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Issn: 2277–4998 IJBPAS, September, 2018, 7(9): 1643-1651 ISSN: 2277–4998 DETERMINATION OF PIGMENTS, ANTIOXIDANT POTENTIAL AND VITAMIN CONTENTS OF BENGUET PINE (Pinus kesiya) POLLEN SHEILA MARIE DE LEON BALURAN1*, LEXTER R. NATIVIDAD2, SOMAR ISRAEL D. FERNANDO3 AND RAVELINA R. VELASCO4 1Senior High School Department, Caanawan High School, San Jose City Division, San Jose City, Nueva Ecija, Philippines 2College of Education, University Science High School, Central Luzon State University, Science City of Muñoz, Nueva Ecija, Philippines 3Department of Biological Sciences, Central Luzon State University, Science City of Munoz, Nueva Ecija, Philippines 4College of Fisheries-Freshwater Aquaculture Center, Central Luzon State University, Science City of Muñoz, Nueva Ecija, Philippines *Corresponding author: Sheila Marie De Leon Baluran: E-mail:[email protected] Received 10th Feb. 2018; Revised 17th March. 2018; Accepted 16th April 2018; Available online 1st September 2018 https://doi.org/10.31032/IJBPAS/2018/7.9.4517 ABSTRACT The study determined the total carotenoid, β-carotene, chlorophyll a and chlorophyll b and total phenolic contents of P. kesiya pine pollen. The study also evaluated the antioxidant property of P. kesiya pollen using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and determined its Vitamin A and Vitamin C contents. Results showed that Benguet (P. kesiya) pine pollen contained 2.90 µg/g total Carotenoid, 0.06 µg/g β-carotene, 409.41 µg/g Chlorophyll A and 704.68 µg/g Chlorophyll B,0.103 µg/g Vitamin A and 0.500 µg/g Vitamin C and displayed an antioxidant component with 271.10 mg/L that can scavenge 50% of radicals. This study is of great potential remedy to reduce antibiotic resistance in fish and provide a plant- based anti-oxidant. Keywords: Pigments, Antioxidant, Vitamin, Benguet pine, Pinus kesiya 1643 IJBPAS, September, 2018, 7(9) Sheila Marie De Leon Baluran et al Research Article INTRODUCTION human body. Pine pollen reportedly The Benguet Pine (Pinus. kesiya) promotes rapid growth, rejuvenation, and also known as Benguet Pine is a conifer healing due to its high vitamins, minerals, species which is considered as one of the and enzymatic contents that nourish the most economically and culturally body at a fundamental level [6]. important species in Benguet province [1]. Preliminary studies on Pinuskesiya of the family Pinaceae are Phytoandrogen components of Pinus common in northern Philippines, kesiya was conducted to screen particularly in highland regions with an phytochemicals and identify the altitude of approximately 1,500 meters that phytoconstituents present in the different provides optimum conditions for its parts of P. kesiya. It was reported that growth [2]. It is commonly found in tannins flavonoids, steroids and alkaloids elevated areas of Baguio City, the are present in branch, needles, pollen, mountain province, Zambales and twigs and roots and that glycosides are Mindoro, Philippines [3].P. kesiya is fast positively detected in the pollen and twigs growing and has the capacity to adapt to but none in the other parts while alkaloids various growing conditions (high and sterols are observed in bark [3]. plasticity), long living and produces high quality and long-fibered pulp [4]. MATERIALS AND METHODS Medicinal plants are of potential as Collection of Pine Pollen immunostimulants fortifying the immune The Department of Environment response of cultured species to effectively and Natural Resources - Cordillera prevent and control occurrence of diseases Administrative Region, Ecosystem caused by different bacterial and viral Research and Development Services pathogens. The role of medicinal plants in DENR-ERDS) facilitated the scouting and disease prevention or control has been collection of pine pollen in Baguio City. attributed to the antioxidant properties of Two boxes of around 518 pieces of catkins their constituents, which are usually of Benguet pine were collected, associated with a wide range of segregated, air-dried and transported to the amphipathic molecules that are broadly laboratory for another week of air drying. referred to as polyphenolic compounds [5]. The granules from each pollen cone from Pine pollen contains an incredibly wide the catkins were removed manually, sieved spectrum and high concentration of unique and the starchy powder pine pollens were and rare nutrients that are essential for the placed in a tightly sealed container. 1644 IJBPAS, September, 2018, 7(9) Sheila Marie De Leon Baluran et al Research Article Analyses of Pigments UV/VIS spectrophotometer at 653 and 666 The method of Tao et al. (2010) nm. was adapted in the analysis of pigment Chlorophyll a (mg/L) = 15.65 Abs666 – 7.340 Abs653 with slight modifications. Five hundred Chlorophyll b (mg/L) = 27.05 Abs – 11.21 Abs (500) mg of the air dried pine pollen was 653 666 placed in a 50 mL centrifuge tube and Determination of Total Phenolic extracted three times with 10 mL ethanol Content using vortex mixer for 1 minute. The The Total Phenolic Content (TPC) mixture was allowed to stand until the was determined using Folin-Ciocalteau particles of pine pollen settled at the Reagent. New samples of pine pollen bottom of the tube. were used and the powdered sample was Supernatants were collected and extracted with 95% ethanol at room measured. The final volume of the extract temperature for 48 hours. The extract was was adjusted to 75 mL by adding 95% separated from the residue by filtering ethanol. The absorbance value of the through an ordinary filter paper. The extract at 450 nm was determined by the crude extract was collected and kept in use of spectrophotometer (A200). dark glass bottles and stored in refrigerator The total carotenoid yield for future use. (microgram/g dried weight) was calculated The determination of TPC was using the formula: carefully done following the existing protocol [23]. About 400 μL (0.4 mL) of each sample extract was added with 0.8 mL distilled water and 1.0 mL diluted Folin-Ciocalteau reagent (1:10). It was allowed to stand for 5 minutes before the addition of 1 mL of 7.5 % sodium carbonate (w/v). After 2 hours, the absorbance of sample was taken at 765 nm wavelength using Unico The concentration of chlorophylls A spectrophotometer 1200. Calibration and B were determined [8]. Methanolic curve of gallic acid was set-up to estimate solutions of plant extracts for chlorophylls the TPC of the samples. The result was a and b at 2 mg/mL were analyzed in a 1645 IJBPAS, September, 2018, 7(9) Sheila Marie De Leon Baluran et al Research Article expressed in milligram of Gallic acid The scavenging activities of the equivalent per gram of dry weight sample. extracts were expressed as EC50 (Effective concentration) values. The EC of the extract required to scavenge 50% of DPPH Evaluation of Antioxidant Property of radical was also calculated from the curves Pine Pollen using 2,2-Diphenyl-1- obtained after applying non-linear Picrylhydrazyl (DPPH) Radical regression (sigmoidal dose-response) to Scavenging Method the data using Graph Pad Prism 7.0. The 2,2-Diphenyl-1- Determination of Vitamin A and C Picrylhydrazyl (DPPH) radical scavenging Dried P. kesiya pine pollen sample activity assay was adopted with of 500 g was packed aseptically and sent modifications [22]. Different dilutions of through courier to SentroTek Corporation the extracts (1000 ppm, 100 ppm, 10 ppm, in Mandaluyong City, Philippines for the 1 ppm, 0.1ppm and 0.01ppm) were evaluation of vitamin A and C contents. prepared. DPPH solution was also prepared by dissolving 6.0 mg DPPH in RESULTS AND DISCUSSION 100 mL methanol. A volume of 1.5 mL of Pigment Analysis of P. kesiya extract from each dilution was added into This is a preliminary information the test tube containing 2.5 mL of DPPH on total carotenoid content of P. kesiya solution. Butylated hydroxylanisole pine pollen. The result of analysis for (BHA) and ascorbic acid were used as pigments such as total carotenoid (TC), β- comparators. The mixture was shaken carotene, chlorophyll a, and chlorophyll b vigorously and left to stand in a dark area contents of pine pollen were presented in for 30 minutes. The absorbance of the Table 1. resulting solution was measured using Small amount of carotenoids at 2.9 spectrophotometry at 517 nm. Triplicate µg/g was observed, β-carotene has mean samples were prepared and the scavenging levels at 0.06 µg/g. Chlorophyll a content activity of each extract of DPPH radical was recorded as 409.41 mg/L and was calculated using the following chlorophyll b at 704.68 mg/L. equation: DPPH scavenging activity = Absblank-Abssample x 100 Absblank 1646 IJBPAS, September, 2018, 7(9) Sheila Marie De Leon Baluran et al Research Article Table 1: Analysis of pigments in P. kesiya convert light energy into chemical energy PIGMENTS LEVELS OF PIGMENTS [15] while Chlorophyll-b acts as accessory Total Carotenoid 2.90 µg/g Β-carotene 0.06 µg/g pigments indirectly in photosynthesis by Chlorophyll a 409.41 mg/L Chlorophyll b 704.68 mg/L transferring the light it absorbs to chlorophyll-a [17]. Chlorophyll, along Carotenoids are secondary with carotenoids, plays an important role metabolites commonly found in fruits and in providing nutrients. vegetables with many medical applications Total Phenolic Content of Pine Pollen [9]. Carotenoids are reportedly effective Expressed as Gallic Acid inhibitor of heart disease and risk-reducer The total phenolic content (TPC) of to some types of cancer [10]; improve eye the Benguet pine (Pinus kesiya) pollen health [11] and have potential expressed in terms of Gallic acid antimicrobial activity [7]. Carotenoids are equivalent (GAE) was presented in Table plant pigments responsible for bright red, 2. yellow and orange hues in fruits and Table 2: Total phenolic content of P. kesiya pine vegetable and one of the carotenoid pollen in comparison with the other pine pollen families include β-carotene [12] and as Concentration Sample source of nutrients [13].
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