Phytochemical Screening And Tyrosinase Inhibition Activity Of Leaves Cassia siamea L

Cassia siamea L. has been used traditionally as medicine because it has chemical constituents such as saponins, anthraquinones, alkaloids, flavonoids, tannins, terpenoids and steroids. Indonesia, which is located in a tropical region with high temperatures and ultraviolet radiation, can cause skin disorders such as hyperpigmentation due to excess melanin synthesis. Tyrosinase enzymes can prevent or inhibit melanin formation. The purpose of this study was to determine the chemical content of Cassia siamea L. leaves extract with various solvents and the inhibitory activity of tyrosinase enzymes. In this study, the leaves of Cassia siamea L. were macerated with methanol as a solvent. Dry methanol extract was fractionated using the liquid-liquid method using aquadest, butanol and chloroform as solvents. The four dried extracts were then phytochemical screening to determine their chemical content. Tyrosinase inhibition test was carried out in vitro with L-Dopa as a substrate using ELISA plate well reader at concentrations of 100, 1000 and 10000 ppm with three repetitions (triplo). Methanol extract had percent inhibition of 19.993±1.125%, 28.984±0.624%, 57.164±0.623%, respectively; butanol fraction 25.914±0.541%, 32.566±0.767%, 52.120±1.616%; chloroform fraction 19.920±0.730%, 26.425±0.937%, 62.865±0.167% and aquadest fraction 23.830±0.879%, 30.885±0.778%, 67.471±0.352%. Kojic acid had tyrosinase activity and acts more active than the other extract and fraction with 91.155±0.228% at 500 ppm. The aquadest fraction had the highest percent inhibition (67.471 ± 0.352%) at 10000 ppm which was thought to be due to the presence of alkaloids, tannins, flavonoids, steroids and terpenoids from the results of the phytochemical screening test.


INTRODUCTION
Indonesia is a country located in a tropical region which is famous for its high temperatures and ultraviolet (UV) radiation at the highest level. Exposure to UV rays for a long time with frequent frequency can cause skin irritation such as darkening of the skin color. The brown color of the skin is the result of excessive melanin formation 1 . Hyperpigmentation is the most common pigment disorder of the skin. An increase in melanin synthesis or an uneven distribution of melanin can cause hyperpigmentation or patches of skin. The process of forming melanin compounds (melanogenesis) occurs with the help of biocatalysts, especially the enzyme tyrosinase 2 .
In the melanogenesis process, tyrosinase acts as a catalyst in two different reactions, namely the hydroxylation of tyrosine to dihydroxy-phenylalanine (L-DOPA) and the oxidation of L-DOPA to dopakuinone. Dopakuinone compounds have very high reactivity so they can polymerize spontaneously to form dopaque which then becomes melanin 3 . Tyrosinase in skin tissue is activated by solar UV radiation, thereby accelerating the process of melanin production. Tyrosinase enzymes can prevent or inhibit melanin formation 1 .
One way to lighten skin color is by inhibiting melanin formation through inhibition of the tyrosinase enzyme. Bleach acts as an inhibitor of melanin production and is known as a competitive tyrosinase inhibitor. Various tyrosinase inhibitors are found in cosmetic ingredients including hyaluronic, arbutin, kojic acid, mercury, and hydroquinone. This compound has a very large bleaching power, but is dangerous because it is carcinogenic 4 . The discovery of natural ingredients that are safe for human health, one of which is looking for tyrosinase inhibitors found in nature.
Cassia siamea L. has been used traditionally as medicine and in previous studies had an IC50 antioxidant activity of 144.12 µg/mL 5 . Cassia siamea L. potential as a medicinal substance is thought to have chemical constituents such as saponins, anthraquinones, alkaloids, flavonoids, tannins, terpenoids and steroids 6 . The total phenolic content (TPC) that has been carried out in previous studies was 88.5 mgGAE/g, while the total flavonoids content (TPC) obtained were 35.6 mgRE/g 7 . Flavonoids with their antioxidant effects play a role in inhibiting the tyrosinase enzyme. The purpose of this study was to determine the chemical content of Cassia siamea L. leaves extract with various solvents and the inhibitory activity of tyrosinase enzymes.

Sample Preparation, Extraction, and Fractionation
Cassia siamea L.dry leaves was ground to obtain 500 gram sample powder to the extraction process. Methanol 75% 5 L was used as the solvent in the maceration extraction of the samples for 3x24 hours. The crude methanol extracts were then dried using a rotary evaporator. Liquid-liquid fractionation was conducted using aquadest, buthanol, chlroform to the methanol extract to obtain fractions with different polarities then dried using a rotary evaporator. The four dried extracts were then phytochemical screening to determine their chemical content and tyrosinase inhibition assay.

Phytochemical Screening
Alkaloids 0.25 grams of sample was added with 0.5 mL of 2N hydrochloric acid and 4.5 mL of distilled water, heated over a water bath for 2 minutes, cooled and then filtered. The filtrate as much as 3 drops was transferred into three test tubes. To tube 1 are added 2 drops of Wagner solution. If there is a brown to black sediment, the powder contains alkaloids. Tube 2 is added with 2 drops of Mayer reagent. If a white precipitate is formed, the powder contains alkaloids. Tube 3 is added 2 drops of Dragendorff reagent. If a brown sediment is formed, the attack contains alkaloids.
Flavonoids 0.5 gram of sample was extracted with 50 mL of hot water, then filtered with filter paper until the filtrate was obtained. 2.5 mL of the filtrate is poured into the test tube. The filtrate was added with 0.5 mL of 5% NaNO2 solution and 0.5 mL of 10% AlCl3 and then shaken. 1 mL of 1N NaOH was added slowly into the filtrate through the test tube wall. The presence of flavonoids indicates a red or orange color Saponins 0.5 gram of sample was extracted with 100 mL of hot water, then filtered with filter paper until the filtrate was obtained. 5 mL of the filtrate is poured into a test tube then shaken vigorously for 10 seconds. If a foam is formed that is stable for not less than 10 minutes and does not disappear with the addition of 1 drop of 2 N hydrochloric acid, it indicates the presence of saponins to diphenolase activity. The assay was carried out using ELISA plate well reader with tyrosinase enzyme, L-DOPA as the substrates, phosfat buffer pH 6.5, with three repetitions (triplo) 17   Phytochemical screening was carried out using the previous method 9 . The most important types of phytochemicals found in extract and fraction of leaves of Cassia siamea L. are alkaloids, flavonoids, tannins, steroids, terpenoids positively detected during phytochemical confirmation as shown in Table   2. Tyrosinase inhibitory activity was evaluated to measure the ability of samples as lightening agents. Tyrosinase inhibition is correlated with the decrease of melanogenesis on the skin since this enzyme is responsible for hyperpigmentation in humans 10 . In this study, using the L-DOPA substrate because this substrate with the tyrosinase enzyme will produce dopaque which can then form melanin to prevent it by tyrosinase inhibitors. The formation of dopaid products is characterized by the appearance of a brown color which can cause inhibition of the substrate-tyrosinase reaction, resulting in reduced dopaque production which is indicated by a decrease in the intensity of the brown color. This dopaque formation uses a microplate reader using 96 wells microplates at a wavelength of 492 nm 11 .

DISCUSSION
Each phytochemical showed potency towards some biological action; for example, flavonoids with their antioxidant effects play a role in inhibiting the tyrosinase enzyme 12 . Tannins also have the ability to act as anti-tyrosinase because they inhibit the biosynthesis process of melanin so that an increase in melanin production does not occur after exposure to UVB rays 13 . Inhibitors of other classes, such as terpenes, steroids, alkaloids, have also been reported in the tyrosinase inhibitory activity 14 .
Investigation of tyrosinase inhibitory activity showed that aquadest fraction had the highest percent inhibition (67.471 ± 0.352% ) at 10000 ppm compared to the other extract and fraction. The presence of alkaloids, tannins, flavonoids, steroids and terpenoids from the results of the phytochemical screening test of aquadest fraction play a role in inhibiting the tyrosinase enzyme. Kojic acid has tyrosinase activity and acts more active than the other extract and fraction with 91.155±0.228% at 500 ppm. This was affected by the extract conditions, which was a crude extract and fraction which had not been purified, so it was suspected there were other compounds that did not act as tyrosinase inhibitors Kojic acid has an inhibitory effect of tyrosinase enzyme and the greatest stability in cosmetic products 15 , but it is carcinogenic and its use in high concentration can damage the skin 16 .