189 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) ISSN (Print) 2313-4410, ISSN (Online) 2313-4402 © Global Society of Scientific Research and Researchers http://asrjetsjournal.org/ A Comparative Study of Peel and Seed Extract of Passion Fruit (Passiflora edulis) as Antihyaluronidase Darwisa, I Nyoman Ehrich Listerb, Edy Fachrialc* aMaster Program of Biomedical Sciences, Faculty of Medicine, Universitas Prima Indonesia, Medan, Indonesia bFaculty of Medicine, Universitas Prima Indonesia, Medan, Indonesia cLaboratory of Molecular Biology, Faculty of Medicine, Universitas Prima Indonesia, Medan, Indonesia cEmail: fachrial_edy@yahoo.co.id Abstract The plant produces various metabolites and one among the benefit is to inhibit the aging process. The aim of the study was to compare the antioxidant activity between the peel and the seeds of P.edulis and also to compare anti-hyaluronidase activity. The antioxidant activity was determined using the ABTS reduction method. The result showed that the reduction percentage of ABTS of seed and peel of P.edulis was 15,58 ± 1,04 % and 27,68± 0,09 % respectively at concentration 50 µg/ml. The inhibition activity of hyaluronidase of seed and peel of P.edulis was 70,96 ± 3,55 % and 61,68 ±4,05 %. The IC50 of anti hyaluronidase activity of seed dan peel extract was 122,70 ± 6,35 and 67,35 ±6,58 respectively. From the result, it can be concluded that the seeds and peel of P.edulis have the potential as a source of anti-aging particularly as anti hyaluronidase. Keywords: antioxidant; anti hyaluronidase; P.edulis; anti-aging; passion fruit. 1. Introduction Aging is a process that cannot be avoided by all living things. In human, the tissue that the most affected by the aging process is skin [1]. Aging is divided into two parts namely intrinsic aging and extrinsic aging. The extrinsic factor of the skin including UV rays and smoking habit, whereas intrinsic factor due to genetic and epigenetic mechanisms [2]. The aging of the skin will lead to changes in the appearance of the skin. The most common changes are dry and scaly skin. Recovery of damaged barrier function occurs more slowly on aging skin, resulting in dryness of the skin [3]. ------------------------------------------------------------------------ * Corresponding author. http://asrjetsjournal.org/ American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 52, No 1, pp 189-196 190 Although extrinsic and intrinsic skin aging is different, there are similarities in molecular mechanisms, for example, reactive oxygen species (ROS), which arise from oxidative cell metabolism. ROS in extrinsic or intrinsic skin aging induce the transcription factor c-Jun via mitogen-activated protein kinase (MAPK), leading to overexpression of matrix metalloproteinase (MMP)-1, MMP-3 and MMP-9 and prevention of the expression of procollagen-1. The degraded collagen and reduced collagen synthesis are pathologies occurring in intrinsically aged as well as photoaged skin [4]. On aging skin, the collagen has been shown irregular, the ratio of Col-3 to Col-1 appears to be increased, due to significant loss of Col-1. Glycosaminoglycan (GAG’s) particularly hyaluronic acid (HA) is a matrix dermis constituent that helps in binding water to keep the skin soft and moist. Total HA levels in aging skin of the dermis intrinsically remain stable, but epidermal HA significantly loss along with the aging process [5]. Plants are one source of antioxidant that has potential as an anti-aging agent, including inhibiting hyaluronidase activity. Plants have been extensively used as ingredients in cosmetics and therapeutics, as beautifying agents and also remedy for the dermatological disorder. These provide largely unexplored sources for the potential development of active ingredients for cosmetic formulations, and also driven by the rising desire of people to maintain healthy skin without using chemicals [6]. P.edulis is a plant that is widespread in the world, particularly in the tropical region. This plant has several health benefits including anti-inflammation, anti-hypertension, antifungal, antitumor and also has high antioxidant levels [7]. Only the fruit is consumed, while the peel and seeds are removed. In this study, the peel and the seed of P.edulis will be investigated for the antioxidant activity using ABTS reduction method, and also its ability to inhibit hyaluronidase enzyme. 2. Experimental Section Materials Materials used in this study are peel and seed of P.edulis, distilled water, ethanol 70%, ABTS, potassium persulfate, phosphate buffer saline, dimethylsulfoxide, sodium phosphate monobasic, hyaluronic acid, hyaluronidase, sodium chloride, bovine serum albumin, sodium acetate, acetic acid, chloride acid Instrumentation Instruments used in this study are macerator, rotary evaporator, analytical balance, multiScan Go reader, micropippete, 96 well plate, falcon tube, vortex, pH meter, incubator, and other glassware. Procedures 2.1 Samples preparation P.edulis were obtained from Sampali village, Percut Sei Tuan Subdistrict, Deli Serdang regency, Medan. The peel of P.edulis was washed and the seeds are taken. These two materials are air dried for 5 days. The peel and the seeds then milled. Drying losses are calculated by the following formula : % drying loss = 𝑑𝑑𝑑𝑑𝑑𝑑 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑤𝑤𝑤𝑤𝑠𝑠𝑤𝑤ℎ𝑡𝑡 (𝑤𝑤) 𝑓𝑓𝑑𝑑𝑤𝑤𝑠𝑠ℎ 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑤𝑤𝑤𝑤𝑠𝑠𝑤𝑤ℎ𝑡𝑡 (𝑤𝑤) x 100% American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 52, No 1, pp 189-196 191 2.2 Sample Extraction 1500 g of peel and 650 g of dried P.edulis seeds are mashed into powder. Each P.edulis’s peel and seeds are then macerated to obtain an extract. Maceration was conducted by soaking the peel and seeds powder with ethanol 70% and then filtered. The filtrate was evaporated using rotary evaporator. 2.3 Antioxidant Activity test with ABTS Method The concentration of extract used in the antioxidant test was 6 concentration variations, namely 500 µg/mL ; 250 µg/mL ; 125 µg/mL ; 31,25 µg/mL ; 15,625 µg/mL. as 2 µL sample was added into the 96-well plate, then added with 198 µL ABTS reagents. At well blank, 200 µL of DMSO was added. In well control, 200 µL reagent was added and then incubated for 6 minutes at 37°C. the absorbance is measured using a microplate reader at λ = 745 nm. 2.4 Anti hyaluronidase activity test The variation of concentration used in this test were 166,67 µg/mL ; 83,33 µg/mL ; 41,67 µg/mL ; 20,83 µg/mL ; 10,42 µg/mL ; 5,21 µg/mL. The inhibition of hyaluronidase enzyme activity was measured based on the method used by Widowati and his colleagues (2016) with slight modification [8]. A mixture of a solution consisting of 25µL samples, 3 µL enzymes hyaluronidase from bovine testes and 12 µL phosphate buffer, incubated at 37°C for 10 minutes. In addition, the mixture of the solution was added as much as 10 µL of the hyaluronic acid substrate and re-incubated at 37 C for 45 minutes. The STOP solution in the form acid albumin was added as much as 100 µL into the solution and left at room temperature for 10 minutes. Absorbance is measured using microplate reader at 600 λ nm. 3. Result and Discussion 3.1 Reduction activity of ABTS Antioxidant test using method 2’-Azino-Bis-3-Ethylbenzothialine-6-Sulfonic Acid (ABTS) was determined based on loss of blue color due to the reduction of ABTS by antioxidants. This blue intensity is measured at a wavelength 745 nm. Reduction reaction of ABTS by antioxidant : H2O2 + ABTS peroxide 2H2O + oxidized ABTS The reduction of ABTS by peel and seed extract of P.edulis was shown at table 1. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 52, No 1, pp 189-196 192 Table 1: Reduction activity of ABTS by peel and seed extract of passion fruit (Passiflora edulis) Concentration (µg/mL) Mean of reduction activity of ABTS (%) by samples Peel extract Seed extract 50.00 27.68 ±0.09d 15.58 ±1.04d 25.00 18.35 ±1.35c 10.65 ±0.35c 12.50 11.74 ±0.67b 9.81 ±0.83a,b 6.25 10.61 ±0.66a,b 7.66 ±0.28a,b 3.13 9.43 ±0.34a 8.52 ±0.67a,b 1.56 7.32 ±0.69a 7.50 ±0.75a Data were presented as mean ± standard deviation. Different small letters in the same column are significant at P < 0.05 (Tukey HSD post hoc test). Table 1 showed that the reduction of ABTS by peel extract was higher than seed extract. The similar report was reported by Orak and his colleagues (2012) which studied the comparison of antioxidant activities of juice, peel, and the seed of Pomegranate (Punica granatum L) using DPPH scavenging activity. It is reported that. The EC50 values of DPPH scavenging activities in peel extracts (PE) had 23,4-fold higher than the juice extracts (JE). The reducing power in peel extracts was found to be 4.7-fold higher than SE and 10.5-fold higher than the JE [9]. Ang and his colleagues (2012) reported papaya peel and seed extracts has potency as an antioxidant source. The antioxidant activities of papaya peel were determined using the ABTS method and the reduction activity was 28.30%, while the antioxidant activity of papaya seed was 11.19% [10]. Antioxidants are compounds that inhibit the oxidation process and protect cells from the harmful effects of free radicals. Previous research reported that phytochemical and antioxidant activity screening has been done to 31 types of fruit peel extract, including P.edulis. The peel extract of P.edulis was shown contained triterpenoid, saponin, and phenolics [11]. The IC50 ABTS reduction of peel and seed extract of P.edulis was shown in table 2 Table 2: The IC50 value of ABTS reduction by peel and seed extract of P.edulis Samples (average) equation R2 IC50 (µg/mL) Peel extract Y = 0.4046x + 7.7976 0,99 104.30 ± 0.68 Seed extract Y = 0.1611x + 7.3042 0,96 268.26 ± 34.65 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 52, No 1, pp 189-196 193 Based on data in table 2, it showed that the antioxidant activity of peel extract was higher than seed extract. IC50 of ABTS scavenging activity is the concentration of sample or standard that can inhibit 50% of ABTS scavenging activity. The lowest IC50 means had the highest antioxidant activity. The IC50 were used to categorize antioxidant activity of a sample that compared to standard. The sample that has IC50 less than 50 µg/mL is a very strong antioxidant, 50-100 µg/mL is a strong antioxidant, 101-150 µg/mL is a medium antioxidant, while IC50 greater than 150 µg/mL is a weak antioxidant [12]. 3.2 Anti hyaluronidase activity of P.edulis The analysis data hyaluronidase inhibition activity of peel and seed extract of P.edulis was shown in table 3 and 4. From the tables above it can be seen that the inhibition of hyaluronidase activity of P.edulis seed extract was almost twice higher than P.edulis peel extract. It means that the hyaluronidase inhibition activity in peel extract was stronger than seed extract. The similar report was reported by Tu and Tawata (2015) which investigated the anti-oxidant and anti-aging effects of essential oils (Eos) from the leaves of Alpinia zerumber (tairin and shima). The result revealed that tairin and shima Eos showed strong anti-oxidant activities against DPPH and nitric oxide. Tairin EO also exhibited strong antiaging activity by inhibiting hyaluronidase (IC50= 83±1,6) [13]. In other study reported that ethyl acetate fraction of Garcinia indica at a concentration as low as 25µg/mL showed significant hyaluronidase inhibition. Garcino and cambogiol present in fruit rinds of Garcinia indica were reported to be good antioxidant ability [14]. The exact mechanism of hyaluronidase inhibition is still obscure, but in general, polyphenols such as tannins, are known to form complexes with a variety of proteins, including hyaluronidase, which may reduce the activity [15]. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 52, No 1, pp 189-196 194 Table 3: hyaluronidase inhibition activity of seed extract Sample Final concentration (ug/mL) Hyaluronidase inhibition activity(%) Average SD RSD IC 50 average R2 IC 50 Average of IC50 1 2 3 1 2 3 Peel extract 166.67 66.35 59.39 59.29 61.68 4.05 6.56 122.37 115.81 123.96 128.33 122.70 83.33 40.61 41.94 41.18 41.25 0.67 1.62 122.70 ± 6.35 41.67 28.31 32.51 22.97 27.93 4.78 17.11 20.83 12.77 16.97 17.92 15.89 2.74 17.24 10.42 12.87 12.20 13.35 12.81 0.57 4.49 5.21 6.48 7.05 7.44 6.99 0.48 6.86 0.97 0.977 0.927 0.971 6.35 Table 4: hyaluronidase inhibition activity of peel extract Sample Final Conc. (ug/mL) Inhibition Activity (%) Average SD RSD Average of IC50 - R² IC50 1st experiment (R2) IC50 2nd experiment (R2) IC50 3rd experiment (R2) IC50 (STDV) 1 2 3 Seed extract 166.67 68.45 69.40 75.02 70.96 3.55 5.01 67.10 71.93 70.30 59.81 67.35 83.33 49.48 55.00 51.86 52.11 2.77 5.32 67.35 ± 6.58 41.67 46.90 43.47 44.61 45.00 1.75 3.88 20.83 47.00 42.99 42.33 44.11 2.53 5.73 10.42 36.03 36.51 40.32 37.62 2.35 6.25 5.21 32.13 32.70 39.18 34.67 3.92 11.30 0.97 0.818 0.9656 0.976 6.58 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 52, No 1, pp 189-196 195 4. Conclusion From the study, it can be concluded that the antioxidant activity of P.edulis peel is higher than the seeds with percentage ABTS reduction is was 15,58 ± 1,04 % and 27,68± 0,09 % respectively at concentration 50 µg/ml. The IC50 of antihyaluronidase activity of seed dan peel extract was 122,70 ± 6,35 and 67,35 ±6,58 respectively. 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