Beta Fulltext view is in preview — article structure may vary. Browse all articles
Contents
Journal of Infectious Diseases & Travel Medicine Research Article 20 min read

Formulation and Pharmaceutical Characterization of Antidermatophyte Creams of Methanol Leaf Extracts of Euphobia Hirta, Jatropha Curcas and Acantus Montanus

Ojiako CM
ISSN: 2640-2653  10.23880/jidtm-16000157  Received: September 20, 2021  Published: January 19, 2022
 — views
 23 references
 7 tables
PDF
Keywords
Dermatophytes Euphorbia Hirta Jatropha Curcas Acantus Montanus Cream Formulations
Abstract

Background: The search for novel natural products from medicinal plants against multidrug fungal strains is very essential to overcome the resistance complication. Many researchers have focused on the antidermatophyte activity of crude extracts from naturally occurring plants. Objective: This study investigates cream formulations of methanolic crude extracts of E.hirta, J. curcas and A. montanus for their pharmaceutical characteristics and antifungal potentials, especially against some drug resistant dermatophytes isolated from work equipment of local hair dressers. Methods: Dermatophytes samples were inoculated into sterile Sabouraud dextrose agar (SDA) broth and incubated. Positive growths were examined macroscopically and microscopically and confirmed with the aid of a fungi atlas. The phytochemical constituents of the plants extracts were also determined. Antifungal susceptibility testing was also carried out using agar diffusion assay. Using the best extract combination ratios obtained from the checkerboard assay, creams were formulated with the methanolic crude extracts and the pharmaceutical properties of the creams were determined. Results: MIC of the individual extracts showed that Trichophyton schoenleinii and Microsporon ferugenium were resistant to all the extracts except J. curcas. The checkerboard assay showed that the combination of J. curcas and A. montanus had the best anti-fungal activity for T. schoenleinii, while the combination of J. curcas and E.hirta also gave an excellent result. All the formulated creams exhibited good pharmaceutical characteristics. Conclusion: The formulated creams showed good anti-fungal and pharmaceutical properties. Overall, the study scientifically validates that methanolic leaf extracts of A. montanus, E. hirta and J. curcas are potent antifungal agents.

Introduction

Dermatophytosis is a fungal infection, which has been very frequent in occurrence among other fungal infections [1]. It is one of the most resistant infections, mainly due to environmental factors of temperature, moisture and phenotype [2]. It affects mainly the skin, scalp (Tinea capitis), fingernails, groin (Tinea cruris) and toe nails (Tinea pedis) and may also affects other parts of the body surfaces (Tinea corporis) [3]. Its prognosis is a high long-term impairment lesion that occurs again over time and progresses to a more complicated secondary infection. It can infect both individuals with high immune system and compromised immune system. The most common dermatophyte implicated belongs to the genera Microsporium, Trichophyton and Epidermophyton. Dermatophytes constitute a non-opportunistic class of fungi which are not life threatening and survive by obtaining nutrients from keratin, which are abundant in tissues of the nails, hair and skin [4]. They are referred to as keratinophillic infectious fungi because keratin is their source of nutrient. The enzyme keratinases, which are specialized keratinolytic/ proteolytic enzymes produced by dermatophytes to degrade the protein keratin found on the body surfaces, nails and hair scalp [5].

In view of the research for newer drugs due to increasing resistance to current ones, natural products continue to provide lead compounds for researchers to finding new drugs. Bioactive molecules from these natural herbs and plants have been chemically modified to produce new drug molecules with diverse activities. Thus, these naturally occurring plants and herbs have been the origin of so many drug molecules used in treatment of varying degrees of diseases and various illnesses owing to their therapeutic value. Many researchers have focused on the antidermatophyte activity of crude extracts from naturally occurring plants, because many plants have the vast ability to synthesize biologically active compounds with different functional groups. Hence, crude extracts provide good basis for the research of more potent, effective and safe to use antifungal agents, particularly dermatophytes which is more resistant to most existing antifungal agents [3]. In this respect, the J. curcas, E. hirta and A. montaneus have been investigated individually [6, 7, 8, 9].

Additionally, plant extracts also contain secondary metabolites, which can target more than one cell, and this is the basis for using multiple plant extracts in disease management in which multiple targets are involved. These secondary metabolites are the defense mechanism used by plants, which are also very useful to humans in terms of nutrition [10]. Secondary metabolites are also involved in activation of signaling compounds that provides protection for the plants from the pathogens or environmental hazards and this aspect of nutritional value of these plant secondary metabolites has been experimented to also have high therapeutic index in treating many diseases and ailments [11]. Thus this study aims at investigating the methanolic crude extracts of J. curcas, E. hirta, and A. montaneus combinations as antidermatophyte, as well as the preparation of various cream formulations of these extracts, their pharmaceutical characterization, and the tests for antifungal properties of the creams using suitable clinical isolates.

Methods

Extraction of Plant Material

A mechanical grinder was used to pulverize air-dried plant samples and 2.5 kg each of the powdered material macerated, with intermittent shaking, in methanol (5 L) for two days. Using a combination of muslin cloth and filtered paper (Whatman No 1), the marcs were filtered, and concentrated using rotary evaporator. Resultant crude extracts were refrigerated until they were ready for further analysis.

Phytochemical Analysis

The standard procedure reported in Trease, et al. [12] was used to determine the phytochemical constituents of the plant extracts. Among the phytochemical screening tests conducted include tests for saponin, flavonoids, steroids, terpenoids (using Salkowski test), and cardiac glycosides (using Keller-Killani test). Other tests include tests for alkaloids, protein, carbohydrate, and reducing sugar.

Collection of Test Organism Samples

Sample collection was preceded by oral assent from the study participants. A total of 45 samples were aseptically collected using sterile swab sticks from the work equipment (combs of various sizes, scissors, hair pins, needles etc.) of the local hair dressers. Samples were transported to the laboratory without delay for mycological analysis and were maintained in the refrigerator at 4°C until they were ready to be analyzed. The antifungal drugs (clotrimazole, ketoconazole and miconazole) that were used for this study were bought from pharmacy premises, also the antidandruff solutions (hair shampoo and hair conditioner) that were used for this study were bought from Eke-Awka Market in Anambra State Nigeria, stored under appropriate conditions until they were ready to be used.

Inoculation and Isolation

After aseptic collection of the samples with the sterile swab, the samples were inoculated in sterile Sabouraud Dextrose Broth (SDB) base (Oxiod, UK) and incubated at 25°C for 5 days. At the end of incubation, the test tubes were visually observed for turbidity (indicating growth). The growing samples were platted out on a freshly SDA (Oxoid, UK) supplemented with chloramphenicol 0.05 g/L (that serves as a broad spectrum antibiotic) and the cultures were incubated aerobically for 7 days at room temperature (25°C). Growing (positive) cultures were examined for both macroscopic and microscopic characteristics as described below. Isolates from the positive cultures were confirmed with the aid of an atlas. Pure colonies were then stored on slants at 4°C for further studies as described by Cheesebrough [13]. At the end of 4 weeks, the absence of any growth in the culture indicates negative. The frequency of occurrence of the isolates was also calculated.

Macroscopic (Colonial) Characterization of Fungal Isolates

The following features were used to macroscopically characterize the pure cultures: colour, texture (colony, velvety, powdery, granular and pasty) and pigment production (seen on the reverse of the medium).

Microscopic Examination and Fungal Culture

This involves the use of direct microscopy. The mixture of 10% KOH and 40% Dimethyl sulphoxide (DMSO), in equal proportion, was used to mount the samples collected and were screened for the presence of fungal elements. Scraped skin and plucked hair samples were placed in the KOH+DMSO drops on the slide and a clean cover slip placed over the samples, pressed to prevent the formation of air bubbles and observed after 8 minutes. The combination of low power (10x) and high power (40x) magnification were used to visualize each slide for the presence of hypae and/ or arthroconidia. Thereafter, the hair and skin scraped specimens were then inoculated into two Petri dishes containing SDA base and Dermasel agar base, both supplemented with chloramphenicol, which serves as a broad spectrum antibiotic, and cycloheximide (for the inhibition of saprophytic fungi). It should be noted that in the absence of heat, the DMSO increases susceptibility of the preparation and softens keratin more quickly than KOH alone. The above fungal cultures were subjected to selective media, which was prepared by adding one vial of Dermasel Selective Supplement SR0075 (Oxoid, UK) to each 500 ml of medium and the cultures incubated aerobically for 7 days at room temperature (25°C). Positive cultures were examined both macroscopic (color of the surface and reverse, topography, and texture) and microscopic characteristics, as before.

Antifungal Susceptibility Testing

The antifungal susceptibility was evaluated using three antifungal agents: clotrimazole (50 μg), miconazole (10 μg) and fluconazole (25μg). The tip of a sterile Pasteur pipette was used to probe dermatophytes colonies from Dermasel/ SDA agar cultures and the mixture of mycelium and conidia obtained were inoculate into a sterile tube containing 1mL distilled water and allowed to sediment for 30 minutes. The innocula suspensions, with the aid of swabs, were streaked uniformly over the surface of Mueller-Hinton (MH) agar (Oxoid, UK) plates, and the plates, with lids left ajar for 3 minutes – to prevent the absorption of excess surface moisture into the agar before the drug-impregnated disks were applied –incubated in a laminar flow cabinet at 25°C for 5 days. At the end of incubation, the zones of inhibition around the disks were measured.

Preparation of McFarland Turbidity Standard

Fungal growth in cultures was evaluated using McFarland turbidity standard, as described by Oli, et al. [14]. Briefly, a 0.5 McFarland standard was prepared by adding 0.5 mL of 0.048M BaCl2 (1.17% w/v BaCl2. 2H2O) to 99.5ml of 0.18M H2SO4 (1% v/v) with continuous stirring. Using matches cuvettes and distilled water as a blank standard, the optical density of barium sulphate precipitate was determined. The standard was compared visually to a suspension of fungal growth in sterile saline. The approximate cell density corresponding to 0.5 McFarland is 1×106 cells/mL.

Standardization of Test Isolates

The test organisms (molds), which were previously grown on SDA for 4 days, were subjected to 10-fold serial dilutions. The 0.1ml of the spore suspension of each isolate was taken from the stock culture and serially diluted. 0.1mL of the suspension was plated out by spread plate method for semi-confluent growth. Dilutions which gave working inoculums of 106 CFU/mL were used. For the yeast isolates, the test organism was inoculated into Sabouraud Dextrose Broth (SDB) and incubated over-night.

Agar Well Diffusion Assay

Sterile SDA plates prepared in accordance with the manufacturer’s specification were inoculated with the test culture by surface spreading method using cotton swab, and wells (8 mm in diameter) made on the plates with the aid of sterile cork borer. Aliquots of 80 μL of each extract diluted in DMSO at concentrations of (50, 25, 12.5 and 6.25 and 3.125) mg/mL were dispensed into each of the wells in the culture plates, and the culture incubated for 48 h at 25°C (clotrimazole (50 µg/mL) served as the positive control against the fungal test organisms. On the other hand, the DMSO served as the negative control). The extent of zone of inhibition around each well was used to determine the antimicrobial potential of the crude extract and triplicate determinations were taken. Each of the sample extracts was tested against all the fungal isolates.

**Synergistic Study**

To determine the interactive synergistic inhibition between the methanolic crude extracts, the Checkerboard assay as describe by Oli, et al. [14] was used. Briefly, it involves preparing the stock solution of each of the extract using the individual MICs. Also, separate solutions of the crude extracts were prepared with DMSO, each solution containing twice the MIC of the extracts. Thereafter, continuous variations model was adopted, in which the solutions were combined in different ratios. Each combination was then diluted two-fold for up to 6 serial dilutions in sterile Pyrex test tubes. An aliquot of 60 µL of the diluted solutions was transferred into a corresponding well in a sterile agar plate, which was previously seeded with 0.5 McFarland standard of the test organism. The plates were incubated at 37°C for 24 hours. The fractional inhibitory concentration (FIC) of each extract is the minimal inhibitory concentration in the combination divided by the independent MIC of the extracts. The sum of the FICs of both extracts gives the FIC index. This is expressed as in Equation 1:

$$FIC \text{ Index} = \frac{A}{A} + \frac{B}{B}$$

Where, and B represent minimal concentrations of extracts A and B having inhibitory effects when acting together, while A and B stand for the respective MICs of the extracts. The FIC Index value of less than 1.0 indicates synergism; equal to 1.0 implies additive; value of more than 1.0 suggests indifference, and more than 2.0 indicates antagonism.

**Method of Cream Formulation**

The herbal creams were formulated by tritrating the crude extracts with a mixture of sorbitol and glycerin (1:5). Tragacanth served as a suspending agent in the presence of selected preservatives, which was added to the wetted crude-extract mass slowly. Finally, the final volumes of different batches of the preparations were made up using purified water.

**Evaluation of the Formulated Creams**

  • Physical evaluations: The following preliminary evaluations of the herbal cream formulations were carried out:
  • pH: To determine how acidic or basic the various formulations were, the Digital pH meter (Digital pH meter 335, Systronics, Noroda, Ahmedabad) was used. A 0.5 g of the weighed formulation was dispersed in 50 mL of distilled water and the pH was measured.
  • Homogeneity: The formulated creams were visually checked for homogeneity. This was done by assessing the creams for the appearance of lumps.
  • Viscosity: This as a measure of the cream formulation’s resistance to motion under an applied force was assessed using Brookfield Viscometer (model LV-DV-II, Helipath spindle type S-96). The viscosity for each formulation was taken in triplicates and the value expressed as mean± standard deviation.
  • Spreadability: The ability of the formulated polyherbal cream to spread was determined using the formula in Equation 2:

$$S = \frac{LM}{T}$$

Where $S$ is spreadability, $M$ is weight tied to upper slide, $L$ is length of glass slides and $T$ is time taken to separate the slides completely from each other.

**Acute Skin Irritation Study**

The ability of cream formulations to cause skin irritation as evidenced by signs of oedema and erythema was assessed using albino rats weighing between 150-200 g, which were obtained from Pharmacology department, Pharmacy school, Nnamdi Azikiwe University, Agulu. Required ethical approval for the use of the animals was obtained from the Animal Use and Care Ethical Committee, Ministry of Health of Anambra state. All the animals were provided standard animal feed and access to water ad libitum. A day prior to the commencement of the study, fur from different sites (e.g. head, tail, right hand (RH), and right leg (RL)) was removed, and the animals kept individually in cages to prevent contact with the other rats. The untreated skin areas served as control. The standard irritant used was aqueous solution of 0.8% formalin. Thereafter, a50mg of each of the formulation containing combinations of J. curcas/A. montanus, E. hirta/J. curcas, and A. montanus/E. hirta, respectively were evenly and gently applied on the test sites.

Stability Studies

The ability of all cream formulations to retain and maintain cream characteristics within the original desirable properties were assessed at different temperature conditions (4, 25 and 37°C) over 90 days in a climatic chamber. The cream formulation properties evaluated at different time intervals (i.e., 15, 30, 60 and 90th day) include pH, viscosity, spread ability, consistency and phase separation.

Results

Percentage Yield of Plant Extracts and their Qualitative Phytochemistry

Table 1 shows the qualitative phytochemistry, as well as the percentage yield of methanolic extracts of the various plants used in this study. Percentage yields for J. curcas, E.hirta and A. montanus are 5.88, 3.57 and 7.92, respectively. The phytochemical analysis shows moderate quantity of tannin in J. curcas and E. hirta. While J. curcas is also rich in protein, E. hirta has appreciable quantities of saponin and flavonoid. Moderate quantities of alkaloid and flavonoid were found in A. montanus. There were no traces of terpenoid, cardiac glycoside, carbohydrate and steroid found in A. montanus. While J. curcas has traces of alkaloid, flavonoid and steroid, some other molecules such as saponin, terpenoid, cardiac glycoside and carbohydrate were completely absent. All the evaluated molecules were found in varied quantities in E. hirta.

PlantsJatropha curcasEuphorbia hirtaAcantus montanus
% yield of herbal extracts5.883.577.92
Qualitative phytochemistry of the herbal extractsAlkaloid++++
Saponin-+++
Tannin+++++
Flavonoids+++++
Steroid++-
Terpenoids-+-
Cardiac Glycoside-+-
Carbohydrate-+-
Protein++++

Table 1: Percentage yield of herbal extracts and their Qualitative phytochemistry.

KEY: += Trace / mildly positive; ++ = Moderately positive; and -= Negative Table 1: Percentage yield of herbal extracts and their Qualitative phytochemistry.

Identification of the Test Isolates

The dermatophytes used for this study were identified as follows: Trichophyton schoenleinii, Microsporongypseum, Microsporonferungineum and Trichophyton mentagrophytes (Table 2).

CodeIsolate
T2Trichophyton schoenleinii
T3Microsporon gypseum
T4Microsporon ferugineum
T5Trichophyton mentagrophytes

Table 2: The identified Dermatophytes.

Antimicrobial Susceptibility Test

Table 3 shows the antimicrobial susceptibility profiles of J. curcas, E. hirta and A. montanus against T. schoenleinii, M. gypseum, M. ferungineum and T. mentagrophytes J. curcas has the highest activity alone against M. gypseum, T. mentagrophytes and M. ferugineum, but has high resistance against T. schoenleinii; while Euphorbia hirta and Acantus montanus showed moderate activities against M. gypseum, T. mentagrophytes and M. ferugineum.

Minimum Inhibitory Concentration

Table 3 shows the minimum inhibitory concentrations of the various extracts against M. gypseum and T. mentagrophytes

PlantsDermatophytes usedExtract Concentration (mg/ml)Control
50 Inhibition zone diameter (mm)2512.56.253.13
Jatropha curcasTrichophyton schoenleinii000000
Microsporon gypseum11.5±0.79.5±0.76.5±0.76±05±015
Microsporon ferugineum5±0.73±02.5±0.701±010
Trichophyton mentagrophytes7.5±0.76.5±0.76±04±1.42.5±0.710
Acantus- montanusTrichophyton schoenleinii000000
Microsporon gypseum8±0.76.5±0.75.5±0.75±0.74.5±0.715
Microsporon ferugineum000007
Trichophyton mentagrophytes7±05.5±0.74±03±02±00
Euphorbia hirtaTrichophyton schoenleinii000000
Microsporon gypseum8±07±06±05.5±0.7015
Microsporon ferugineum0000010
Trichophyton mentagrophytes4.5±0.74±03.5±0.72±02±00

Table 3: Inhibition zone diameter (mm) of the plants extract against test dermatophytes.

Note: Control is 1% Clotrimazole Table 3: Inhibition zone diameter (mm) of the plants extract against test dermatophytes.

Antidermatophyte Combination Assay

Tables 4 and 5 show the effect of combination of (J. curcas and A. montanus); (J. curcas and E.hirta); and (A.montanus and E.hirta) against drug resistant M. ferugineum and T. schoenleinii, respectively. At the ratios of 5:5 and 6:4 the combinations showed synergism against all test isolates, while at other ratios synergism, additive, indifference and antagonism effects were observed. The checkerboard assay carried out against M. ferugineum and T. Schoenlenii showed resistance to the individual extracts alone. The combination of J. curcas and A. montanus at equal concentration has the highest activity at the ratio of 8:2, 7:3, 6:4 and 5:5 against T. schoenleinii while the activity reduced when the concentration of J. curcas was reduced to 25mg. Combination of J. curcas and E. hirta at equal concentration (50mg) also gave an excellent result at the ratio of 8:2 and 7:3 against T. schoenleinii.

Combination RatiosM. ferugeniums
JC : AMJC : EHAM : EH
FICInterpretationInterpretationFICInterpretation
10:00-----
9:011.83Indifference > 1Indifference > 11.44Indifference > 1
8:021.46Indifference > 1Indifference > 12.16Antagonism >2
7:030.76Synergism < 1Indifference > 11.41Indifference > 1
6:040.62Synergism < 1Synergism ˂ 10.89Synergism < 1
5:050.89Synergism < 1Synergism ˂ 10.78Synergism˂1
4:061Additive =1Indifference > 10.7Synergism˂ 1
3:071.46Indifference > 1Indifference˃11.38Indifference > 1
2:081.97Indifference > 1Indifference ˃12.26Antagonism >2
1:092.48Antagonism >2Antagonism> 21.24Indifference >1
0:10-----
Combination RatiosT. schoenlein
JC : AMJC : EHAM : EH
FICInterpretationInterpretationFICInterpretation
10:00-----
9:011.48Indifference ˃1Indifference˃11.4Indifference˃1
8:021.08Indifference˃1indifference˃12.24Antagonism˃2
7:031.24Indifference˃1Indifference˃11.3Indifference˃1
6:040.68Synergism˂1Synergism˂10.98Synergism˂1
5:050.92Synergism˂1Synergism˂11Additive=1
4:060.84Synergism˂1Indifference1˃1.41Indifference˃1
3:070.88Synergism˂1Antagonism ˃21.33Indifference˃1
2:080.6Synergism˂1Antagonism˃22.36Antagonism˃2
1:091.14Indifference˃1Indifference˃12.27Antagonism˃2
0:10-----

Table 4: Checkerboard assay of the extracts against M. ferugenium.

Inhibition Zone Diameter(IZD) of Polyherbal Formulations Against Test Isolates

The activities of the polyherbal combined extracts formulations in comparison with standard drug are clearly illustrated in Tables 4-6. All the combinations showed zero activity against Trichophyton mentagrophytes. The IZD of J. curcas and A.montanus against M.gypseum was larger than the standard drug (1 % clotrimazole).

Inhibition zone diameter (mm)
Test IsolatesJ. curcas + A. montanusE. hirta+ J. curcasA. montanus+ E. hirta
Trichophyton mentagrophytes7.34.54.5
Microsporon ferugineum10.554.5
Microsporon gypseum6.557
Trichophyton schoenleinii000

Table 5: Inhibition zone diameter (mm) of the Polyherbal formulations against test isolates.

Pharmaceutical Characteristics of the Polyherbal Formulation

The Pharmaceutical characteristics of the polyherbal formulation are represented in Table 7. The average speed of all the 3 formulations is 6rpm. The viscosity ranges from 8467.50 mp as to 15494.67 mp as while percentage of torque is between 42.37 and 77.47 as shown in Table 7. The pH values of the combined extracts were fairly stable even three months after formulations. The average pH of the formulation is 6.5. The spreadability of the cream is 2.

Acute Irritation Study

Table 8 showed the result of the acute skin irritation test of the J. curcas and A. montanus combined formulation. There was mild irritation only on the day 3 of treatment. There was no other irritation with the other treatment as shown in Table 7.

Evaluation TimeFormulationsTorque
(%)		Viscosity (mpas)pHSpreadability
(cm)		Speed (rpm)
1 hr. after formulationA41.350.78252.7101387011.86.76.486.6223.46
B65.253.41304210671116906.296.756.5225.26
C72.8851456416992149286.596.436.6523.66
1 month afterA66.865.61795.6168011786.46.526.556.36ND6
B49.350.21524.61447.11425.76.626.376.73ND6
C64.972.31582.71615.31886.46.646.586.72ND6
3 months afterA40.752.61462908780485.736.425.68ND6
B63.152.71286710692115906.275.796.49ND6
C83.771.81204614565148996.346.725.8ND6

Table 6: Pharmaceutical characteristics of the formulation.

A = J. curcas and A.montanus; B = E.hirta and J. curcas; C = A.montanus and E.hirta; ND = Not done Table 7: Pharmaceutical characteristics of the formulation.

Observation Time (hrs)1247296120144168
CLTrtCLCLTrtCLTrtCLTrtCLTrtCLTrt
Diameter of inflammation (mm)
Head (JC /AM)000030302.30200
Tail ( EH/JC)0000000000000
Right hand (AM/EH)0000000000000

Table 7: Score of erythema and edema after application of the antidermatophyte polyherbal cream.

Right leg wasused as control (CL); Trt = Treatment; JC = J. curcas; AM = A.montanus; EH = E.hirta Slight irritation was seen on the head on day 3 to 5 but started diminishing gradually Table 8: Score of erythema and edema after application of the antidermatophyte polyherbal cream.

Discussion

Dermatophytosis is one of the most commonly occurring fungal infections. Also, it has a problem of increase in the level of resistance to antifungal drugs which could be attributed to several factors. This has led to various researches which are focused on the synthesis of newer, more potent, more effective and safer antifungal agents. Naturally occurring plans have been the source of many drug molecules which are used in the treatment of various diseases including fungal infections. The bioactive molecules which are obtained from these natural plants are modified to obtain newer drug molecules with various activities [15, 16, 17, 18, 19]. The assessment of the antifungal effect of A. montanus, E. hirta and J. curcas_as seen in Table 3 showed that the methanolic leaf extracts demonstrated moderate antifungal activity against clinically isolated strains of _T. schoenleinii, M. gypseum and M. ferugineum, the causal organisms mostly implicated in skin structure infections. The effect of the extract was higher against M. gypseum, followed by T. schoenleinii and M. ferugineum in a concentration dependent pattern. When used alone, J. curcas showed the highest activity against 3 of the organisms except T. schoelenleinii as compared to the other two extracts. On the other hand, E. hirta was active against T. mentagrophytes (Table 3) as a single extract. Syngergistic effect of the combination of two extracts exhibited better result as compared to the use of a single extract. For instance, combining A. montanus and J. curcas showed a good activity against T. schoenleinii, and J. curcas and E. hirta even showed a better activity against T. schoenleinii. This could be as a result of the increase in the level and types of phytochemicals which are present in the combined extracts. Although the control drug (1% clotrimazole) showed strong activity against all the four isolates, none of the extract had effect against T. mentagrophytes.

Additionally, pronounced variability has been observed for the in vitro sensitivity of the three pathogenic fungi tested (M. gypseum, T. schoenleinii and M. ferugineum), to various plant extracts (Table 5). At the concentration used, the order of effective control of the growth of M. gypseum by the methanolic extracts is as J. curcas > A. montanus > E. hirta. This finding is in agreement with Field, et al. [20]; Rongai, et al. [21]; Elad, et al. [22] and Giordani, et al. [23], indicating that the presence of different phytochemical compounds, either acting alone or in synergy, could be responsible for the observed antifungal effect of these crude extracts.

The result of the checkerboard assay (Table 4) showed that the combination of J. curcas and A. montanus is synergistic at the ratio of 7:3, 6:4, 5:5 and 4:6 while J. curcas and E.hirta have activity at 6:4 and 5:5, A. montanus and E. hirta showed activity at 6:4, 5:5 and 4:6. The cream formulations of these extracts showed promising antifungal activity that is comparable to the standard drug – 1% clotrimazole and they all have good pharmaceutical properties, with very mild irritation on third to fifth day of application which eventually weaned off gradually. The combination of J. curcas and A. montanus showed significant activity against M. ferugineum, while the combination J. curcas and E. hirta, and the combination of A. montanus and E. hirta are effective against M. gypseum and T. schoenleinii, respectively.

Conclusion

The methanolic extracts of the natural plants used in this study have been shown to be effective against the clinical isolates used. The implication of this finding is that these plants could serve as potential raw material for the development of plant-based products which could be used in the cure and management of the various dermatophytosis infections. All things taken together, this study scientifically validates the use of the methanolic leaf extracts of Acantus montanus, Euphorbia hirta and Jatropha curcas as potent antifungal agents, and their cream formulations have good pharmaceutical characteristics.

Recommendations

More research should be carried out on the elucidation of the bioactive components of natural plants and the assessment of their pharmaceutical abilities.

References

  1. Araya S, Tesfaye B, Fente D (2020) Epidemiology of Dermatophyte and Non-Dermatophyte Fungi Infection in Ethiopia. Clin Cosmet Investig Dermatol 13: 291-297.
  2. Sardana K, Kaur R, Arora P, Goyal R, Ghunawat S (2018) Is Antifungal Resistance a Cause for Treatment Failure in Dermatophytosis: A Study Focused on Tinea Corporis and Cruris from a Tertiary Centre? Indian Dermatol Online J 9(2): 90-95.
  3. Graciliana L, Andrade PB, Valentão P (2015) Screening of a marine-Algal extract or Antifungal Activitie. Methods Mol Biol 1308: 411-420.
  4. Rossi NMM, Rossi A, Peres NTA (2008) Antifungal Resistance mechanisms in Dermatophytes. Mycopathologia 166(5-6): 369-383.
  5. Kaushik N, Pujalte GGA, Reese ST (2015) Superficial Fungal Infections. Prim Care 42(4): 501-516.
  6. Basma AA, Zuraini Z, Sasidharan S (2011) A transmission electron microscopy study of the diversity of Candida albicans cells induced by _Euphorbia hirta_ L. leaf extract in vitro. Asian Pac J Trop Biomed1(1): 20-22.
  7. Okoli CO, Akah PA, Onuoha NJ, Okoye TC, Nwoye AC, et al. (2008) _Acanthus montanus_: an experimental evaluation of the antimicrobial, anti-inflammatory and immunological properties of a traditional remedy for furuncles. BMC Complement Altern Med 8: 27.
  8. Esimone CO, Nworu CS, Jackson CL (2008) Cutaneous wound healing activity of a herbal ointment containing the leaf extract of _Jatropha curcas L.(Euphorbiaceae_). Int J Appl Res Nat Prod 1(4): 1-4.
  9. Singh G, Kumar P (2013) Phytochemical study and screening for antimicrobial activity of flavonoids of Euphorbia hirta. Int J Appl Basic Med Res 3(2): 111-116.
  10. Erb M, Kliebenstein DJ (2020) Plant Secondary Metabolites as Defenses, Regulators, and Primary Metabolites: The Blurred Functional Trichotomy. Plant Physiol 184(1): 39-52.
  11. Umashankar DD (2020) Plant secondary metabolites as potential usage in regenerative medicine. _The_ Journal of Phytopharmacology 9(4): 270-273.
  12. Trease GE, Evans WC (2002) Textbook of Pharmacognosy. 16th (Edn.), Bailliere Tindall, London UK.
  13. Cheesebrough M (2006) District laboratory practices in tropical countries. Part 2. 2nd (Edn.), Cambridge university press. New York.
  14. Oli AN, Obaji M, Enweani IB (2019) Combinations of Alchornea cordifolia, Cassytha filiformis and Pterocarpus santalinoides in diarrhoegenic bacterial infections. BMC Res Notes 12(1): 649.
  15. Kalimuthu K, Vijayakumar S, kumar R (2010) Antimicrobial activity of the biodiesel plant, _Jatropha_ _curcas L_. Int J Pharm Bio Sci 1(3): 1-5.
  16. Rahman MM, Ahmad SH, Mohamed MTM, Rahman MZA (2014) Antimicrobial compounds from leaf extracts of _Jatropha curcas_, _Psidium guajava_, and _Andrographis_ _paniculata_. Scientific World Journal 635240.
  17. Tomar NS, Sharma M, Agarwal RM (2015) Phytochemical analysis of Jatropha curcas L. during different seasons and developmental stages and seedling growth of wheat (Triticumaestivum L) as affected by extracts/leachates of _Jatropha curcas L._ Physiol Mol Biol Plants21(1): 83-92.
  18. Adamu M, Oshadu OD, Ogbaje CI (2010) Anthelminthic efficacy of aqueous extract of _Acanthus montanus_ leaf against strongy lid nematodes of small ruminants. Afr J Tradit Complement Altern Med 7(4): 279-285.
  19. Prasad DMR, Izam A, Khan MR (2012) Jatropha curcas: Plant of medical benefits 6(14): 2691-2699.
  20. Field B, Jordán F, Osbourn A (2006) First encounters- deployment of defence-related natural products by plants. New Phytol 172(2): 193-207.
  21. Rongai D, Milano F, Sciò E (2012) Inhibitory effect of plant extracts on conidial germination of the Phytopathogenic fungus _Fusarium oxysporum_. American Journal of Plant Sciences 3(12): 1693-1698.
  22. Elad Y, Yunis H, Katan T (1992) Multiple fungicide resistance to benzimidazoles, dicarboximides and diethofencarb in field isolates of _Botrytis cinerea_ in Israel. Plant Pathology 41(1): 41-46.
  23. Giordani R, Hadef Y, Kaloustian J (2008) Compositions and antifungal activities of essential oils of some Algerian aromatic plants. Fitoterapia 79(3): 199-203.
More from this journal

Cite this article

BibTeX
APA
RIS
@article{ojiako2022,
  title   = {Formulation and Pharmaceutical Characterization of Antidermatophyte Creams of Methanol Leaf Extracts of Euphobia Hirta, Jatropha Curcas and Acantus Montanus},
  author  = {Ojiako CM},
  journal = {Journal of Infectious Diseases & Travel Medicine},
  year    = {2022},
  volume  = {6},
  number  = {1},
  doi     = {10.23880/jidtm-16000157}
}
Ojiako CM (2022). Formulation and Pharmaceutical Characterization of Antidermatophyte Creams of Methanol Leaf Extracts of Euphobia Hirta, Jatropha Curcas and Acantus Montanus. Journal of Infectious Diseases & Travel Medicine, 6(1). https://doi.org/10.23880/jidtm-16000157
TY  - JOUR
TI  - Formulation and Pharmaceutical Characterization of Antidermatophyte Creams of Methanol Leaf Extracts of Euphobia Hirta, Jatropha Curcas and Acantus Montanus
AU  - Ojiako CM
JO  - Journal of Infectious Diseases & Travel Medicine
PY  - 2022
VL  - 6
IS  - 1
DO  - 10.23880/jidtm-16000157
ER  -