Formulation & Evaluation of Antifungal Activity of Polyherbal Ointment in Animal Model

Polyherbal ointments are topical preparations that combine multiple medicinal herbs for enhanced therapeutic action. They target specific conditions like wound healing, pain relief, or skin care. Common types include wound healing ointments containing herbs like aloe vera, calendula, and turmeric, known for their antimicrobial and anti-inflammatory properties. Pain relief ointments use herbs like arnica, ginger, and eucalyptus, providing analgesic and anti-inflammatory effects. Skin care ointments may include neem, lavender, and chamomile, which can soothe and nourish the skin, potentially helping with conditions like eczema or acne. Anti-inflammatory ointments target inflammation and pain, while antifungal ointments treat fungal infections (1, 2). Polyherbal ointments offer synergistic effects, reduced medication costs, and a natural alternative to conventional medications. However, it is important to be aware of potential allergies or sensitivities to specific herbs, consult with a healthcare professional before using a polyherbal ointment, and ensure the ointment is sourced from a reputable manufacturer and high-quality ingredients. The benefits of polyherbal ointments include synergistic effects, reduced medication costs, and a natural alternative to conventional medications (3, 4). The study aims to assess the antifungal activity of a poly-herbal blend of Aloe vera leaves, cinnamon bark, and ginger rhizome using experimental animals. The objectives include conducting a literature survey, procuring and authenticating plant material, preparing the formulation, and evaluating its antifungal properties.

2. MATERIALS AND METHODS

2.1 Selection, Collection, authentication and processing of the Crude Drugs

All the crude drugs were collected from Chikhli, Maharastra in the month of July-August when the plants bear flower and authenticated from the Shri Shivaji Science and Arts College, Chikhli, Buldana, Maharastra. The collected plant materials were shade dried. The dried materials were coarsely powdered individually by means of mechanical grinder. Then they were passed though sieve no.40 (aperture size-425mm) to get moderately coarse powder. The resulting powdered materials were used for further studies.

2.2 Preparation of the Ointment

Polyherbal ointment (PHEO) of 5% was prepared by incorporating a sufficient quantity of simple ointment in the melted state to other ingredients of the formulation. The powdered components were previously reduced to fine powders and a small portion of the powder is mixed with a portion of the base until a uniform mixture is obtained. The process is continued until all portions of the powder and the base are combined and thoroughly and uniformly blended. We selected 2.5:2.5:2.5 ratio of methanolic extracts of leaves of Aloe vera, bark of cinnamon and rhizome of ginger for polyherbal syrup. The remaining sweetening agents were added and mixed thoroughly. Adjust the pH between 5.5 and 7.0 with, if necessary (5-7).

2.3 Pharmacological Activity

2.3.1 In vitro method for the Evaluation of Antifungal Activity

The predetermined procedure was followed for performing the assay. In this experiment, groups containing 105CFU/ml of fungal microbe inoculums (McFarland standard) suspension, FLC (1 μg/ml), and PHE ointment (PHEO) at 1% and 2% w/v were incubated at 35°C with continuous shaking (200 rpm). The drug-free group functioned as a control growth group. Colony counting techniques were then used to determine the number of living cells at predetermined intervals (0, 6, 12, 24, and 48 hours) following incubation (8-10).

2.3.2 Acute Dermal Toxicity Study

Total 6 rats of 10-12 weeks age were selected (IAEC referral number: 751/PO/Re/S/O 3/CPCSEA/20 25/1-4) and randomly divided into 2 groups. Group I was vehicle control group which received vehicle (gum acacia 1% w/v in distilled water) while group II was test group that received Polyherbal Ointment (PHEO). Each group consisted of 3 animals (females). Females were nulliparous and non-pregnant. Dose selected for the present study is limit test dose as mentioned in the guidelines. The starting dose for limit test 2000 mg/kg was selected on the basis of dose suggested in OECD guideline number 402 (11).

2.3.3 In vivo for the Evaluation of Antifungal Activity

Total animals will be divided among 5 groups of 6 rats each and these groups will be assigned to various treatment groups for duration of study of 30 days (Table 1) 751/PO/Re/S/O 3/CPCSEA/20 25/1-4. Normal saline will be used as the sole vehicle for Group 1 (Healthy Control). Group 2 will be used as a experimental control and receive fungal infection with no treatment. Group 3 will take Ketoconazole 20 mg/ kg, BW orally (standard drug) after fungal infection. Groups 4 and 5 will then receive fungal infection and PHE ointment (PHEO) at 1% and 2% w/v, respectively. On the back of each animal, 4 cm2 areas were cleaned and depilated. The fungal inoculum was prepared from 7-day-old cultures of C. albican, suspended in sterilized physiological saline containing 0.1% Tween 80. Following filtration through four layers of sterile gauze to remove hyphal fragments and agar flicks, the final conidial suspension was adjusted to 107 conidia/ml for use as the inoculum. The conidia were counted using a hemocytometer under a microscope. The inoculum was applied on the back of the animals immediately after depilation and left for 3 days. The establishment of active infection was confirmed on day 4 by isolation of the pathogens from skin scales cultured from infected loci on SDA plates containing 100 units/ml penicillin and streptomycin. Infections were also confirmed by visual examination of the animals on days 8–10. In the animals in which active infections were confirmed, treatment was initiated on day 20 post inoculation and continued for next 15 days from infection was achieved. The commercial fungicide Ketoconazole was used as a control. The treatments were applied once daily, and the infected areas were scored visually for inflammation and scaling (12, 13).

3. RESULTS AND DISCUSSION

3.1 Acute Dermal Toxicity Study

The Polyherbal Ointment (PHEO) was found to be safe at a dose level of 2000 mg/kg, BW of the animals and no clinical signs were observed other than normal activity. On conclusion, we can say that Polyherbal Ointment (PHEO) so prepared is safe for the further animal study.

3.2 Invitro Assessment of Antifungal Activity by using Time Killing Curve Assay

The results showed that, in the presence of high dose level of the samples, a significant enhancement in the degree of antifungal activity was observed after 6 h, and there was a 1.92/2.08 log10 CFU ml−1 decrease at 24/48 h time point compared with the control group (Figure 1).

Figure 1: Invitro Assessment of Antifungal Activity by using Time Killing Curve Assay

3.3.1 Skin Lesion Score

For the experiment in vivo and inducing experimental dermatomycoses we used the most frequent dermatomycete T. mentagrophytes, which is common in rodents but also in human. The first symptoms on the infected rats inoculated with T. mentagrophytes were observed on 3th day of the experiment in the form of well defined clinical parameters for this species (small inflammatory vesicles). After 3 days from erythematous lesions on the skin symptoms goes to the mycotic foci well developed with ulceration, which later (day 20) on resulted in wounds. The treatment started on 5th day of the experiment. Animals treated with Polyherbal Ointment (PHEO) at both doses were completely cured after 15 days of treatment. For Polyherbal Ointment (PHEO) tested there no visually observed symptoms at the end of the treatment and the culture reinoculated were negative (Figure 2).

Fungal infection causes the inflammation to the skin area which can be confirmed by the rise in the levels of the pro-inflammatory cytokines (IL-6 and TNF-α). IL-6 is a major mediator of inflammation and is involved in B-cell maturation, acute inflammatory responses, and CNS functions. TNF-α is another important pro-inflammatory cytokine that induces IL-6 release, is involved in B-cell differentiation, and is associated with catabolism and atherosclerosis. It can be seen that the animals with fungal infection has raised levels of these cytokines, which is found to be reduced in treatment groups (Figure 3 and 4).