World Journal of Pharmaceutical Sciences ISSN (Print): 2321-3310; ISSN (Online): 2321-3086 Published by Atom and Cell Publishers © All Rights Reserved Available online at: http://www.wjpsonline.org/ Original Article Antioxidant capacity of piper longum and piper nigrum fruits grown in Bangladesh Proity Nayeeb Akbar1,2, Ismet Ara Jahan1*, M. Hemayet Hossain1, Rajib Banik1, Husna Purvin Nur1 and Md. Tofazzal Hossain1 1Bangladesh Council of Scientific and Industrial Research (BCSIR), Dr. Quadrat-E-Khuda Road, Dhanmondi, Dhaka-1205 2Randolph College, 2500 Rivermont Avenue, Virginia, USA Received: 13-07-2014 / Revised: 06-08-2014 / Accepted: 27-08-2014 ABSTRACT The study is performed to determine and compare the antioxidant capacity of water and ethanol extracts of Piper longum and Piper nigrum fruits grown in Bangladesh. DPPH, ABTS and nitric oxide free radical scavenging activity, FRAP, superoxide dismutase, Fe2+ ion chelating ability, total antioxidant capacity, reducing power assay, total flavonoid, and total phenolic content determination assays were used for evaluation of antioxidant capacity. The ethanol extracts of P. longum (long pepper from Dhaka, long pepper from Rajshahi) and P. nigrum (white pepper, black pepper) fruits showed significant activities compared to the water extracts in most of the antioxidant assays in a dose dependent manner. The total phenolic and flavonoid contents ranged between 32.83 to 174.92 mg of GAE/g of dry extract and 33.44 to 172.98 mg of QE/g of dry extract, respectively. In total antioxidant capacity and FRAP assay, the activity of the extracts ranged from 9.05 to 199.17 mg AAE/g extract and 5.32 to 18.33 mg FeSO4E/g extract. In DPPH scavenging and SOD assay, the percent inhibitions were found to be similar. However, ABTS, Fe2+ scavenging, and reducing power assay showed better results for ethanol extracts, while nitric oxide scavenging activity showed better activity for water extracts. Keywords: Piper Longum, Piper Nigrum, Antioxidant Activity, Total Phenolic, Total Flavonoid. INTRODUCTION and Srilanka and is cultivated almost everywhere in the tropical regions especially for its fruits, which Many plant-derived molecules have shown a are used as spices. P. nigrum, also known as black promising effect in therapeutics [1]. Spices and Pepper or in many cases white pepper, is produced herbs are recognized as sources of natural from the still-green drupes of the pepper plant. antioxidants and thus play an important role in the Once dried, the spice is called black pepper. White chemoprevention of diseases and aging. Among the pepper, however, consists of the seed of the pepper plants investigated to date, one showing enormous plant alone, with the darker-colored skin of the potential is the pepper family otherwise known as pepper fruit removed. It has a slightly different Piperaceae [2]. Piper longum and Piper nigrum are flavor than black pepper, due to the lack of certain the two flowering vine in the family Piperaceae. P. compounds present in the outer fruit layer of the longum, also known, as Long Pepper is one of the drupe, but not found in the seed. Externally, both most widely used Ayurvedic herbs original to black and white pepper are used for its rubefacient northeastern and southern parts of Bangladesh, and as a local application for relaxed sore, throat India and Srilanka. It is used for the treatment of and some skin disorder. It has anti-microbial [5] respiratory tract diseases like cough, bronchitis, and anti-mutagenic properties [6]. Inhalation of asthma, cold, as counter-irritant and analgesic [3]. black pepper oil also increases the reflexive It is applied locally for muscular pain and swallowing movement [7]. In addition, an animal inflammation and used internally as a carminative study showed that piperine, a constituent of black in conditions such as loss of appetite and and white pepper beneficially improved the plasma sleeplessness [4]. P. nigrum is another well-known levels of insulin [8]. climbing vine native to Bangladesh, southern India *Corresponding Author Address: Ismet Ara Jahan, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dr. Quadrat-E- Khuda Road, Dhanmondi, Dhaka-1205, Bangladesh; E-mail: [email protected] Akbar et al., World J Pharm Sci 2014; 2(9): 931-941 The major chemical constituents of the plants are stored in an airtight container and kept in a cool volatile oil, resin and alkaloids viz. piperine, place until used for the current research purpose. piperlongumine, piperlonguminine, pipperin, pippalartin, piplartine etc [9]. It also contains Extraction of Plant Materials: 25 g of each of the polyphenols, which includes flavanols, flavonoids, powdered samples (two sets) were taken and flavandiols and phenolic acids and amides soaked in 250 ml of 95% ethanol and distilled including N-isobutyleicosa-2,4-dienamide, N- water (250 ml) separately. The samples were Isobutyleicosa-2,4,14-trienamide, N- shaken for 24 hours using an orbital shaker (VRN- Isobutylocatadeca-2,4,14-trienamide, piperine, 480, Gemmy, Taiwan). The solutions were then guineensine etc [10]. Excessive generation of filtered through Whatman filter paper (Bibby reactive oxygen species (ROS) and other radicals RE200, Sterilin Ltd., UK) under reduced pressure. can damage proteins, carbohydrates, Finally, the filtrates were concentrated separately polyunsaturated fatty acids, and DNA, and may by a rotary vacuum evaporator (R-215, Buchi, thus lead to oxidative stress and to a variety of Switzerland) at low temperature and then freeze degenerative processes and diseases such as aging, dried. The dried ethanol and water extracts of P. immunodeficiencies, neurologic disorders, longum, Dhaka (2.42 g & 1.94 g); P. longum, inflammation, arthritis, ischemia, arteriosclerosis, Rajshahi (2.58 g & 2.01 g); P. nigrum (black, 2.95 coronary heart disease, stroke, diabetes mellitus, g & 2.15 g) and P. nigrum (white, 2.73 g & 2.08 g) Parkinson’s disease, Alzheimer’s disease and were obtained respectively. The dried extracts were certain cancers [11, 12, 13] ROS are continuously stored in a cool place for further research. produced during normal physiologic events and removed by antioxidant defense mechanisms [14]. Preliminary Phytochemical Screening: Therefore, the great interest has been recently Phytochemical tests were performed on the ethanol focused on the natural foods, medicinal plants and and water extracts of P. longum and P. nigrum for phytoconstituents due to their well known abilities different chemical groups as alkaloids, flavonoids, to scavenge free radicals (i.e. antioxidant power). gums, reducing sugars, saponins, steroids and As a part of our ongoing research on investigation tannins following the standard methods (Evans, of the antioxidant capacity of medicinal plants and 1989). spices grown in Bangladesh, we have performed a study on the antioxidant capacity of ethanol and Determination of Total Phenolic Content: The water extracts of P. longum from two cultures total phenolics were determined by the modified region Dhaka and Rajshahi, and P. nigrum (white folin-ciocalteu method [15]. 1 ml of the aqueous pepper and black pepper) from Dhaka, Bangladesh, and ethanolic extracts was collected in two 10 ml volumetric flasks separately. To each flask, 5 ml of MATERIALS AND METHODS Folin-Ciocalteu reagent (1: 10 v/v distilled water) and 4ml (75 g/L) of sodium carbonate were added. Chemicals: DPPH, ABTS, ascorbic acid, gallic The solutions were Vortex-ed for 15 seconds and acid, quercetin, BHA, folin-ciocalteu’s phenol allowed to stand for 30 min at 40°C for color reagent, griess reagent, NBT, NADH, PMS, TPTZ, development. The absorbance was measured Trolox, and ferrozine were purchased from Sigma against the blank in a double beam UV/Visible Chemical Co. (St. Louis, MO, USA). Ammonium spectrophotometer (Analykjena, Model 205, Jena, molybdate, sodium acetate, glacial acetic acid, Germany) at absorption maxima 765 nm. Three FeCl3.6H2O, FeCl2.4H2O, potassium ferricyanide, readings were taken per solution to get trichloro acetic acid, phosphate buffer pH 6.6, reproducible results. The total phenolic content was sodium nitroprusside, phosphate buffer saline pH determined and expressed as mg Gallic acid 7.4, aluminium chloride, potassium persulfate, equivalents per gram of dry extract using the Tris-HCL buffer, and the solvents were of equation obtained from a standard Gallic acid analytical grade and obtained from SD Fine Chem. calibration curve, y = 6.2548x - 0.0925, R2 = Ltd. (Biosar, India). 0.9962. Plant Material: Seeds of P. longum and P. nigrum Determination of Total Flavonoid Content: were collected from Dhaka and Rajshahi, Aluminium chloride colorimetric method was used Bangladesh during April 2013, and identified by for the determination of total flavonoid the experts of BCSIR. concentration of the extracts [16]. 1 ml of the extracts was individually mixed with 1.5 ml of Sample Preparation: The fruits of P. longum and methanol, 0.1 ml of 10% aluminum chloride, 0.1 P. nigrum were cleaned, dried and coarsely ml of 1 M potassium acetate and 2.8 ml of distilled powdered. The dried powdered samples were water. The solution was allowed to stand for 30 min at room temperature and the absorbance of the 932 Akbar et al., World J Pharm Sci 2014; 2(9): 931-941 reaction mixture was measured at 415 nm with a Nitric Oxide radical Scavenging activity: Nitric double beam Analykjena UV/Visible oxide (NO) scavenging activity was measured spectrophotometer (Model 205, Jena, Germany). following the method of Dinis and co-workers [19]. The total flavonoid content was determined as mg Sodium nitroprusside (5 mmol) in phosphate of Quercetin equivalent per gram using the buffered saline was mixed with different equation obtained from a standard Quercetin concentrations of the different extracts (10– calibration curve, y = 4.7385x + 0.0355; R2 = 500µg/ml) dissolved in ethanol and incubated at 0.9993. 25°C for 30 min. A control without the test compound but with an equivalent amount of Methods for Evaluation of Antioxidant capacity ethanol was taken.