Agaricus bisporus Mushrooms

Amongst the candidates for tyrosinase inhibitors are mushrooms. Mushrooms have been long known to be

• Rich in bioactive agents, such as phenolic compounds

• Appreciated for their antioxidant activity, primarily free radical scavenging effects

• Valuable natural sources for pharmaceutical and nutritional purposes

Agaricus bisporus, commonly known: the white button mushroom, are the most conventional, cultivated species of mushrooms.

UVR and Melanogenesis

Research of NASA scientists have evidenced that ultraviolet radiation (UVR) reaching the Earth’s surface has increased significantly over the past few decades. UVR’s wavelengths are shorter and contain more energy, relative to visible light, enabling UVR to penetrate the human skin. Moderate exposure to UVR is required by humans to promote conversion of 7-dehydrocholesterol => provitamin D => vitamin D.

However, excessive amounts of UVR may may trigger

1.  Overproduction of the reactive oxygen species (ROS)

2.  DNA damage

This damage then cause adverse effects, such as abnormal transformation of skin cells and immunosuppression towards malignant cells. The typical phenotypic changes vary from benign changes such as wrinkles to fatal conditions such as blindness and the development of tumors and/or cancerous cells.

In response to UV radiation, the human skin employs endogenous mechanisms to prevent and/or cure the radiative effects. Examples of the mechanisms include apoptosis of mutated cells, overexpression of antioxidant genes, rapid repair of DNA, thickening of the epidermis and, most importantly, increased melanogenesis.

Melanogenesis = a biosynthetic pathway responsible for the production of melanin in the epidermal layer

Melanin produced = UVR absorbent, antioxidant, ROS scavenger. Thus, increased melanogenesis is essential to photoprotection against the chronic effects of UVR including photo-aging, photodamage and photo-carcinogenesis (skin cancer).

Overaccumulation of melanin however is also unfavourable as pheomelanin is especially vulnerable to photodegradation, generating hydrogen peroxide, superoxide anions hence causes mutations in melanocytes, hyperpigmentation, neurodegeneration and eventually may induce the apoptosis of cells. Tight regulation of skin melanin levels is thus crucial since both abnormally high and low melanin levels pose a potential danger for human well-being.

Tyrosinase Inhibition

Regulation of melanin levels can be achieved indirectly through adjustments of tyrosinase activity. Tyrosinase is a rate-limiting enzyme responsible for the first step of melanin synthesis - conversion of tyrosine into L- 3,4-dihydroxyphenylalanine (DOPA). Overproduction of melanin is almost always due to overexpression of tyrosinase. The search for tyrosinase inhibitors is thus of utmost importance and has been an attractive endeavour for the cosmetics, food and medical industry.

Sample Preparation and Extraction Procedure

1. Finely chop Agaricus bisporus

2. Freeze dry for 72 hours

3. Grind using dry spice grinder into fine powder form

4. 25g of freeze-dried powder extracted with 250g ethanol absolute

5. Maceration left to sit for 24 hours at RT

6. Supernatant filtered through Whatman filter paper in vacuum pump

7. Rotary evaporator used to remove ethanol from recovered supernatant (after filtration)

SPF Test

The absorption characteristics of the sunscreen agents against UVB (290-320 nm) can be determined in vitro by utilizing UV spectrophotometry with the equation:

SPFspectrophotometric: 

CF * UVB spectrum (290-320nm) EE(λ) * I (λ) * Abs (λ)

where: EE (l) – erythemal effect spectrum; I (l) – solar intensity spectrum; Abs (l)- absorbance of sunscreen product ; CF – correction factor (= 10).

STEPS:

1. Sample was dissolved in ethanol to a concentration of 5,000 ppm

2. The absorbance of the sample solution was then read at wavelengths 290 - 400 nm with 5 nm interval. Ethanol was used as the blank, negative control

3. To obtain the SPF value, the absorbances of the sample at various wavelengths are obtained and then plugged into the equation. Their summation gives the total SPF value.

Total Phenoliv and Flavonoid Content

Total phenolic content in A.bisporus extracts determined using the Folin-Ciocalteu reagent.

• Standard curve = Folin-Ciocalteu stock solution + gallic acid + Na2CO3 solution

• Absorbance of sample measured against Folin reagent, by spectrophotometer at 765 nm wavelength.

Phenolic compounds are indicative of and closely related with antioxidant activities.

A.bisporus = ~1143 mg GAE/100g.

Flavonoids are renowned for a broad spectrum of favourable health-promoting benefits. This can be attributed to their “anti-oxidative, anti-inflammatory, anti-mutagenic and anti-carcinogenic properties.”

The determination of the total flavonoid content in Agaricus bisporus was carried out with ethanol extracted samples using quercetin as standard.

Total flavonoid content of A.bisporus mushrooms = 0.759 mgQE/g

Mushrooms do not contain flavonoids and cannot actually synthesize flavonoids as they lack the required enzymes.

DPPH Radical Scavenging Assay

Aim: investigate the free radical scavenging activity of antioxidants towards the purple-coloured DPPH.

Samples were prepared by serial-dilution method to yield 2, 4, 6, 8, and 10 ppm concentrations.

Antioxidant assay is performed by adding combining 0.2 mM DPPH + ethanol (et-OH).

The absorbance of each sample and control was measured against 2mL of ethanolic DPPH as blank on spectrophotometer at wavelength 517nm.

%RSA = [A(control) - A (sample)] / A (control) *100

The assay relies on measuring the absorbance of the naturally purple-coloured DPPH with respect to how much the sample can quench it, rendering the solution colourless. The antioxidant compound donates a hydrogen atom to the DPPH radical  solution which is then reduced. The absorption of the hydrogen by the antioxidant, namely the A bisporus given that it contains the bioactive compounds for the mushroom to act as an antioxidant, is in stoichiometric ratios with respect to the level of reduction and the DPPH remaining after the radical-scavenging effect. Upon measurement following a set amount of time, the absorption/reduction of the hydrogen is inversely related with the radical scavenging capability of the antioxidant

IC50 value = the concentration required to achieve 50% RSA => A bisporus ethanolic extract presents IC50 = ~ 5456 μg/mL.

Tyrosinase Inhibition Assay

1. Sample was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10,000 ppm and then diluted in potassium phosphate buffer (50 mM, pH 6.8) to 0.05 ppm.

2. Samples with different concentration (70 µl) was incubated with 30 µl of the tyrosinase enzyme (333 U/ml in phosphate buffer, pH 6.8) for 5 min, followed by the addition of 2 mM of the substrate, L-tyrosine (110 µl) and further incubated for 30 min at RT, ensuring that effect of light is limited.

3. Kojic acid was used as positive control. Absorbance reading was performed at 475nm. Percentage of tyrosinase inhibition calculated using equation:

%inhibition: [A(control) - A(sample)} / A (control) *100

Tyrosinase is an oxidase comprised of copper and an enzyme that catalyses the synthesis of melanin. tyrosine => dihydrophenylalanine (DOPA) => dopaquinone.

Melanin inherently is a natural mechanism of the human body however the over-secretion of melanin from melanocytes is what becomes problematic, caused by external factors such as exposure to UVR and release of α-melanocyte-stimulating hormones.

The overexpression of tyrosinase therefore leads to the overproduction of melanin associated with HYPERPIGMENTATION, FRECKLES, MELANOMA. Kojic Acid, a synthetic tyrosinase inhibitor widely utilized in skin-whitening agents, is used as a positive control in the current study.

However, these synthetic compounds are associated with side effects such as ERYTHEMA or CONTACT DERMATITIS

=> increasing interest towards natural, biocompatible agents. Through this study, discovered that Agaricus bisporus ethanolic extract exhibits an exceptionally high tyrosinase inhibiting activity. IC50 of 3.68 ppm or μg/mL.

Qualitative Phytochemical Screening

The phytochemical screening is a qualitative test of the chemical contents of plants in order to determine the class of compounds contained in the extracts derived from plants.

The screening analyses the presence of secondary metabolites like alkaloids, flavonoids, terpenes, tannins, saponins, terpenoids, glycosides, quinones and anthraquinones in the extracts. The extraction was carried out with absolute ethanol. The ethanolic extract in our work shows only the presence of terpenoid

• The presence of terpenoid indicates that A. bisporus has another potential application in the field of therapeutics medicine. Indeed, there appears to be renewed interest in the study of mushrooms as a natural source of terpenoids. Research suggests that terpenoids are effective for therapeutic treatment of neurodegenerative conditions and as antiviral agents.

• The presence of terpenoid could also be bolstering the high tyrosinase inhibition activity in the Agaricus bisporus. Both phenolic and terpenoid compounds have been found to directly contribute to tyrosinase inhibiting activities.

Conclusion

In conclusion, the common button mushroom, species Agaricus bisporus has exhibited viable as a

1. Minor antioxidant agent with %RSA IC50 of ~5456 μg/mL

2. Moderate sun protecting agent with SPF value of ~ 5.355 at 5000 ppm concentration, prior to supplementation with chemical UV filters such as titanium oxide or zinc

3. Exceptionally high tyrosinase inhibiting capability of the Agaricus bisporus ethanolic extract with IC50 of merely 3.69 μg/mL. This is seen to be likely attributable to the phenolic contained in the mushrooms, found to have a value of ~ 1143 mg GAE/100g and possibly partially attributable to the terpenoid present in our sample

Overall, through this assessment, it is clear that Agaricus bisporus presents a really exciting outlook for the cosmetic industry and the field of pharmaceuticals where the search for natural compounds over synthetic products has been burgeoning with the rising concerns and prevalent health-issues related with UV radiation and exposure to free-radicals.