniculata was found to ascend with the augmentation in concentration. Though all the samples showed somewhat less radical scavenging activity at a lower concentration, the very significant antioxidant potential was demonstrated compared to standard compound when the concentration was greater with the highest activity at 500 µg/ml. Among the extracts of A. paniculata, maximum inhibition of DPPH free radicals at 500 µg/ml was observed (91.78, 92.67 and 86.57%) for methanol, acetone, and water; whereas the standards ascorbic acid and butylated hydroxyanisole both at 500 µg/ml displayed 97.42 and 94.62% inhibition, respectively. The experiment also revealed that the DPPH radical quenching ability of these extracts is concentrationdependent, and scavenging capability is enhanced with gradual increase in concentration.

Reducing power assay of various solvent extracts
Antioxidant potential can also be evaluated by quantifying the reducing power of a substance where a rise in the absorption of a substance is an indicator of heightened reducing power (Li et al., 2017). Increasing antioxidant activity would reduce the cycle of lipid peroxidation and thus protect cell damage (Uddin et al., 2017;Adnan et al., 2019;Amin et al., 2020). With the continued elevation in concentration, reducing the power of methanol, acetone, and water extract of A. paniculata was found to climb upward. Figure 2 depicts the comparative reducing power of various extracts and fractions of A. paniculata and standard compounds (ASA and BHA). The methanol extract showed noticeably pronounced reducing power than acetone and water extract when compared to selected standards.

Total antioxidant capacity for different solvent extract
Total antioxidant capacity in various solvent extracts and fractions of the A. paniculata was also explicated. Total antioxidant capacity was denoted as ascorbic acid equivalents by reference to a standard curve (y = 0.0026x  (Table 2). Methanol and acetone extracts contained a similar amount of total antioxidant capacity 83.35 ± 0.13% and 82 ± 0.02% (Ascorbic acid equivalent per gram of dry extract) respectively but water extract displayed relatively lower total antioxidant capacity (51.4 ± 0.01%).

ABTS scavenging activity of different solvent extracts
The ABTS scavenging assay is another well-grounded approach taken up to establish antioxidant activity. A blue or green chromophore of ABTS .+ is generated as a result of oxidation between potassium persulfate and ABTS. The reduction of ABTS .+ formed during the interaction of these two chemicals is assessed with the existence of antioxidants having hydrogen donating property at 734 nm by using a spectrophotometer. The ABTS scavenging is not only an indicator of the antioxidant potential hydrogen donating antioxidants but also chain-breaking antioxidants (Li et al., 2017). The ABTS radical activity of methanol, acetone, and water extracts of A. paniculata was seen to rise with the rise in concentration (Figure 3). The IC 50 value was also computed ( Table 3). The scavenging power of methanol and acetone extract provided excellent result but water extract showed weak results when the comparison was made with standard ascorbic acid.

Nitric oxide (NO) scavenging activity of different solvent extracts
NO extricated from sodium nitroprusside gives rise to NO + which in turn can modulate the cellular function through the alteration of cellular structure. NO can be even more toxic by reacting with the superoxide and constituting potent oxidant peroxynitrite anion that eventually can decay later to yield both . OH and NO 2 (Awah and Verla, 2010). Nitric oxide scavengers work by  Values are expressed as mean ± SD (n=3) and same column with a different letter are significantly different (P < 0.05).
competing with oxygen and thereby resulting in the synthesis of reduced NO 2. The NO scavenging activity was seen to enhance with the increase in the concentration of methanol, acetone, and water extract which showed good activity in comparison to standard ascorbic acid. Figure 4 and Table 4 delineate the comparative analysis of different extracts and IC 50 among the extracts of A. paniculata.

Total phenolic and flavonoid content of different solvent extracts
Polyphenols are the significant plant compounds that confer antioxidant potential. Typical phenolics are known as phenolic acids and flavonoids (Demiray et al., 2009). The antioxidant potential of the plant varies due to phenolics. Studies have been able to shed light upon the  Values are expressed as mean ± SD (n=3) and same column with a different letter are significantly y different (P < 0.05).
evidence that flavonoids being phenolic acids are accountable for the antioxidant potential of various medicinal plants (Esmaeili et al., 2015) and viable scavengers of the majority of oxidizing molecules, for instance, a wide range of free radicals linked with numerous ailments, and singlet oxygen (Bravo, 1998). Total phenolic content and flavonoids of various extracts were also determined. Acetone extracts of A. paniculata were observed to contain the maximum amount of total phenolic content (80.66% mg Gallic acid equivalent per gram of dry extract) than methanol and water extracts. Methanol extracts of A. paniculata samples were reported to contain the maximum amount of flavonoid content (592.34% mg Quercetin equivalent per gram of dry extract) than acetone and water extracts. Figure 5 illustrates the amount of total phenolic content and total flavonoids content in different solvent extracts of the three samples.  Values are expressed as mean ± SD (n=3) and same column with a different letter are significantly different (P < 0.05).

Conclusion
In this study, the antioxidant potential of different extracts of A. paniculata was determined with three different solvents. Different solvent extracts showed potential antioxidant activity through the evaluation of antioxidant tests. Methanol is identified as the most appropriate extraction solvent for A. paniculata because it showed significant antioxidant power and the highest flavonoid contents than acetone and water solvent extracts. Further scientific studies are suggested to identify the exact compounds exploiting antioxidant activity and to understand its mechanism for such activity. co-operation to conduct the research work.