INTRODUCTION:

The growing demand for herbal formulations with fewer side effects has created an opportunity for novel herbal formulations in cosmeceuticals. Across the board, Asian and American populations have been striving for a natural glowy, brightened skin tone. Over the last few decades, embracing the type of skin and addressing contributing factors to breakouts and hyperpigmentation have become increasingly popular. People over the age of 50 are more likely to suffer from skin hyperpigmentation, age spots, melasma, acne spots and other UV-induced skin conditions.¹

Melanin synthesis is one of the major reasons for darkening of skin. Human skin colour is one of the most distinguishable phenotypic characteristics, owing to the type and quantity of melanin synthesized within melanosomes, as well as the distribution of melanosomes within melanocytes². The melanin synthesis occurs from tyrosine amino acid which on oxidation and further polymerization give rise to melanin which is natural skin pigment and this process is known as melanogenesis. The tyrosinase enzyme plays a crucial role in the synthesis of melanin.

Among the three types of melanin, there are eumelanin, which gives black-brown tones, pheomelanin, which gives yellow-red tones, and neuromelanin, which gives white tones to the skin. Melanocytes are found in the epidermis, hair follicles, inner ear and eye are responsible for darker colour to these parts. Melanin plays a key role in defence against the harmful UV rays. However, increased melanin synthesis may cause other skin conditions like hyperpigmentation, age spots, melasma, actinic keratosis, freckles etc. Therefore, there is a need for formulations that prevent melanin synthesis.^3,4,5

The recent market is in search for non-toxicskinhypo-pigmentation agents. Although various chemical skin whitening agents are available, the major problem is to use a safer agent. According to literature studies done in the course of search for better alternatives for hydroquinone has been a targetas it may contribute to various skin diseasessuc has skin cancer paving way for herbal formulations⁶. The further challenges faced are with respect to solubility problem, penetrability and effectiveness of the formulation.

The Zebrafish model was used in this study as they are recognized as a highly dominant vertebrate model system for estimating anti-melanogenic activity since they have similar organ systems and gene sequences to humans. Various advantages of using the zebrafish model include rapid pigmentation, permeability to tiny compounds, simplicity of handling, capacity to absorb bathing media through the skin and gills⁷. Late-stage zebrafish, on the other hand, absorb the chemicals orally rather than through the skin (from 7 days after fertilisation (pdf) through adulthood). As a result, using early-stage larvae to investigate the percutaneous effects of pharmacological and/or cosmetic chemicals has another advantage, making it an excellent model for studying melanogenesis about skin whitening. Moreover, zebrafish have melanin pigments on their body surface, allowing easy observation of the effect of extract on pigmentation without the need for extensive experimental procedures.

Tyrosinase is an enzyme present in cells that assists in the synthesis of melanin pigment in the cells. Anti-tyrosinase activities of some herbal formulations produce skin whitening effect by enzyme inhibition of the enzyme tyrosinase which in-turn in hibits the synthesis of melanin. This results in skin whitening leading to better skin tone or complexion. Another mechanism involved in skin whitening is by dissolving the melanin pigment resultingin skin whitening effect of herbals and conversion of dopaquinone to DOPA^8,9. Glycyrrhiza glabra L and Citrus sinesis L. are evaluated as one such herbal used for their skin whitening activity on zebrafish models. Liquorice root (Glycerrhiza glabra L.) belonging to family Leguminosae⁹. Glycyrrhiza glabra possess antitumor, antimicrobial, antiviral, anti-inflammatory, immunoregulatory, and several other activities that contribute to the recovery and protection of the nervous, alimentary, respiratory, endocrine, and cardiovascular systems^10,11. Orange (Citrus sinensis, family Rutaceae) contains orange peel (op) which is important by product. The orange peel structure consists of an external coloured surface refer to as flavedo and an inner white spongy structure named albedo. Orange peel contain many nutritional values that make it an excellent diet for keeping body feet and healthy. Peeling have about the same nutritional value as flesh itself¹².

MATERIALS AND METHODS:

Experimental animal:

Zebrafish embryos (Danio rerio).

Chemicals and mediums:

Egg medium, Stock Solution, DMSO (Dimethyl sulfoxide), Phenylthiourea (PTU).

Instruments and Equipment:

Labomed Digital Microscope, Biological Oxygen Demand (BOD) Incubator, Aczet Ultra Sonicator, Petri dish, Beaker, Measuring Cylinder, Pipette.

Zebrafish Embryos collection and Maintenance:

The zebra fishes were procured from a pet shop in Mumbai. All fishes were bred and eggs were collected as per standard protocol. The research plan was approved by the IAEC (Ref.No: AIKTC/SoP/IAEC/2021/02).

Plant material and extraction:

Liquorice root: Plant material was purchased from Chheda Ayurved (Thane, West) and sun-dried for 1-3 days before being pulverized and sifted using a fine mesh.100g Liquorice powder was macerated kinetically in 1L of 96% ethanol for 24hrs. The filtrate produced was evaporated in a rotary evaporator at 40°C, 180 mmHg pressure and stored in a desiccator to yield Liquorice root ethanol extract. The dried extract powder was then kept in vacuum to prevent the moisture from seeping back into the extract powder.

Orangepeel: Orange peel obtained from the local market, it was then peeled and then sliced into little pieces and dried for 48hours in a hot air oven set to 50^oC.Dried orange peel was pulverized and soakedin 95% ethanolina1:3ratios before being incubated and filtered in an incubator shaker at 25^oC. The filtrate was pressure evaporated by a rotary evaporator to yield crude extract, which was stored in a -4^oC refrigerator untiluse.

Preparation of egg medium: Stock Solution for egg medium is prepared by taking 0.4gm of instant sea salt in 10ml of Distilled water. 1.5ml of this stock solution is then added to 1000ml of distilled water to prepare egg medium.

Preparation and Dilutions of Tests Extracts: The extract was prepared by dissolving 45mg of extract powder in 30ml of water and placing it in a sonicator for 5-10 minutes at 45^oC. For treatment purposes, this concentration mustbe diluted. The extracts were diluted in egg solution to 6.25, 12.5, 25, 50, 100, 200, 400, and 800 ug/ml concentrations before being transferred to labelled petri-plates. 0.1% DMSO was added to enhance permeation of theextract.

Acute Toxicity Test of extracts on zebrafish/ fish embryo toxicity (FET) OECD TG 236:

For the toxicity test, the embryonic test system was studied in a zebrafish model treated with both extracts. Test was conducted as per OECD TG No.236.Initial concentration ranges in the experiment taken (6.25-800ug/ml)n=10. The test was performed at 9 hpf^13,14.

Method:

Ten embryos were chosen and transferred into Petri-plates labelled with varying concentrations. The control group received 30ml of Egg Medium that contained only 0.1% DMSO. Incubate in a BOD incubator at 27^oC for 24 hours. Aftera five-day exposure (96hpf), the following development end points were examined on both embryos and larvae: egg coagulation, absence of heartbeat in larvae, tail separation, delayed growth, limited movement, abnormal head- trunk angle, scoliosis, and yolk sac edema. First, the experiment was performed to determine the toxicity of herbal extracts containing melanogenic inhibitors by measuring the survival rate of zebrafish at 96hpf.

Table 1: Data for determination of MTD

Group	No of zebrafish taken (n)	Concentration
Group 1- Control group	10	Egg medium only
Group 2 - Liquorice extract	10	6.25mcg, 12.5mcg, 25mcg, 50mcg, 100mcg, 200mcg, 400mcg and 800mcg
Group 3 - Orange peel extract	10	6.25mcg, 12.5mcg, 25mcg, 50mcg, 100mcg, 200mcg, 400mcg and 800mcg

Hatchability Rate Evaluation:

Ruptured chorion leads to release of zebrafish larvae from the egg (embryo stage). At this stage zebrafish embryo is regarded as hatched. Percentage hatchability was calculated after 72 hpf using formula:

No. of Hatched embryo

% Hatchability = -------------------------------------- x 100

Initial no. of embryos

Developmental abnormalities:

Morphological abnormalities such as egg coagulation, delayed growth, tail separation, scoliosis, abnormal head-trunk angle were examined and noted from the images taken at 24, 48, 72, 96 hpte.

Determination of LC10, MNLC:

At 72 hpf to 120 hpf zebrafish larvae were treated with different amounts of liquorice extract and orange peel extract to determine LC10 and MNLC. The mortality rate of zebrafish larvae was reported. Under a microscope, larvae with no heartbeat were reported dead. LC10 and MNLC were determined, and two doses for anti-melanogenic action were chosen based on MNLC.

Evaluation of Anti Melanogenic activity in zebrafish model:

Zebrafish embryos were collected from the institute and maintained in a healthy aquatic environment (26-30°C). 9-hour post fertilisation (hpf) embryos will be placed into 96 well plates (eight embryos per well), treated with the calculated dose of extract dissolved in 1% DMSO, and incubated at 28°C for 48hrs (total time: 57hpf). At 5dpf the digital image was taken through a Labomed digital microscope at a total magnification of 4 X. The melanin content of a zebrafish larvae and embryos will be observed by viewing pixel intensity by the Image J software¹⁵.

Table 2: Anti-melanogenic activity on zebrafish

Group	Treatment
Normal Control	Normal Egg Water
Standard Group	0.2 mM 1-Phenyl-2-Thiourea (PTU)
Test group-I	Eggs water + Liquorice extract (1/1.5th of MNLC, 25 ppm)
Test group-II	Eggs water + Liquorice extract (1/3th of MNLC, 12.5 ppm)
Test group-III	Eggs water + Orange peel extract (1/1.5th MNLC, 25 ppm)
Test group-IV	Eggs water + Orange peel extract (1/3th MNLC, 12.5 ppm)
Test group-V	Egg water + Orange peel extract(12.5ppm) + liquorice extract (12.5ppm)

RESULTS:

Fig. 1: The curve of Log 50 concentration vs probit of Liquorice and Orange peel extract Y=1.866*x+1.9 , R²= 0.7720

Fig 2: Representative image of the teratogenic effect of Orange peel extract

Fig. 3: Representative image of teratogenic effect of Liquorice extract

Table 3: Teratogenic malformation of zebrafish (LQ extract)

Concentration of LQ	Type of malformation
12.5 ppm	Slightly curved malformation
800 ppm	Pericardial edema
800 ppm	Coagulated embryos
200, 400, 800 ppm	Death of fish after 96hpte

Table 4: Teratogenic malformation of zebrafish (OP extract)

Concentration of OP	Type of malformation
12.5 ppm	Limited growth
50 ppm	Yolk sac edema
200, 400 ppm	Death of larvaes
800 ppm	Coagulated eggs

Fig. 4: Comparative effect of extract on % hatchability in zebrafish embryo at varying concentration. Values are expressed as mean ± SD (n =10 embryos), p < 0.0014 for LQ and p< 0.0011 for OP.

Fig 5: Comparative effects of extract on % mortality in zebrafish embryos at varying concentration values are expressed as mean± SD (n =10 embryos), p < 0.0014 for LQ and p< 0.0011 for OP.

Fig 6: Effect of different conc of LQ and OP on the melanin content of Zebrafish. Increasing conc. of Herbal extract with respect to skin pigment (melanocytes)

Fig 7: Integrated density vs concentration graph indicating reduction in melanin content

DISCUSSION:

As indicated in Figure 1, LC50 and MNLC of Liquorice and Orange peel were calculated based on zebrafish larvae mortality. MNLC and LC50 of Liquorice and orange peel were found to be 34.67mg/L and 56.23 mg/L respectively and 1/1.5th of MNLC (25ppm), 1/3rd MNLC (12.5ppm) were selected for further studies of its anti-melanogenic activity.

The abnormalities in the embryo and larvae stages were examined and noted for the teratogenic evaluation with respect to increasing doses.At lower concentrations, the zebrafish embryos have shown 100% hatchability in both LQ and OP.As the concentration of herbal extract increases and time increases (i.e hours post fertilization), structural abnormalities are observed in zebra larvae. Lower concentration has shown normal development in embryos. At higher concentrations (ppm) of LQ and OP extract, death of many embryos and zebrafishes has been observed after 96 hpte showing egg coagulation and morphological abnormalities such as delayed growth, yolk sac edema, detachment of tail, venous congestion, abnormal head trunk, and pericardial edema¹⁶. Some larvae also showed limited movement compared to the control group. The results are shown in figure - 2 and 3 and table- 3 and 4.

As shown in figure-4, At 72 hpf, the hatchability was examined showing a significant (p < 0.0014) decrease in the rate of hatchability at higher concentrations of both extracts. The % Hatchability was related to the higher concentration of extract with the presence of a slimy covering that might possibly be the cause of delayed hatching of embryos compared to that in the control groups.

The mortality rate was observed in zebrafish larvae post 72hpte on the basis of the parameters showing lethality. As the concentration of liquorice and orange peel extract increases the death rate also increases which was used in determining the maximal tolerable dose of the extract (MTD). The mortality rate also helps in determining the LD50 value which defines 50% death of the population. The concentration which shows the minimum mortality rate is used for further studies as shown in figure-5. After the calculation of MTD, studies to determine the skin whitening effect by reducing the biosynthesis of melanin pigment were done. The embryos were determined for the decreased melanin with increased concentration. The effect of LQ and OP extract on tyrosinase enzyme was determined at a concentration of 12.5 and 25 ppm respectively. At the above concentration anti melanogenic effect was observed in a group of 20 embryos with the synergistic effect having a 50:50 ratio concentration of LQ and OP extract.

Visual assessment of anti-melanogenic activity was seen using the Labomed Microscope at 4x magnification. The reduction in melanin content is determined by measuring the integrated density of images taken on a Labomed digital microscope on ImageJ software.

The synergistic activity of the herbal extract was compared using the ImageJ software by estimation of integrated density as shown in figure-6and7.The synergistic effect of herbal extracts was found to be more effective in reducing the melanin content. The phytochemicals work by inhibiting the tyrosinase enzyme which is predominantly responsible for melanin biosynthesis¹⁷.

In current research studies, we have determined that the phytochemicals present in herbal extracts of liquorice and orange peel have shown an Anti-melanogenic activity on zebra fish by inhibiting the tyrosinase enzyme which is responsible for melanin Biosynthesis. Visual parameters of skin whitening activity such as decrease pigmentation, less accumulation of melanin, whitish texture of skin indicates that the herbal extract is reducing darkness of skin.

ACKNOWLEDGEMENTS:

The author wishes to thank Dr. Ramjan Khatik , Director, AI-Kalsekar Technical Campus and Dr. Shariq Syed, Dean, School of Pharmacy, AI-Kalsekar Technical Campus for providing facilities to carry out this work.

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Received on 14.02.2023 Modified on 29.08.2023

Research J. Pharm. and Tech 2024; 17(2):659-664.

DOI: 10.52711/0974-360X.2024.00102