Background
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 [
1].
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 [
2]. 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 [
3].
O. europaea is the most abundant phenolic compound (up to 14% of the dry weight) with numerous health benefits attributed to it [
4]. 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) [
7] 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 [
8]. 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 [
12]. 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 [
13]. 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 [
14].
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.
Results and discussion
In present study the preliminary qualitative and quantitative analyses of the
Olea europaea L. leaves extracts (methanol, ethanol, acetone and aqueous) were executed to analyze antibacterial and antioxidant properties against
P. acnes and
S epidermidis. The results of our study clearly portray significant antibacterial and antioxidant properties with reference to the MICs as well as IC
50 (mg/ml) values through 96 well microtitre plates (CLSI recommended broth micro dilution method). The leaves extracts were found to be more effective against anaerobic bacteria
P. acnes (methanolic extracts MIC: 0.933/IC
50:0.636, ethanolic extracts MIC: 1.050/IC
50: 1.040, aqueous MIC: 2.263/IC
50:1.626 and acetone MIC: 2.534/IC
50:2.500) as compared to aerobic bacteria
S. epidermidis (methanolic extracts MIC: 1.031, IC
50:0.670, ethanolic extracts MIC: 1.502/IC
50: 1.234, aqueous extracts MIC: Range/IC
50: Range and acetone extracts MIC: Range/IC
50:1.890). These quantitative value were compared by standard (tetracycline) against
P.acnes (MIC: 0.028, IC
50: 0.013) and
S. epidermidis (MIC: 0.159, IC
50:0.106) (Table
1).
Table 1
Antibacterial activity of Olea europaea leaf extracts of different solvent along with standard against P. acnes and S. epidermidis
P. acnes
|
Olea europaea
| Standard drug (Tetracycline) |
Aqueous | Methanolic | Ethanolic | Acetone |
IC50 | MIC | IC50 | MIC | IC50 | MIC | IC50 | MIC | IC50 | MIC |
1.626 | 2.263 | 0.636 | 0.933 | 1.040 | 1.050 | 2.500 | 2.534 | 0.013 | 0.028 |
S. epidermidis
| Range (NA) | Range (NA) | 0.670 | 1.031 | 1.234 | 1.502 | 1.890 | Range (NA) | 0.106 | 0.159 |
The
Olea europaea L. leaves have a tremendous history of traditional therapeutic uses (antimicrobial and antioxidant) [
21]. Olive leaves are acknowledged for the secondary metabolites such as oleacein oleuropein [
22,
23] Flavonoids and tannins etc. were among the major phenolic contents present in plant extracts [
24]. The phenolic radicals were testified to be less reactive and retain lower electron reduction potential than the oxygen radicals [
25]. Owing to these properties, the phenolic compounds are reflected as excellent radical scavengers. Thus our study, correspondingly evaluates the total phenolic content and flavonoid content along with radicals scavenging activity by DPPH assay. Our data have validated that the mean phenolic content (PC) in olive leaf extracts in terms of mg dispense from 16.9 to 25.6 mg and flavonoid content (FC) from 9.5 to 24.1 respectively (Table
2). Our results showed that methanolic, ethanolic and acetone extracts had the highest amount of phenolic content as well as flavonoid than aqueous extract [
26,
27]. In general, the obtained data showed a statistical significant correlation between leaves extracts (of all the solvents) phytoconstituents analyses,
i.e. total phenols and total flavonoids.
Table 2
Phenolic content, Flavonoid content and DPPH radical scavenging activity of Olea europaea L. leaf extracts along with standard
Methanolic extracts | 25.6 | 24.1 | 14.7 | 23.5 | 66.1 | 75.2 | 92.5 |
Ethanolic extracts | 24.0 | 19.5 | 14.1 | 22.9 | 64.6 | 70.2 | 86.8 |
Acetone extracts | 24.8 | 17.3 | 11.9 | 17.6 | 45.2 | 60.5 | 80.8 |
Aqueous extracts | 16.9 | 9.5 | 10.5 | 11.6 | 24.3 | 35.2 | 70.5 |
Standard | | | | | | | 91.2 |
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 [
28]. 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 [
29]. 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 [
32]. Studies have demonstrated that the phenolic compounds may also stimulates anti-inflammatory effects of lipoxygenase activity, leukotriene B4 production [
33] and hindering biosynthesis of pro-inflammatory cytokines [
34] or tempering inflammatory parameters [
35]. 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.
Acknowledgement
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.