Myanmar Health Sciences Research Journal, Vol. 31, No. 2, 2019 Determination of Total Phenolic Contents and Antioxidant Activity of the Roots of Pimpinella candolleana Wight & Arnott Kyi Kyi Oo1*, Ei Ei Thant1, Khin Myo Oo3, Swe Swe2, May Thandar Tun4, Soe Myint Aye5, Win Aung4, Theim Kyaw1 & Yi Yi Myint2 1University of Traditional Medicine, Mandalay 2Department of Traditional Medicine, Nay Pyi Taw 3University of Pharmacy, Mandalay 4Department of Medical Research (POLB) 5Department of Botany, University of Mandalay Pimpinella candolleana Wight & Arnott belonging to family Apiaceae is a valuable medicinal plant. The plant specimens were collected from Pindaya Township, Southern Shan State in July 2014. The total phenolic contents in the aqueous, ethanolic and methanolic extracts of the roots of Pimpinella candolleana Wight & Arnott were determined at Food and Drug Administration Department, Mandalay by Folin-Ciocalteu colorimetric method using gallic acid as the standard. The antioxidant activity of the different concentrations (100 µg/ml, 200 µg/ml, 300 µg/ml, 400 µg/ml and 500 µg/ml) of three extracts was evaluated at Department of Medical Research (Pyin Oo Lwin Branch) by DPPH free radical scavenging method. The total phenolic contents of aqueous, ethanolic and methanolic extracts of roots were 83.63 mg GAE/g extract, 93.87 mg GAE/g extract and 118.85 mg GAE/g extract, respectively. The IC50 values of aqueous, ethanolic and methanolic extracts were 69.183 µg/ml, 63.096 µg/ml and 31.622 µg/ml, respectively. The results showed that there is a positive correlation between free radical scavenging effect and total phenolic contents. Thus, this study scientifically proved that this resource sample is rich in total phenolic contents and significantly has antioxidant activity. Keywords: Pimpinella candolleana roots, Folin-Ciocalteu, Phenolic, Antioxidant, DPPH INTRODUCTION In China, the subterranean part's decoction or infusion of P. candolleana Wight & Arnott Pimpinella candolleana Wight & Arnott is used for treatment of cold and flu, muscu- belonging to family Apiaceae is a valuable loskeletal system disorders, digestive system medicinal plant. This family consists of 400-450 disorders, urticarial, rheumatism and in skin genera and 3500-3700 species and is found in care products for its anti-inflammatory and most parts of the world mainly in the Northern whitening properties.5, 6 It was also proposed Hemisphere.1 There are 150 species in the genus that the ethyl acetate and methanolic extracts of Pimpinella distributed in Africa, Asia, and aerial parts of P. candolleana Wight & Arnott Europe. This species is grown in the pinus had shown the α-glucosidase inhibitory and forest margins, among shrubs, grassy slopes, antioxidant activities in vitro. The luteolin streamsides, 1300-3500 m above sea level in and isovitexin isolated from the ethyl acetate China, South India and Southeast Asia and extracts showed α-glucosidase inhibitory native is peninsular India.2 Certain members of activity and luteolin had antioxidant activity.7 Apiaceae, 28 genera and 48 species and, It had been indicated that different extracts 26 genera and 40 species were described in of the Pimpinella species such as P. anisum L.,8 Myanmar plant checklists3, 4 but P. candolleana _______________________________________ Wight & Arnott has not been reported. *To whom correspondence should be addressed. In Myanmar, this species is grown in temperate Tel: +95-0943014497 and hilly region 1100 m above sea level E-mail: [email protected] especially Southern Shan State. DOI: https://doi.org/10.34299/mhsrj.00934 120 P. tragioides (Boiss) Benth.,9 P. brachycarpa University of Mandalay according to the method (Kom.) Nakai.,10 P. tirupatiensis Bal. & Subr., of Menglan, et al.2 The root pieces of had antioxidant activity.11 P. candolleana Wight & Arnott (100 g) were mixed with 1000 ml of distilled water and It had been reported that medicinal plants rich heated by reflux extractor at 80°C for 3 hours. in flavonoids and phenolic acids could be They were then allowed to cool at room a good source of natural antioxidants.12 temperature, filtered and evaporated to dry It was described that the various bioactivities under reduced pressure at 50°C by rotary of phenolic compounds are responsible for evaporator and made into powder by freeze drier their chemopreventive properties such as anti- (FD 1). For ethanolic and methanolic extracts, oxidant, anticarcinogenic and anti-inflammatory the crude pieces (100 g) were mixed with activities.13 It was considered that the cut-off 1000 ml of 95% ethanol and methanol, point for antioxidant activity as IC 50 µg/ml. 50 = respectively, and agitated and sonicated for IC >50 µg/ml was as moderately active and 50 3 hours. The extracted liquid was filtrated and IC <50 µg/ml was judged as high antioxidant 50 evaporated to dry under reduced pressure at capacity.14 50°C by rotary evaporator and dried in water In Myanmar, local people in Southern Shan bath until constant weight was achieved. State use the leaves of P. candolleana Wight & Determination of total phenolic contents in Arnott as vegetables and the root as tonic. various solvent extracts of roots Annually, about 11000 kg of the roots of this plant have been traded as a commercial raw The total phenolic contents of the aqueous, material for traditional crude drug by ethanolic and methanolic extracts of roots were local people. It is needed to identify for determined by Folin-Ciocalteu colorimetric standardization on the quality and authenticity method using gallic acid as the standard.15 The of raw materials used as commercial raw absorbance was measured at 765 nm by UV material in Myanmar traditional crude drug spectrophotometer 1601. markets. However, there is a lack of scientific studies about these roots distributed in Table 1. Absorbance of standard compound (gallic Southern Shan State of Myanmar. Therefore, acid) this study was conducted to determine total Concentration (μg/ml) Absorbance (Mean)λmax=765 nm phenolic contents and to evaluate the free 5 0.359 10 0.745 radical scavenging activity of the aqueous, 20 1.4135 ethanolic and methanolic extracts of roots of 30 2.2853 P. candolleana Wight & Arnott. 40 3.2148 3.5 MATERIALS AND METHODS Absorbance 3 This study was carried out as a laboratory-based experimental design. The sample was extracted 2.5 at Research Division, University of Traditional 2 Medicine, Mandalay. The total phenolic con- tents and the antioxidant activity of various 1.5 extracts of roots were determined at Food and (nm) Absorbance 1 Drug Administration Department, Mandalay and y=0.080x-0.094 0.5 R²=0.996 at the Department of Medical Research (Pyin Oo Lwin Branch), respectively. 0 0 100 2020 3030 40 40 50 50 Preparation of sample ConcentrationConcentration (µg/ml) (µg/ml) The plant specimens were collected from Fig. 1. Standard calibration curve of different con- Pindaya Township, Southern Shan State in July centrations of gallic acid 2014. Pindaya is located between North latitude 20º 42’ and 21º 13’ and East longitude 96º 25’ A standard calibration curve was prepared and 96º 57’and the altitude is above 1100 meter. using different concentrations of gallic acid in The plant specimens were identified by ethanol (5, 10, 20, 30 and 40 μg/ml). In a 20-ml a taxonomist from Department of Botany, test tube, 1 ml gallic acid of each concentration 121 Absorbance Absorbance was added to 5 ml of 10% Folin-Ciocalteu - reagent and 4 ml of 7% Na CO to get a total Scavenging of control of sample 2 3 activity = X 100% volume of 10 ml. The blue colored mixture was Absorbance of control shaken well and incubated for 30 minutes at 40°C in a water bath. The average absorbance Absorbance Absorbance of 60 µM DPPH solution values obtained at different concentrations of of control = gallic acid were used to plot the calibration Absorbance = Absorbance of test sample solution curve (Table 1 & Fig. 1). of sample Total phenolic contents of the three extracts Statistical analysis were calculated from the regression equation of calibration curve (y=0.080x-0.094, R2=0.996) All the experiments were carried out in and expressed as mg gallic acid equivalent triplicates and data were reported as mean. For (GAE) per gram of sampleas gallic acid the total phenolic content, the linear correlation equivalent by the following equation: coefficient was calculated by using Microsoft Office Excel (2007). The concentrations of cV extracts were calculated from this regression C = equation. The total phenolic content of each m extract at that concentration was calculated. For antioxidant activity, the mean, standard C = Total phenolic content mg GAE/g dry deviation (SD) and the linear regression of extract percent inhibition of different extracts were c = Concentration of gallic acid obtained from analyzed by using the computer package SPSS calibration curve in mg/ml version 20 software. The 50% inhibitory V = Volume of extract in ml concentration (IC ) of free radical scavenging m = Mass of extract in gram 50 activity was calculated from linear regression line according to log concentration. Determination of antioxidant activity of the root extracts RESULTS The antioxidant activity of the aqueous, ethanolic and methanolic extracts of the In the extractability, the yield percentages of roots was determined by using the DPPH aqueous, ethanolic, and methanolic extracts of (1,1-diphenyl-2-picrylhydrazyl). The stock solu- the root of P. candolleana Wight & Arnott were tion was prepared by dissolving root extract 33.4%, 18.8% and 8.98%, respectively. with 95% ethanol at concentration of 1 mg/ml. Total phenolic contents in the various extracts From this stock solution, different concen- of roots trations (100, 200, 300, 400 and 500 µg/ml) were prepared. The sample solutions were The total phenolic content in aqueous, ethanolic prepared by mixing thoroughly with 1 ml of and methanolic extracts of roots were 83.63 mg 60 µM DPPH solution (2.36 mg of DPPH in GAE/g extract, 93.87 mg GAE/g extract and 100 ml of 95% ethanol) and 1 ml of different 118.85 mg GAE/g extract, respectively.