Comprehensive evaluation of pharmacological properties of Olea europaea L. for Cosmeceuticals prospects
© The Author(s). 2017
Received: 8 November 2016
Accepted: 24 May 2017
Published: 4 July 2017
Propionibacterium acnes (anaerobic bacteria) and Staphylococcus epidermidis (aerobic bacteria) have been acknowledged as key comedone forming pathological factor, eliciting an inflammation in acne. The present study was conducted to evaluate antibacterial and antioxidant activities of Olea europaea leaves extracts (OLE) of different solvents (methanol, ethanol, deionized water, and acetone) against etiologic pathogens of acne vulgaris.
The antibacterial testing against the selected pathogen viz., P. acnes and S. epidermidis were evaluated using broth micro dilution method recommended by CLSI, in duplicate. Correspondingly the total phenolic content and flavonoid content along with radicals scavenging activity by DPPH assay were also evaluated. The data of antibacterial assay demonstrated that these plant extracts differ quantitatively in their activity against the tested pathogens.
The results (mg/ml) exhibited that Olea europaea leaves extracts (MIC:2.263/IC50:1.626, MIC:0.933/IC50:0.636, MIC:1.054/IC50:1.040, MIC:2.534/IC50:2.500 of aqueous, methanol, ethanol, acetone extracts respectively) are more effective against growth of P.acnes as compared to S. epidermidis (MIC: Range (Not active at particular concentration), IC50: Range, MIC:1.031, IC50: 0.670, MIC:1.502, IC50:1.234, MIC: Range, IC50:1.890 mg/ml aqueous, methanol, ethanol, acetone extracts respectively). The readings were statically analyzed and also compared with standard drug tetracycline.
The current findings suggested Olea europaea L. as a promising source of potential antioxidants and antibacterial activity against P.acnes and S. epidermidis that may be an efficient therapeutic agent in the pathogenesis of Acne vulgaris and proves a potential source of Cosmeceuticals.
KeywordsPropionibacterium acnes Staphylococcus epidermidis CLSI Antibacterial activities MIC DPPH
Natural drug resources with their varied biological and pharmacological properties (due to the presence of phenolic acids, flavonoids, tannins, vitamins and terpenoids) represent a treasure for researchers, to combat problem concerning treatment of health disorders or dermal infections. In the last few years, with the increasing doses of conventional drugs, multidrug resistance of pathogens develops. To overcome these persistent dilemmas of conventional treatments an increasing interest in herbal therapy has emerged. The herbal formulations are a viable option that could be useful in reducing the side effects associated with synthetic antibiotic treatment. Emphasis has been mainly on the antibacterial, anti-inflammatory and antioxidant properties of herbal extract .
Olea europaea L. leaves are a sort of waste product, this waste product is not profitable; olive leaves are often used as animal feed or simply burned with excess branches gathered . The concern in olive leaves grew in the last few years due to its high pharmacologicalproperties, presence of phenolic compound flavonoids, tannins, vitamins C and terpenoids and high concentration of phenolic compounds . O. europaea is the most abundant phenolic compound (up to 14% of the dry weight) with numerous health benefits attributed to it . It has been found to have potent antioxidant and radical scavengers with anti-tumor and anti-inflammatory, antimicrobial properties and anti-atherogenic, hypoglycemic, hepatic, cardiac and neuro-protective effects [5, 6]. Oleuropein has a protective effect in counteracting low-density lipoprotein (LDL) oxidation, validated through the estimation of the decreased formation of thiobarbituric acid-reactive substances (TBARS are naturally present in organic specimens, include lipid hydroperoxides and aldehydes which increase in concentration as a response to oxidative stress)  and malondialdehyde (MDA, compound that results from the decomposition of polyunsaturated fatty acid lipid peroxides) and 4-hydroxynonenal (4-HNE) as lipid peroxides by-products . Its anti-tumour activity has shown to inhibit proliferation and migration of a number of advanced grade human tumors cell lines in a dose dependent manner [9–11]. Its anti-inflammatory activity is also remarkable, demonstrated by decreasingthe production of monocytic inflammatory mediators, decreasing in the production of IL-1β in human whole blood cultures stimulated with monocytes-triggered by LPS . Interestingly, olive oil phenolic compounds reduce the circulating concentrations of IL-6, a pro-inflammatory agent that stimulates inflammation in several pathologies. Acne vulgaris is a chronic inflammatory disorder of pilosebaceous unit that affects more than 85% of adolescents and young adults . The increase sebum production, hypercornification of the pilosebaceous follicle, an abnormality of the microbial flora (P. acnes and S. epidermidis), and the production of inflammation are the main triggering cause of Acne vulgaris .
The present study intends to evaluate the antimicrobial properties of different solvent extracts of Olea europaea L. leaves (olive leaf) against most common but ticklish anaerobic bacteria of human dermal pathogen i.e. Propionibacterium acnes and aerobic bacteria Staphylococcus epidermidis, causative agent of acne vulgaris.
Olive leaves were received as a gift from Riyadh, Saudi Arabia. All the chemicals and reagents used were of analytical grade, and were either purchased from Himedia.
Preparation of extracts
The test organisms
The test organisms Propionibacterium acnes (MTCC 1951) and Staphylococcus epidermidis (MTCC 435) were procured from Microbial Type Culture Collection, Chandigarh, India and the media were procured from Hi-Media. The culture of P. acnes (anaerobic bacteria) was maintained on Anaerobic Blood Agar Medium supplemented with freshsheep blood. The proper anaerobic environment was provided to the bacterial culture by the Anaxomate advance instrument after which the culture was incubated for 48 h at 37 °C in CO2 incubator to provide optimum temperature for bacteria growth (Fig. 1). The culture of aerobic bacteria was maintained on Nutrient Agar and incubated in BOD incubator for their appropriate growth (Fig. 1).
Phenolic content: 20 μl of each extract solution and standard (tannic acid) were mixed with 1 ml of ddH2O and 100 μl of Folin-Ciocalteu reagent, followed by the addition of 300 μl of 20%Na2CO3 solution after 1 minutethe resulted mixture now incubated in shaker incubator (temp 40 °C, 30 min). The phenolic content was determined as milligram tannic acid equivalents (TAE)/g of dry weight powder (DW) .
Flavonoidscontent: 4 ml of ddH2O was mixed with1ml of each olive extract. Subsequently, 5% sodium nitrite solution (0.3 mL) and 10% aluminum chloride solution (0.3 mL) was added and incubated at room temp for 5-10 min. Then 2 mL of 1 M NaOH was added to the mixture, the volume makes upto 10 ml with ddH2O and subjected to vortex thoroughly. The pink colour developed and show absorbance at 510 nm. Total flavonoids content was determined as mg catechin equivalents per g of dry weight powder (mg CE g-1 DW) .
DPPH: it is a method to measure the antioxidant/free radical scavenging activity of extracts. The antioxidant activity/radical scavenging of leaf extracts were evaluated using 1, 1-Diphenyl-2-Picrylhydrazyl. For this purpose 0.1 mM solution of DPPH in methanol was prepared. 30 μL of extracts in different concentration (10, 50, 100, 500, and 1000 μg/mL) were mixed with DPPH solution and thoroughly vortex and incubated for 30 min at 25 °C, decrease in absorbance was measured at A = 517 nm against blank. The standard (butylhydroxyl toluene) was used as synthetic antioxidant positive control. The scavenging ability of the plant on DPPH was calculated using the equation:
Where Abs control is the absorbance of DPPH + methanol; Abs sample is the absorbance of DPPH radical + sample extract or standard .
Determination of minimum inhibitory concentrations (MICs) and IC50
All experiments were carried out in duplicate. The data were analyzed using analysis of variance (ANOVA) and significant differences (p < 0.05) among means were determined by R commander software.
Results and discussion
Antibacterial activity of Olea europaea leaf extracts of different solvent along with standard against P. acnes and S. epidermidis
(IC50 and MIC mg/ml)
Standard drug (Tetracycline)
Phenolic content, Flavonoid content and DPPH radical scavenging activity of Olea europaea L. leaf extracts along with standard
DPPH radical scavenging activity
Higher the antioxidant concentration (in solvent) higher will be the percentage of DPPH scavenging activity of a compound and better will be the antioxidant activity. Further, DPPH radical scavenging activity was found to agree with total phenolics and total flavonoids outcomes. The present data revealed methanolic extracts of olive leaves exhibited appreciative higher free radical scavenging activity, followed by ethanolic ≥ acetone ≥ aqueous respectively, using DPPH. Free radical scavenging DPPH assay data dispensed from 14.7 to 92.5 (Table 2). These values were compared to the value obtained using standard (91.8 μg/ml). These data were also in statistical significant correlation with the data of total phenols and flavonoids. The hight free radical scavenging activity would be due to the high content of phenolic compounds in methanolic extractof O. europaea leaf especially oleuropein and hydroxytyrosol . The reducing properties of polyphenols as hydrogen or electron-donating agents relay their potential for free-radical scavengers (antioxidants). Polyphenols holds an ideal chemical structure for free radical-scavenging activities and most of them have been shown to be strong antioxidants in vitro than vitamin E . It has been demonstrated that hydroxytyrosol is empowered with a potent antioxidant activity due to the ortho diphenol function. Thus, the high antioxidant activities of oleuropein can be described by the presence of hydroxytyrosol unit in its structure [30, 31].
Regarding antimicrobial properties, OLE exhibits high antibacterial activity against anaerobic and aerobic bacteria causing acne vulgaris, this activity isalso in correlation with the data of above discussed results. The presence of phenolic content confers O. europaea L. natural resistance to microbe (Gram negative, Gram positive) outbreak . Studies have demonstrated that the phenolic compounds may also stimulates anti-inflammatory effects of lipoxygenase activity, leukotriene B4 production  and hindering biosynthesis of pro-inflammatory cytokines  or tempering inflammatory parameters . Likewise COX-2, an enzyme involved in the generation of some inflammatory mediators (TNF-αand IL-1β mediated enzyme) and the expression of these inflammation inducing enzymes, interleukins and tumor necrosis factors were reported to be attenuated significantly with the treatment of Olive-derived phenolic compounds [36, 37]. All these activities of OLE confer antibacterial activity against pathogens of Acne vulgaris.
The developing natural therapies encase naturally derived drugs from active plant extracts, essential oilsand phytomolecules. The antibacterial and antioxidant potential of the olive leaf extracts can be attributed to its high contents of phenols, flavonoids and vitamin C that act synergistically. Although there is an observed significant variation in chemical constituents and biological activities of olive leaf extracts treated with different solvents, the current findings support that this medicinal plant Olea europaea L. is a promising source of potential antibacterial and antioxidants that may be efficient therapeutic agent in the pathogenesis of acne vulgaris and proves potential source of Cosmeceuticals.
The authors would like to expresstheir gratitude to Head, Department of Botany, and University of Allahabad to providing research facilities, UGC, New Delhi for financial support, Mr. Rick Z for providing anaerobic jar and Moti Lal Nehru Medical College, Allahabad for providing the anaerobic culturing facilities.
AQ conducted the experiments and design conception and conduction of research. Data interpretation and analysis were done by MP participated in drafting the manuscript revised the manuscript critically for important intellectual content. RK critically reviews the manuscript. AD made the necessary corrections in the write up and gave final approval for the submission of revised version. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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