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 Table of Contents  
Year : 2019  |  Volume : 11  |  Issue : 1  |  Page : 16-22  

High-performance liquid chromatographic analysis explores the potential antioxidative agents of Argyreia argentea ARN. EX CHOISY extract

1 Institute of Food Science and Technology, Bangladesh Council of Scientific and Industrial Research, Dhaka 1205, Bangladesh
2 Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong 4331, Bangladesh

Date of Web Publication12-Feb-2019

Correspondence Address:
Dr. Md. Atiar Rahman
Associate Professor, Department of Biochemistry & Molecular Biology, University of Chittagong, Chittagong-4331
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpbs.JPBS_252_15

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Background: Antioxidative properties of medicinal plants play the key role in plant defense mechanism. Argyreia argentea is an evergreen shrub which is used in the treatment of boils, gastric ulcers, tumor, marasmus, paralysis and spermatorrhea, rheumatoid arthritis, cold, painful sensation, and fever. Aims: This research investigates the phytochemical contents and antioxidative effects of optimized crude methanol extract of A. argentea. Materials and Methods: Crude methanol extract of A. argentea prepared in an optimized procedure has been analyzed by high-performance liquid chromatography to quantitatively determine the phytochemical contents. Tannin content of the extract was determined by established method. The extract was also analyzed for in vitro antioxidative actions by spectrophotometric analysis using 2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) method, N, N-dimethyl-1,4-diaminobenzene (DMPD) free radical scavenging method, superoxide radical scavenging method, and nitric oxide scavenging method. Results: The experimental results showed a high amount of catechin hydrate (348.62mg/100g of dry extract) and moderate amount of gallic acid, p-coumaric acid, and rutin hydrate in the methanol extract of A. argentea. Tannin content was found to be 29.66mg/g tannic acid equivalent. Scavenging effects expressed as inhibition concentrations (IC50) for ABTS assay, DMPD assay, superoxide assay, and NO assay were 1148.3 µg/mL ± 7.32 µmol ascorbic acid/g, 1017.68 µg/mL, 1116.89 µg/mL, 1835.23 µg/mL, respectively. All the values were compared with their respective standards. No β-carotene was detected in the extract. Conclusions: Use of A. argentea extract as a source of functional food as well as an antioxidative agent could be considered with further confirmation.

Keywords: Antioxidant and IC50 value, Argyreia argentea, catechin, phytochemicals

How to cite this article:
Uddin MN, Rahman MA, Hossain H, Khan TA, Akter R. High-performance liquid chromatographic analysis explores the potential antioxidative agents of Argyreia argentea ARN. EX CHOISY extract. J Pharm Bioall Sci 2019;11:16-22

How to cite this URL:
Uddin MN, Rahman MA, Hossain H, Khan TA, Akter R. High-performance liquid chromatographic analysis explores the potential antioxidative agents of Argyreia argentea ARN. EX CHOISY extract. J Pharm Bioall Sci [serial online] 2019 [cited 2020 Nov 27];11:16-22. Available from:

Argyreia argentea is an evergreen shrub which is locally known as Bitarak, Ludi, Rupar tola, Naiprabong, or Kajinganj. In Bangladesh, it is available in Chittagong, Jessore, Mymensingh, Noakhali, Sylhet, and Tangail. It is also distributed in Eastern India, Bhutan, and Nepal. The plant is vastly used by the local tribes of Chittagong region for their treatment of boils, gastric ulcers, tumor, marasmus, paralysis, and spermatorrhea.[1] The plant has long been used in the treatment of rheumatoid arthritis, cold, and painful sensation. Chung et al. reported the antipyretic effect of its petroleum ether and chloroform fractions of root extract.[2] Recently, the antinociceptive and anti-inflammatory effects of A. argentea methanol extract have also been studied.[1] Since antioxidative effects of plants are getting much concern as the central mechanism of disease management, we have investigated the antioxidative properties of A. argentea extract, analyzing its phytochemical contents by high-performance liquid chromatography (HPLC).

   Materials and Methods Top


Gallic acid (GA), (+)-catechin hydrate (CH), vanillic acid (VA), caffeic acid (CA), (−)-epicatechin (EC), p-coumaric acid (PCA), rutin hydrate (RH), ellagic acid (EA), and quercetin (QU) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile, methanol, acetic acid, and ethanol were obtained from Merck (Darmstadt, Germany). DPPH, ABTS, and N, N-dimethyl-1,4-diaminobenzene (DMPD) were also purchased from Merck India.

Collection of plant materials

Whole A. argentea plants were collected from Chittagong University Campus in August, 2012. The plant was taxonomically identified and authenticated by Dr. Sheikh Bokhtear Uddin, Professor and Taxonomist, Department of Botany, University of Chittagong, Bangladesh. A voucher specimen (accession no is 34198) of the sample has been preserved at the Bangladesh National Herbarium, Mirpur, Dhaka.

Preparation of plant extract

The fresh and cleaned A. argentea was chopped into small pieces, air dried under shade, and ground into coarse powder to store in an airtight container. Plant powder (900g) was soaked in methanol (98%) for 7 days at room temperature (23±1°C) with occasional stirring. Methanol extract was filtered off through a cotton plug and then with a Whatman No. 1 (Grade 589/2) filter paper. The extract was concentrated under reduced pressure below 50°C through rotary vacuum evaporator (RE 200, Bibby Sterling Ltd., England). The concentrated extract was collected in a  Petri dish More Details and air dried at room temperature. The whole process was repeated 3 times obtaining the crude extract (60g blackish-green, yield 6.6%), which was stored at 4°C until further use.

Optimization of the extraction procedure

Established procedure was applied to optimize the crude extraction.[3] Briefly, the dried plants were put through a process of extraction with methanol 98% to 23±0.5°C. A central composition design was done to evaluate the effect of the progress variables: Extract time (T: 36–72h) and solvent-seed ratio (SSR: 2/1–4/1) expressed as milliliter of solvent per gram of dry sample. The response variable: Crude extract yielded from each gram of dry powder. The extract was filtered using a filter paper (Whatman, Grade 589/2) and the solvent was eliminated using a vacuum evaporator® (RE 200, Bibby Sterling Ltd., England).

High-performance liquid chromatography system and chromatographic conditions

Chromatographic analyses were carried out on a Thermo Scientific Dionex UltiMate 3000 Rapid Separation LC system (Thermo Fisher Scientific Inc., MA, USA), coupled to a quaternary rapid separation pump (LPG-3400RS), Ultimate 3000RS autosampler (WPS-3000), and a rapid separation diode array detector (DAD-3000RS). Phenolic compounds were separated on an Acclaim® C18 (4.6mm × 250mm; 5 µm) column (Dionex™, USA), which was controlled at 30°C using a temperature-controlled column compartment (TCC-3000). Data acquisition, peak integration, and calibrations were performed with Dionex™ Chromeleon software (Version 6.80 RS 10). The phenolic composition of the ethanolic leaf extract of A. argentia was determined by HPLC, as described by Sarunya and Sukon with some modifications.[4] The mobile phase consisted of acetonitrile (solvent A), acetic acid solution pH 3.0 (solvent B), and methanol (solvent C) in a gradient composition summarized in [Table 1]. There was a 5min post run at initial conditions for equilibration of the column. The flow rate was kept constant throughout the analysis at 1mL/min and the injection volume was 20 µl. For DAD detection, the wavelength program was optimized to monitor phenolic compounds at their respective maximum absorbance wavelengths as follows: λ 280nm held for 18.0min, changed to λ 320nm, and held for 6min, and finally changed to λ 380nm, held for the rest of the analysis, and the DAD was set at an acquisition range from 200nm to 700nm. The wavelength for detection and quantification of GA, CH, VA, CA, and EC was 280nm, for PCA, RH and EA was 320nm and for QU was 380nm.
Table 1: Distribution of solvent gradient in different columns on specific run-time

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Standard and sample preparation

A stock standard solution (100 µg/mL) of each phenolic compound was prepared in methanol by weighing out approximately 5mg of the analyte into 50mL volumetric flask. The mixed standard solution was prepared by diluting the mixed stock standard solutions in methanol to give a concentration of 20 µg/mL for each polyphenols except CA (8 µg/mL) and QU (6 µg/mL). All standard solutions were stored in dark at 5°C and were stable for at least 3 months. The calibration curves of the standards were made by serial dilution of the stock standards (five set of standard dilutions) with methanol to yield 1.25–20 µg/mL for GA, CH, VA, EC, PCA, RH, and EA; 0.5–8.0 µg/mL for CA, and 0.375–6.0 µg/mL for QU. The calibration curves were constructed from chromatograms as peak area versus concentration of standard. A solution of ethanolic extract of A. argentea at a concentration of 5mg/mL was prepared in ethanol by vortex mixing (Branson, USA) for 30min. The samples were stored in dark at low temperature (5°C). Spiking the sample solution with phenolic, standards were done for additional identification of individual polyphenols. Prior to HPLC analysis, all solutions (mixed standards, sample, and spiked solutions) were filtered through 0.20 µm nylon syringe filter (Sartorius, Germany) and then degassed in an ultrasonic bath (Hwashin, Korea) for 15min.

Tannin content determination

Quantitative determination of tannin was carried out using the modified Vanillin-HCL method.[5] Vanillin reagent (0.5%, 5mL) was added to the extract (1mL) and the absorbance of the color developed after 20min at 30°C was read at 500nm. A standard curve was prepared expressing the results as catechin equivalents, i.e., amount of catechin (mg/100g) which gives a color intensity equivalent to that given by proanthocyanidins after correcting for blank. Tannin content was calculated as catechin (mg/100g) using the following equation based on the calibration curve: Y = 1299x + 2.944, R2 = 0.996, where x is the absorbance and y is the catechin concentration.

Scavenging assay of 2,2’- azino-bis(3 ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt radical cation

ABTS assay was carried out according to the established method suggested by Sochor et al. with some modifications.[6] Briefly, the ABTS radical scavenging method is monitored spectrophotometrically by the change of the absorption spectrum. Seven mmol/L ABTS and 4.95 mmol/L potassium peroxodisulfate are mixed and dissolved in distilled water. The solution was diluted with distilled water in a 1:9 v/v ratio (10mL is quantitatively transferred into 100mL calibrated flask and diluted). The solution was incubated for 12h in dark and the reagent was used for 7 days if stored in dark at 4°C. The reaction mixture (3.5.0mL) consists of 3.0mL of ABTS and 0.50mL of various concentrations of methanol extract and it was incubated for 10min in dark to measure the absorbance at 734nm against blank distilled water. GA was used as positive control in this assay.

The percentage of inhibition can be calculated using the formula:

Inhibition (%) = [(A0−A1)/A0] ×100.

Where; A0 is the absorbance of control and A1 is the absorbance of test.

N, N-dimethyl-1,4-diaminobenzene free radical scavenging assay

DMPD assay was carried out by following the method of Sochor et al. with minor modifications.[6] Briefly, the compound N, N-dimethyl-1,4-diaminobenzene (DMPD) was converted into solution to a relatively stable and colored radical form by the action of ferric salt. After addition of a sample containing free radicals, these were scavenged and the colored solution was decolorized.[7],[8] Sodium acetate buffer (1) in distilled water (0.2mol/L, pH 5.25 adjusted with concentrated acetic acid) and 0.74 mmol/L ferric chloride (2) in distilled water were prepared. A 36.7 mmol/L DMPD (3) is dissolved in distilled water and solution was prepared. DMPD solution must be prepared at the time of use due to its low stability. These three solutions (solutions No. 1, 2, and 3) are mixed in a 20:1:1 (v/v/v) ratio. The reaction mixture (4.0mL) consists of 3.0ml of DMPD and 1.0mL of various concentrations of methanol extract and it was incubated for 6min to measure the absorbance at 505nm against distilled water as a blank and control was prepared by DMPD reagents and sodium acetate buffer in the place of sample extract. In this assay, the positive control was GA and ascorbic acid.

The percentage of inhibition can be calculated using the formula:

Inhibition (%) = [(A0 − A1)/A0] × 100.

Where; A0 is the absorbance of control and A1 is the absorbance of test.

Superoxide radical scavenging assay by alkaline dimethyl sulfoxide method

Superoxide radical inhibition was assayed using the protocol established by Laloo and Sahu, 2011.[9] In alkaline dimethyl sulfoxide (DMSO) method, superoxide radical was generated by the addition of sodium hydroxide to air saturated DMSO. To the reaction mixture containing 0.1mL of NBT (1mg/ml solution in DMSO) and 3ml of the various concentrations of the extract, 1ml of alkaline DMSO was added to give a final volume of 4.1mL and the absorbance was measured at 560nm. The same procedure was repeated for the standard GA. The percentage inhibition was calculated using the following equation.

Inhibition (%) = (A0 – A1/A0) × 100.

Where; A0 is the absorbance of control and A1 is the absorbance of test.

Nitric oxide scavenging activity

Nitric oxide scavenging effect was assayed using the protocol proposed by Kumar et al., 2008.[10] Various concentrations of the extract were mixed with 2.5mL of sodium nitroprusside (SNP) and made up to 3.0mL with Phosphate buffer solution. Then the mixture was incubated for 15min at 25°C. After incubation, 0.5mL of the reaction mixture was removed and 0.5mL of the Griess reagent was added. Then, the absorbance was measured at 546nm. The percentage inhibition was calculated by comparing the results of the test with those of controls not treated with the extract, as per the following formula:

Scavenging activity (%) = [1−(A1 − A2)/A0] × 100.

   Results Top

Identification and quantification of individual phenolic compound in the extract of A. argentea were analyzed by HPLC. The chromatographic separations of polyphenols in methanol extract are shown in [Figure 1]. The content of each phenolic compound was calculated from the corresponding calibration curve and presented as the mean of five determinations as shown in [Table 2].
Figure 1: HPLC chromatogram of AA leaf extract. Peaks: 1, gallic acid; 2, (+)-catechin; 3, p-coumaric acid; 4, rutin hydrate

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Table 2: Contents of polyphenolic compounds in the ethanol extract of Argyreia argentea (AA) leaf (n = 5)

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The experimental results showed that A. argentea extract contained an especially high concentration of CH (348.62mg/100g of dry extract). It was also shown that GA, PCA, and RH were found in the extract as moderate concentration (26.10, 21.77, and 23.56mg/100g of dry weight, respectively).

Tannin content of A. argentea extract was 29.66mg/g tannic acid equivalent [Figure 2].
Figure 2: Linear regression analysis to determine the tannin content in the extract of A. argentia

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In ABTS assay, the activity was found to be increased in a dose-dependent manner at a concentration of 0.2mg/mL to 1mg/mL. The concentration needed for 50% ABTS radical scavenging activity (IC50) was found to be 4.91 µg/mL for GA and 1148.3 µg/mL for A. argentea extract [Table 3] and [Figure 3].
Table 3: Inhibition concentration (IC50) of different antioxidative models

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Figure 3: Comparative inhibitions of A. argentia and gallic acid in 2,2’- azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt assay

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The A. argentea extract (0.2–1.0mg/mL), analyzed for the ability to quenching the DMPD radicals, was found to increase the discoloration of the DMPD solution with an increase in the concentration of the extract. The DMPD•+ radical cation solution shows a maximum absorbance at 505nm. The half maximal inhibition concentration (IC50) value was found to be 32.93 µg/mL for GA and 1017.68 µg/mL for A. argentea extract [Table 3] and [Figure 4].
Figure 4: Percentage (%) inhibition effects of gallic acid and A. argentia extract in N, N-dimethyl-1,4-diaminobenzene assay

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The A. argentea extract was found to scavenge the superoxide generated by photoreduction of riboflavin. The concentration needed for 50% scavenging of superoxide (IC50) was found to be 75.54 µg/mL for GA and 1116.89 µg/mL for A. argentea extract [Table 3] and [Figure 5].
Figure 5: Percentage (%) inhibition effects of gallic acid and A. argentia extract in superoxide scavenging assay

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Nitric oxide is a very unstable species under aerobic condition. It reacts with O2 to produce the stable product nitrates and nitrite intermediates through NO2, N2O4, and N3O4. It is estimated by using the Griess reagent. In the presence of scavenger compound, the amount of nitrous acid decreases. The extent of decrease reflects the extent of scavenging; the % inhibition showed that IC50 values of catechin and A. argentea extract were 497.10 µg/mL and 1835.23 µg/mL, respectively [Table 3] and [Figure 6].
Figure 6: Percentage (%) scavenging effects of reference standard catechin and A. argentia extract in nitric oxide scavenging assay

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   Discussion Top

Phenolic compounds are known to be powerful chain-breaking antioxidants.[11] It had been reported that the antioxidant activity of plant materials is well correlated with the content of their phenolic compounds.[12]A. argentia ethanol extract was analyzed by HPLC and the phenolic compounds of the extract were identified. Due to the antioxidative role of the phenolic compounds, various phenolic compounds such as CH, GA, PCA, and RH which are usually used as reference antioxidants were analyzed.

The tannin compounds are widely distributed in many species of plants, where they play a role in protection from predation, and perhaps also as pesticides and in plant growth regulation.[13] The astringency from the tannins is what causes the dry and puckery feeling in the mouth following the consumption of unripened fruit or red wine.[14] Likewise, the destruction or modification of tannins with time plays an important role in the ripening of fruit and the aging of wine.

In this research, four antioxidative models were chosen for evaluating the scavenging effect of A. argentea. ABTS assay, the first model, is an excellent tool for determining the antioxidant activity of hydrogen-donating antioxidants and of chain-breaking antioxidants.[15] ABTS assay is often used in evaluating total antioxidant power of single compound and complex mixtures of various plants.[16],[17],[18] Therefore, the ABTS radical scavenging activity of the extract of A. argentea indicates its ability to scavenge free radicals, thereby preventing lipid oxidation via a chain-breaking reaction.

DMPD method is suitable for fast and sensitive measurement of antioxidant activity of hydrophilic compounds. The assay based on the discoloration of the DMPD solution was found to increase with an increase in the concentration of the extract. Dark color of DMPD•+ radical cation solution becomes lighter and absorbance of solution becomes lower in the presence of an antioxidant compound. Antioxidant compounds which are hydrogen donors to DMPD•+ quench the color of DMPD•+ solution.[18]

Superoxide anion is also very harmful to cellular components. Robak and Gryglewski (1988) reported that flavonoids are effective antioxidants mainly because they scavenge superoxide anions.[18],[19],[20],[21] As shown in [Figure 3], the superoxide radical scavenging activities of the plant extract and the reference compounds are increased markedly with increasing concentrations. The results suggest that the plant extract is a more potent scavenger of superoxide radical than the standard quercetin.

Nitric oxide is a potent pleiotropic inhibitor of physiological processes such as smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation, and regulation of cell-mediated toxicity. It is a diffusible free radical that plays many roles as an effector molecule in diverse biological systems including neuronal messenger, vasodilation, and antimicrobial and antitumor activities.[8],[22] Suppression of released NO may be partially attributed to direct NO scavenging, as the A. argentea extract decreased the amount of nitrite generated from the decomposition of SNP in vitro. The scavenging of NO by the extract was increased in concentration-dependent manner.

   Conclusions Top

A new HPLC method to analyze nine polyphenolic compounds (GA, catechin, VA, CA, (−)-EC, PCA, RH, EA, and quercetin) simultaneously has been developed and validated for linearity, accuracy, stability, and precision. This HPLC procedure provided excellent identification and quantification of these nine phenolic compounds presented in the A. argentea extract within a short analysis time (30min). Since the phenolic compounds have been of interest of health benefits, the present HPLC study could be a potential application to identify and quantify the polyphenolic compounds in any medicinal plant extracts.


The authors wish to acknowledge the Bangladesh Council of Scientific and Industrial Research for providing the laboratory facilities to carry on the research.

Financial support and sponsorship

Research support from the Bangladesh Council of scientific and Industrial Research, BCSIR, Dhaka.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2], [Table 3]


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