INTRODUCTION:

Kajal is worn for a variety of reasons including tradition, beautification, to ward off the “evil eye,” the widespread belief that kajal is medically beneficial for the eyes. Kajal is reported for improvement of vision, strengthening and keeping the eyes healthier. One of the most striking properties of kohl has been observed effect of UV rays emerging fromthe sun and dust of the desert^1.Firstly, blushes in Arabia were made of antimony trisulfide and the ore stibnite wascalled ‘ethmid’.

As this was scarce and expensive, it was slowly replaced over the years by galena (lead sulfide) which has the same grey-black color and shiny appearance like stibnite². Leadintoxicationfollowing operation of camouflage performing from galena “camouflage- gravestone” has been a majorarea of review. On the contrary there are studies which are of a view thatlead is not absorbed through trans corneal route and thusshould not be linked or blame for increased blood leadlevel and lead poisoning after its application³. However kohl is unapproved for cosmetic use in the United States since there have been reports linking the use ofkohl with lead poisoning in children⁴. The herbs used for formulation of herbal Kajal were Ocimum sanctum, commonly known as holy basil, tulasi or tulsi, is an aromatic perennial plant in the family Lamiaceae. It's native to the Indian key and wide as a cultivated factory throughout the Southeast Asian tropics^5,6. Some of the phytochemical ingredients of tulsi are oleanolic acid, ursolic acid, rosmarinic acid, eugenol⁷, carvacrol, linalool8, β-caryophyllene^8,9. Tulsiessential canvas consists substantially of eugenol (70%) β-elemene (110%), β-caryophyllene (8%) and germacrene (2%), with the balancebeingmade up of colorfultrace composites, substantially terpenes. Eugenol (l-hydroxy-2-me-thoxy-4-allylbenzene), the active constituent present in Ocimum sanctum L., has been plant to belargelyresponsible for the remedial capabilities of Tulsi¹⁰. The leaf juice of Ocimum sanctum L. along with Triphala is used in Ayurvedic eye drop preparations recommended for glau-coma, cataract, chronic conjunctivitis and other painful eye diseases¹¹.

Mentha piperita Linn, commonly known as peppermint,brandy mint or candy mint (Vilayatipodina in Hindi), be-longing to family lamiaceae, is a popular medicinal plant in many traditional systems of medicines. The principal active constituents of Mentha piperita are the essential oils, which comprise about 1% of the herb¹². The oils are domi-nated by monoterpenes, mainly menthol, menthone, and their derivatives (e.g., isomenthone, neomenthol, acetyl-menthol, pulegone). These essential oils dilate peripheral blood vessels and inhibit bacteria. Its oils especially men-thol have a broad spectrum antibacterial activity since Gram +ve and Gram -ve bacteria were found susceptible to the oils¹³.

Almond oil is the fatty oil obtained from cold expression of ripe seeds of Prunuscommunis belonging to family Rosaceae. It finds mention in many traditional practices for treating eye diseases¹⁴.

Yastimadhu: consists of dried, unpeeled, stolon and root of GlycyrrhizaglabraLinn., Fabaceae, a tall perennial herb, upto 2 meters high, distributed in the sub-tropical and warm temperate regions of the world. Further Different workers have stated that Yastimadhu has anti-inflammatory, strengthening and regenerative properties. The anti-inflammatory property is credited to cortisone- such like substance present in the medicine that helps reduction in inflammation. It was noticed that Kemicitine succinate, in spite of its antibacterial property, had not been able to show better results than Yastimadhu in the cases studied. Further it has also been observed that Daruharidra (BerberisaristataDC)¹⁵ and Yastimadhu (Glycyrrhizavglabra Linn.) appeared to show beneficial effects in removing the acute inflammatory features better than Kemictine succinate¹⁶

Anethumgraveolens L. (dill) dill has beenused in ayurvedic drugs since ancienttimes and it's a popular condiment extensively used as a spice and also yields essential canvas. There are colorful unpredictable factors of dill seeds and condiment; carvonebeing the predominant odorant of dill seed and α-phellandrene, limonene, dill ether, myristicin are the most important odorants of dill condiment. Ayurvedic parcels of shatapushpa are katutikta rasa, usnavirya, katuvipaka, laghu, tiksna and snigdhagunas. It cures‘vata’,‘kapha’, ulcers, abdominal pains, eye conditions and uterine pains¹⁷.

Castor oil is a vegetable oilpressed from castor beans. The name presumably comes from its use as a relief for castoreum. Castor canvas is a colourless to veritably pale unheroic liquid with a distincttaste and odor. Castor canvas encourages lipid product in the eyes and inhibits the evaporation of gashes. It contains a range of important adipose acids and other antioxidants that can profit your eyes, and is antibacterial, antifungal and antiviral^18,19.

Vitamin E:

Vitamin E is found naturally in some foods, added to others, and available as a dietary supplement.Several studieshaveshown substantiation of the positive part vitamin E can play in perfecting vision. Vitamin E is believed to cover eyecells from unstable motes calledfree revolutionaries, which break down healthyeye towel. This is what may lead to the conformation of cataracts or age- related macular degeneration (AMD).

Rose canvas (rose incense, balm of rose, balm of roses or rosesubstance) is the essentialcanvasuprooted from the petals of colorfultypes of rose. Rose aromas are uprooted through brume distillation, while rose absolutes are attained through solvent birth, the absolutebeingusedmoregenerally in perfumery. Indeed with their highprice and the arrival of organic conflation, rosecanvases are stillmaybe the most extensively used essential canvas in perfumery.

MATERIALS AND METHODS:

Materials: Formulation of Herbal Kajal:

The product was formulated by using following ingredients:

Coconut oil, Almond oil, Castor oil, Mentha extract, Ocimum sanctum extract, Yasthimadhu, Vitamin E, Rose oil, Anethrumgraveolens, Vanspati ghee (Q.S) (Table 1).

Methods:

Method 1:

The coconut oil and almond oil was burnt and its carbon black was collected. The hydroalcoholic extraction of fresh leaves of Ocimumsantum, Mentha piperita and rhizomes of Yasthimadhu was done by maceration method. Plant extracts were incorporated dropwise in the coconut oil, almond oil, castor oil, rose oil and vitamin E. The seeds of Anethumgraveolens was also used in small quantities during burning. Lastly vegetable ghee was used in desired proportion to make the formulation viscous.

Method 2:

The coconut oil and almond oil was burnt and its carbon black was collected. The hydro alcoholic extraction of fresh leaves of Ocimumsantum, Mentha piperitaand rhizomes of Yasthimadhu was done by microwave assisted extraction. Plant extracts were incorporated dropwise in the coconut oil, almond oil, castor oil, rose oil and vitamin E. The seeds of Anethumgraveolens was also used in small quantities during burning. Lastly vegetable ghee was used in desired proportion to make the formulation viscous.

Table 1 Composition of herbal kajal:

Sr No	Ingredient	Quantity
1	Coconut oil soot	1gm
2	Almond oil soot	3gm
3	Ocimumsantum	5 drops
4	Mentha piperita	5 drops
5	Yasthimadhu	5 drops
6	Anethumgraveolens	5 drops
7	Castor oil	3 drops
8	Rose oil	2 drops
9	Vit E	2 drops
10	Vegetable ghee	Qs
Total		7gm

Evaluation of Herbal Kajal:

Evaluation of Kajal Products: (Table 2)

Both the formulated products were evaluated for Physical evaluation, pH determination and viscosity determination.

Physical evaluation parameters were color, odor, texture and consistency. PH was determined by using a pH meter. Viscosity was determined by using a Brookfield Viscometer.

Table 2 Evaluation of herbal kajal

Sr No	Parameter	MAEK	TMEK
1	Physical Evaluation Color Odor Texture Consistency	shiny black color Characteristic non-gritty and smooth Semisolid	black color Characteristic non-gritty and less smooth semisolid
2	PH	7.2	7.2
3	Viscosity	6.2	6.0

Evaluation of Base: (Table 3)

Vegetable ghee was used as a base and was evaluated for acid, saponification, and ester values, vegetable ghee as per I.P. 1996.

Acid Value: Acid value = 5.61 × n/w, where n is the number of ML of 0.1 M KOH required and w is the weight in grams of the solvent

Saponification Value: Saponification value = 28.05 (b − a)/w, where w is the weight in grams of the substance, b is the blank solution reading, and a is the sample solution reading

Ester value equals the saponification value minus the acid value.

Table 3: Evaluation of Base

Sr No	Parameter	Vegetable ghee
1	Acid value	1.122 (n=0.4; w=2.0)
2	Saponification value	269.28 (b=23; a=3.8; w = 2.0)
3	Ester value	268.158

Microbial Contamination:

Microbial tests were performed to check the quality of the kajal products to determine the microbial, total fungal, and Escherichia coli countsby acylindrical plate method. Nutrient agar (for total bacterial count), MacConkey agar (for total E. coli count), and Sabouraud dextrose agar (for fungal and yeast count) were incubated at 37°C overnight, 43°C overnight, and 25°C for 4 days, respectively. Finally, the growth was observed and, by a colony counter, counted in per volume plated. (Table 4)

Table 4: Antimicrobial activity

Sr No	Plant material	Dose (ug/cup)	*Zone of inhibition (diameter in mm)**
			Gram +ve				Gram -ve
			S.p.	*B.c*	*B.p*	*S.a*	*E.c*	*E.a*	*P.a*	*S.m*
1	Kajal Formulation
	MAEK	100	19	21	23	18	16	21	15	18
	TMEK	100	11	13	10	12	09	-	14	-
2	Standard
2	Chloramphenicol	30	20	27	28	19	18	23	17	22
3	Vehicle
3	Water		-	-	-	-	-	-	-	-

S.p=Streptococcus pneumoniae; B.c=Bacillus cereus; B.p=Bacillus pumilis; S.a=Staphylococcus aureaus; E.c=Escherichia coli; E.a=Enterobacter aerogens; P.a=Pseudomonas aeruginosa; S.m=Streptomyces marienensis; - =No activity

*Values are the average of triplicate; cup diameter (6mm)

Effect on Protein Denaturation:

The process of protein denaturation was as follows:

The reaction mixture (5mL) consisted of 0.2mL of 0.5% egg albumin, 4.78mL of phosphate buffered saline (PBS, pH 6.4), and 0.02mL of formulations, and the mixture was mixed, and was incubated in a water bath (37⁰C) for 15 min, and then the reaction mixture was heated at 70⁰C for 5 min. After cooling, the turbidity was measured at 660 nm using a UV/VIS spectrometer (Optima, SP 3000, Tokyo, Japan). Phosphate buffer solution was used as the control. The percentage inhibition of protein denaturation was calculated by using the following formula:

% inhibition of denaturation =

100 x(1 - A2/A1) (Table 5)

where

A1 = absorption of the control sample, and A2 = absorption of the test sample.

Table 5: inhibitory effect of MAEK and TMEK at different concentrations (25–100 μg/mL) on protein denaturation

Formulation	Concentration (ug/ml)	% inhibition of protein denaturation (mean of triplicate reading)
MAEK	25	58
	50	60
	75	71
	100	76
TMEK	25	53
	50	57
	75	63
	100	65

RESULT:

Herbal kohl MAEK and TMEK were formulated by using same formula of ingredients (Table 1) but the methods of extraction of active constituents was different and evaluated on various parameters. The pH, viscosity (Table 2) followed expected significance. The base satisfied the evaluated parameter values (Table 3) and physi-cal evaluation was suggestive of a cosmetically appealing product.

A control was formulated for antimicrobial studies was conducted to evaluate the comparative antimicrobial potential of the MAEK, TMEK, Standard (Chloramphenicol) and distilled water. Composition of nutrient agar I.P and cylinder plate method was employed. MAEK showed higher level of zone of inhibition in microbial contamination as compared with TMEK (Table 4).

MAEK was able to inhibit protein denaturation in a concentration-dependent manner as compared with TMEK. Inhibition % of protein denaturation of the formulations was within the range from 53.0% to 76.0% at the concentration range of 25–100ug/ml. MAEK exhibited a significantly higher level of inhibition compared to TMEK showed the lowest inhibition levels (Table 5).

DISCUSSION:

Maximal antimicrobial response was shown by the control formulation containing chloramphenicol (1500mg) which is a nitrobenzene derivative with broad spectrum of activ-ity. MAEK formulation prepared by microwave assisted extraction method showed significant antimicrobial potential which was less-er than control but greater than the TMEK formulation prepared by traditional methods of extraction. pH value satisfied the pH requirement for opthalmics. Viscosity and physical evaluation complied with cosmetic acceptability.MAEK will be cosmetically acceptable as indicated by pH, viscosity, particle size analysis, physical evaluation²⁰ parameters. Natural formulations are safe over the synthetic remedies as well as having very less side effects^21-24. Microbial infections can be treated with the use of herbal formulations^25-27. Formulation was known to promote wound healing process due to its antioxidant and antimicrobial activities^28-30. MAEK formulation prepared by microwave assisted extraction method showed significant in vitro anti-inflammatory activity than the TMEK formulation prepared by traditional methods of extraction. Green chemistry and microwave assisted extraction was used to obtain high yield, short extraction time, reducing waste and promoting efficiency in extraction³¹. Topical antimicrobial agents are the first line of treatment in mild to moderate acne vulgaris. So antimicrobial agents have anti-acne properties. In vitro tests revealed that the product formulated by microwave extraction possesses more significant antimicrobial efficacy, anti-inflammatory property and also can be used to treat acne in eye than the product formulated by traditional methods of extraction process.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 24.02.2022 Modified on 29.08.2022

Research J. Pharm. and Tech 2023; 16(7):3295-3299.

DOI: 10.52711/0974-360X.2023.00543