Formulation and Evaluation of Novel Herbal Ointment for the Treatment of Fungal Infection

Muthukumar. S¹*, Noori Irfana Parvin M. K¹, Shobana. S¹, Vimala. N¹, Vinesha. R¹, Sundaraganapathy. R²

¹Department of Pharmaceutics, KMCH College of Pharmacy, Coimbatore, Tamil Nadu, India.

(Affiliated to The Tamil Nadu Dr. M.G.R. Medical University)

²Faculty of Pharmacy, Karpagam Academy of Higher Education, Coimbatore.

*Corresponding Author E-mail: pharmmuthu@gmail.com

ABSTRACT:

Clove (Eugenia caryophyllus) and orange peel oil are most valuable spices that have been used for centuries for many medicinal purposes. It is effective in reducing fungal infection such as athlete’s foot. (a contagious fungal skin infection caused by a species of trichophyton or epidermorphyton (tinea pedis) that usually affects the foot). Pre formulation studies were done for antifungal activity of clove and orange oil. (clove oil and orange oil mixture was taken and soaked in a sterile disc and those disc were placed in the medium and incubated, zone of inhibition was identified). FTIR was done for the determination of constitute present in the orange oil and clove oil. there were no additional peaks were found when both the oil was mixed for preparation of formulation it was confirmed with the IR interpretation. The herbal ointment was formulated by using clove oil and orange peel oil with simple ointment bases. The extracts of orange peel oil and clove oil were incorporated into simple ointment base by fusion method. The physiochemical parameters such pH, viscosity, spreadability, washability, homogeneity was evaluated. The optimized formula (F1 &F3) was concluded based on evaluation parameters.

KEYWORDS: Clove Oil, Orange Peel Oil, Antifungal Activity, Fusion and Trituration Method.

INTRODUCTION:

Overview of Traditional Herbal Medicine¹:

India is a birth place of indigenous medicine such as Siddha, Ayurveda, and Unani where many herbs have been used for treatment of human ailments. About 65% of total global population remains dependent on traditional medicines for their primary healthcare. Herbs are occupying a comeback and an ‘Herbal Renaissance’ is blooming across the world. They have been evidently prized for their medicinal, flavoring and aromatic qualities for centuries, yet for a while they were over shadow by synthetic products of modern civilization.

The World Health Organization (WHO) defines traditional medicine as “the health practices, approaches, knowledge and beliefs incorporating plant, animal and mineral-based medicines, spiritual therapies, manual techniques and exercise, applied singularly or in combination to treat, diagnose and prevent illnesses or maintain well-being.” Once having realized their sources and adverse effects, people are going back to nature with hopes of safety and security. The rich treasure of herbal drugs is forming a boon for our society. Plant derived compounds, apart from their nutritive values, could serve as important therapeutic weapons to fight various human and animal diseases, thereby making them indispensable in traditional medicine for treating a number of diseases.

OINTMENT²:

An ointment is a homogeneous, viscous, semi-solid preparation, most commonly greasy, thick oil (oil 80% - water 20%) with a high viscosity which is intended for external application to the skin or mucous membranes. Ointments have a water number that defines the maximum amount of water that it can contain. They are used as emollients or for the application of active ingredients to the skin for protective, therapeutic, or prophylactic purposes and where a degree of occlusion is desired.

There are various parts of the body surfaces, skin and mucous membranes where ointment is applied for curing certain skin or disease conditions. Ointment is applied on hands, legs, face, eyes, ears, vagina, anus, throat etc. There are various problems when an ointment is suggested for treatment such as Ointment for burns, Ointments for cuts, Ointments for pain, Ointments for itching, Ointments for inflammation and pain, Ointments for boils and scars, Ointments for skin problems like eczema, dermatitis and psoriasis.

FUNGAL INFECTION:

Fungal infection characterized by nodular lesions--first in the lungs and spreading to the nervous system. candidiasis, monilia disease, moniliasis. an infection caused by fungi of the genus Monilia or Candida (especially Candida albicans). Fungi can live in the air, soil, water, and plants. There are also some fungi that live naturally in the human body. Like many microbes, there are helpful fungi and harmful fungi. When harmful fungi invade the body, they can be difficult to kill, as they can survive in the environment and re-infect the person trying to get better. The symptoms of a fungal infection will depend on the type, but common symptoms include Skin changes, like red and possibly cracking or peeling skin, Itching.

Tinea pedis or athlete's foot is a common fungal infection that affects the foot. Athlete's foot is commonly associated with sports and athletes because the fungus grows perfectly in warm, moist environments, such as socks and shoes, sports equipment, and locker rooms. In reality, anyone may be affected by athlete's foot. It is most common in warmer climates and summer months, where it can quickly multiply.

VOLATILE OILS:

CLOVE OIL³:

Cloves are the aromatic flower buds of a tree in the family Myrtaceae, Syzygium aromaticum. Active Constituents of Clove. Eugenol (up to 90%), acetyl eugenol, betacaryophyllene and vanillin; crategolic acid; tannins, gallotannic acid, methyl salicylate, the flavonoids eugenin, kaempferol, rhamnetin, and eugenitin; triterpenoids like oleanolic acid, stigmasterol and campesterol; and several sesquiterpenes. Flavonoids as kaempferol, quercetin and its derivates (glycosilated) are also found in clove in lower concentrations. Concentrations up to 18% of essential oil can be found in the clove flower buds. Roughly, 89% of the clove essential oil is eugenol and 5% to 15% is eugenol acetate and β-cariofile. Clove oil contains a chemical called eugenol, which acts as an anesthetic and antibacterial agent. Clove oil is anti-inflammatory and antifungal as well.

ORANGE OIL⁴:

It is an essential oil produced by cells within the rind of an orange fruit (Citrus sinensis fruit). In contrast to most essential oils, it is extracted as a by-product of orange juice production by centrifugation, producing a cold-pressed oil. Used in aromatherapy applications, Orange Essential Oil is known to have a pleasant scent that has a cheerful and uplifting yet simultaneously relaxing, calming effect, as it helps reduce pulse rate. Its sedative properties make it a natural aphrodisiac, which is ideal for relieving the symptoms of low libido in both men and women, if used recurrently and systematically. Used cosmetically or topically in general, Orange Oil is known to be beneficial for maintaining the health, appearance, and texture of skin by promoting clarity, radiance, and smoothness. In doing so, it reduces the signs of acne and other uncomfortable skin conditions.

MATERIALS AND METHODS:

INGREDIENTS USED:

Orange oil, Clove oil, Hard paraffin, Yellow soft paraffin, Wool fat, Cetostearyl alcohol.

INSTRUMENTS USED:

Electronic Balance (Shimadzu, Japan), FTIR (JASCO FTIR 4100), PH meter (Elico, L1120), Brookfield viscometer (Precision Scientific Company /LV -11 pro)

FORMULATION OF HERBAL OINTMENT:

1. Preparation of ointment by fusion method^{6 7}:

The components are melted in the decreasing order of their melting point. i.e. the higher melting point substance should be melted first, the substances with next melting point and so on. the Medicament is added slowly in the ingredients and stirred thoroughly until the mass cools down and homogenous product is formed.

Advantages: This will avoid over heating of substances having low melting point⁸

2. Preparation of ointment by Trituration method:

This method is applicable in the base or liquid present in small amount. The oil mixture is taken on an ointment slab and triturated with a small amount of the base. A steel spatula with long, broad blade is used. To this add additional quantities of the base are incorporated and triturated until the medicament is mixed with the base. Finally, liquid ingredients are incorporated. To avoid loss from splashing, a small volume of liquid is poured in a depression in the ointment an thoroughly incorporated before more is added in the same way. Splashing is more easily controlled in a mortar than on a tile.⁹

PREFORMULATION STUDIES:

Compatibility Study⁵:

IR Spectroscopy:

Procedure:

Turn on IR spectrometer and allow it to warm up. Obtain an unknown sample from the instructor and record the letter and appearance of the sample. Collect the background spectrum. Using a metal spatula, place a small amount of sample under the probe. Twist the probe until it looks into place. Record the IR spectrum of the antifungal ointment. Repeat if necessary to obtain a good quality spectrum. Record the absorption frequencies indicative of the functional qroup present. Clean the probe with acetone. Turn of the spectrometer. Analyse the obtained spectrum.

Post formulation Studies:

Zone of Inhibition anti fungal screening by disc Diffusion method:

TABLE 1: List of fungi used in the study

SL. NO	ORGANISM
1.	Candida albicans
2.	Aspergillus niger

The inoculums for the experiment were prepared in fresh sabouraud’s broth from preserved slant culture. The inoculums were standardized by adjusting the turbidity of the culture to that of McFarland standards. The turbidity of the culture may be adjusted by the addition of sterile saline or broth. The standardized inoculums is inoculated in the plates prepared earlier (aseptically) by dipping a sterile in the inoculums removing the excess of inoculums by passing by pressing and rotating the swab firmly against the side of the culture tube above the level of the liquid and finally streaking the swab all over the surface of the medium 3 times rotating the plate through an angle of 60º after each application. Finally pass the swab round the edge of the agar surface. Leave the inoculums to dry at room temperature with the lid closed. Each petri dish is divided into 3 parts, in 2 parts extract discs such as Control and MeOH HMS (100mcg) discs, (discs are soaked overnight in extract solution) and one quadrant for standard clotrimazole 10mcg, are placed in each quadrant with the help of sterile forceps. Then petri dishes are placed in the refrigerator at 4ºC or at room temperature for 1 hour for diffusion. Incubate at room temperature for 24 - 48 hours. Observe the zone of inhibition produced by different samples. Measure it using a scale or divider or venire calipers and record the average of two diameters of each zone of inhibition.

PHYSICAL EVALUATION¹⁰:

Preliminary evaluation of formulation at different concentrations was carried out as follows.

1. Organoleptic Parameters:

Organoleptic parameters like colour, odour of the formulation was carried out by visual examination.

2. pH:

The pH of various formulation was determined by using digital PH meter. The 0.5g of the weighed formulation was dispersed in 50ml of distilled water and the PH was noted.

3. Homogeneity:

All the developed ointment was tested for homogenecity by visual inspection. They were tested for their appearance with no lumps.

4. Viscosity¹¹:

The measurement of viscosity of prepared ointment was carried out with Brookfield Viscometer. the values of each formulation were done in triplicate and average values were depicted.

5. Spreadability¹²:

Spreadability was determined by apparatus which consist of a wooden block, which was provided by a pulley at one end. By this method spreadability was measured on the basis on slip and drag characteristics of ointments. An excess of ointment (about 2gm) under study was placed on the ground slides. The ointment was then sandwiched between this slide and another glass slide having the dimension of fixed ground slide and provided with the hook. A one kg weighted was placed on the top of slides for 5 mins. to expel air and to provide a uniform film of the ointment between the slides. Excess of the ointment was scrapped off from the edges. The top plate was then subjected to pull of 80 gm. With the help of string attached to the hook and the time (in sec) required by the top slide to cover a distance of 7.5 cm be noted. A shorter interval indicates better spreadability. Spreadability was determined by using the formula

S=M x L/T

S=spreadability M=weigh tied to upper slide L=length of glass slide T=time taken to separate the slides completely from each other.

6. Washability:

0.5 gm of prepared formulation was applied on the skin. And it was washed with luke warm water. The time taken for removal of preparation was noted.

RESULTS AND DISCUSSION:

Preformulation studies:

COMPATABILITY STUDIES:

IR ANALYSIS FOR VOLATILE OIL:

IR spectrum of clove oil, orange peel oil and sample (Antifungal ointment) were carried out using FTIR by ATR (attenuated total reflection) sampling method.

1. IR spectrum of clove oil:

FIG 1: IR spectrum of clove oil

2. IR spectrum of orange peel oil:

FIG 2: IR spectrum of orange peel oil

3. IR spectrum antifungal ointment:

A.0.5% Concentration:

FIG 3: IR spectrum of herbal ointment of 0.5% concentration

B. (1.5% concentration):

FIG 4: IR spectrum of herbal ointment of 1.5% concentration

Antifungal Activity:

Determination of zone of inhibition:

Zone of inhibition produced by sample is shown below.

Aspergillus niger:

FIG: 5 : ZOI produced by (0.5% conc) the Ointment

Candida albicans:

FIG:6 : ZOI produced by (1.5% conc) the Ointment

Physical evaluation results:

Table no 2: Evaluation results for herbal ointment

Evaluation Test	F1(0.5%)	F2(1%)	F3(1.5%)	F4(2%)
pH	6.2	7.1	6.6	6.9
Viscosity (centipoise)	9300	6103	9800	8256
Spreadability (seconds)	15	20	16	19
Washability (seconds)	6	13	8	12
Homogeneity	No lumps	No lumps	No lumps	No lumps

DISCUSSION:

IR spectrum of antifungal formulation (sample) was compared with IR spectrum of clove oil and orange peel oil. From results obtained by IR spectroscopy, it was concluded that there was no significance change in IR spectrum of antifungal ointment. No additional peaks were observed and compatible with each other. So prepared formulations showed compatibility between ingredients

Prepared formulation was evaluated for antifungal properties. The significant zone of inhibitions was found. (When the sterile disk is soaked in the formulation inoculated and incubated for 1 day.) They were comparable with standard (fluconazole) zone of inhibition.

So that the formulation which contain clove and orange oil may be inhibited the growth of fungi such as candida albicans and Aspergillus niger. (Shown in figure No-5,6).

The various physicochemical parameters were utilized to evaluate the prepared ointment formulation. The rheological behaviour of the different formulations of ointments in rotational Brookfield viscometer indicated that when speed of spindle increased viscosity decreased. (Shown in table No- 2). Spreadability denote the extent of area to which the prepared formulation readily spreads on application to skin or affected part and homogeneity confirms no lumps. A comparative study of viscosity and spreadability showed that increasing viscosity of the formulations decreased spreadability.

CONCLUSION:

From this study we concluded that optimized formula F1 and F3 (Shown in table no 2) showed significant results based on evaluation parameters.

It was showing expected physiochemical parameters.

Herbal ointment showed significant antifungal activity.

Initial screening showed promising potential for herbal products

This work is considerable for further development.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of KMCH College of Pharmacy, Coimbatore for the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 19.10.2019 Modified on 14.12.2019

Research J. Pharm. and Tech 2021; 14(3):1459-1464.

DOI: 10.5958/0974-360X.2021.00325.5