In Vitro Antioxidant Activity and Gc-Ms Analysis of Peel and Pulp
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IJBPAS, June, 2020, 9(6): 1269-1283 ISSN: 2277–4998 IN VITRO ANTIOXIDANT ACTIVITY AND GC-MS ANALYSIS OF PEEL AND PULP EXTRACTS OF DIMOCARPUS LONGAN ALFIA BEGAM S1, SHEILA JOHN1*, SARAH JANE MONICA2, PRIYADARSHINI. S1, SIVARAJ C3 AND ARUMUGAM P3 1Department of Home Science, Women’s Christian College (Autonomous) Chennai, Tamilnadu, India 2Department of Nutrition, Food Service Management and Dietetics, Ethiraj College for Women (Autonomous) Chennai, Tamilnadu, India 3Armats Biotek Training and Research Institute, Chennai, Tamilnadu, India *Corresponding author: Dr. Sheila John: Associate Professor and Head, Department of Home Science, Women’s Christian College (Autonomous), Chennai: 600 006, E Mail id: [email protected]; Phone: 9444904638 Received 2nd March 2020; Revised 30th March 2020; Accepted 7th April 2020; Available online 1st June 2020 https://doi.org/10.31032/IJBPAS/2020/9.6.5071 ABSTRACT Waste substrates such as peels and seeds are accumulated with several bioactive compounds that impart various pharmacological properties. Longan is a sub tropical fruit that is either consumed as fresh, frozen, canned or dried. In spite of being rich in several bioactive and non-nutritive compounds, the peels and seeds of this fruit is usually discarded. The objective of the present study was to compare the antioxidant activity of peel and pulp extracts of Dimocarpus longan. Total phenol and flavonoid content was estimated using Folin – Ciocalteau and Aluminum Chloride method respectively. Antioxidant activity was determined using DPPH, 1269 IJBPAS, June, 2020, 9(6) Alfia Begam S et al Research Article FRAP and Phosphomolybdenum assay. All the assays were carried out in triplicates. Bioactive compounds were analyzed using Gas Chromatography-Mass Spectrometry. Results indicated that the peel extract exhibited greater potential to scavenge DPPH free radicals when compared to pulp extract thereby indicating the use of waste substrates as free radical inhibitors.IC50 value of peel and pulp extracts were found to be 57.07µg/mL and 256.21µg/mL. Results of reducing power assays showed that peel extract exhibited greater reducing capacity than the pulp extract. Gas Chromatography-Mass Spectrometry analysis showed the presence of 14 bioactive compounds in peel extract and 12 bioactive compounds in pulp extract. Findings of the study indicate that peel extract possessed relatively higher antioxidant activity thereby highlighting the possibility of utilizing fruit peels as a natural source of antioxidant. Keywords: fruit peel, waste substrates, polyphenols, antioxidant activity INTRODUCTION Fruit is one of the basic food groups that maturity [2]. It is consumed in different provide essential micronutrients such as forms such as fresh, canned, frozen and vitamins, minerals, dietary fibre and dried. Longan fruit is used to treat stomach phytochemicals. Fruit industry is one of the disorders, help to relieve insomnia, functions leading industries in terms of production, as an antipyretic agent, act as a vermifuge consumption, export and expected growth. and possess anticancer activity [3, 4]. Peels Tropical and sub tropical fruits are rich in and seeds are the major waste by-products non-nutritive components, essential organic obtained during processing of fruits and acids, pectin, cellulose and dietary fibre that vegetables. Exploiting the usage of fruit peels impart several nutritional and therapeutic in different industries has gained importance health benefits [1]. Longan (Dimocarpus due to the presence of various natural Longan L.) is a subtropical fruit that is native bioactive compounds that is responsible for to southern China and belongs to the family pharmacological and therapeutic properties Sapindaceae. In India, it is widely distributed [5]. Yang et al. [6] reported that longan in south western parts such as Assam and pericarp and seed as by-products account for Bengal. The fruit weighs about 5-20g and is 16-40% of the whole fruit that can be either conical or spherical in shape. The potentially utilized as a readily accessible colour of the fruit varies from greenish source of natural antioxidant. The hazardous yellow to yellowish brown depending upon effects of synthetic antioxidants have revived 1270 IJBPAS, June, 2020, 9(6) Alfia Begam S et al Research Article the search for natural sources that have Hundred μL of the extracts (1mg/mL) were potent antioxidant properties. Studies made up to 1 mL using methanol and mixed focusing on antioxidant activity of biological with 1 mL of Folin Ciocalteu reagent (1:10 waste substrates such as peels, seed residues diluted with distilled water). After 5 minutes, and pericarp of longan fruit, particularly 1 mL of 20% sodium carbonate (Na2CO3) peels are limited. Based on this, the objective solution was added. The mixture was of the present study was to compare the incubated at room temperature for 30 antioxidant activity of peel and pulp extracts minutes and the absorbance was measured at of longan fruit and to exploit the use of these 760 nm using a UV visible waste fractions in different industries. spectrophotometer. A calibration curve was MATERIALS AND METHODS prepared using gallic acid as standard and the Preparation of extract: The peels were total phenolic content was expressed in terms separated manually and shade dried for 4-5 of gallic acid equivalent (μg/mg). days at room temperature. The dried peels Estimation of total flavonoid content: Total were cut into small pieces approximately 1x1 flavonoid content was determined using the cm. The dried peels (50g) and the fresh pulp Aluminium Chloride method [11]. The (50g) was soaked in 100 mL of ethanol extracts (500μg/mL) were made up to 1 mL separately for 72 hours by maceration using methanol and was mixed with 0.5 mL technique. The supernatant obtained from of 5% sodium nitrate (NaNO2) solution and both the extracts were filtered and allowed to stand for 5 minutes. Later, 0.3 mL concentrated using rotary evaporator. The of 10% aluminium chloride (AlCl3) solution dry residue was preserved at 5°C until further was added and the mixture was allowed to use. stand for 5 minutes. Finally, 1 mL of 1M Screening of phytochemicals: The extracts sodium hydroxide (NaOH) solution was were screened for the presence of various added, and the volume was brought to 5 mL phytochemicals such as alkaloids, glycosides, using distilled water. The mixture was saponins, phenols, flavonoids, terpenoids, incubated for 15 minutes at room steroids, quinones and tannins [7-9]. temperature and the absorbance was Estimation of total phenol content: The measured at 510 nm using UV visible total phenolic content was estimated using spectrophotometer. A calibration curve was Folin-Ciocalteau reagent method [10]. prepared using quercetin as standard and the 1271 IJBPAS, June, 2020, 9(6) Alfia Begam S et al Research Article total flavonoid content was expressed in power was expressed as the absorbance value terms of quercertin equivalent (μg/mg). at 700 nm [13]. Antioxidant activity Phosphomolybdenum reduction assay: DPPH assay: DPPH radical scavenging Various concentrations of the extracts were activity was determined by following the mixed with 1 mL of reagent solution (0.6 M method described by Blois [12]. Briefly, 1 sulphuric acid, 28 mM sodium phosphate and mL of 0.1 mM of DPPH solution (dissolved 4 mM ammonium molybdate). The tubes in methanol) was added to various were capped and incubated in a water bath at concentrations of the extracts. The setup was 95°C for 90 minutes. The tubes were cooled left in dark at room temperature and the to room temperature and the absorbance was absorption was measured after 30 minutes. measured at 695 nm against blank. The blank Absorbance was read at 517nm using UV – solution contained 1 mL of the reagent visible spectrophotometer. The results were solution along with 1mL of the solvent used expressed in terms of percentage inhibition for dissolving the sample was also incubated of DPPH using the following formula:- under the same condition. The reducing power was expressed as the absorbance value at 695 nm [14]. Gas Chromatography – Mass Absorbance control was the absorbance of DPPH and methanol Spectrometry (GC-MS): The extracts were Absorbance sample was the absorbance of DPPH and the test sample. injected into a HP-5 column (30 m X 0.25 mm id with 0.25 μm film thickness), Agilent FRAP assay: Various concentrations of the technologies 6890 N JEOL GC Mate II GC- extracts were mixed with 1 mL of phosphate MS model. The following chromatographic buffer (0.2 M, pH 6.6) and 1mL of 1% conditions were used. Helium was used as potassium ferricyanide. The mixture was the carrier gas at a constant flow rate of 1 o incubated at 50 C for 20 minutes. Later, 1 mL/min and the injector was operated at mL of 10% trichloroacetic acid was added to 200°C. The column oven temperature was the mixture. Then 1 mL of 0.1% of freshly maintained at 50-250°C at a rate of 10°C/min prepared ferric chloride was added and the injection mode. Following MS conditions absorbance was measured at 700nm using were used: ionization voltage of 70 eV, ion UV visible spectrophotometer. The reducing source temperature of 250°C, interface 1272 IJBPAS, June, 2020, 9(6) Alfia Begam S et al Research Article temperature of 250°C and mass range of 50- when compared to pulp extract thereby 600 mass units. The database of National indicating the use of biological waste Institute Standard and Technology (NIST) substrates as natural free radical inhibitors. having more than 62,000 patterns was used IC50 value of peel and pulp extracts were for the interpretation on mass spectrum of found to be 57.07µg/mL and 256.21µg/mL GC-MS. The mass spectrum of the unknown respectively. The IC50 value for ascorbic acid component was compared with the spectrum was 2.89µg/mL.