Galenic Formulation of an Antibacterial Ointment Based on Derivatives of Coriaria myrtifolia and Pistacia lentiscus
- *Corresponding Author:
- Kawtar Fikri-Benbrahim
Laboratory of Microbial Biotechnology
Sidi Mohamed Ben Abdellah University
P.O. Box 2202, Fez, Morocco
Faced to therapeutic limits of chemical drugs, research development programs on medicinal plants are oriented toward the obtaining of phytomedicines. In this context, this work aims to develop a dermal ointment intended for external use. In order to achieve this objective, the derivatives of two studied plants: Pistacia lentiscus (100 μl of Essential oil) and Coriaria myrtifolia (1 g of the ethyl acetate extract) are incorporated in 100 g of excipient (40 g of shea butter and 60 g of laboratorys white petrolatum "vaseline"). The quality control of this ointment has focused on the evaluation of organoleptic properties (color, odor), homogeneity, consistency or spread ability, pH and antibacterial activity. The observation has indicated that the ointment is of dark green color, homogeneous, and ensures a good adhesion to the skin. The assessment of its antibacterial power by the disk diffusion method has indicated that this ointment has an important antibacterial effect against Staphylococcus aureus and Bacillus sp. This activity is translated by inhibition zone diameters of 18.3 and 15.6 mm respectively, hence it can be concluded that the derivatives of the plants studied in galenic form retain their biological effect.
Pistacia lentiscus, Antibacterial activity, Coriaria myrtifolia, Dermal ointment, Formulation.
Morocco offers a great ecological and floristic diversity, because of its biogeographical position . As everywhere in Africa, a strong ethnomedicinal tradition is still alive throughout all the country , particularly in the north of Morocco. Since these popular practices are not totally devoid of scientificity , it is therefore necessary to rehabilitate the medicine and the traditional toxicology in Morocco, this work falls within this context.
Pistacia lentiscus is known for its medicinal properties since the Antiquity. The aerial part is traditionally used in the treatment of arterial hypertension thanks to its diuretic properties [4,5]. The leaves are provided with anti-inflammatory, antibacterial, antifungal, antipyretic, astringent, hepatoprotective, expectorant and stimulating activities [6-11]. They are also used in the treatment of eczema, oral infections, diarrhea, nephrolithiasis, jaundice, headache, asthma and respiratory problems [7,12,13]. Finally, the lentisque’s essential oil is known for its therapeutic virtues with regard to lymphatic and circulatory problems .
Materials and Methods
P. lentiscus was collected from Taounate (Altitude: 475 m, north: 34° 35.203', West: 004°38.533'; and C. myrtifolia from Bab Berred (altitude: 1290 M, N 35° 00' 979'', W 004° 58' 092''); in the north of Morocco. The identification and conservation of both plants were carried out at the National Institute of aromatic and medicinal plants of Taouante (Morocco).
Preparation of derivatives of the two plants
The essential oil of P. lentiscus was obtained by hydrodistillation on a Clevenger type device  (100 g of fresh leaves were treated with hydrodistillation during three hours), then the essential oil (E. O.) was stored in optimal conditions (4°C in the dark in the presence of anhydrous sodium sulfate) .
After drying the C. myrtifolia sample in open air, the leaves were separated from the stem and crushed until obtaining a degree of particle size adapted to an optimal dissolution. The extract preparation was carried out by Soxhlet: 45 g of powder first undergoes a delipidation by 200 ml of hexane; then extraction was carried out by the addition of 200 ml of ethyl acetate to the delipidated and dried powder. The Extract was recovered after vacuum evaporation of the solvent by using a rotary evaporator (90 rpm /min, 40°C) .
The ointment preparation
In order to prepare an ointment at 1%, 1 g of the ethyl acetate extract of C. myrtifolia, 100 μl of the foliar EO of P. lentiscus and 100 g of the excipient (40 g of the shea butter and 60 g of laboratory white petrolatum "vaseline") were used. Firstly, the extract, measured by a precision balance, was placed in a previously cleaned and dried porcelain mortar. The EO and the excipient were then gradually added while grinding until obtaining a homogeneous mixture . The ointment thus obtained was conditioned thanks to a spatula in a glass pot, which was then hermetically closed and labeled .
Quality control of the ointment
The control has focused on the evaluation of organoleptic properties (color, odor), homogeneity, consistency or spreadability, pH and antibacterial activity of the formulated ointment.
The ointment color and odor were assessed in relation to those of the pure excipient .
The homogeneity was verified by observing the excipient’s dispersal; hence, after spreading a thin layer on a rigid flat surface using a spatula, regular or non-regular distribution of the extract was noted .
The pH measurement of a tenfold dilution of the ointment in hot distilled water was carried out by using a pH meter .
The disk diffusion method was used to evaluate the antibacterial activity of the formulated ointment . The objective of this analysis was to ensure that derivatives of the studied plants put under galenic form retained their activity. Several concentrations were tested (1 g/ml, 500 mg/ml, 250 mg/ml and 125 mg/ml) against two bacterial strains responsible of dermal infections, Staphylococcus aureus and Bacillus sp. (Laboratory of Microbial Biotechnology, Faculty of Science and Technology, Fez, Morocco).
The results were expressed as the average and standard deviation. The comparison of the averages was performed by Student test and was considered significant at a value of p<0.05.
Results and Discussion
The results of our previous investigations on the chemical composition and the antibacterial effects of C. myrtifolia and P. lentiscus [11,15,16], along with those of the scientific literature, have encouraged us to the exploitation and valorization of these plants by the formulation of a dermal ointment intended for external use. The quality control of the so-called ointment consists on the evaluation of the macroscopic characters (color, odor), homogeneity, pH and antibacterial activity.
The prepared ointment is of dark green color, homogeneous and soft. Characterized by a smell of lentisque and a pH of 6.5, it is stable to the usual conservation conditions and ensures a good adhesion to the skin.
The results of Table 1 indicate that the ointment has an important antibacterial effect against the tested strains, which results in inhibition diameters of 18.3 and 15.6 mm respectively against S. aureus and Bacillus sp. (1000 mg/ml).
|T||1000 mg/ml||500 mg/ml||250 mg/ml||125 mg/ml|
|Staphylococcus aureus||7 ± 0||18.3 ± 0.4||14 ± 1.3||11 ± 1.1||8.3 ± 0.4|
|Bacillus sp.||6.6 ± 0.4||15.6 ± 0.4||14 ± 0.2||10.6 ± 0.8||6.3 ± 0.4|
T: Blank (ethyl acetate and excipient)
Table 1: The inhibition zones diameters of different concentrations of the formulated ointment
The inhibition zones diameter is expressed in (mm), and represents the average of three repetitions.
This antibacterial activity can be explained by the presence of P. lentiscus essential oil and Coriaria myrtifolia’s ethyl acetate extract in this ointment. In fact, the antibacterial activity of these two plants has been attributed to their richness in biologically active secondary metabolites. Hence, Phytochemical screening indicated that several active compounds are present in the ethyl acetate extract of C. myrtifolia which in addition to polyphenols (480 ± 4.80) and flavonoids (17 ± 0.54) , contains tannins, steroidal heterosides and heterozoic triterpenes (Table 2) .
|Chemical families||Test Results|
+ presence/- absence
Table 2: Chemical families’ revelation results of the ethyl acetate extract of C. myrtifolia from Bab Berred 
It is worth noting that polyphenols are recognized as secondary metabolites synthesized by higher plants in response to external aggressions, and have several roles in plant physiology and have potential healthy properties on human organism . This, widely distributed, group of natural products have been shown to exert antioxidant, antiinflammatory, anticancer and antibacterial effects. Indeed, hundreds of publications reporting the antimicrobial activity of polyphenols have been recently published .
Moreover, polyphenols are known to be highly susceptible to auto-oxidation which induces formation of high molecular weight polymers. Indeed, polyphenols molecular weight is an important factor that governs their antimicrobial activity . Because monomers are too small to establish enough hydrogen bridges while high molecular weight polymers are too large to cross the bacterial wall, so the ideal molecular weight would be that of oligomers . It has been demonstrated that mechanisms of toxicity against microorganisms include either the deprivation of metal ions such as iron and non-specific interactions such as the establishment of hydrogen bonds with cell wall proteins (Adhesins) or enzymes .
While, the chromatographic analysis of P. lentiscus E. O. revealed that the major components were: myrcene (25.3%), limonene (15.7%), terpinene-4-ol (9.2%), β-gurjunene (2.6%),D-germacrene (2.3%) and α-pinene (1.6%), accounting for approximately 56.7% of its chemical composition (Table 3) ; beside other components such as 1,8-cineole, α-terpineol, linalool which also have a significant antibacterial activity.
RI: Retention index, t : Traces (< 0.1%.)
Table 3: Volatile components of the essential oil from leaves of Pistacia lentiscus 
Hence, 1,8-cineole is very active against some bacterial strains (Salmonella sp., Staphylococcus sp., Bacillus sp.) [27,28]. Moreover, the linalool-which is a terpene alcohol-is known for its inhibitory activity against Enterococcus sp. and Escherichia coli . Furthermore, being among the major compounds of several medicinal plants such as Cymbopogon citratus , myrcene have been reported as antibacterial terpene . Several other works have also demonstrated an antibacterial power for limonene [32,33], α-terpineol [34,35] and β-Ocimene  against several bacterial strains.
It should be noted that even if the ointment is diluted, the antibacterial effect is maintained (the inhibition diameter of the concentration 250 mg/ml against S. aureus is 11 mm). So, it can be concluded that the derivatives of the studied plants put under galenic form retain their effect.
Based on our previous studies results, exploitation of derivatives of P. lentiscus and C. myrtifolia was necessary. Hence the formulation of dermal ointment (based on these derivatives) intended for external use and having an important antibacterial effect was investigated. The galenic preparation and its quality control allowed obtaining of a dark green, homogeneous ointment, providing good adhesion to the skin and having an important antibacterial effect. In fact, inhibition zone diameters of 18.3 and 15.6 mm were obtained respectively against S. aureus and Bacillus sp. Hence, the objective of this work is therefore reached, and the valorization of the studied plants is performed.
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