Philippine Journal of Science 150 (2): 397-406, April 2021 ISSN 0031 - 7683 Date Received: 27 Jul 2020 Evaluation of the Bioactivities of Natural Phenolics from Mango (Mangifera indica Linn) Leaves for Cosmetic Industry Applications Arsenia B. Sapin*, Maria Katrina N. Alaon, Fides Marciana Z. Tambalo, Rodney H. Perez, and Arra Gaylon National Institute of Molecular Biology and Biotechnology University of the Philippines Los Baños, Laguna 4031 Philippines Mango is one of the most important crops in the Philippines but there had been no local study on the possible utilization of its non-food parts such as the barks and leaves, which were reported to be rich in compounds with biological activities that are of significance in the development of cosmetic products (Masibo and He 2008; DA 2018). In this study, aqueous acetone extracts from leaves of the Philippine mango cultivars (“carabao,” “pico,” “apple mango, “sinaging,” and “sipsipin”) were investigated for their total phenolics content (TPC), phenolics composition, and biological activities – specifically, antioxidant as well as tyrosinase and elastase inhibitory properties. Results show that all mango leaf extracts had significant levels of TPC. Phenolic compounds such as mangiferin, gallic acid, quercetin 3-β-D-glucopyranoside, and kaempferol were all found to be present in the extracts with mangiferin as the predominant compound. All the extracts exhibited greater antioxidant capacity than the standard ascorbic acid, implying greater protection against skin damages due to free radicals. Also, all extracts exhibited greater inhibition on elastase than tocopherol, suggesting a greater anti-aging property. While only some extracts showed greater inhibition on tyrosinase than ascorbic acid, it did not surpass but gave comparable inhibitory activity with that of kojic acid. This implies that the extracts and kojic acid have comparable whitening capacities. Overall, the results affirm the great potential of the extracts of the local mango leaves as a cosmetic active ingredient. Keywords: antioxidant, elastase, mango leaves, phenolics, tyrosinase INTRODUCTION breeding, fruit production, and processing. There were only a few works exploring the potentials and utilization Mango (Mangifera indica L.) is one of the top three of the non-fruit parts of the mango tree such as the bark produced and exported crops in the Philippines, with fruit and leaves. In other countries, this area of study is much production reaching 737.44 metric tons in 2019 (Briones explored and given importance. et al. 2013; PSA 2019a, b, c, d). But for more than a decade now, the mango industry had been experiencing Studies on foreign mango cultivars reveal that mango setbacks due to various factors (DA 2018). Locally, many leaves are an excellent source of polyphenolic compounds, studies had been conducted to continuously increase the which have numerous health benefits such as antioxidation, industry’s profitability, but they are strongly focused on antidiabetic, anticancer, and anti-inflammatory, among others (Masibo and He 2008). Further studies proved *Corresponding Author: [email protected] their appropriate applications in food, pharmaceuticals, 397 Philippine Journal of Science Sapin et al.: Evaluation of the Bioactivities of Natural Vol. 150 No. 2, April 2021 Phenolics from Mango Leaves and cosmetics (Charrier et al. 2006; Masibo and He 2008; would then not only establish the potential of the Morsi et al. 2010; Mohan et al. 2013). The products mentioned cultivars as sources of cosmetic ingredients Vimang® (a popular drug in Cuba) and Zynamite® (a but would also increase the value of the unpopular mango commercial dietary supplement in Spain) are extracts cultivars. Moreover, this could provide consumers from mango barks and leaves, respectively (Garrido et effective nature-based cosmetic ingredients as a al. 2001; Gelabert-Rebato et al. 2019). There were also replacement to the synthetic ones that are used at present approved patents on the extraction and application of to promote safer products for healthier and beautiful skin. polyphenols from mango leaves for cosmetic use such as that of Loreal’s (Deng et al. 2019; Telang et al. 2013). Considering the economic limitations and gains and the great demand for organic cosmetic products in the country MATERIALS AND METHODS at present, it would be beneficial to explore the potential application of mango leaf extracts in cosmetic products. Collection and Preparation of Samples Extracts from mango leaves could qualify as a cosmetic Fresh leaves of mango cultivars (carabao, apple mango, ingredient for their reported antioxidant properties against pico, sinaging, and sipsipin) were collected from Santos free radicals, which cause oxidative damage on skin cells Farm in San Miguel Bulacan, Philippines (15.14°N, leading to premature skin aging and skin damages (Barreto 121.05°E). The selection of these cultivars was based et al. 2008; Pan et al. 2018). It was also reported that the on their availability at the sampling site. Mature leaves extract could reduce metal ions that catalyze the formation (which appear dark green in color) and young leaves of radicals (Barreto et al. 2008). Mango leaf extract was (which were lighter in color) were collected from carabao also found to exert an inhibitory effect on the enzymes and pico cultivars. Young leaves from other cultivars elastase and tyrosinase that cause aging and darkening were not sampled due to their unavailability during the of the skin, respectively (Ochocka et al. 2017; Shi et al. time of sampling. Both leaf types were considered to 2019). No similar work has been done yet for the leaves investigate which could be a better source of phenolics of the Philippine mango cultivars. with better bioactivities. The leaves were immediately washed, oven-dried at 60 °C (fabricated air drier oven), The Philippines grows various mango cultivars wherein ground (Retsch® knife mill GRINDOMIX GM 200), carabao mango is the most popular. Other lesser popular and sieved (Tyler® Mesh No. 60) to produce the mango cultivars are pico, apple, and other cultivars that are not leaves powder (MLP). commonly grown but are found in mango farms – namely, sinaging and sipsipin. All these mango trees similarly Meanwhile, the leaf extracts were prepared by mixing grow abundant leaves guaranteeing an adequate source 0.2 g of the MLP with 5 mL 50% aqueous acetone for of raw materials for possible phenolic extraction. each cultivar. The mixtures were centrifuged to recover the phenolic-rich leaf extracts, which were determined In this study, the potential of the extracts from the leaves by their total phenolic content. The extracts were diluted of the local mango cultivars (carabao, pico, apple, with water to obtain the needed concentrations in each sinaging, and sipsipin) for cosmetic applications was antioxidant and enzyme inhibition assays. explored. Specifically, the polyphenolic compounds present in the leaves were determined, the same with the antioxidant capacity and the inhibitory effect Quantification of Phenolic Compounds against elastase and tyrosinase. The antioxidant capacity The TPC was determined based on the method of Nuñez- was evaluated in terms of the three most common Selles et al. (2002). An aliquot of 0.5 mL of the diluted in vitro antioxidant assays – DPPH (2,2-diphenyl- sample extract was mixed with 0.5 mL of 1N Folin- 1-picrylhydrazyland) radical scavenging, ABTS Ciocalteu’s phenol reagent and 0.5 mL of 10% Na2CO3. (2,2′-azino-di-(3-etylobenzotizoline-6-sulfonate) radical After standing for 5 min, 5 mL of distilled water was added scavenging, and copper reduction antioxidant capacity and the absorbance at λ = 720 nm was determined with (CUPRAC). These antioxidant assays are commonly the reagent solution as the blank. The TPC was computed used to assess the antioxidant activity of cosmetic using a standard curve with gallic acid as reference. The TPC values were expressed as gallic acid equivalents products because of their simplicity, reproducibility, and their applicability in both hydrophilic and hydrophobic (GAE). samples (Ratz-Lyko et al. 2011). Their antioxidant Some of the phenolic compounds were also quantified capacities were compared with that of the commercial using Shimadzu HPLC Model Prominence with UV-Vis antioxidants such as vitamins C and E, while their detector. The column used was Inertsil ODS-3 column inhibitory capacities were compared with commercial (GL Sciences, Inc. Tokyo, Japan; 150 mm x 4.0 mm inhibitors such as kojic acid. The results of this study I.D., 5 µm particle diameter).The peak identification 398 Philippine Journal of Science Sapin et al.: Evaluation of the Bioactivities of Natural Vol. 150 No. 2, April 2021 Phenolics from Mango Leaves of each polyphenol was based on the comparison of the and left to stand for 20 min, after which 0.6 mL of water relative retention time, peak area percentage, and spectra was added. The absorbance was determined at λ = 450 nm, data with polyphenol standards. For the mangiferin with the reagent solution without the antioxidant as the determination, the sample extracts and the standard blank. The CUPRAC was quantified by the absorbance were eluted using 2% aqueous acetic acid and methanol reading, wherein greater absorbance corresponded to (60:40 v/v) at 1mL/min and λ = 254 nm while for relatively greater reduction. quercetin-3-β-D-glucopyranoside and kaempferol, the sample extracts and the standards were eluted using 0.1% Determination of Tyrosinase Inhibition aqueous phosphoric acid (A) and methanol (B) at binary The method used was based on that of Hapsari et al. gradient condition: 0–50% B at 0–2.5 min, 50–100% (2012). A solution of 40 µL 5mM DOPA (3,4-dihydroxy- B for 2.5–14.5 min, and 0–100% B at 14.5–16.0 min; L-phenylalanine, Sigma D-9628), 40 µL 0.1M potassium and at λ = 369 nm. For gallic acid, the sample extracts phosphate buffer pH 6.5, an aliquot of 40 µL extract (or and the standards were eluted using water-phosphoric buffer for control), and 40 µL mushroom tyrosinase (250 acid-methanol (79.9:0.1:20 v/v) mobile phase system at units/mL, Sigma T- 3824) were added consequentially.