Compounds with Tyrosinase Inhibition, Elastase Inhibition and DPPH Radical

Scavenging Activities from the Branches of Distylium racemosum Sieb. Et Zucc

Dahal Ram HariKyonggi University

Laboratory of Ecology of Microorganisms

ContentsAbstractIntroductionMaterials and methods Results & Discussions Conclusion

AbstractTwenty compounds (1-20) were isolated

from the ethanol extract of Distylium racemosum branches.

Compounds (8, 11, 13, 17) showed higher activities for the study of tyrosinase inhibition.

Compound 1 showed greater activity than control for the elastase inhibition test.

For the studies on DPPH radical scavenging activities, five compounds ( 11, 12, 13, 14, 15) showed higher activities compared to ascorbic acid.

IntroductionIn human epidemic layer, melanin pigment

is synthesized and protects the inner skin from damage caused by UV rays.

Over production or accumulation of melanin can induce epidermis pigmentation disorders

Freckles

Melasma

Elastin, as a collagen, is an extracellular matrix protein produced by the fibroblasts.

Inhibition of the elastase activity maintains the elasticity.

Skin aging is reported to be associated with free radical formations.

The reactive oxygen species (ROS) like, super oxides, hydroxyl, peroxide, nitric oxides cause extensive oxidative damage to cells leading to age related degenerative disease including cellular aging, mutagenesis, carcinogenesis, coronary heart disease, diabetes, neurodegeneration.

Distylium racemosum Sieb. Et Zucc (Korean: Jokoknamu) has anti elastase, antioxidation and tyrosinase inhibitory effect.

The ethanol extract of this plant led to isolation of:

1. dibenzofurans (1-4)2. Abscisic acid (5)3. 6’-O-galloylsalidroside (6)4. Catechin derivatives (7-11)5. Gallic acid derivatives (12-14)6. Tyrosol (15)7. Flavonoids (16-18)8. Lupeol (19)9. 1,2,3,6-tetragalloylglucose (20).

Materials and MethodsExtraction and isolation of plant material.

The dried branches of Distylium racemosum were cut into small pieces and extracted with 80% ethanol.

The ethanol extract were partitioned between water & n-hexane, and the aqueous fraction further portioned between ethyl acetate (EtOAc) and water, and then n-butanol and water.

The EtOAc-soluble extract were fractionated using Celite chromatography, eluting with n-hexane, methylene chloride, chloroform, diethyl ether, ethyl acetate and acetone.

Tyrosinase inhibition assayThe reaction mixture was prepared with

total 1 mL of phosphate buffer (0.1M, pH 6.8) containing various amount of the sample.

To this was added 0.5mL of L-tyrosine (0.1M) with 2100unit/mL of mushroom tyrosinase.

The test mixture (1.5mL) was incubated for 10 min at 37˚C.

Absorption at 480 nm due to the formation of dopachrome were measured.

Inhibition %=1-Abs sample – Abs

blank Abs control

X 100

Elastase inhibition assayProcine pancreatic elastase (PPE) inhibition

was done with N-Succ-(Ala)3-p-nitroanilide (SANA) as substrate.

In the reaction mixture were 0.2M Tris-HCL buffer (pH 8.0), 1.0µg/mL elastase, 0.8mM SANA as the substrate, and different inhibitors.

The absorbance were measured at 410 nm. Inhibition % = 1-BA

X 100

DPPH free radical scavenging assayThe free radical scavenging activity was

assyed using the 2,2-dipehenyl-1-picrylhydrazyl (DPPH).

Sample stock solutions (1.0 mg/mL) were diluted to final concentrations of 100, 50, 10 and 5 µg/mL in 70% ethanol or DMSO.

1.0mL sample solutions of different concentrations was added 0.5mL DPPH ethanol solution.

The absorbance at 525 nm were measured. Scavenging activity %=1-Abs sample – Abs

blank Abs control

X 100

Results and Discussions

Conclusion Inhibition of tyrosinase activity can comprise

an important method for treatment of epidermis hyperpigmentation in medicinal and cosmetic applications.

Inhibition of elastase activity in the dermis could be used as a method to maintain skin elasticity.

Free radical scavenging compounds also be used as a cosmetic ingredient to relieve skin aging.

From this experiment, solvent fractions and the isolated compounds were examined for tyrosinase inhibition, elastase inhibition and radical scavenging activities.

Some of isolated compounds exhibited strong activities on these activity tests, so this may prove to have practical values as future cosmetic ingredients.

References

Ko, R. K., Kim, G.-O., Hyun, C.-G., Jung, D. K. & Lee, N. H. (2011). Compounds with tyrosinase inhibition, elastase inhibition and DPPH radical scavenging activities from the branches of Distylium racemosum Sieb. Et Zucc. Phytother. Res. 25, 1451-1456.