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A Study on Antioxidant and Anti Tyrosinase Activities of Coconut

  • Category: Health
  • Subcategory: Human Body
  • Topic: Skin
  • Pages: 7
  • Words: 3080
  • Published: 14 May 2019
  • Downloads: 51
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Human Skin

The human skin being the largest organ and outermost tissue of the body has multifunctional characteristics such as thermoregulation, metabolism, sensation and protection in addition to the psychological effect it imposes on humans due to variations in skin colour and type. The skin is mainly divided into three, the epidermis, dermis and hypodermis. The epidermis is the outermost layer of the skin which acts as a waterproof barrier and generates skin colour due to the presence of the colour determining pigment, melanin. The dermis lies beneath the epidermis and contains sweat glands, hair follicles and connective tissues. The hypodermis is made of fat and connective tissues.

Melanin

Skin pigmentation is caused by the formation of the pigment melanin. Melanin is biosynthesized in the basal layer of the epidermis, within melanocyte cells through a free radical mechanism. Melanocytes are transported to the outer layer of the skin, where melanin is distributed to surrounding keratinocytes. Melanin comes in two forms, lighter, yellowish pheomelanin or darker, brown eumelanin. Skin colour is determined by the type and amount of melanin synthesized and distributed on the epidermis.

Apart from expressing skin colour, melanin functions as a protective barrier against UV radiations, reactive oxygen species and scavenges toxic chemicals in the skin. Some factors that increase the activity of melanocytes are exposure to sun (UVA, UVB, and UVC), post inflammatory hyperpigmentation which favours the free radical chain reaction resulting in an uneven accumulation of melanin which may lead to skin issues such as freckles, melisma and hyperpigmentation.Hyperpigmentation is the darkening of areas of the skin due to increased melanin production, and this can be reduced if the activity of the tyrosinase enzyme is controlled.

Tyrosinase Inhibitory Activity

Acting as a key enzyme for synthesis of melanin pigments, tyrosinase (Polyphenol Oxidase) has proven to be an essential enzyme in the melanin synthesis cycle in humans and plants. It causes hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and the oxidation of L-DOPA to dopaquinone. Compounds displaying anti tyrosinase activity are capable of terminating the cycle by inhibiting the enzyme tyrosinase which is active at the mentioned conversions.

Compounds that inhibit tyrosinase tend to reduce browning of food and melanisation of human skin which gives these compounds importance in the cosmetic and food industries. Apart from synthetic agents, natural compounds can be derived from plants which show tyrosinase inhibitory activity which are inexpensive and easily accessible. Currently in the cosmetic industry compounds such as kojic acid, arbutin and azelaic acid are being used as a complexion enhancing agents which too are obtained from natural sources. However, these fail in terms of important requirements such as low toxicity, good skin penetration and high stability.

Anti Tyrosinase Assay

The anti tyrosinase assay is used to identify the tyrosinase enzyme inhibiting ability of natural products. Oyster mushroom is used to extract the tyrosinase enzyme as it is the most similar in nature to the human enzyme. In this assay we are concerned about inhibiting the action of the enzyme in the conversion of L-DOPA to Dopaquinone. On reaction of the plant extract with mushroom tyrosinase enzyme a brick red colour develops, a higher colour intensity signifies a weaker inhibitory activity. Quantitative results are obtained by measuring the absorbance values at 475 nm using the UV/Vis spectrometer.

Anti-Oxidant Activity

Antioxidants play a vital role in food preservation and human health. Preservation of food is brought about by slowing down the process of discolouration and rancidity that occurs due to oxidation caused by light, heat and some metals.3 Preventing harm to human health is carried out by acting against reactive oxygen species (ROS) which are free radicals produced by the human body as byproducts of oxygen metabolism, some examples being the superoxide radical and hydroxyl radical which are highly reactive, unstable, very toxic and damages living cells. They often lead to various pathological diseases such as cancer, atherosclerosis, cardiovascular diseases and gastrointestinal inflammatory diseases.4 When an imbalance is present between the amount of ROS generated and the amount of antioxidants available, oxidative damage could occur which leads to the above mentioned diseases.

Free radical formation is triggered by environmental factors such as pollution and radiation which cause oxidative damage to lipids, proteins and nucleic acids in cells.6 Compounds that can fight against free radicals are called antioxidants. Antioxidants have excess electrons which can be donated to free radicals to stabilize the unpaired electrons found in them.

Some important chemicals found in plants that scavenge free radicals are phenols and flavonoids (a group of polyphenolic compounds) which have the ability of donating hydrogen atoms owing to the phenolic hydroxyl groups present.

There are various experimental methods available for determination of antioxidant activity of natural products, some of which are the DPPH radical scavenging assay, Ferric Reducing Antioxidant Power assay (FRAP), ABTS free radical scavenging activity and measurement of lipid peroxidation inhibition.

DPPH Assay

The DPPH assay is a very commonly used method to identify the free radical scavenging activity of natural products as it measures the antiradical power of the compounds. The free radical used is the stable DPPH radical (DPPH = 1,1 – Diphenyl – 2 – picrylhydrazyl).

This method is used to identify the hydrogen donating abilities of compounds by observing the extent to which the purple colour of DPPH solution gets discoloured and quantitative results are obtained by using a UV/Vis spectrometer to measure the absorbance values at 517 nm.

FRAP Assay

The Ferric Reducing Antioxidant Power assay (FRAP) measures the electron donating ability of compounds, by assessing the extent to which the compound causes the ferricyanide complex to be reduced to its ferrous form.

Fe(CN)63- + e Fe(CN)64-

Fe(CN)63- which is a yellow colour solution develops a Prussian blue colour on conversion to Fe(CN)64- by the abstraction of an electron, quantitative results are obtained by measuring absorbance at 700 nm using a UV/Vis spectrometer.

Cocos nucifera

The species Cocos nucifera L. belonging to the family Arecaceae, are considerably tall trees which commonly grown in tropical countries.

The coconut fruit structurally consists of an epicarp (outer layer), mesocarp (fibrous husk), endocarp (layer surrounding the seed), and an endosperm.

Coconut contains two distinct endosperms, the liquid endosperm (nut water) and the solid kernel.8 As the nut matures, there is an increase in the nut water-holding capacity until the kernel begins to form a jelly inside the cavity of the fruit. Then, the water volume decreases as it is gradually used by the fruit to form the kernel, that is the water within the coconut shell grows into the kernel (endosperm) upon maturity.9 Studies have shown that in 180 days after pollination (DAP) the meat appears to be a thin, transparent, jelly like layer which upon maturity, at 190 DAP, becomes more thick. The meat grows to be hard and opaque at 225 DAP, along with a significant change in the chemical composition, i.e the total phenolic and flavonoid content increased from 180 to 190 DAP but decreased by the 225 DAP. This in turn resulted in a variation of the antioxidant activity of the endosperm accordingly. 10

The main phenolic compounds found in the coconut meat are gallic acid, caffeic acid, salicylic acid and p-coumaric acid.10

Coconut water is packed with antioxidants, amino acids, enzymes, B-complex vitamins, vitamin C and minerals like iron, calcium, potassium, magnesium, manganese and zinc. Coconut water is said to help rehydrate the body, lower blood pressure, promote weight loss and treat headaches.

Classification of coconut

Coconut is a hardy fruit with a green colour outer appearance, growing mostly in tropical countries. Coconut is subdivided in to 3 major varieties namely Typica, Nana and Aurantiaca, which are further classified into 15 different forms based mainly on its height and breeding patterns.11 The main differences between the major varieties are as follows:

Typica (Tall) – Forms that belong to Typica variety have a tall stature and are cross pollinating plants, they start flowering in 5-6 years after planting, and does so continuously there on, producing fruits with well spread crowns and a root bole. Navasi, Gon thembili, Ran thembili, Pora pol, Bodiri, Kamandala and Dikiri are the different forms of the variety Typica.

Nana (Dwarf) – Starts flowering in 3-4 years after planting, but bears fruits seasonally. Variety Nana has a short stature and is a self-pollinating (inbreeding) plant type. Green dwarf (pumila), yellow dwarf (eburnea), red dwarf (regia) and brown dwarf (braune) are the different forms of the variety Nana.

Aurantiaca – Having an intermediate stature and being a self-pollinating (inbreeding) variety, forms that belong to it flower in 6-7 years after planting and the ability to bear fruit is seasonal. It produces fruits with a medium spread crown and a root bole, and includes the forms King coconut (Thembili), Rathran thembili and Navasi thembili.

In this study the antioxidant and anti tyrosinase activities of the endsoperms from two forms of coconut, namely Gon thembili and King coconut (thembili) belonging to the varieties Typica and Aurantiaca respectively, at two different maturity stages have been compared. The main difference between the two forms is that one is outbreeding whereas the other is inbreeding.

King coconut is a form of coconut that is native to Sri Lanka. The nuts consist of a bright orange epicarp (outer layer), inflorescence and upper surface of leaf rachis, a creamy white mesocarp which is a fibrous husk, a very thin endocarp that surrounds the seed and a thin, hard endosperm which is the solid meat and the liquid nut water which is sweet and refreshing and is being used as an ayurvedic medicine to expel heat from the body.12

King coconut water contains nutrients such as sucrose, fructose and glucose. It also contains bioactive enzymes which helps in metabolism and digestion while the electrolytes present helps to refresh and rehydrate the body.

Gon Thembili

The form Gon thembili is also found in the Philippines and in Malaysia, it bears a nut which appears to consist of an ivory yellow epicarp and rachis of leaf. Its water however lacks flavor and cannot be consumed unlike king coconut. It has a creamy white mesocarp, a thin endocarp and a thick, hard endosperm.12

Research problem

According to literature, coconut meat has shown to have a significantly high content of active compounds such as phenols and flavanoids which leads to its efficient radical scavenging activity and inhibition of the melanin cycle.

Thembili and Gon Thembili shows similar structural features to coconut whilst belonging to the same family, Arecaceae. Therefore, Thembili and Gon Thembili kernels are tested for the bioactivites namely, antioxidant and anti tyrosinase activities.

Antioxidants helps to fight free radicals formed in the skin by preventing harmful diseases from occurring while tyrosinase inhibitors interrupt the melanin cycle thus preventing melanin formation in the skin resulting in a fairer complexion.

The search for natural alternatives for skin whitening and radical scavenging agents remain important due to the harmful effects caused by synthetic agents.

Objective of the research

Determination of antioxidant and anti tyrosinase activities of the young and matured kernels of two varieties of coconut, namely ‘Thembili’ and ‘Gon Thembili’.

Materials and Methodology

Sample collection and identification

The kernels of the two varieties of coconut, Thembili and Gon Thembili were obtained from fruits plucked from trees in the area Bokundara, Western province.

The young kernels were obtained from fruits of age 180 days and the matured kernels from fruits of 210 days from first appearance of fruit.

For identification purposes, dried leaf samples of each variety was handed over to the National Herbarium, Peradeniya.

Reagents

Hexane (C6H14), Ethyl Acetate (C4H8O2), Methanol (CH3OH) solvents were distilled before use.

Anhydrous Sodium Sulphate (Na2SO4), Disodium Hydrogen Phosphate (Na2HPO4), Potassium Dihydrogen Phosphate (KH2PO4), 1,1 – Diphenyl – 2 – picrylhydrazyl (DPPH), Butylated Hydroxy Toluene (BHT), Potassium Ferricyanide (K3[Fe(CN)6]), Ferric Chloride (FeCl3), Trichloro Acetic Acid (C2HCl3O2), L-3,4-dihydroxyphenylalanine (L-DOPA), Kojic Acid.

Instrumentation

C2 Platform shaker, Heidolph, Laborata 4000 Rotary evaporator, Hitachi U-2910 UV-Visible spectrometer, sonicator, laboratory blender, analytical balance, pH meter.

Sequential extraction procedure

The fresh kernels (young and matured) of both varieties were scraped off of the fruit, the young were cut into small pieces and the matured were cut and homogenized using a blender in order to increase surface area.6 Approximately about 100 g of each was weighed and transferred into conical flasks followed by the addition of 250 ml of distilled hexane after which they were placed in the shaker for 24 hours at 160 rpm.

The mixtures were then filtered using a cheesecloth into a beaker, and anhydrous Na2SO4 was added to the filtrate to remove any water present. The filtrate was then decanted and solvent was evaporated using a rotary evaporator at 160 rpm, 0-20 °C. The extracts were collected into cleaned, labeled, pre weighed vials and nitrogen gas was purged to remove any remaining solvent. The vials containing the sample crudes were then placed in the fridge for later use in bioassays.

The plant residue obtained after filtration, was collected and sequential extraction was carried out by the addition of 250 ml of distilled ethyl acetate followed by 250 ml of distilled methanol.

Antioxidant assays

DPPH radical scavenging assay

2.3.1.1 Preparation of DPPH solution

A 0.05 mg/ml DPPH solution was prepared by dissolving 5 mg of DPPH in 100.00 ml of distilled methanol, in a volumetric flask covered in aluminium foil. The solution was then sonicated to ensure the solid has completely dissolved and the flask was placed inside a dark cupboard.

2.3.1.2 Preparation of standard, test samples and control

Butylated Hydroxyl Toluene (BHT) was used as the positive standard.

A 1.0 mg/ml stock solution of the standard/sample was prepared by dissolving 10 mg of solid in 10 ml of methanol, from which the standard/sample series was made of the following concentrations – 0.02, 0.06, 0.10, 0.18, 0.26, 0.34 mg/ml.

A 1.50 ml volume of each different concentration of standard/sample was mixed with 0.50 ml of methanol and 2.00 ml of 0.05 mg/ml DPPH solution, in test tubes.

A control was prepared by mixing 2.00 ml of methanol and 2.00 ml of DPPH solution.

The test tubes were then placed in the dark for 10 minutes and the extent to which the sample/standard is able to scavenge DPPH radicals was measured by obtaining the absorbance at 517 nm wavelength using UV/VIS spectrometer and applying in to the following equation.13

% Scavenging Activity=[(A-B)/A]*100%

A – Absorbance for the control

B – Absorbance for the standard/sample

The standard/sample concentration series was prepared and absorbance was measured, in triplicates.

Ferric Reducing Antioxidant Power assay

2.3.2.1 Preparation of reagents

1% Potassium ferricyanide solution was prepared by dissolving a 1 g mass of solid in 100 ml of deionized water.

1% w/v Ferric chloride solution was prepared by dissolving a 1 g mass of solid in 100 ml deionized water.

10% w/v Trichloro acetic acid solution was prepared by dissolving a 10 g mass of solid in 100 ml deionized water.

Phosphate buffer of pH 6.6 was prepared by adding a 0.2 M KH2PO4 solution to 0.2 M Na2HPO4 solution. The pH was adjusted to pH 6.6 using a pH meter.

2.3.2.2 Preparation of standard and test samples

Butylated Hydroxyl Toluene (BHT) was used as the positive standard.

A 0.1 mg/ml stock solution of the standard/sample was prepared by dissolving 1 mg of solid in 10.00 ml of methanol, from which a series of the following concentrations were prepared – 0.01, 0.015, 0.02, 0.025, 0.03, 0.035 mg/ml.

A 0.25 ml volume of each different concentration was added to test tubes followed by 1.0 ml of phosphate buffer and 1.00 ml of 1% potassium ferricyanide solution.

The solution mixtures were incubated at 50 °C for 20 minutes, after which 1.25 ml of trichloro acetic acid and 0.25 ml of ferric chloride was added.

The mixtures were left to stand for 20 minutes and absorbance was measured at 700 nm wavelength using the UV/VIS spectrometer.

The standard/sample concentration series was prepared and absorbance was measured, in triplicates.13

Anti tyrosinase assay

Preparation of reagents

2.6.1.1 Preparation of Phosphate buffer solution (50 mM, pH 6.5)

A 50 mM phosphate buffer of pH 6.5 was prepared by dissolving 1.70 g of KH2PO4 solid in 250 ml of deionized water. The solution was placed in an ice bath and pH was adjusted to pH 6.5 using the pH meter.

2.6.1.2 Preparation of L-DOPA solution (12 mM)

12 mM solution of L-DOPA was prepared into a conical flask covered with aluminium foil by dissolving 50 mg of solid in 25.00 ml of deionized water.

Extraction of tyrosinase enzyme

Figure 2 1 Oyster mushroom used for anti tyrosinase assay

A 100 g mass of freshly plucked oyster mushroom was cut and transferred into a blender followed by 100 ml of 50 mM phosphate buffer. The mixture was filtered using a cheesecloth into a beaker immersed in an ice bath.

The filtrate was centrifuged for 3 mins at 30 x 100 speed, after which the supernatant was transferred into a beaker, covered and was stored in an ice bath.

Preparation of standard, test samples and control

Kojic acid was used as the positive standard.

A 4.0 mg/ml stock solution of the standard/sample was prepared by dissolving 8 mg of the solid in 2.0 ml of 50 mM phosphate buffer solution from which a series of the following concentrations were prepared – 0.80, 0.60, 0.40, 0.20, 0.10, 0.05 mg/ml.

A 0.70 ml volume of each different concentration was added to test tubes followed by 1.00 ml of tyrosinase enzyme. Similarly, a control was prepared by using 0.70 ml of phosphate buffer in place of the standard/sample. The solution mixtures were incubated at room temperature for 5 minutes after which 2.10 ml of L-DOPA was added.

The mixtures were incubated at 15 °C for 30 minutes and the amount of dopachrome produced was measured by obtaining the absorbance at 475 nm wavelength using the UV/VIS spectrometer. The percentage tyrosinase inhibition by kojic acid and test samples were then calculated by applying the absorbance values into the following equation.13

% Inhibition =[(A-B)/A]*100%

A – Absorbance for the control

B – Absorbance for the standard/sample

The standard/sample concentration series was prepared and absorbance was measured, in triplicates.

Results and discussion

Sequential extraction

Sequential extraction was carried out in solvents of varying polarity in order to extract the nonpolar, moderately polar and polar compounds of the samples into them, therefore hexane (nonpolar solvent) was used in the extraction of nonpolar compounds, ethyl acetate (medium polar solvent) for moderately polar compounds and methanol (polar solvent) for polar compounds.

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