Total antioxidant capacity and profiling of polyphenolic compounds in jute leaves by HPLC-DAD

*Ali, M.M., Ahmed, K.S., Hossain, H., Roy, B., Rokeya, B., Rahman, M.T., Jahan, I.A. and Rahman, M.M. Department of Biochemistry, Bangladesh Jute Research Institute (BJRI), Dhaka-1207, Bangladesh Chemical Research Division, BCSIR Laboratories, Dhaka, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka-1205, Bangladesh Department of Chemistry, Hajee Mohammad Danesh Science & Technology University, Dinajpur, Bangladesh Department of Pharmacology, Bangladesh University of Health Science, Mirpur-1, Dhaka, Bangladesh. Jute Seed Production and Research Centre, BJRI, Nashipur, Dinajpur, Bangladesh Institute of Food Science and Technology (IFST), Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka-1205, Bangladesh


Introduction
Jute (Corchorus spp.) is a cash crop in Bangladesh being cultivated in 10% of agricultural land area (Islam, 2019). It is cultivated in many other countries like as India, Myanmar, Nepal, China, Taiwan, Thailand, Vietnam, Cambodia, Brazil, etc. C. olitorius jute namely is Tossa Jute and C. capsularis jute namely is White Jute (Islam, 2013).
Demand for medicinal plants is increasing in both developed and developing countries due to the growing recognition of natural products being equally effective, safe, non-narcotic, affordable and has no side effects.
One such medicinal plant part is jute leaves (Islam, 2013). Young shoots and leaves are eaten as vegetable and food ingredients and have long been used as medicinal folk remedies in East Asia and Africa. Healthflourishing effects of plant-derived secondary metabolites in human health, including antioxidative, anticarcinogenic, antibiotic, and pharmacological effects, are well documented (Lee et al., 2015). C. olitorius leaves are used in the treatment of fever, tumors, pectoral pains, dysentery, aches, enteritis, cystitis, piles and dysuria (Adegoke and Adebayo-Tayo, 2009). C. capsularis leaves are also used in ayurvedic for ascites, piles, cystitis, dysuria, fever and gonorrhoea (Islam et al., 2013).
Reactive oxygen species (ROS) could be characterized as signaling molecules and lead to oxidative-induced damage to cell membranes, protein denaturation, DNA mutations and lipid peroxidation (Beckers and Spoel, 2006), which are related to some chronic diseases, like cancer, inflammation, cardiovascular diseases and others (Pietta, 2000). Antioxidants may be defined as complex determined compounds that function as defensive shields against several diseases (Nath et al., 2013). Phenolic compounds are an important group of plant-based biologically active compounds that strengthen the organism and prevent disease (Sun et al., 2002;Gharras, 2009). Plant polyphenols are secondary metabolites characterized by one or more hydroxyl groups binding to one or more aromatic rings (Zhou et al., 2019). Phenolic compounds have a particularly strong antioxidant effect (Hider et al., 2001;Scalbert et al., 2005;Pandey and Rizvi, 2009). Numerous epidemiologic literature has verified an important correlation between the consumption of phenolic compound-rich food and a decreased risk for developing cardiovascular and other diseases (Weichselbaum et al., 2010;Spencer, 2010).
Hence, in this study, we attempted to investigate the total antioxidant capacity and HPLC profiling of bioactive polyphenolic compounds in 80% ethanol extract of two varieties of jute leaves growing in Bangladesh.

Plant material
The two varieties of jute Leaves, C. olitorius (O-9897) and C. capsularis (CVL-1) were collected from Jute Seed Production and research centre, Bangladeh Jute Research Institute, Nashipur, dinajpur during June 2017. The leaves were properly washed to remove dirt and other impurities. After that, the leaves were dried under the shade. The dried leaves were powder by pulverizes. The sample was then saved in an airtight container and storage in the refrigerator until extraction.

Extraction
The two varieties of shade dried leaves were extracted in an orbital shaker with 80% ethanol for 24 hrs at room temperature to obtain ethanol extract of jute leaves. The extract was initially filtered in a cotton plug to get rid of the plant debris and next through Whatman filter paper no 1. The solvent was removed using a rotary vacuum evaporator (R-215, Buchi, Switzerland) under reduced pressure. The concentrated filtrates were kept in the bottle at -20°C prior to further analysis.

Chemicals and reagents
All the standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and reagent was collected from Scharlau (Spain) and Merck (Germany).

Total antioxidant capacity
The total antioxidant capacity of the C. olitorius and C. capsularis leaves sample extract were evaluated by the phosphomolybdenum assay method (Prieto et al., 1999) which is based on the reduction of Mo (VI) to Mo (V) and the subsequent formation of a green phosphate-Mo (V) complex in acidic condition. 0.3 mL of each extract (1 mg mL -1 ) was allowed to mix with 3.0 mL of the reagent solution (0.6 M H 2 SO 4 , 28 mM Na 3 PO 4 , 4 mM ammonium molybdate). This reaction mixture was incubated at 95 o C for 90 mins. After letting the solution cool back to room temperature, the absorbance was measured at 695 nm with a double beam UV/Visible spectrophotometer (Specord 205, Analytikjena, Germany) against a blank solution. The total antioxidant capacity was determined and expressed as mg ascorbic acid equivalents per gram of dry extract using the equation obtained from a standard ascorbic acid calibration curve.

Identification and quantification of polyphenolic compounds by HPLC
Identification and quantification of selected phenolic compounds in the 80% ethanol extract were determined by HPLC-DAD analysis as described by Hossain et al. (2016) with some modifications. It was carried out on a Dionex UltiMate 3000 system equipped with quaternary rapid separation pump (LPG-3400RS) and photodiode array detector (DAD-3000RS). The separation was performed using Acclaim® C 18 (5 µm) Dionex column (4.6 x 250 mm) at 30°C with a flow rate of 1 mL/min and an injection volume of 20 µL. The mobile phase consisted of acetonitrile (solvent A), acetic acid solution pH 3.0 (solvent B), and methanol (

Total antioxidant capacity
Phosphomolybdate assay is an easy and well-known technique based on the reduction of Molybdenum (VI) to Molybdenum (V) by forming phosphate-Molybdenum (V) complex which is green in color with maximal absorption at a wavelength of 695 nm. Total antioxidant capacity of C. olitorius and C. capsularis is illustrated in Table 1. Results showed that C. capsularis leaves contained high total antioxident capacity (214.32±1.95 mg of ascorbic acid/g of dry extract) then C. olitorius (165.66±1.30 mg of ascorbic acid/g of dry extract). Total antioxidant capacity is an analyte frequently used to assess the antioxidant status of biological samples and can evaluate the antioxidant response against the free radicals produced in a given disease (Rubio et al., 2016).

Identification and quantification of polyphenols in Corchorus olitorius and Corchorus capsularis leaves
Identification and quantification of individual polyphenolic compounds in the 80% ethanolic extracts of C. olitorius and C. capsularis were analysed by HPLC. The chromatographic separations of polyphenols in standard and 80% ethanolic extracts of C. olitorius and C. capsularis are shown in Figures 1, 2 and 3 respectively. The content of each polyphenolic compound found in the 80% ethanolic extracts of C. olitorius and C. capsularis was calculated from the corresponding calibration curve and presented as the mean of five determinations as shown in Tables 2 and 3.
The analysis of the results of HPLC-DAD allowed the detection of six and eight polyphenolic compounds from C. olitorius and C. capsularis leaves respectively. The experimental results indicated that 80% ethanolic extract of C. olitorius leaves contained an especially high concentration of rutin hydrate, ellagic acid, and quercetin hydrate (152.17±0.51, 143.27±0.58, and 292.83±0.73 mg/100 g of dry extract, respectively) than that of C. capsularis (32.16±0.08, 53.65±0.11, and 46.17±0.09 mg/100 g of dry extract, respectively). It was also shown that caffeic acid and vanillin were detected both in the 80% ethanol extract of C. olitorius and C. capsularis but the concentration of caffeic acid was at the moderate amount (51.06±0.11 and 55.93±0.13 mg/100 g of dry extract) and vanillin at a lower amount (5.18±0.04 and 1.04±0.01 mg/100 g of dry extract) shown in Tables 2  and 3. It was also found that vanillic acid, trans-ferulic acid and rosmarinic acid (13.28±0.05, 58.02±0.18 and 3.54±0.02 mg/100 g of dry extract, respectively) were detected only in the 80% ethanol extract of C. capsularis leaves and on the other side kaempferol (13.32±0.07 mg/100 g of dry extract) was detected only in the 80% ethanol extract of C. olitorius at a lower concentration shown in Tables 2 and 3. The major identified polyphenolic compounds of C. olitorius were rutin hydrate, ellagic acid and quercetin hydrate. These compounds display interesting biological properties, such as antioxidant as well as anti-inflammatory and anticancer activities (Selloum et al., 2003;Vattem and Shetty, 2005;Anand David et al., 2016;Ganeshpurkar and Saluja, 2017). The previous study reported the presence of ethanol, ethanol: water (50:50) and water extract of C. olitorius leaves contain caffeic acid (229.56, 146.02 and 306.43 mg/kg), quercetin (52.01, 35.26 and 3.13 mg/kg) and kaempferol (18.28, 29.39 and 16.24 mg/kg) etc (Ben Yakouba et al., 2018). Which is lower than our present study. In C. capsularis leaves, the most abundant polyphenolic compounds were caffeic acid, trans-ferulic acid, rutin hydrate, ellagic acid and quercetin hydrate. These compounds have also a noticeable Pharmacological propriety such as antioxidant, anti-inflammatory and anticancer activities (Selloum et al., 2003;Vattem and Shetty, 2005 Ganeshpurkar and Saluja, 2017;Espíndola et al., 2019). In this context, Mosihuzzaman et al. (1986) showed that p-coumaric, caffeic, vanillic, ferulic acid and phydroxybenzoic acids were present in the 80% aqueous ethanol extract of C. capsularis in unretted bark and stem of jute. The antioxidant is generally used to evaluate the total antioxidant power of single compounds and complex mixtures of different plants (Huang et al., 2008). Antioxidant activity depends on the present of polyphenolic compounds (Materska and Perucka, 2005). Therefore, significant results of total antioxidant capacity of both extracts may be due present of different polyphenolic compounds.

Conclusion
The results of the present study indicated that, C. olitorius and C. capsularis leaves exhibit a significant amount of total antioxidant capacity and polyphenolic compounds.

Conflict of interest
The authors declare no conflict of interest.