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 ( 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 (“,” “,” “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 ( 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 , 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 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. 1 mL/min and λ = 210 nm. The samples were extracted The solution was mixed and left to stand for 15 min. The using 100% methanol with sonication except for the absorbance was measured at λ = 490 nm using a microplate gallic acid determination, which was done using 15% reader, with the reagent solution without the enzyme as aqueous methanol. The extracts were filtered through a blank. The tyrosinase inhibition was calculated according 0.20-µm Nylon membrane (Ciro Syringe Filters). The to the formula: same conditions were used for their polyphenol standards [mangiferin, quercetin, and kaempferol (Sigma-Aldrich); Ac − As − Ab % inhibition = x 100 (2) quercetin-3-β-D-glucopyranoside (Carbosynth)]. (Ac)

where A is the absorbance of the control, A is the Determination of Antioxidant Properties c b absorbance of the blank, and As is the absorbance of the DPPH radical scavenging activity. The method used was sample. Kojic acid was used as a positive control. The based on that of Ribeiro et al. (2008). An aliquot of 100 inhibition of the leaf extracts from the cultivars carabao, µL of the samples of different concentrations (4, 8, 12, pico, and apple mango was determined at concentrations 16, and 20 µg GAE) was added each to 5 mL of 0.1mM 5, 10, 15, 20, and 25 µg GAE; it was likewise done for DPPH in methanol. The solutions were stirred and left to cultivars sinaging and sipsipin (at 6, 12, 18, 24, and 30 stand for 20 min. The absorbance was determined at λ = µg GAE) and for kojic acid (at 2, 4, 6, 8, and 10 µg). 517 nm with distilled water as blank. The DPPH reagent in methanol served as control and DPPH inhibition was calculated using the formula: Determination of Elastase Inhibition The method used was based on that of Moon et al. (2010). Acontrol − Asample % DPPH inhibition = x 100 (1) Briefly, 40 µL of N-succinyl-(ALA)3-p-nitroanilide, 40 (Acontrol) µL of the sample (or buffer for control), 1 mL of phosphate buffer, and 40 µL of elastase were mixed consequentially. ABTS radical scavenging capacity . The method used The solution was left to stand for 20 min and 2 mL TRIS- was based on that of Re et al. (1999). An aliquot of 40 HCl buffer was added. The absorbance at λ = 490 nm was µL of the samples of different concentrations (0.5, 1.0, measured, with reagent solution without enzyme as blank. 1.5, 2.0, and 2.5 µg GAE) were added each to 3-mL Elastase inhibition was calculated with the same formula ABTS radical solution with an initial absorbance of 0.72 as that of tyrosinase inhibition. Tocopherol was used as ± 0.05 at λ = 734 nm. The solutions were stirred, left to a positive control. stand for 5 min, and had the absorbance determined at λ = 734 nm with distilled water as blank. The ABTS with 40 µL of 90% methanol served as control and the ABTS Statistical Analysis inhibition was calculated using the same formula as in The data statistical analysis was carried out using the DPPH inhibition. Minitab software. One-way and two-way analyses of variance (ANOVA) were utilized for the appropriate data Copper reducing antioxidant capacity . The method used with Tukey’s honestly significant difference (HSD) test was based on that of Alpinar et al. (2009). An aliquot of for further mean comparisons. The reported values were 0.5 mL of the samples of different concentrations (2, 4, 6, the mean of measurements from duplicate assays and 8, and 10 µg GAE) were added each to the mixture of 1.0 pooled samples. All analyses were done at a 5% level of mL of 0.01M CuCl2, 1.0 mL of 1.0 M NH4CH₃COOH, and significance. 1.0 mL of 7.5 mM neocuproine. The solutions were stirred

399 Philippine Journal of Science Sapin et al.: Evaluation of the Bioactivities of Natural Vol. 150 No. 2, April 2021 Phenolics from Mango Leaves

RESULTS AND DISCUSSION of carabao and pico also contained greater concentrations of mangiferin and quercetin-3-β-D-glucopyranoside than the mature leaves. However, for the other two phenolic Quantification of Polyphenolic Compounds compounds, it is otherwise. Polyphenolic compounds Table 1 shows that the TPC of the extracts from the leaves are secondary metabolites produced by plants, which is of the different mango cultivars are significantly different, confirmed to exhibit biological activities important in wherein the younger leaves of pico contained the greatest skincare such as antioxidant and inhibition on skin enzymes TPC while its mature leaves contained the least value such as tyrosinase, elastase, cellulase, and hyaluronidase (p < 0.05). Similarly, the young leaves of the carabao (Ko et al. 2011; Zilich et al. 2015). The succeeding contained greater TPC than its mature leaves. This has also discussion will affirm the antioxidant properties of these been observed by Barreto et al. (2008) in their study of phenolic compounds. several mango cultivars in Brazil. However, for potential industrial-scale extraction of phenolic compounds, the mature leaves are to be considered instead of the young Determination of Antioxidant Properties leaves. Among the samples of mature leaves, the leaves The antioxidant properties of the various mango leaf of sipsipin had the greatest TPC. Meanwhile, the mature extracts and the commercial antioxidant ascorbic acid were leaves of the commonly grown carabao cultivar had evaluated specifically for their radical scavenging and greater TPC than the leaves of apple mango and pico. copper reducing properties at varied TPC concentrations. Their antioxidant strengths were also evaluated through It is also useful to determine the type of phenolic compounds their EC50 values or the effective concentration to effect present in the extracts to understand the reason for their 50% inhibition or reduction. The antioxidant strengths bioactivities and as a basis for health claims and future of the pure phenolic compounds were also determined. studies. The result of the HPLC analysis (Table 1) reveals that the extract from mango leaves contains mangiferin, For the free radical scavenging property, the inhibitory quercetin, gallic acid, and kaempferol. Studies of Barreto effect of the samples on the stable free radical DPPH and et al. (2008), Saleem et al. (2013), Pan et al. (2018), and the cation radical ABTS were evaluated at varied total Lauloo et al. (2018) also reported the presence of the said polyphenol contents. The DPPH inhibition assay is limited phenolic compounds in the leaves of varied mango cultivars to phenolic compounds that are soluble in organic solvents from Brazil, China, Pakistan, and Mauritius, respectively. especially alcohols while the ABTS inhibition assay The data also shows that mangiferin is predominant in all includes both the hydrophilic and hydrophobic phenolic cultivars, with the pico mature leaves containing the greatest compounds (Ratz-Lyko et al. 2011). This suggests then amount (p < 0.05). Barreto et al.(2008) also reported that that ABTS assay gives a better estimate of the overall mangiferin was the predominant phenolic compound in free radical scavenging capacity. But nevertheless, DPPH the leaves of the mango cultivar “embrapa-141-roxa” and is equally useful since many phenolic compounds are second predominant in the cultivar “.” In addition, alcohol soluble. among the extracts from mature leaves, the extract from pico leaves was also richest in quercetin-3-β-D-glucopyranoside Results of the two-way ANOVA and regression show while the extract from the leaves of apple mango is richest in a linear dose-dependent inhibition of DPPH in all the gallic acid and kaempferol. The extract of the young leaves samples at all total polyphenol contents considered (p

Table 1. TPC and concentration of some polyphenolic compounds found in the mature leaves of different Philippine mango varieties. Phenolic contents from HPLC analysis (mg/g) Cultivar Total phenolic content Quercetin 3-β-D- (mg GAE/g) Mangiferin Gallic acid Kaempferol glucopyranoside Apple 81.14F 18.09E 1.37A 1.27B 0.281A Sinaging 90.08D 50.05C 0.29F 1.11C 0.252BC Sipsipin 100.6C 40.52D 0.73C 0.84D 0.248C Carabao 84.16E 38.60D 0.34E 1.10C 0.255BC Pico 75.27G 52.85A 0.38D 2.14A 0.259B Y. carabao* 116.00B 50.25BC 0.97B 0.80D 0.251BC Y. pico* 128.00A 52.24AB 1.34A 2.12A 0.259B Rows with similar superscript letters are not significantly different at 5% level of significance; analyzed using one-way ANOVA with Tukey’s HSD test; n = 2 *Y – young leaves

400 Philippine Journal of Science Sapin et al.: Evaluation of the Bioactivities of Natural Vol. 150 No. 2, April 2021 Phenolics from Mango Leaves

< 0.05). Figure 1 shows that the extract from the young Results of the two-way ANOVA and regression also show a leaves of pico had the greatest inhibitory effect on DPPH linear dose-dependent inhibition of ABTS in all samples at at TPC of 8–20 µg GAE, suggesting the greatest free all total polyphenol contents considered (p < 0.05). Figure 2 radical scavenging activity by the alcohol-soluble phenolic shows that the extract from leaves of apple mango exhibited compounds compared to the other samples. The extract of the greatest inhibitory effect on ABTS at 1.5–2.5 µg GAE, both young leaves of pico and carabao exhibited greater implying the greatest free radical scavenging capacity by free radical scavenging capacity compared to their mature both the hydrophilic and hydrophobic phenolic compounds leaves. Table 2 also shows that the former are more potent compared to the other samples. Table 2 also shows the free radical scavengers than the latter based on their EC50 said extract was the most potent free radical scavengers values. But among the samples of mature leaves, the among all the samples. The table also shows that all mango extract from sinaging leaves and carabao leaves had the leaves extracts were more potent free radical scavengers greatest free radical scavenging activity at 12–20 µg GAE almost twice than the ascorbic acid. Meanwhile, the free and are the most potent free radical scavengers. Compared radical scavenging activity of younger leaves of carabao to ascorbic acid, all the mango leaves extract exhibited and pico compared to that of the mature ones did not show greater potency – almost twice in scavenging free radicals. a significant difference.

Figure 1. Inhibitory effect of different mango cultivar leaf extracts on DPPH at varied total polyphenol levels. Means with Figure 2. Inhibitory effect of different mango cultivar leaf extracts similar letters are not significantly different within on ABTS at varied total polyphenol levels. Means with total polyphenol content. Y means young leaves (two- similar letters are not significantly different within total way ANOVA with Tukey’s HSD test, p < 0.05; linear polyphenol content. Y means younger leaves (two- regression, p < 0.05; n = 2). way ANOVA with Tukey’s HSD test, p < 0.05; linear regression, p < 0.05; n = 2).

Table 2. Antioxidant strength (EC50) of various mango cultivar leaf extracts and some phenolic standards.

Mango cultivar/standard DPPH EC50 ABTS EC50 CUPRAC EC50 (µg GAE) (µg GAE) (µg GAE) Apple mango 11.27C 1.91E 8.17B Carabao 10.81D 2.10CD 8.18B Pico 15.39B 1.98DE 8.30B Sinaging 10.80D 2.27B 8.40B Sipsipin 11.14CD 2.17BC 7.76B Y. carabao* 9.61E 2.07CD 7.74B Y. pico* 9.45E 1.97DE 7.96B Ascorbic acid** 19.85A 5.92A 23.15A Mangiferin** 31.20 4.28 13.70 Gallic acid** 8.34 1.67 6.44 Quercetin-3-β-D- 18.31 7.67 14.05 glucopyranoside** Kaempferol** 38.88 4.24 ND Rows with similar superscript letters are not significantly different at 5% level of significance; analyzed using one-way ANOVA with Tukey’s HSD test; n = 2 *Y – young leaves ** In μg

401 Philippine Journal of Science Sapin et al.: Evaluation of the Bioactivities of Natural Vol. 150 No. 2, April 2021 Phenolics from Mango Leaves

Meanwhile, in both assays, gallic acid exhibited the greatest substitute ascorbic acid as an antioxidant in cosmetic free radical scavenging strength among the standard products. Meanwhile, comparing the matured leaves phenolic compounds, even greater than all the samples. from different mango cultivars, the extracts from apple However, the EC50 values of other phenolic compounds mango and pico leaves were better free radical scavengers were much greater than that of the samples especially while the extract from sipsipin leaves was a better mangiferin, which was predominant in the leaves of all copper ion reducer. Also, the extracts from sinaging and cultivars. The low EC50 values of the samples then reflect carabao leaves were better free radical scavengers when the synergistic effect of the polyphenolic compounds. Ko considering the organic-soluble phenolic compounds only. et al. (2011) also reported that gallic acid and its derivatives The free radical scavenging and reducing properties of the were the most potent free radical scavengers compared to phenolic compounds can be attributed to their chemical quercetin and other phenolic compounds. structure (Zilich et al. 2015). Apart from phenolic compounds, other compounds such as chlorophyll and Meanwhile, CUPRAC was quantified by the measured carotenoids that are normally found in leaves may have absorbance at 450 nm. Greater absorbance corresponded possibly contributed to the antioxidant activity of the to a relatively greater reduction. The results of two-way extracts (Allalou et al. 2019). ANOVA showed a linear dose-dependent reduction of copper ions (Cu2+) by all the samples at all TPCs considered (p < 0.05). Figure 3 shows that the extract Determination of Whitening Property of the young leaves of carabao had the greatest copper The formation of melanin, which is the skin’s dark reducing power at TPC 2–10 µg GAE compared to other pigment, is catalyzed by the enzyme tyrosinase. Although samples. Also, the extracts from the young leaves of the this pigment protects the skin from the harmful effect of carabao and pico had greater reducing power compared ultraviolet radiation, its excessive activity can lead to severe to the extract from mature leaves. Among the samples health conditions (Shi et al. 2019). Also, inhibition of of mature leaves, the extract from the leaves of sipsipin tyrosinase corresponds to skin whitening. In this study, the inhibitory effect of the mango leaf extracts was determined. The result of the two-way ANOVA reveals that the extract from leaves of the cultivars apple mango, carabao, and pico exhibited a linear dose-dependent inhibitory effect on tyrosinase at TPC of 5–25 µg GAE (p < 0.05). Figure 4 shows that the extract from young leaves of pico and carabao exhibited the greatest inhibitory effect on

Figure 3. CUPRAC of the different mango cultivar leaf extracts at varied total polyphenol levels. Means with similar letters are not significantly different within total polyphenol content. Y means young leaves (two-way ANOVA with Tukey’s HSD test, p < 0.05; linear regression, p < 0.05; n = 2). exhibited the greatest reducing power at the mentioned TPC levels. The EC50 values, however, showed that the samples exerted similar copper ion reducing strength but much lesser than that of the ascorbic acid, implying that all the mango leaf extracts were more potent copper ion reducers than the commercial antioxidant. Again, the phenolic compound gallic acid exhibited the greatest copper ion reducing strength among all the standard phenolic compounds and the samples. Figure 4. Inhibitory effect on tyrosinase of different mango cultivar Overall, all the extracts from the different mango cultivar leaf extracts at varied total polyphenol levels. Means leaves are better free radical scavengers and copper ion with similar letters are not significantly different within reducers compared to the commercial antioxidant ascorbic total polyphenol content. Y means young leaves (two- acid, suggesting greater protection against skin damages way ANOVA with Tukey’s HSD Test, p < 0.05; linear due to free radicals. Thus, the extracts can potentially regression, p < 0.05; n = 2).

402 Philippine Journal of Science Sapin et al.: Evaluation of the Bioactivities of Natural Vol. 150 No. 2, April 2021 Phenolics from Mango Leaves tyrosinase, suggesting the greatest whitening potential have shown that natural phenolic compounds from plants compared to the other extracts. But among the mature exhibit inhibitory activity on the mentioned enzymes leaves, the extract from the leaves of apple mango (Thring et al. 2009). In this study, the elastase inhibitory exhibited the greatest inhibitory effect on tyrosinase at effect of the mango leaves extract was evaluated. TPC 20–25 µg GAE. On the other hand, different TPC levels were considered for sinaging and sipsipin leaf The result of the two-way ANOVA reveals a linear dose- extracts, and results show that the former had a greater dependent inhibitory effect of all mango leaves extract on inhibitory effect on tyrosinase compared to the latter at elastase at all TPCs considered (p < 0.05). Figure 5 shows 6–39 µg GAE (p < 0.05). that the extract from the leaves of apple mango exhibited the greatest inhibitory effect at TPC 2–10 µg GAE Table 3 shows that among all the samples, the extract compared to other leaves extract, suggesting the greatest from young leaves of pico and carabao were most potent anti-aging effect. Also, the extracts from young leaves of in inhibiting tyrosinase while among the extracts from carabao and pico showed a greater inhibitory effect than the mature leaves, that of the apple mango, exhibited the the extract of their mature leaves. Their EC50 values also greatest potency. These extracts also had greater potency compared to commercial antioxidant ascorbic acid but less potent compared to the commercial whitening agent kojic acid. This suggests that extracts from the leaves of apple mango and young leaves of pico and carabao are better whitening agent than the ascorbic acid but not kojic acid. Meanwhile, in a similar study of mango leaves, Shi et al. (2019) identified 36 polyphenolic tyrosinase inhibitors in the leaves extract, which includes mangiferin, gallic acid, quercetin, kaempferol, and their derivatives.

Table 3. Effective inhibitory concentration on tyrosinase and elastase Figure 5. Inhibitory effect of extract from the leaves of different by the mango leaves extract and some standard. mango cultivars on elastase at varied total polyphenol Tyrosinase IC Elastase IC levels. Means with similar letters are not significantly Mango variety 50 50 (µg GAE) (µg GAE) different within total polyphenol content. Y means young leaves (two-way ANOVA with Tukey‘s HSD test, p < Apple mango 18.92DE 2.64D 0.05; linear regression, p < 0.05; n = 2). Carabao 29.14A 9.75A Pico 22.75BC 9.27A Sinaging 24.77B 5.74C showed that the extract from apple mango leaves was the most potent elastase inhibitor that was two times to four Sipsipin 27.62A 7.46B times more potent than the other extracts. In comparison Y. carabao* 16.77EF 5.69C to the standard tocopherol, the mango leaf extracts were Y. pico* 15.54F 5.79C probably 10 times more potent, since even when the Ascorbic acid** 20.62CD ND concentration of the former was increased 10-fold than Kojic acid** 4.39G ND that of the TPC of the extracts, the inhibition had still not exceeded 50%. This suggests that the mango leaf extract Tocopherol** ND >100 was a more potent elastase inhibitor than tocopherol, Rows with similar superscript letters are not significantly different at 5% level suggesting a better anti-aging property. Meanwhile, it was of significance; analyzed using one-way ANOVA with Tukey’s HSD test; n = 2. *Y – young leaves reported that elastase is inhibited by mangiferin through **In µg non-competitive inhibition (Ochocka et al. 2017).

Determination of Anti-aging Property Elastin and collagen are the two important structural proteins that maintain the elasticity and firmness of the CONCLUSION skin together with hyaluronic acid. But in the presence The aqueous acetone extracts from leaves of carabao, of high levels of free radicals due to excessive ultraviolet pico, apple mango, sinaging, and sipsipin Philippine exposure, these are broken down due to increased activities mango varieties exhibited significant levels of TPC. of the enzymes elastase, collagenase, and hyaluronidase. Analysis using HPLC revealed that the extracts contained This results in loss of strength and flexibility of skin, the phenolic compounds mangiferin, quercetin 3-β-D- which manifests as wrinkles (Zilich et al. 2015). Studies glucopyranoside, gallic acid, and kaempferol – with

403 Philippine Journal of Science Sapin et al.: Evaluation of the Bioactivities of Natural Vol. 150 No. 2, April 2021 Phenolics from Mango Leaves mangiferin as the predominant phenolic in all extracts. Capacities of Some Food Plants Wildly Grown in Among all the extracts from mature leaves, the extract Ayvalik of Turkey. Food Sci Technol Res 15(1): 59–64. of sipsipin leaves had the greatest TPC while the extract BARRETO JC, TREVISAN MTS, HULL WE, ERBEN of pico leaves contained the greatest concentration of G, DE BRITO ES, PFUNDSTEIN B, WURTELE mangiferin and quercetin 3-β-D-glucopyranoside; the G, SPIELGELHALDER B, OWEN RO. 2008. extract from apple mango leaves contained the highest Characterization and Quantitation of Polyphenolic level of gallic acid and kaempferol. Meanwhile, the Compounds in Bark, Kernel, Leaves, and Peel of young leaves of carabao and pico contained higher TPC, Mango (Mangifera indica L.). J Agric Food Chem mangiferin, and gallic acid than the mature leaves. 56: 5599–5610. Also, all extracts were better free radical scavengers BRIONES RM, TURINGAN PA, RAKOTOARISOA and copper ion reducers compared to the commercial MA. 2013. Market Structure and Distribution of antioxidant ascorbic acid, suggesting that the extracts Benefits from Agricultural Exports: The Case of the can potentially substitute ascorbic acid as an antioxidant Philippine Mango Industry. FAO Commodity and in cosmetic products. Among the matured leaves, the Trade Policy Research Working Paper No. 42. Food extracts from apple mango and pico leaves were better free and Agriculture Organization of the United Nations. radical scavengers while the extract from sipsipin leaves was a better copper ion reducer. In addition, the extracts CHARRIER L, POIRIER F, MAILLET G, LUBRANO from sinaging and carabao leaves were better free radical C. 2006. Cosmetic Use of Mangiferin. US Patent scavengers when considering the organic-soluble phenolic 2006/0088560. compounds only. Meanwhile, the extract from apple mango [DA] Department of Agriculture. 2018. Philippines leaves exhibited the greatest effect and potency in inhibiting Mango Industry Roadmap 2017–2022. Office of the tyrosinase and elastase among all the leaves samples and Undersecretary High Value Crops and Rural Credit, greater than the ascorbic acid and tocopherol, respectively. Department of Agriculture, Quezon City. But kojic acid was a more potent tyrosinase inhibitor than all the extracts. Overall, the results show the great potential DENG Y, PAN Z, BERNARD AS, WU S. 2019. of the phenolic extract (using aqueous acetone) from the Aqueous Compositions with Mangiferin for Cosmetic leaves of local mango cultivars as a cosmetic ingredient Applications. US Patent 20190159989. with good antioxidant, whitening, and anti-aging properties. GARRIDO G, GONZALEZ D, DELPORTE C, BACKHOUSE N, QUINTERO G, NÚÑEZ-SELLÉS AJ, MORALES MA. 2001. Analgesic and Anti- inflammatory Effects of Mangifera indica L. extract ACKNOWLEDGMENTS (Vimang). Phytother Res 15(1): 18–21. The authors acknowledge with thanks and appreciation GELABERT-REBATO M, MARTIN RINCON M, the funding from the Department of Agriculture – Bureau GALVAN-ALVAREZ V, GALLEGO- SELLÉS A, of Agricultural Research for this study and the assistance MARTINEZ-CANTON M, VEGA-MORALES of Jessica Juarez and Roland Martinez during the T, WIEBE J, FERNÁNDEZ-DEL CASTILLO C, experimentation. CASTILLA-HERNANDEZ E, DIAZ-TIBERIO O, CALBET J. 2019. A Single Dose of the Mango Leaf Extract Zynamite® in Combination with Quercetin Enhances Peak Power Output During Repeated Sprint REFERENCES Exercise in Men and Women. Nutrients 11(11): 2592. ALLALOU H, BOULEKBACHE-MAKHLOUF L, Retrieved on 25 Jul 2020 from https://www.mdpi. MAPELLI-BRAHM P, SABRINA S, TENORE com/2072-6643/11/11/2592/htm GC, BENMEZIANE A, KADRI N, MADANI HAPSARI R, ELYA B, AMIN J. 2012. Formulation and K, MARTÍNEZ A. 2019. Antioxidant activity, Evaluation of Antioxidant and Tyrosinase Inhibitory carotenoids, chlorophylls and mineral composition Effect from Gel Containing the 70% Ethanolic from leaves of Pallenis spinosa: an Algerian medicinal Pleurotus ostreatus Extract. International Journal of plant. Journal of Complementary and Integrative Medicinal and Aromatic Plants 2(1): 135–140. Medicine. DOI:17.10.1515/jcim-2017-0081 MASIBO H, HE Q. 2008. Major Mango Polyphenols ALPINAR K, OZYUREK M, KOLAK U, GUCLU and their Potential Significance to Human Health. K, ARAS C, ALTUN M, CELIK S, BERKER K, Comprehensive Reviews in Food Science and Food BEKTASOGLU B, APAK R. 2009. Antioxidant Safety 7: 309–319.

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APPENDIX

Appendix I. Representative samples of mature (left) and young (right) carabao mango leaves.

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