42 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 Anti Collagenase Surya Hartantoa, 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 Natural plant compound is known to have various effects that are beneficial to human health including as antioxidant and anti-aging. The aim of this study was to investigate the antioxidant activity of passion fruit peel and seeds an the activity in inhibiting collagenase. The ethanolic extract of seed and peel of P.edulis was determined by antioxidant activity using DPPH scavenging method and continued with the investigation of anticollagenase activity. The result showed that the IC50 of peel extract and seed extract of P.edulis were 56.11 ± 2.26 µg/mL and 88.25 ± 14.31 µg/mL respectively. The IC50 of anti-collagenase activity of peel and seed extract of P.edulis were 82.53 ± 1.42 µg/mL and 225.40 ± 9.12 µg/mL respectively. From the result, it concluded that peel and seed extract of P.edulis possesses remarkable potency as anti-collagenase activity and have the potential to be developed as anti-aging skin nutraceutical. Keywords: Passiflora edulis; antioxidant; anti collagenase; DPPH. 1. Introduction The aging process has occurred since we were born. The skin is the outermost organ and directly exposed to environmental changes, making us aware that skin aging occurs in every minute. The desire of humans all over the world for a long time is to live longer and stay young, or at least look younger, so the study in related with anti-aging has become a special concern by a researcher [1]. Aging is defined as a physiological genetic process that associated with changes in function and morphology of cellular and extracellular components aggravated by injury throughout life and resulting in a progressive imbalance of the control regulatory systems of the organism, including hormonal, autocrine, neuroendocrine and immune homeostatic mechanisms [2]. ----------------------------------------------------------------------- * Corresponding author. http://asrjetsjournal.org/ American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 42-48 43 There are various ways to inhibit the skin aging process, one of them by inhibiting the free radical activity. materials that can be used to inhibit free radical activity are called antioxidant [3]. Many plants can be used as treatment and prevent diseases. Natural plant compound is showing a wide range of activities including anti- aging [4]. Tight and firmness of the human skin is due to elastin and collagenous present in its dermis. Collagen is synthesized and secreted by the fibroblast cells of the dermis and its rate of synthesis decreases with aging. With the increase in age and particularly due to overexpression to sunlight, collagen deteriorates resulting in skin wrinkling [5]. Passiflora spp, popularly known as passion fruit, is a medicinal plant and is recognized by several associations including Brazilian Pharmacopoeia and British Herbal. This plant is rich in flavonoid, phenols, and alkaloids which acts as an antioxidant [6]. This fruit is usually consumed freshly, the skin and seeds are removed. The aim of this study was to investigate the antioxidant activity of passion fruit peel and seeds and the activity in inhibiting collagenase. 2. Experimental Section 2.1 Samples Preparation P.edulis obtained from Sampali village, Percut Sei Tuan sub-district, the regency of Deli Serdang. P.edulis is peeled and separated between the peel and the seeds and then weighed. The wet weight of the peel was 1500 gram and the wet weight of the seeds was 650 gram. The peel and the seeds the milled and wind dried for 14 days in room temperature. The drying losses are calculated by the formula below : % drying loss = 𝑑𝑑𝑑𝑑𝑑𝑑 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑤𝑤𝑤𝑤𝑠𝑠𝑤𝑤ℎ𝑡𝑡 (𝑤𝑤) 𝑓𝑓𝑑𝑑𝑤𝑤𝑠𝑠ℎ 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑤𝑤𝑤𝑤𝑠𝑠𝑤𝑤ℎ𝑡𝑡 (𝑤𝑤) x 100% 2.2 Sample Extraction as much as 240 gram of peel and 200 gram of seeds that have dried, mashed into powder. Each P.edulis’s peel and seeds are then macerated to obtain an extract. The peel and the seeds powder are macerated using 70% ethanol. Every 24 hours the filtrate was collected. Maceration is repeated until the ethanol filtrate became colorless. The filtrate was evaporated using a rotary evaporator 2.3 Determination of antioxidant activity The concentration of each extract of P.edulis peel and seed were 200 µg/mL; 100 µg/mL ; 50 µg/mL ; 25 µg/mL µg/mL; 12,5 µg/mL ; and 6,25 µg/mL. 50 µL samples (peel and seed extract) were pipetted into 96-well plates. As much as 0.077 mmoles DPPH was added as 200 µL well plate containing the samples. DMSO was used as a blank, and DPPH alone was used as a control. The well plate was incubated for 30 minutes in dark condition. Absorbance was measured using microplate reader at λ = 517 nm. % scavenging activity = 𝑠𝑠𝑐𝑐𝑐𝑐𝑡𝑡𝑑𝑑𝑐𝑐𝑠𝑠 𝑠𝑠𝑎𝑎𝑠𝑠𝑐𝑐𝑑𝑑𝑎𝑎𝑠𝑠𝑐𝑐𝑠𝑠𝑤𝑤−𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑤𝑤𝑠𝑠 𝑠𝑠𝑎𝑎𝑠𝑠𝑐𝑐𝑑𝑑𝑎𝑎𝑠𝑠𝑐𝑐𝑠𝑠𝑤𝑤 𝑠𝑠𝑐𝑐𝑐𝑐𝑡𝑡𝑑𝑑𝑐𝑐𝑠𝑠 𝑠𝑠𝑎𝑎𝑠𝑠𝑐𝑐𝑑𝑑𝑎𝑎𝑠𝑠𝑐𝑐𝑠𝑠𝑤𝑤 x 100% 2.4 Anti collagenase activity test American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 42-48 44 The concentration of samples used in this test varied between 250 µg/mL ; 125 µg/mL ; 31.25 µg/mL ; 15.625 µg/mL ; and 7.81 µg/mL. 10µL collagenase enzyme was pipetted into each control-well, samples-well and blank-well. Respectively, 90 µL; 60 µL; and 80 µL was added into control-well, samples-well, and blank-well. 30 µL samples were added into samples-well and control well. The 20 µL FALGPA (N-[3-(2-furyl)acryloyl]- leu-gly-Pro-Ala) was added into each sample well and control well. Absorbance was measured using microplate reader at λ = 335 nm 3. Result and Discussion 3.1 DPPH Scavenging Activity The result of DPPH scavenging activity was shown in table.1. the Table 1: DPPH scavenging activity of peel and seed of P.edulis extract Concentration (μg/mL) Average of DPP scavenging activity (%) Peel extract of P.edulis Seed extract of P.edulis 200 95.61 ±0.62f 71.55 ±0.20c 100 71.06 ±0.56e 51.50 ±3.50b 50 52.57 ±1.18d 42.83±3.79a,b 25 40.58 ±1.02c 38.00 ±3.94a 12.5 32.73 ±0.42b 35.00 ±3.09a 6.25 30.07 ±1.54a 33.96 ±3.75a 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 DPPH scavenging activity of P.edulis peel extract was higher than P.edulis seed extract. The DPP scavenging activity also increased with the increase in concentration. Irawan and his colleagues ( 2017) reported that the DPPH free radical scavenging activity of Pometia pinnata extract varied widely increased with the increase of concentration. Among parts of Pometia pinnata fruit, the peels had higher antioxidant activity compared to the seed extract [7]. Our result is in agreement with the findings of Jalal and his colleagues [8] who reported the antioxidant activity of pomegranate peel and seed powder extracts, and in their reported, it mentioned that the DPPH scavenging activity of pomegranate peel powder was significantly higher (P<0,05) than the pomegranate seed powder. DPPH test which is based on the ability of DPPH, a stable free radical, to decolorize in the presence of antioxidants, is a direct and reliable method for determining radical scavenging action. The DPPH radical contains an odd electron which is responsible for the absorbance at 515- 517 nm. when DPPH accepts and electron donated by an antioxidant compound, the DPPH is decolorized, which can be quantitatively measured from the changes in absorbance [9]. The IC50 value of DPPH scavenging activity of P.edulis seed extract and P.edulis peel extract was shown in table 2. The test was carried out in triple American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 42-48 45 Table 2: The IC50 value of DPPH scavenging activity od P.edulis seed extract and peel extract samples IC50 (µg/mL) Average IC50 (µg/mL) P.edulis peel extract (1st ) 56,34 P.edulis peel extract (2nd) 58,25 P.edulis peel extract (3rd) 53,75 56,11 ±2.26 average 56,15 P.edulis seed extract (1st) 71,73 P.edulis seed extract (2nd) 96,49 P.edulis seed extract (3rd) 96,53 88,25 ± 14,31 average 89,12 From the table above it showed that the value of IC50 in P.edulis peel extract is lower than P.edulis seed extract. The similar result reported by Kanatt [10] which reported peel extract of pomegranate showed a concentration- dependent DPPH radical scavenging activity with an IC50 of 4.9 µg/mL. peel extract scavenged the DPPH radical more efficiently than seed extract, indicating seed extract was lacked hydrogen donating capacity. Benites and his colleagues [11] reported the pulp of A.sylvatica showed a lower value of IC50 as 695.61±6.67 µg/mL than the seeds as 724±17.79 µg/mL. P.edulis is known to have a strong antioxidant activity. In the study reported by Ramaiya [12] the highest phenolic content was observed in vine-ripened purple and yellow P.edulis at 362.00±4.68 mg GAE/L and 361.73±3.99 mg GAE/L respectively. Similarly, the strongest antioxidant activity was observed in vine-ripened P.edulis (purple) at 547 ± 3.08 µmol Trolox/L and P.edulis (yellow) at 524 ± 1.96 µmol Trolox/L. in general, a higher TPC value gave a stronger antioxidant activity. the antioxidant activity exhibited by plant extracts obtained from the activity is suspected of secondary metabolites present in the plant. Secondary metabolites are produced or synthesized compounds on cells and certain taxonomic groups in the rate of growth or particular stress [13]. 3.2 Anticollagenase activity of peel and seed extract of P.edulis The collagenase inhibition activity of peel and seed extract of P.edulis was shown in table 3 Table 3: Anti-collagenase activity of peel and seed extract P.edulis Concentration (µg/mL) Average of Anticollagenase Activity (%) Peel extract Seed extract 250 76.18 ±1.40e 51.84 ±1.72e 125 60.74 ±0.39d 37.99 ±0.27d 62.5 50.07 ±1.27c 31.86 ±0.97c 31.25 41.09 ±0.87b 28.10 ±2.04b 15.625 36.64 ±0.81a 21.46 ±3.13ab 7.8125 34.78 ±1.30a 17.60 ±2.25a American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 42-48 46 (*data are presented in the form of averages ± standard deviations. Differences in lowercase letters in the same column show the significance of data P < 0.05 (Tukey HSD Post Hoc test) From the data above it can be seen that the higher the extract concentration, the higher the percentage of collagenase inhibition. Mansauda and his colleagues [3] reported that 50 µg/ mL S.plagyophyllum extract inhibited 54.46±0.37 %activity of collagenase enzyme. The higher concentration of S.plagyophyllum, the higher activity of anti-collagenase. Table 4: IC50 of anti-collagenase activity of P.edulis peel and seed extract The analysis of IC50 of anti-collagenase activity using P.edulis peel and seed extract was conducted in the triple. Based on the data, it showed that the peel extract of P.edulis has lower IC50, this means the anti-collagenase activity of P.edulis peel extract is higher than P.edulis seed extract. MMPs or collagenase is a group of zinc- containing proteinases. MMP-1 or interstitial collagenase initiates the breakdown mostly type I, II and III collagens which are the abundant interstitial collagens in the dermis, while MMP-2 is responsible for breakdown of type I-III, IV and VII collagens in which the latter two are most abundant in the dermal-epidermal junction. The inhibition effect of peel and seed extract of P.edulis might involve several mechanisms. Hydroxyl groups of polyphenols could interact with the backbone or other functional groups side chain of collagenase. In addition, hydrophobic interaction between the benzene ring of polyphenols and collagenase could also result in the conformational changes leading to unfunctioned enzyme. Another mechanism involves the Zn ion active site on collagenase. Collagenase contains structural Zn ions at its active site which plays a major role in facilitating interaction with an inhibitor [14]. 4. Conclusion that the IC50 of peel extract and seed extract of P.edulis were 56.11 ± 2.26 µg/mL and 88.25 ± 14.31 µg/mL respectively. The IC50 of anti-collagenase activity of peel and seed extract of P.edulis were 82.53 ± 1.42 µg/mL and 225.40 ± 9.12 µg/mL respectively. From the result, it concluded that peel and seed extract of P.edulis Sample Equation R2 IC50 (µg/mL) IC50 (µg/mL) P.edulis peel extract (1st) Y = 0.1612x+36.709 0.97 82.45 82.53±1.42 P.edulis peel extract (2nd) Y = 0.1778x+35.563 0.97 81.15 P.edulis peel extract (3rd) Y = 0.1728x+35.486 0.96 83.99 Average Y = 0.1706x+35.919 0.97 82.54 P.edulis seed extract (1st) Y = 0.1418x+19.455 0.97 215.41 225.40 ±9.12 P.edulis seed extract (2nd) Y = 0.1303x+19.605 0.92 234.51 P.edulis seed extract (3rd) Y = 0.1269x+21.126 0.92 227.53 Average Y = 0.1330x+20.062 0.95 225.63 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 42-48 47 possesses remarkable potency as anti-collagenase activity and have potential to be developed as anti-aging skin nutraceutical References [1] I. Binic, V. Lazarevic, M. Ljubenovic, J. Mojsa, and D. Sokolovic, “Skin Ageing : Natural Weapons and Strategies,” Evidence-Based Complement. Altern. Med., vol. 2013, pp. 1–10, 2013. [2] S. Singh and S. Mishra, “Aging and Nutrition : A Review Article,” IOSR J. Nurs. Heal. Sci., vol. 1, no. 4, pp. 43–47, 2013. [3] K. Lifie, R. Mansauda, E. Anwar, T. Nurhayati, and E. Anwar, “Antioxidant and Anti-Collagenase Activity of Sargassum plagyophyllum Extract as an Anti-Wrinkle Cosmetic Ingredient,” Pharmacogn J, vol. 10, no. 5, pp. 932–936, 2018. [4] S. Sharafzadeh, “Medicinal Plants as Anti-Ageing Materials : A Review,” Glob. J. Med. Plant Res., vol. 1, no. 2, pp. 234–236, 2013. [5] W. D. Ratnasooriya, W. P. K. M. Abeysekera, G. A. S. Premakumara, C. D. T. Ratnasooriya, and S. G. Ratnasooriya, “In vitro anti-collagenase collagenase activity of Sri Lankan low grown orthodox Orange Pekoe grade black tea ( Camellia sinensis L .),” Res. J. Chem. Sci., vol. 7, no. 7, pp. 11–15, 2017. [6] T. C. Colomeu, D. De Figueiredo, R. D. L. Zollner, L. Maria, and M. Meletti, “Comparison of Antioxidant and Ant proliferative Effect among Four Passiflora Spp .,” J. Agric. Life Sci., vol. 4, no. 2, pp. 1–8, 2017. [7] C. Irawan, L. Sulistiawaty, and H. Rochaeni, “Evaluation of DPPH free radical scavenging activity of Pometia pinnata from Indonesia,” Pharma Innov. J., vol. 6, no. 8, pp. 403–406, 2017. [8] H. Jalal, M. A. Pal, H. Hamdani, and M. Rovida, “Antioxidant activity of pomegranate peel and seed powder extracts,” J. Pharmacogn. Phytochem., vol. 7, no. 5, pp. 992–997, 2018. [9] S. M. R. Hasan, M. Hossain, R. Akter, and M. Jamila, “DPPH free radical scavenging activity of some Bangladeshi medicinal plants,” J. Med. Plant Res., vol. 3, no. 11, pp. 875–879, 2009. [10] S. R. Kanatt, R. Chander, and A. Sharma, “Antioxidant and antimicrobial activity of pomegranate peel extract improves the shelf life of chicken products Original article Antioxidant and antimicrobial activity of pomegranate peel extract improves the shelf life of chicken products,” Int. J. Food Sci. Technol., vol. 45, pp. 216–222, 2010. [11] RSR. Benites. ASN. Formagio. EJS Argandona. CRF. Volobuff. LNF. Trevizan. MC. Vieira. MS. Silva, “Contents of constituents and antioxidant activity of seed and pulp extracts of Annona coriacea and Annona sylvatica,” Brazilian J. Biol., vol. 74, no. 3, pp. 685–691, 2015. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 42-48 48 [12] S. Ramaiya, J. S. Bujang, M. H. Zakaria, M. Arif, and S. Sahrir, “Sugars , ascorbic acid , total phenolic content and total antioxidant activity in passion fruit ( Passiflora ) cultivars Shiamala Devi Ramaiya , a ∗ Japar Sidik Bujang , a Muta Harah Zakaria , b Wong,” J Sci Food Agric, vol. 93, pp. 1198–1205, 2013. [13] I. Ahmad, R. Sulistiarini, and L. Rijai, “Antioxidant Activity of Some Selected East Borneo Plants,” Int. J. Publich Heal. Sci., vol. 4, no. 1, pp. 58–62, 2015. [14] S. Pientaweeratch, V. Panapisal, and A. Tansirikongkol, “activities of Phyllanthus emblica , Manilkara zapota and silymarin : an in vitro comparative study for anti-aging applications,” Pharm. Biol., vol. 54, no. 9, pp. 1865–1872, 2016.