Development of dermopharmaceutical forms based on the fruit of Alchornea cordifolia (Euphorbiaceae) for the treatment of dermatophytes.

Our work was to develop an ointment and a lotion from the fruit juice of Alchornea cordifolia for the treatment of dermatophytes. The juice from the cold pressing of the fruit pulp of A. cordifolia was lyophilized. The ointments and lotions obtained from the lyophilizate have been evaluated in vitro on different Trichophyton species (Trichophyton rubrum, Trichophyton interdigitale, Trichophyton soudanense, and Trichophyton mentagrophytes) according to two techniques, incorporation and direct connection. In vivo tests on white mice were performed after T. mentagrophytes infestation on scarified skin parts. The reference substance was griseofulvin. The 125 mg/ml lyophilizate solution of the fruit pulp showed an activity identical to that of the 125 mg/ml solution of griseofulvin. The lotions were made with ethanol at different degrees and at concentrations of 1/50 and 1/100. Ointments at different concentrations of the lyophilizate (20%, 33%, and 50%) were made with different types of excipients. All lotions and ointments showed in vitro antifungal activity identical to that of griseofulvin up to 16 and 30 days of incubation. The ointments showed better in vivo activity compared to lotions, and the 50% vaseline-based ointment revealed an activity identical to that of griseofulvin, with a healing time of 8 days. The lyophilizate of A. cordifolia fruit juice was formulated as an ointment and lotion, maintained a good antifungal activity in vitro and in vivo on Trichophyton species, compared to griseofulvin. Copyright:

INTRODUCTION

Dermatophytes are conditions caused by microscopic filamentous fungi that have an affinity for keratin (epidermis, nails, and hair). They cause in humans and animals superficial lesions called dermatophytes: epidermophytes (epidermis), intertrigo (folds), onyxis (nails), ringworm (hair), and folliculitis (hair). Fungal infections are common reasons for consultation in dermatology, particularly in developing countries.[12] As symptoms, they present polycyclic lesions, pruritus, inflammatory lesions, superinfections, cracking at the bottom of interdigital folds, and broken hair and nails lose their glow. These symptoms cause a permanent gene, a bad smell, and sometimes, deep scars. Although the global prevalence is 20%–25%,[3] there is very little research and development for the development of new antifungal drugs. However, there are many plants with excellent antifungal activities; but their traditional use in the form of decoctions, juices, extracts, and other primary forms does not allow efficient activity and daily use. The objective of this work is to produce dermopharmaceutical forms from an extract from the fruit juice of Alchornea cordifolia and to demonstrate that the antifungal activity is preserved.

MATERIALS AND METHODS

Plant material

A. cordifolia of the Euphorbiaceae family is a shrub up to 3 m tall with many thick, tender, flared branches with alternating, whole, sinuate leaves [Figure 1]; these initially green fruits turn yellow to orange [Figure 2]. It is very abundant in all the secondary formations in Côte d'Ivoire's forests of the South than the Center. It is the fruit that is taken; it guarantees the sustainability of these plants. The plant and plant parts have been identified in the Botanical Garden of the University Felix Houphouet Boigny-Abidjan.

Figure 1

The shrub Alchornea cordifolia about 3 m high; sheets alternate, whole, twisted

Figure 2

The fruit of Alchornea cordifolia: Capsule in two subsphériques changing rooms, sometimes three. Initially of green color and becomes yellow in orange

Raw materials

The raw materials used were pharmaceutical grade; these are 95° ethanol for lotions; medicinal Vaseline, cold cream, lanolin, polyethylene glycol 400 (PEG 400), wheat starch, and glycerol were used as bases for the formulation of ointments. Vaseline oils, sweet almond, paraffin, and osmosis water were used as wetting agents.

Harvesting fruits

The fruits were harvested in January in a secondary school in the region of Bingerville, Côte d'Ivoire. They were harvested in the ripening period where the pulp is easily removed while leaving the red seed in the center. The identification was carried out by a botanist from the Swiss Center for Scientific Research.

Fruit processing

The fresh pulp separated from the seeds will be used to prepare the juice. They were ground in a porcelain mortar until it became a paste. The paste was then pressed with a mechanical press to get the juice. The juice distributed in balloons was frozen and freeze-dried in a “Christ Alpha” lyophilizer. The lyophilizate obtained was the subject of organoleptic, physicochemical, and textural characterizations.

Preparations of ointments

Anhydrous ointments and hydrophilic ointments were made; they differed in nature as far as the base excipient and the lyophilizate concentration (20%, 33%, and 50%) are concerned. The different formulae are summarized in Tables 1 and 2. For the anhydrous ointments, the lyophilizate was dispersed in the wetting agent and then introduced with stirring into the base excipient. For hydrophilic ointments, the lyophilizate was dissolved in the osmosis water and then was mixed in the hydrophilic base excipient. All the stable formulae were stored for 1 month in the laboratory at a temperature of 25°C to ensure the preservation of their stability. Stable formulae after 1 month of storage have been evaluated in vitro and in vivo.

Table 1

Formulae of anhydrous ointments

	Vaseline ointment	Cold cream ointment	Lanolin ointment
Active substance	Lyophilizate (20%; 33%; 50%)
Base excipient	Vaseline	Cold cream	Lanolin
Wetting agent	Vaseline oil	Almond oil	Paraffin oil

Table 2

Formulae of hydrophilic ointments

	PEG 400 ointment	Glycerol starch ointment
Active substance	Lyophilizate (20%; 33%; and 50%)
Base excipient	PEG 400	Glycerol starch (wheat starch, water, glycerin)
Wetting agent	Water	Water

PEG 400: Polyethylene glycol 400

Preparation of lotions

Ethanol was used at different degrees, 20°, 30°, and 60°. For each alcoholic degree, three lotions with different proportions of lyophilizate were made, 1/5th, 1/50th, and 1/100th [Table 3]. The method of preparation is a simple dissolution with the agitation of lyophilizate in ethanol. These lotions were also tested for 1 month prior to their in vitro and in vivo evaluations.

Table 3

Lotions formulae

	20° lotion	30° lotion	60° lotion
Active substance	Lyophilizate (1/5; 1/50; and 1/100)
Vehicle (° alcohol)	20	30	60

Physicochemical and textural evaluation of formulations

The pH of ointments and lotions was determined. The consistency of the ointments was evaluated with the texture analyzer TA-XT2.

In vitro evaluation of antifungal activity

Four species of dermatophytes belonging to the genus Trichophyton were used. They were provided by the Parasitology Department of Institute Pasteur of Abidjan. These are Trichophyton rubrum, Trichophyton interdigitale, Trichophyton soudanense, and Trichophyton mentagrophytes. The tests were carried out on glucose agar media consisting of biothone, biotrypcase, yeast and malt extracts, glucose, monopotassium phosphate, disodium phosphate, agar, chloramphenicol, and actidione.

Two techniques were used, the incorporation technique and the connection technique.

The incorporation technique

The agar medium of Sabouraud was liquefied at the time of use by heating in a heating water bath. 2 ml of the product to be tested was introduced at the lowest possible temperature before solidification of the agar (40°C). After an inversion shaking with hand, half part was left at laboratory temperature for solidification. 2 ml of vehicle used for the preparation was incorporated under the same conditions into a tube which served as a control. At the platinum loop, some mushroom fragments were removed from the culture medium and were seeded by touching on the slope of the Sabouraud medium containing the product to be tested or controlled. Incubation was carried out in an oven at 27°C. In general, if there is growth, the colonies appear from the 4th day and the incubation is prolonged for at least 16 days. The reading, performed every 4 days, was both qualitative (absence or presence of shoot) and quantitative (shoot speed). This incorporation technique was not possible with the aqueous lyophilizate solution and with the ointments. With the lyophilizate solution, half part of Sabouraud agar remained liquid after incorporation. With ointments, there was a miscibility problem with liquefied agar.

The direct connection technique

This technique was possible with the aqueous solution of lyophilizate, lotions, and ointments.

For the lotion and the aqueous lyophilizate solution at 125 mg/ml, 2 ml was placed in a hemolysis tube. Two mushroom culture fragments were removed from the platinum loop and were introduced into the hemolysis tube containing the test preparation. The tube was capped with carded cotton and was put in an oven of 27°C for 24 h. The controls (distilled water, hydro alcoholic solution, and aqueous solution of griseofulvin of 125 mg/ml) were carried out under the same conditions. Transplanting was carried out 24 h later, on half of the agar Sabouraud nine. Each of the two culture fragments was placed on the slope of the agar plate. Incubation was done in an oven at 27°C. In general, if it grows, the cultures would be visible from the 4th day and the incubation is prolonged for at least 16 days.

As for the ointments, they were spread in a thin layer in a clean and sterile petri dish. Two pieces of mushroom culture were collected from the platinum loop and were placed on the ointment spread in the petri dish. The petri dish was then put in an oven at 27°C for 24 h. The control boxes (griseofulvin ointment, excipient + preservative) were carried out under the same conditions. Transplanting, incubation, and reading were done under the same conditions as with liquid preparations.

In vivo evaluation of the antifungal activity[4]

This protocol was authorized by The National Committee of Etique of Côte d'Ivoire on number 395. The selected white laboratory mice were weighed and then grouped in pairs according to weight. They were anesthetized with ether and the hairs were cut flush on a plate at least 4 cm in diameter on a flank. The skin was then irritated by a few razor shaves or scarified with vaccineostyle. On this irritated skin, a fungal paste comprising a recent culture of T. mentagrophytes mixed in 2 ml of physiological saline was applied using a wooden tongue depressor. After plating, a bandage with compresses and perforated plaster was performed. Ten days later, the bandage was defeated to notice the infestation, which is manifested by erythema and desquamation invading the hair. After making the infested mice sleep again, the preparations were applied to the lesions. The treatment was renewed once daily until definitive disappearance of the signs observed. One group of control mice was treated with griseofulvin ointment and another control group was not treated. The treatment was done twice, that is to say, the same product was used on two mice. In vivo tests were performed with 50% ointments and 1/50 lotions for ethical reasons. The results were expressed in an average healing time which was the time taken for the lesions to disappear completely. To ensure the effective healing of the animals, the remains of scales were sown on Sabouraud and the incubation time was up to 16 days. To verify the effect of the lyophilizate concentration on the antifungal activity, the most effective ointment at the concentration of 50% was selected and the in vivo activities at different concentrations 20%, 33%, and 50% of this formulation have been compared.

RESULTS

Obtaining and characterizing lyophilizate of Alchornea cordifolia fruit

The lyophilizate was a brown semi-fine powder, extracted at 20% complying with European Pharmacopoeia criteria 9.4.

Galenic formulations

The organoleptic, physicochemical, and textural characteristics of the most stable formulations are summarized in Tables 4 and 5.

Table 4

Characteristics of the most stable anhydrous and hydrophilic creams

	Texture	Stability	pH	Consistency (N.mm)
Vaseline ointment (20%, 33%, and 50%)	Not translucent brown color	Stable over (1 month) observation	5.6±0.2	0.042±0.005 (good spread)
Cold cream ointment (20%, 33%, and 50%)			5.9±0.3	0.047±0.004 (good spread)
Lanolin ointment (20%, 33%, and 50%)				0.045±0.005 (good spread)
PEG 400 ointment (20%, 33%, and 50%)	Translucent			0.68±0.05 (average spread)
Starch glycerin ointment (20%, 33%, and 50%)			5.7	0.070±0.01 (average spread)

Table 5

Characteristics of hydroalcoholic lotions

	Lyophilizate concentration in Aptenia cordifolia juice
	1/5	1/50	1/100
60° lotion	Unstable (two-phase separation, overlaying substance, and deposit)	Stable and homogeneous	Stable and homogeneous
30° lotion
20° lotion

Result of antifungal reaction during in vitro evaluation

For the 125 mg/ml lyophilizate solution, tempted over 4–16 days of incubation, no Trichophyton growth was recorded for the four species. The same report has recorded with the 125 mg/ml griseofulvin solution. However, with distilled water, the growth was observed from day 4 and got more and more important over time.

With both techniques, lyophilizate lotions at different degrees (20°, 30°, and 60°) and at both concentrations (1/50 and 1/100) showed inhibition of all four Trichophyton species from day 4 to 16 of incubation. However, with alcoholic excipients at different degrees, progressing growth was recorded over incubation period.

All hydrophilic and anhydrous ointments whatever their concentration (20%, 33%, and 50%) inhibited growth on the four Trichophyton species; with identical reactions recorded with griseofulvin ointment. However, with basic excipients (i.e., Vaseline, cold cream, lanolin, PEG 400, and starch glycerol), progressing growth was recorded over incubation period.

Result of antifungal reaction during in vivo evaluation

Results on all 50% creams are summarized in Table 6.

Table 6

Evaluation of 50% ointment antifungal reactions on mice infested with Trichophyton mentagrophytes

Tagged animals	Weight (g)	Ointment	Average healing period (days)
PAD: Front right paw	22	50% cold cream ointment	9±2
PAG: Front left paw	21.3	50% cold cream ointment
PPD: Back right paw	23	50% starch glycerin ointment	14±1
PPG: Back left paw	23	50% starch glycerin ointment
B: White	23.8	50% lanolin ointment	11±2
T: Head	24	50% lanolin ointment
D: Back	25	50% PEG 400 ointment	12±3
Q: Tail	25.2	50% PEG 400 ointment
FD: Right side	25.8	50% Vaseline ointment	8±1
FG: Left side	26	50% Vaseline ointment
TD: Head-back	27	Griseofulvin ointment	8±2
TQ: Head-tail	27	Griseofulvin ointment
DQ: Back-tail	27.5	Laboratory rat: With no treatment	22±4
TDQ: Head-back-tail	28	Laboratory rat: With no treatment

PEG 400: Polyethylene glycol 400

All creams used for the experience showed antifungal reaction on T. mentagrophytes in vivo. Vaseline ointment came out as most effective with quite identical result to griseofulvin ointment. Reaction of the three concentrations at 20%, 33%, and 50% of the Vaseline ointment was therefore compared to assess influence of lyophilizate concentration [cf. Table 7]. All three concentrations showed antifungal reactions, but only 20% ointment reaction was lower compared to the other two concentrations.

Table 7

Evaluation of antifungal reaction of the three concentrations of Vaseline cream on Trichophyton mentagrophytes-infested mice

Tagged animals	Weight (g)	Vaseline cream (%)	Average healing period (days)
PAD: Front right paw	22	20	14±2
Q: Tail	25.2	33	10±3
OD: Right ear	29	50	9±1

To guarantee effective healing of animals, remaining scales collected after the treatment with all cream were cultivated. After 16 days of incubation, no growth was recorded.

Results on lotions are summarized in Table 8.

Table 8

Evaluation of lotions’ antifungal reaction on Trichophyton mentagrophytes-infested mice

Tagged animals	Weight (g)	Lotions to 1/50 (°)	Average healing period (days)
OD: Right ear	29	20	15±3
OG: Left ear	29	30	14±3
QR: Stripped tail	30	60	11±2

All lotions demonstrated antifungal reaction with a better healing period recorded with 60° lotions. The remaining scales collected after the treatment were cultivated on Sabouraud. After 16 days of incubation, no growth was recorded.

DISCUSSION

Recorded antifungal reactions of lyophilizate solution A. cordifolia fruit juice comply with the work of Ake.[5] The lotions antifungal reaction was reinforced with 60° alcohol, which could be explained by the intrinsic antiseptic reaction that the alcohol possesses. This property has been through comparison of alcohol-based controls at 20°, 30°, and 60°. Indeed, with 60° alcohol control, an inhibition of dermatophytes development from the 1st day of incubation was recorded. For lotion concentrations, up to day 16 of incubation, or even a month, no proof could help to justify possible difference in reaction between the 1/50th lotion and the 1/100th lotion.

Although all ointments made caused sufficient in vitro antidermatophytic reaction, it has been recorded that for each type of excipient, increasing the amount of excipient reduces the effectiveness of cream. This was indirectly revealed by the presence of yeast contamination in culture materials corresponding to these types of cream. In all cases, stains observed by electron microscopic were not dermatophytes.

Anti-dermatophytic creams showed more important reactions in vivo than lotions. This could be explained by the volatile nature of alcohol used as an excipient for lotions. Indeed, alcohol burns off very quickly, and the drug does not stay long on lesions, while the cream remains several hours in contact with the parts to be treated. To have a healing period similar to that observed with ointments, lotions should be applied at least twice a day.

The results have demonstrated that Vaseline ointments come out with the best in vivo reaction. However, it would be difficult for us to stand that Vaseline ointments were more effective because it all depends on the degree of infestation treated on the animal prior to the treatment. However, based on Vaseline spreading ability, could it be recorded as better than all other excipients used, to explain this improvement in reaction recorded with Vaseline ointments?

It has been recorded that anhydrous creams have a better reaction than hydrophilic creams. It has been demonstrated that that an active substance of hydrophilic nature is more easily released from the cream when combined with a lipophilic base excipient, and vice versa. In our formulations, lyophilizate is hydrophilic in nature; therefore, it should be more easily released from lipophilic excipients than hydrophilic excipients and thus causes better anti-dermatophytic reaction.

About 33% and 50% Vaseline ointments recorded roughly the same reaction. However, with our methods used, it would be difficult to say that the 33% cream is as effective as the 50% cream because it all depends on the degree of infestation treated before applying any of the two ointments. It will be therefore recommended to elaborate two formulae and keep the 50% ointment for much larger lesions.

CONCLUSION

Lyophilizate of A. cordifolia fruit juice retained its antifungal reaction when it has been presented both as solution and lotion or ointment. The reaction was identical to that of griseofulvin, especially under the 50% Vaseline form. Methods of in vitro reaction evaluation did not favor efficient comparison of effectiveness between the two forms on the one hand (cream and lotion), and on the other hand, between different formulae within a given form. In vivo evaluation methods allowed differences in anti-dermatophytic reactions. This work should be complemented with 6-month conservation studies and membrane passage studies to guarantee safe use.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.