Antioxidant and tyrosinase inhibitory activity of face serum containing cocoa pod husk phytosome (Theobroma cacao L.)

Reagents

Ethanol (Bratachem, Indonesia), Propylene glycol (Bratachem, Indonesia), Phosphatidylcholine (Lipoid, Switzerland), L-DOPA (Sigma-Aldrich, United State), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma-Aldrich, United State), Mushroom Tyrosinase (Sigma-Aldrich, United State), and Viscolam MAC 10 (Lamberti, Italy).

Plant material

The cocoa pod were collected from the cocoa plantation in December 2018, West Java, Indonesia. The sample was identified in Herbarium Bandung Institute of Technology Indonesia.

Preparation and phytochemical screening of cocoa pod extract

Cocoa pod husk were dried (at 40^oC) and powdered before extraction. Simplicia powder then extracted by maceration using ethanol 70% at comparison 1:3 for 24 hours (three repetitions). Rotatory vacuum evaporator was used to drying the extract (Rachmawati et al., 2018). Phytochemical screenings were conducted in plant simplicia and extract using the standard procedure to detect alkaloids, saponins, quinones, polyphenol, tannin, terpenoid, and steroid. (Franswort, 1966)

Determination total polyphenol content of cocoa pod extract

Polyphenol content was determined using the Folin-Ciocalteu Assay. 1 mL of extract solution (1,000 ppm) and gallic acid standard solution (30, 40, 50, 80, and 100 ppm) in methanol were added with 5 ml Folin-Ciocalteu solution 7.5% then preincubated in room temperature for 8 minutes. After preincubation, 4 ml of NaOH 1% solution was added to the mixture and incubated for 1 hour. The absorbance against the reagent blank was determined at 550 nm with an UV-Visible spectrophotometer. The absorbance of extract solutions was converted to polyphenol content using the regression equation of standard gallic acid. Total phenolics content was expressed as mg gallic acid equivalents (GAE) (John et al., 2014).

Antioxidant and inhibitor tyrosinase activity test of cocoa pod extract

Antioxidant activity tests were conducted by DPPH radical scavenging assay. 2 ml of extract solution in ethanol (5, 10, 15, 20, and 25 ppm) were added by 2 ml DPPH solution (60 ppm). The control solution was prepared by mixing ethanol (2 ml) and DPPH radical solution (2 ml). The mixture then mixes vigorously using vortex for 1 minute. The mixture solution incubated for 30 minutes at room temperature. The absorbance was determined at 517 nm. Percent inhibition of DPPH by the extract was then calculated and converted to inhibitory concentration 50 (IC₅₀) using regression equation (Boulanouar et al., 2017).

Inhibitor tyrosinase tests were conducted by a colorimetric enzymatic assay using a spectrophotometer. 20 μl of extract solution in dimethyl sulfoxide (6.25, 12.5, 25, 50, 100, 200, and 400 ppm) transfer to 96 well plate. 138 μl phosphate buffer (50 mM) and 2 μl tyrosinase enzyme (2,500 U/ml in phosphate buffer pH 6.5) were added to each plate. Finally, 40 μl L-DOPA (2.5 mM at phosphate buffer). The mixture then incubated for 10 minutes at 37^oC. Absorbance in each well was read at 475 nm using microplate reader. The same procedure was conducted to arbutin as a reference.

% inhibition of the samples was calculated using the following equation:

% inhibition = [(A− B) / A] ×100 %

where A is the absorbance of mixture at 475 nm without inhibitor and B is the absorbance of mixture at 475 nm with inhibitor. % inhibition value then converts to inhibitory concentration 50 (IC₅₀) using regression equation (Karim et al., 2014; Wang et al., 2018, Wuttisin et al., 2017).

Formulation and characterization of cocoa pod phytosome

Phytosome complex formation was carried out by thin-layer method by combining cocoa pod extract and phosphatidylcholine with weight comparison (1:1) in ethanol. The mixture then sonicated for 30 minutes, followed by the evaporation process using rotary vacuum evaporator at 40 rpm (40^oC), and resulting in a thin layer on the bottom of the flask. After overnight storage, the thin layer than solve by double distilled water using rotary vacuum evaporator at 45^oC. To decrease the particle size of phytosome system, it was then ultrasonicated for 120 minutes (Maryana et al., 2016; Tahir, 2016).

The phytosome suspension then centrifuged at 12,000 rpm for 60 minutes to determine the entrapment efficiency (EE). Polyphenol content of the supernatant was determined using the same procedure with the extract (Folin-Ciocalteu Assay). The EE then calculated with the following equation with Qt is the total polyphenol content in phytosome suspension and Qs is the polyphenol content in the supernatant (Tripathy et al., 2018).

Particle size distribution (mean diameter and polydispersity index) of the phytosome was measured by Particle Size Analyzer based on Dynamic Light Scattering (Beckman colter; United State).

Formulation and physical characterization of cocoa pod serum

Formula of face serum was presented in Table 2. The gel was prepared with disperse viscolam on distilled water with adding triethanolamine (TEA) using magnetic stirrer at 300 rpm. Ascorbic acid that has been dissolved in distilled water and methylparaben and propylparaben in propylene glycol were added to the previous mixture. The phytosome complex suspension was then added to the gel. The final mixture was homogenized using magnetic stirrer at 300 minutes for 10 minutes. (Surini et al., 2018).

The face serum then submitted to the cycling test. The preparations were placed at 4^oC for 24 hours then moved to 40^oC storage condition. The process was repeated in six cycles. Physical characteristics of the gel including organoleptic properties, homogeneity, pH, spreadability, viscosity, and rheological properties were compared before and after cycling test procedure. (Krongrawa et al., 2018)

Antioxidant and tyrosinase inhibitory activity tests of cocoa pod serum

The antioxidant activity test was carried out with a similar procedure for the extract. Diluted face serum (1,000 times) was submitted to the DPPH radical scavenging assay. Percent inhibition of the face serum was calculated and compared with a marketed cosmetic product containing arbutin, ascorbic acid, and hyaluronic acid as reference. The tyrosinase inhibition was carried out with a similar procedure for the extract (enzymatic colorimetric assay) to dilute face serum containing cocoa pod phytosome (5,000 times). The percent of enzyme inhibition of cocoa pod serum was compared with the marketed product (Hadalabo ultimate whitening milk, Rohto, Indonesia) (Zhao et al., 2015).

Figure 2. Linear regression curves of % inhibition. [Click here to view]

Preparation and phytochemical characterization of cocoa pod husk extract

Maceration proses using ethanol 70% was done to extract the active compound of cocoa pod husk (semi polar–polar). The yield of the extraction process is 10.25%. Phytochemical screening of herbal simplicia and extract give a positive result for alkaloids, flavonoids, saponins, terpenoids, tannins, quionone, polyphenol, monoterpene, and sesquiterpene.

Determination polyphenol content of cocoa pod husk extract

Since polyphenol is the constituent that gives antioxidant and inhibitor tyrosinase activities, polyphenol content of the extract must be determined. Our study shows that the polyphenol content of the cocoa pod extract from cocoa plantation in Indonesia is 15.56% or 155.6 mg GAE/g. This result was similar with the previous study which states that polyphenol content of cocoa pod extract is 153.51 mg GAE/g (Teboukeu et al., 2018). While, in another study, the polyphenol content of cocoa pod extract using ethanol 80% as a solvent is 49.54 mg GAE/g (Karim et al., 2014). All the results show that more polar solvent (ethanol 70%) could extract polyphenol compounds better than ethanol 80%. Previous study conducted by Karim et al. (2014) using LC-MSMS showed that polyphenol compound of cocoa pod extract contains phenolic acid (protocatechuic acid, salicylic acid), flavonols (kaempferol), and flavons (linarin, apigenin, luteolin), stilbenoid (resveratrol), and crysoplenol (terpenoid).

Antioxidant and inhibitor tyrosinase activity test of cocoa pod husk extract

Cocoa pod extract will develop as an active agent in cosmetic preparation for treatment of hyperpigmentation. Inhibitor tyrosinase and antioxidant compound could prevent melanin production by inhibiting the enzymatic and oxidative reaction, respectively. Inhibitor tyrosinase activity was conducted by a colorimetric enzymatic assay using a spectrophotometer. Tyrosinase is a key enzyme in melanin biosynthesis. Inhibition of the enzyme could decrease the production of DOPA-chrome from L-DOPA as substrate. Decrease of red color intensity of DOPA-chrome shows the inhibition of the enzyme by the extract (Chang, 2009; Di Petrillo et al., 2016). Antioxidant activity was determined by DPPH radical scavenging assay. DPPH is a stable free radical α-diphenyl-β-picrylhydrazyl. The assay based on the measurement of the scavenging capacity of antioxidants toward DPPH. The odd electron of a nitrogen atom in DPPH is reduced by receiving a hydrogen atom from antioxidants. Decrease color intensity (violet) of DPPH shows free radical inhibition (Kadare and Singh, 2011). Correlation between percent inhibition (tyrosinase inhibitor and antioxidant) with extract concentration shown at Figure 2. Our study shows that cocoa pod extract has antioxidant and tyrosinase inhibitory activities due to high polyphenol content (Table 1). Study conducted by Karim et al. (2014) shows that cocoa pod extract (ethanol 80%) have antioxidant and tyrosinase inhibitory activities with IC50 26.1 ppm and 357.95 ppm, respectively. Our recent study shows stronger antioxidant and tyrosinase inhibitory activities of cocoa pod extract. Based on our analysis, the difference of solvent used at the extraction process causing differences of extract activity. Presence of phenolic acid, citric acid, malic acid, and other polyphenols in cocoa pod husk extract contribute very strong antioxidant activity of it (IC50 < 50 ppm). Stilbenoids, Flavonols, Flavanols, and phenolic acid in cocoa pod extracts contributed to tyrosinase inhibitory activity (Karim et al., 2014). The activity of the extract was weaker compared with ascorbic acid and alfa-arbutin due to the purity of extract is lower than references as pure active compounds.

Table 1. Inhibitory concentration (IC50) of cocoa pod extract. [Click here to view]

Table 2. Physical characterization of cocoa pod phytosome. [Click here to view]

Figure 3. Particle size analyzer result. [Click here to view]

Preparation and evaluation of cocoa pod phytosome

Phytosome formulation of the extract was carried out to improve the skin penetration of the active compounds (polyphenol); that is the reason causing phytosome technology to be widely used in cosmetics preparation (Awasthi et al., 2011). In phytosome complex, phytoconstituents are surrounded by phospholipid molecules. Since phosphatidylcholine, being an important component of the cell membrane, phytosome complex regulates the physiology of skin composition without damaging the epidermis and enhance skin penetration (Mazumder et al, 2016). Physical characteristic of phytosome was shown in Table 2. Result shows that EE of polyphenol compound in phytosome complex was 90.2%, which means the majority of polyphenol constituent has entrapped in phytosome complex. The result of particle size analysis shows that cocoa pod phytosome was categorized as a fine particle (100–2,500 nm) with narrow particle size distribution since PDI < 0.5 (Fig. 3) (Shailaja and Sugunthan, 2016).

Preparation and evaluation of cocoa pod phytosome serum

For topical application, cocoa pod phytosome was then formulated as face serum using viscolam as a gelling agent. Formulation of the serum was described in Table 3. Physical stability of cocoa pod serum was determined by the cycling test. Physical characteristics of the serum were compared before and after cycling test period. The results show that cocoa pod serum has good physical stability at the change of storage temperature (Table 4). The preparation revealed non-Newtonian behavior, pseudoplastic (Fig. 4). Pseudoplastic is a desirable property of topical semisolid preparation since it should thin during application and thicken otherwise. (Shahin et al., 2011)

Tyrosinase inhibitory and antioxidant activities test of cocoa pod phytsome serum

Activity test was conducted to cocoa pod serum and compared with the marketed product (Hadalabo ultimate whitening milk, Rohto, Indonesia) containing arbutin, vitamin C, and hyaluronic acid. The result shows that the face serum of cocoa pod phytosome has antioxidant and tyrosinase inhibitory activities that relatively equal with the marketed product (Fig. 5). The cocoa pod face serum contains ascorbic acid that also contributes to antioxidant and tyrosinase inhibitory activities. From this final result, it can be concluded that cocoa pod extract has very potential to develop as an active ingredient of a cosmetic product. For the future, in vivo anti melanogenic activity will be done to the extract and the formulated serum.

Table 3. Formulation of face serum. [Click here to view]

Table 4. Result of cocoa pod serum physical evaluation. [Click here to view]

Figure 4. Rheologycal properties of cocoa pod serum. [Click here to view]

Figure 5. Cocoa pod serum activities. [Click here to view]