Anti-Elastase, Antioxidant, Total Phenolic and Total Flavonoid Content of Wuru Ketek (Myrica javanica Reinw. ex Bl.) from Tangkuban Perahu, West Java-Indonesia

Aging is a common process by which humans gradually lose skin elasticity and collagen fibers. Over the age of 20 years, aging manifestations appear as collagen content per unit area begins to decrease, there is a 1% reduction in collagen content per unit area of the skin each year.1 Skin aging is determined by a consolidation of intrinsic factors (genetics, cellular metabolism, and hormones) and extrinsic (continual light risk, pollution, ionizing radiation, chemicals, poisons). The subject of most of the research on antiaging and efforts for aesthetic-antiaging strategies related to the skin are the three main structural components of the dermis, namely collagen, elastin, and Glycosaminoglycans (GAGs).2


INTRODUCTION
Aging is a common process by which humans gradually lose skin elasticity and collagen fibers. Over the age of 20 years, aging manifestations appear as collagen content per unit area begins to decrease, there is a 1% reduction in collagen content per unit area of the skin each year. 1 Skin aging is determined by a consolidation of intrinsic factors (genetics, cellular metabolism, and hormones) and extrinsic (continual light risk, pollution, ionizing radiation, chemicals, poisons). The subject of most of the research on antiaging and efforts for aesthetic-antiaging strategies related to the skin are the three main structural components of the dermis, namely collagen, elastin, and Glycosaminoglycans (GAGs). 2 Skin elasticity and firmness are obtained in the presence of collagen and elastin in the skin layers. Collagen and elastin are broken down by the presence of MMP-1 and elastase enzymes which are increased due to excessive sun exposure. The absorbed UV light causes an increase in oxygen radical species (ROS) and induces oxidative stress. A high amount of ROS increases elastase activity. Expanded elastase enzyme activity triggers different diseases such as rheumatoid arthritis, cystic fibrosis, chronic respiratory diseases, psoriasis, deferred wound healing and premature skin aging with wrinkle formation. Therefore, antioxidants and anti-elastase can potentially be cosmetic in the fight against skin aging. 3,4 Plants from the genus Myrica such as Myrica esculenta, Myrica faya, and Myrica gale have antioxidant activities because they contain secondary metabolites such as polyphenols (such as phenolic acids and flavonoids), [5][6][7] and several research results show that polyphenol compounds and flavonoids have strong activity in inhibiting the activity of enzymes and antioxidants. [8][9][10] Some compounds contained in the genus Myrica reported to have elastase inhibitory activity including myricetin, quersetin. 11 Based on this, the Myrica javanica plant, which is also a plant from of the Myricaceae family might possible to have pharmacological activities that are no less as potential as the other Myrica genus, especially for its antiaging activities, as well as their use in making cosmetic preparations in the development of antiaging cosmetics.
The purpose of this study was to determine the potential anti-elastase and antioxidant of Myrica javanica and analysed the TPC and their TFC.  Extraction 50 gram of Myrica javanica leaves, stems and fruit of each plant was extracted by 3 times maceration method using 96% ethanol (500ml), then stirred and allowed to stand for 24 hours. The extracts were concentrated with a rotary vacuum evaporator and dried over a water bath, stored in a closed container.

Anti-elastase assay
Anti-Elastase assay was determined according to the method of Wittenauer, et al. with modifications. 12 Pre-incubation of 20 µl extract sample in DMSO solution, 130 µL buffered Tris-HCl pH 8.0 with PE (Porcine Elastase) 25 µl for 15 minutes at 25ºC and the reaction was started with the addition of SANA 20 µl substrate for 15 minutes on 96-well microplate (NUNC). Extracts and substrate solutions without enzymes are used as control. The absorbance was measured at 401 nm using a microplate reader. The inhibitory effect of the sample on antielastase activity was calculated as: After obtaining the percentage of inhibition of each concentration, then the equation y = bx+a was determined by calculating a linear regression curve where x is the concentration (µg/mL) and y is the percentage of inhibition (%). Anti-elastase activity is expressed by 50% Inhibition Concentration (IC50), which is a sample concentration that can reduce enzyme elastase by 50%. Myricetine was used as positive control.

DPPH assay
DPPH free radical scavenging activities of tested extracts were determined based on a protocol modified from Bobo-Garcia, et al. A total of 0.3 mM DPPH solution in absolute methanol was available. Samples with different concentrations in absolute methanol were evenly mixed with methanol DPPH solution to purchase a total of 200 µL. After a 30 min incubation period in room temperature and the dark condition, the absorbance was detected at 517 nm by using the microplate reader. Quercetin was used as the positive control.

IC50 antioxidant
IC50 values were calculated based on the presentation of inhibition of DPPH radicals from each concentration of the sample solution with the formula: % inhibition = (Absorbance sample/Absorbance control) x 100% After obtaining the percentage of inhibition of each concentration, then the equation y = bx+a was determined by calculating a linear regression curve where x is the concentration (µg/mL) and y is the percentage of inhibition (%). Antioxidant activity is expressed by 50% Inhibition Concentration (IC50), which is a sample concentration that can reduce DPPH radical by 50%.

FRAP assay
The FRAP radical method (Tomasina, et all) was a little modified in order to estimate the antioxidant effect of the samples. The FRAP reagent was ready freshly by mixing 10 mL of acetate buffer 300 mM, 1 mL of ferric chloride hexahydrate 20 mM dissolved in distilled water, and 1 mL of 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) 10 mM dissolved in HCl 40 mM. 20 µL sample were mixed with 280 µL FRAP reagent then incubate for 30 min at 37°C. The absorbance was measured at 593 nm with microplate reader.

Total phenolic content (TPC) assay
An amount of 20 µl of methanol extract was added 100 µl of FCR solution shaken for 60 seconds in a 96-well microplate (NUNC). Incubated at room temperature for 5 minutes and then added 80 µl sodium carbonate solution. Shake again for 1 minute and incubate at room temperature for 2 hours. Uptake was measured on a microplate reader at 720 nm. Control solution is made in the same way replaces the extract with distilled water. Calculation of total polyphenol content by means of a conversion on the calibration curve of gallic acid solution 4-24 µg/mL.

Total flavonoid content (TFC) assay
A total of 20 µl of methanol extract was added 20 µL of aluminium chloride solution and 20 µL of sodium acetate solution and 180 µL of distilled water in 96-NUNC well microplate. The mixture was incubated at room temperature for 30 minutes and measured the absorption with microplate reader at 415 nm. Calibration solution curves used quercetin in methanol (4,6,8,10,12, and 14 µg/mL) were used to calculate total flavonoid contents.

Statistical analysis
All statistics analyses were achieved using SPSS 24.0 for Windows. The data was analysed by Pearson's Correlation. It is technique for investigating the relationship between two quantitative and finding association between two variables.

Elastase assay
Elastase is the one of the main components of extracellular matrix and thus the main to loosening of skin resulting in skin aging. The observations showed anti-elastase activities in some parts extract of Myrica javanica. Anti-elastase activity of the Myrica javanica was found to be highest in LE, SE showed the second highest anti-elastase activity while FE did not show any anti-elastase activity. Elastase inhibition results for the three extracts are below when compared with myricetine. The results are shown in (Figure 1).

Antioxidant assay
DPPH assay DPPH radical scavenging activity was determined by using quercetin as standard (R 2 = 0.9908) (Figure 2). Researces showed presence of potent DPPH radical scavenging activity in all part of Myrica javanica extracts (Table 1).

FRAP assay
The anti-oxidant ability of the Myrica javanica extracts from the three different parts to scavenge radical FRAP in vitro was analysed by microplate reader. The results are shown in Table 2.

Total phenolic content (TPC) and total flavonoid content (TFC)
TPCs of Myrica javanica extracts were determined by using gallic acid as standard (R 2 = 0.9964) (Figure 3). It was highest in SE and lowest in fruit extract (Table 3). TFC were determined by using quercetin as standard (R2 = 0.997) (Figure 4). LE showed highest TFC and FE showed lowest TFC (Table 3).   TPC was showed a very strong relationship with DPPH and FRAP (Table 4). Due to the same principle that utilizes an electron-transfer mechanism, extract with high TPC contribute to express high DPPH scavenging activity. 17

CONCLUSION
Among the test samples, anti-elastase and antioxidant activity are influenced by differences in the content of compounds in the extract. In the elastase inhibition and reduction of Fe ions, the compounds that play a role are the flavonoid group. As for the inhibition of free radicals are a phenolic group. Therefore, it is hoped that further research into a main compound that has the role of inhibiting the antiaging process.

CONFLICTS OF INTEREST
The authors have not declared any conflict of interest.