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

Evaluation of the Bioactivities of Natural Phenolics from Mango (Mangifera indica Linn) Leaves

for Cosmetic Industry Applications

Keywords: antioxidant, elastase, mango leaves, phenolics, tyrosinase

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.

*Corresponding Author: absapin@up.edu.ph

INTRODUCTIONMango (Mangifera indica L.) is one of the top three produced and exported crops in the Philippines, with fruit production reaching 737.44 metric tons in 2019 (Briones et al. 2013; PSA 2019a, b, c, d). But for more than a decade now, the mango industry had been experiencing setbacks due to various factors (DA 2018). Locally, many studies had been conducted to continuously increase the industry’s profitability, but they are strongly focused on

breeding, fruit production, and processing. There were only a few works exploring the potentials and utilization of the non-fruit parts of the mango tree such as the bark and leaves. In other countries, this area of study is much explored and given importance.

Studies on foreign mango cultivars reveal that mango leaves are an excellent source of polyphenolic compounds, which have numerous health benefits such as antioxidation, antidiabetic, anticancer, and anti-inflammatory, among others (Masibo and He 2008). Further studies proved their appropriate applications in food, pharmaceuticals,

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and cosmetics (Charrier et al. 2006; Masibo and He 2008; Morsi et al. 2010; Mohan et al. 2013). The products Vimang® (a popular drug in Cuba) and Zynamite® (a commercial dietary supplement in Spain) are extracts from mango barks and leaves, respectively (Garrido et al. 2001; Gelabert-Rebato et al. 2019). There were also approved patents on the extraction and application of 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 at present, it would be beneficial to explore the potential application of mango leaf extracts in cosmetic products.

Extracts from mango leaves could qualify as a cosmetic ingredient for their reported antioxidant properties against free radicals, which cause oxidative damage on skin cells leading to premature skin aging and skin damages (Barreto et al. 2008; Pan et al. 2018). It was also reported that the extract could reduce metal ions that catalyze the formation of radicals (Barreto et al. 2008). Mango leaf extract was also found to exert an inhibitory effect on the enzymes elastase and tyrosinase that cause aging and darkening of the skin, respectively (Ochocka et al. 2017; Shi et al. 2019). No similar work has been done yet for the leaves of the Philippine mango cultivars.

The Philippines grows various mango cultivars wherein carabao mango is the most popular. Other lesser popular cultivars are pico, apple, and other cultivars that are not commonly grown but are found in mango farms – namely, sinaging and sipsipin. All these mango trees similarly grow abundant leaves guaranteeing an adequate source of raw materials for possible phenolic extraction.

In this study, the potential of the extracts from the leaves of the local mango cultivars (carabao, pico, apple, sinaging, and sipsipin) for cosmetic applications was explored. Specifically, the polyphenolic compounds present in the leaves were determined, the same with the antioxidant capacity and the inhibitory effect against elastase and tyrosinase. The antioxidant capacity was evaluated in terms of the three most common in vitro antioxidant assays – DPPH (2,2-diphenyl-1-picrylhydrazyland) radical scavenging, ABTS (2,2′-azino-di-(3-etylobenzotizoline-6-sulfonate) radical scavenging, and copper reduction antioxidant capacity (CUPRAC). These antioxidant assays are commonly used to assess the antioxidant activity of cosmetic products because of their simplicity, reproducibility, and their applicability in both hydrophilic and hydrophobic samples (Ratz-Lyko et al. 2011). Their antioxidant capacities were compared with that of the commercial antioxidants such as vitamins C and E, while their inhibitory capacities were compared with commercial inhibitors such as kojic acid. The results of this study

would then not only establish the potential of the mentioned cultivars as sources of cosmetic ingredients but would also increase the value of the unpopular mango cultivars. Moreover, this could provide consumers effective nature-based cosmetic ingredients as a replacement to the synthetic ones that are used at present to promote safer products for healthier and beautiful skin.

MATERIALS AND METHODS

Collection and Preparation of SamplesFresh leaves of mango cultivars (carabao, apple mango, pico, sinaging, and sipsipin) were collected from Santos Farm in San Miguel Bulacan, Philippines (15.14°N, 121.05°E). The selection of these cultivars was based on their availability at the sampling site. Mature leaves (which appear dark green in color) and young leaves (which were lighter in color) were collected from carabao and pico cultivars. Young leaves from other cultivars were not sampled due to their unavailability during the time of sampling. Both leaf types were considered to investigate which could be a better source of phenolics with better bioactivities. The leaves were immediately washed, oven-dried at 60 °C (fabricated air drier oven), ground (Retsch® knife mill GRINDOMIX GM 200), and sieved (Tyler® Mesh No. 60) to produce the mango leaves powder (MLP).

Meanwhile, the leaf extracts were prepared by mixing 0.2 g of the MLP with 5 mL 50% aqueous acetone for each cultivar. The mixtures were centrifuged to recover the phenolic-rich leaf extracts, which were determined by their total phenolic content. The extracts were diluted with water to obtain the needed concentrations in each antioxidant and enzyme inhibition assays.

Quantification of Phenolic CompoundsThe TPC was determined based on the method of Nuñez-Selles et al. (2002). An aliquot of 0.5 mL of the diluted sample extract was mixed with 0.5 mL of 1N Folin-Ciocalteu’s phenol reagent and 0.5 mL of 10% Na2CO3. After standing for 5 min, 5 mL of distilled water was added and the absorbance at λ = 720 nm was determined with the reagent solution as the blank. The TPC was computed using a standard curve with gallic acid as reference. The TPC values were expressed as gallic acid equivalents (GAE).

Some of the phenolic compounds were also quantified using Shimadzu HPLC Model Prominence with UV-Vis detector. The column used was Inertsil ODS-3 column (GL Sciences, Inc. Tokyo, Japan; 150 mm x 4.0 mm I.D., 5 µm particle diameter).The peak identification

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of each polyphenol was based on the comparison of the relative retention time, peak area percentage, and spectra data with polyphenol standards. For the mangiferin determination, the sample extracts and the standard were eluted using 2% aqueous acetic acid and methanol (60:40 v/v) at 1mL/min and λ = 254 nm while for quercetin-3-β-D-glucopyranoside and kaempferol, the sample extracts and the standards were eluted using 0.1% aqueous phosphoric acid (A) and methanol (B) at binary gradient condition: 0–50% B at 0–2.5 min, 50–100% B for 2.5–14.5 min, and 0–100% B at 14.5–16.0 min; and at λ = 369 nm. For gallic acid, the sample extracts and the standards were eluted using water-phosphoric acid-methanol (79.9:0.1:20 v/v) mobile phase system at 1 mL/min and λ = 210 nm. The samples were extracted using 100% methanol with sonication except for the gallic acid determination, which was done using 15% aqueous methanol. The extracts were filtered through a 0.20-µm Nylon membrane (Ciro Syringe Filters). The same conditions were used for their polyphenol standards [mangiferin, quercetin, and kaempferol (Sigma-Aldrich); quercetin-3-β-D-glucopyranoside (Carbosynth)].

Determination of Antioxidant PropertiesDPPH radical scavenging activity. The method used was based on that of Ribeiro et al. (2008). An aliquot of 100 µL of the samples of different concentrations (4, 8, 12, 16, and 20 µg GAE) was added each to 5 mL of 0.1mM DPPH in methanol. The solutions were stirred and left to stand for 20 min. The absorbance was determined at λ = 517 nm with distilled water as blank. The DPPH reagent in methanol served as control and DPPH inhibition was calculated using the formula:

(1)x 100% DPPH inhibition = Acontrol − Asample

(Acontrol)

ABTS radical scavenging capacity . The method used was based on that of Re et al. (1999). An aliquot of 40 µL of the samples of different concentrations (0.5, 1.0, 1.5, 2.0, and 2.5 µg GAE) were added each to 3-mL ABTS radical solution with an initial absorbance of 0.72 ± 0.05 at λ = 734 nm. The solutions were stirred, left to 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 inhibition was calculated using the same formula as in DPPH inhibition.

Copper reducing antioxidant capacity . The method used was based on that of Alpinar et al. (2009). An aliquot of 0.5 mL of the samples of different concentrations (2, 4, 6, 8, and 10 µg GAE) were added each to the mixture of 1.0 mL of 0.01M CuCl2, 1.0 mL of 1.0 M NH4CH₃COOH, and 1.0 mL of 7.5 mM neocuproine. The solutions were stirred

and left to stand for 20 min, after which 0.6 mL of water was added. The absorbance was determined at λ = 450 nm, with the reagent solution without the antioxidant as the blank. The CUPRAC was quantified by the absorbance reading, wherein greater absorbance corresponded to relatively greater reduction.

Determination of Tyrosinase InhibitionThe method used was based on that of Hapsari et al. (2012). A solution of 40 µL 5mM DOPA (3,4-dihydroxy-L-phenylalanine, Sigma D-9628), 40 µL 0.1M potassium phosphate buffer pH 6.5, an aliquot of 40 µL extract (or buffer for control), and 40 µL mushroom tyrosinase (250 units/mL, Sigma T- 3824) were added consequentially. The solution was mixed and left to stand for 15 min. The absorbance was measured at λ = 490 nm using a microplate reader, with the reagent solution without the enzyme as blank. The tyrosinase inhibition was calculated according to the formula:

(2)% inhibition = x 100Ac − As − Ab

(Ac)

where Ac is the absorbance of the control, Ab is the absorbance of the blank, and As is the absorbance of the sample. Kojic acid was used as a positive control. The inhibition of the leaf extracts from the cultivars carabao, pico, and apple mango was determined at concentrations 5, 10, 15, 20, and 25 µg GAE; it was likewise done for cultivars sinaging and sipsipin (at 6, 12, 18, 24, and 30 µg GAE) and for kojic acid (at 2, 4, 6, 8, and 10 µg).

Determination of Elastase InhibitionThe method used was based on that of Moon et al. (2010). Briefly, 40 µL of N-succinyl-(ALA)3-p-nitroanilide, 40 µL of the sample (or buffer for control), 1 mL of phosphate buffer, and 40 µL of elastase were mixed consequentially. The solution was left to stand for 20 min and 2 mL TRIS-HCl buffer was added. The absorbance at λ = 490 nm was measured, with reagent solution without enzyme as blank. Elastase inhibition was calculated with the same formula as that of tyrosinase inhibition. Tocopherol was used as a positive control.

Statistical AnalysisThe data statistical analysis was carried out using the Minitab software. One-way and two-way analyses of variance (ANOVA) were utilized for the appropriate data with Tukey’s honestly significant difference (HSD) test for further mean comparisons. The reported values were the mean of measurements from duplicate assays and pooled samples. All analyses were done at a 5% level of significance.

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RESULTS AND DISCUSSION

Quantification of Polyphenolic CompoundsTable 1 shows that the TPC of the extracts from the leaves of the different mango cultivars are significantly different, wherein the younger leaves of pico contained the greatest TPC while its mature leaves contained the least value (p < 0.05). Similarly, the young leaves of the carabao contained greater TPC than its mature leaves. This has also been observed by Barreto et al. (2008) in their study of 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 leaves. Among the samples of mature leaves, the leaves of sipsipin had the greatest TPC. Meanwhile, the mature leaves of the commonly grown carabao cultivar had greater TPC than the leaves of apple mango and pico.

It is also useful to determine the type of phenolic compounds present in the extracts to understand the reason for their bioactivities and as a basis for health claims and future studies. The result of the HPLC analysis (Table 1) reveals that the extract from mango leaves contains mangiferin, quercetin, gallic acid, and kaempferol. Studies of Barreto et al. (2008), Saleem et al. (2013), Pan et al. (2018), and Lauloo et al. (2018) also reported the presence of the said phenolic compounds in the leaves of varied mango cultivars from Brazil, China, Pakistan, and Mauritius, respectively. The data also shows that mangiferin is predominant in all cultivars, with the pico mature leaves containing the greatest amount (p < 0.05). Barreto et al.(2008) also reported that mangiferin was the predominant phenolic compound in the leaves of the mango cultivar “embrapa-141-roxa” and second predominant in the cultivar “van Dyke.” In addition, among the extracts from mature leaves, the extract from pico leaves was also richest in quercetin-3-β-D-glucopyranoside while the extract from the leaves of apple mango is richest in gallic acid and kaempferol. The extract of the young leaves

Table 1. TPC and concentration of some polyphenolic compounds found in the mature leaves of different Philippine mango varieties.

Cultivar Total phenolic content (mg GAE/g)

Phenolic contents from HPLC analysis (mg/g)

Mangiferin Gallic acid Quercetin 3-β-D-glucopyranoside Kaempferol

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

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 compounds, it is otherwise. Polyphenolic compounds are secondary metabolites produced by plants, which is confirmed to exhibit biological activities important in skincare such as antioxidant and inhibition on skin enzymes such as tyrosinase, elastase, cellulase, and hyaluronidase (Ko et al. 2011; Zilich et al. 2015). The succeeding discussion will affirm the antioxidant properties of these phenolic compounds.

Determination of Antioxidant PropertiesThe antioxidant properties of the various mango leaf extracts and the commercial antioxidant ascorbic acid were evaluated specifically for their radical scavenging and copper reducing properties at varied TPC concentrations. Their antioxidant strengths were also evaluated through their EC50 values or the effective concentration to effect 50% inhibition or reduction. The antioxidant strengths of the pure phenolic compounds were also determined.

For the free radical scavenging property, the inhibitory effect of the samples on the stable free radical DPPH and the cation radical ABTS were evaluated at varied total polyphenol contents. The DPPH inhibition assay is limited to phenolic compounds that are soluble in organic solvents especially alcohols while the ABTS inhibition assay includes both the hydrophilic and hydrophobic phenolic compounds (Ratz-Lyko et al. 2011). This suggests then that ABTS assay gives a better estimate of the overall free radical scavenging capacity. But nevertheless, DPPH is equally useful since many phenolic compounds are alcohol soluble.

Results of the two-way ANOVA and regression show a linear dose-dependent inhibition of DPPH in all the samples at all total polyphenol contents considered (p

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

Mango cultivar/standard DPPH EC50 (µg GAE)

ABTS EC50(µg GAE)

CUPRAC EC50(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-glucopyranoside** 18.31 7.67 14.05

Kaempferol** 38.88 4.24 NDRows 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

< 0.05). Figure 1 shows that the extract from the young leaves of pico had the greatest inhibitory effect on DPPH at TPC of 8–20 µg GAE, suggesting the greatest free radical scavenging activity by the alcohol-soluble phenolic compounds compared to the other samples. The extract of both young leaves of pico and carabao exhibited greater free radical scavenging capacity compared to their mature leaves. Table 2 also shows that the former are more potent free radical scavengers than the latter based on their EC50 values. But among the samples of mature leaves, the extract from sinaging leaves and carabao leaves had the greatest free radical scavenging activity at 12–20 µg GAE and are the most potent free radical scavengers. Compared to ascorbic acid, all the mango leaves extract exhibited greater potency – almost twice in scavenging free radicals.

Results of the two-way ANOVA and regression also show a linear dose-dependent inhibition of ABTS in all samples at all total polyphenol contents considered (p < 0.05). Figure 2 shows that the extract from leaves of apple mango exhibited the greatest inhibitory effect on ABTS at 1.5–2.5 µg GAE, implying the greatest free radical scavenging capacity by both the hydrophilic and hydrophobic phenolic compounds compared to the other samples. Table 2 also shows the said extract was the most potent free radical scavengers among all the samples. The table also shows that all mango leaves extracts were more potent free radical scavengers almost twice than the ascorbic acid. Meanwhile, the free radical scavenging activity of younger leaves of carabao and pico compared to that of the mature ones did not show a significant difference.

Figure 1. Inhibitory effect of different mango cultivar leaf extracts on DPPH 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).

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

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Meanwhile, in both assays, gallic acid exhibited the greatest free radical scavenging strength among the standard phenolic compounds, even greater than all the samples. However, the EC50 values of other phenolic compounds were much greater than that of the samples especially mangiferin, which was predominant in the leaves of all cultivars. The low EC50 values of the samples then reflect the synergistic effect of the polyphenolic compounds. Ko et al. (2011) also reported that gallic acid and its derivatives were the most potent free radical scavengers compared to quercetin and other phenolic compounds.

Meanwhile, CUPRAC was quantified by the measured absorbance at 450 nm. Greater absorbance corresponded to a relatively greater reduction. The results of two-way 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 of the young leaves of carabao had the greatest copper reducing power at TPC 2–10 µg GAE compared to other samples. Also, the extracts from the young leaves of the carabao and pico had greater reducing power compared to the extract from mature leaves. Among the samples of mature leaves, the extract from the leaves of sipsipin

substitute ascorbic acid as an antioxidant in cosmetic products. Meanwhile, comparing the matured leaves from different mango cultivars, the extracts from apple mango and pico leaves were better free radical scavengers while the extract from sipsipin leaves was a better copper ion reducer. Also, the extracts from sinaging and carabao leaves were better free radical scavengers when considering the organic-soluble phenolic compounds only. The free radical scavenging and reducing properties of the phenolic compounds can be attributed to their chemical structure (Zilich et al. 2015). Apart from phenolic compounds, other compounds such as chlorophyll and carotenoids that are normally found in leaves may have possibly contributed to the antioxidant activity of the extracts (Allalou et al. 2019).

Determination of Whitening PropertyThe formation of melanin, which is the skin’s dark pigment, is catalyzed by the enzyme tyrosinase. Although this pigment protects the skin from the harmful effect of ultraviolet radiation, its excessive activity can lead to severe health conditions (Shi et al. 2019). Also, inhibition of 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 4. Inhibitory effect on tyrosinase of 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).

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.

Overall, all the extracts from the different mango cultivar leaves are better free radical scavengers and copper ion reducers compared to the commercial antioxidant ascorbic acid, suggesting greater protection against skin damages due to free radicals. Thus, the extracts can potentially

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tyrosinase, suggesting the greatest whitening potential compared to the other extracts. But among the mature leaves, the extract from the leaves of apple mango exhibited the greatest inhibitory effect on tyrosinase at TPC 20–25 µg GAE. On the other hand, different TPC levels were considered for sinaging and sipsipin leaf extracts, and results show that the former had a greater inhibitory effect on tyrosinase compared to the latter at 6–39 µg GAE (p < 0.05).

Table 3 shows that among all the samples, the extract from young leaves of pico and carabao were most potent in inhibiting tyrosinase while among the extracts from the mature leaves, that of the apple mango, exhibited the 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.

have shown that natural phenolic compounds from plants exhibit inhibitory activity on the mentioned enzymes (Thring et al. 2009). In this study, the elastase inhibitory effect of the mango leaves extract was evaluated.

The result of the two-way ANOVA reveals a linear dose-dependent inhibitory effect of all mango leaves extract on elastase at all TPCs considered (p < 0.05). Figure 5 shows that the extract from the leaves of apple mango exhibited the greatest inhibitory effect at TPC 2–10 µg GAE compared to other leaves extract, suggesting the greatest anti-aging effect. Also, the extracts from young leaves of carabao and pico showed a greater inhibitory effect than the extract of their mature leaves. Their EC50 values also

Table 3. Effective inhibitory concentration on tyrosinase and elastase by the mango leaves extract and some standard.

Mango variety Tyrosinase IC50(µg GAE)

Elastase IC50(µg GAE)

Apple mango 18.92DE 2.64D

Carabao 29.14A 9.75A

Pico 22.75BC 9.27A

Sinaging 24.77B 5.74C

Sipsipin 27.62A 7.46B

Y. carabao* 16.77EF 5.69C

Y. pico* 15.54F 5.79C

Ascorbic acid** 20.62CD ND

Kojic acid** 4.39G ND

Tocopherol** ND >100

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

Determination of Anti-aging PropertyElastin and collagen are the two important structural proteins that maintain the elasticity and firmness of the skin together with hyaluronic acid. But in the presence of high levels of free radicals due to excessive ultraviolet exposure, these are broken down due to increased activities of the enzymes elastase, collagenase, and hyaluronidase. This results in loss of strength and flexibility of skin, which manifests as wrinkles (Zilich et al. 2015). Studies

Figure 5. Inhibitory effect of extract from the leaves of different mango cultivars on elastase 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).

showed that the extract from apple mango leaves was the most potent elastase inhibitor that was two times to four times more potent than the other extracts. In comparison to the standard tocopherol, the mango leaf extracts were probably 10 times more potent, since even when the concentration of the former was increased 10-fold than that of the TPC of the extracts, the inhibition had still not exceeded 50%. This suggests that the mango leaf extract was a more potent elastase inhibitor than tocopherol, suggesting a better anti-aging property. Meanwhile, it was reported that elastase is inhibited by mangiferin through non-competitive inhibition (Ochocka et al. 2017).

CONCLUSIONThe aqueous acetone extracts from leaves of carabao, pico, apple mango, sinaging, and sipsipin Philippine mango varieties exhibited significant levels of TPC. Analysis using HPLC revealed that the extracts contained the phenolic compounds mangiferin, quercetin 3-β-D-glucopyranoside, gallic acid, and kaempferol – with

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mangiferin as the predominant phenolic in all extracts. Among all the extracts from mature leaves, the extract of sipsipin leaves had the greatest TPC while the extract of pico leaves contained the greatest concentration of mangiferin and quercetin 3-β-D-glucopyranoside; the extract from apple mango leaves contained the highest level of gallic acid and kaempferol. Meanwhile, the young leaves of carabao and pico contained higher TPC, mangiferin, and gallic acid than the mature leaves.

Also, all extracts were better free radical scavengers and copper ion reducers compared to the commercial antioxidant ascorbic acid, suggesting that the extracts can potentially substitute ascorbic acid as an antioxidant in cosmetic products. Among the matured leaves, the extracts from apple mango and pico leaves were better free radical scavengers while the extract from sipsipin leaves was a better copper ion reducer. In addition, the extracts from sinaging and carabao leaves were better free radical scavengers when considering the organic-soluble phenolic compounds only. Meanwhile, the extract from apple mango leaves exhibited the greatest effect and potency in inhibiting tyrosinase and elastase among all the leaves samples and greater than the ascorbic acid and tocopherol, respectively. But kojic acid was a more potent tyrosinase inhibitor than all the extracts. Overall, the results show the great potential of the phenolic extract (using aqueous acetone) from the leaves of local mango cultivars as a cosmetic ingredient with good antioxidant, whitening, and anti-aging properties.

ACKNOWLEDGMENTSThe authors acknowledge with thanks and appreciation the funding from the Department of Agriculture – Bureau of Agricultural Research for this study and the assistance of Jessica Juarez and Roland Martinez during the experimentation.

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

APPENDIX

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