In Vitro Antioxidant and Antimicrobial Properties of Murraya Paniculata L. Extracts as well as Identification of Their Active Secondary Metabolites by HPLC-ESI-MS

Introduction

Infectious diseases are the world’s leading cause of premature deaths and led to killing many people every day. Morbidity and Mortality due to diarrhea are continuous to be a major problem in many developing countries, especially amongst children due to infections with a variety of bacterial agents [1]. Although the antibiotics have been proved to be an excellent antimicrobial agent the resistant to these antibiotics is an increasing public health problem [2]. In addition, in developing countries, the synthetic drugs are not only expensive and inadequate for the treatment of diseases but also often with adulterations and side effects [1]. Therefore, there is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-emerging infectious diseases [2]. Medicinal plants have played an important role in the healthcare system since the dawn of human civilization and remain one of the major sources of drugs in modern and in traditional medicine because it contains different bioactive compounds [3]. These bioactive compounds such as terpenoids (essential oils and carotenoids), vitamins, and phenolic compounds have a medical important such as antimicrobial and antioxidants [4].

The Murraya (family rutaceae) is genus made up of 14 species. Murraya paniculata L. Jack is a small tropical evergreen shrub, native to the tropical and subtropical parts of the world [5]. For many years, M. paniculata has been used as a medicinal plant and used in traditional medicine [6,7]. Previous reports have shown that the extracts from bark and leaf of this plant had antioxidant, anti-inflammatory, antidiabetic, antimalarial, antidiarrheal, antifungal and antibacterial activities [8-10]. M. paniculata has been investigated for its bioactive ingredients such as coumarins, indole, alkaloids, phenolic acids, flavonoids, and terpenoids. These compounds serve as the scientific evidence for the traditional usage of M. paniculata [11,12].

The aims of this study were to evaluate in vitro the antioxidant and antimicrobial properties of the methanolic of M. paniculata and certain fractions derived from it as well as identify their chemical composition using High-Performance Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry (HPLC-ESI-MS).

Materials and Methods

Reagents and standards

DPPH (1, 1-diphenyl-2-picrylhydrazyl) and Folin- Ciocalteu’s reagent were purchased from Sigma -Aldrich (Steinheim, Germany). Ammonium molybdate, sodium carbonate and aluminum chloride were purchased from Merck (Darmstadt, Germany). Gallic acid, rutin and ascorbic acid were purchased from Sigma-Aldrich (St. Louis, USA). All solvents used for HPLC analysis and biological experiments are analytical grade.

Plant materials

The leaves of M. paniculata were collected from Giza Governorate, Egypt. The plant under study was identified by Prof. Dr. Rim Samir Hamdy Professor of Plant Taxonomy, Faculty of Science, Cairo University. The voucher specimen was stored in Medicinal Chemistry laboratory, Theodor Bilharz Research Institute, Giza, Egypt. The leaves of the plant were washed with distilled water and air dried at room temperature. The dried leaves were ground using an electric mill and the dry powers of the leaves were stored in dried vials.

Preparation of plant extract

The dried leaves powder of M. paniculata (700 g) was extracted several times with 80% methanol. The methanolic (MeOH) extract was filtered and concentrated by the rotatory evaporator (BUCHI, Switzerland) until drying then kept in a desiccator. The methanolic was defatted using petroleum ether. The defatted methanol extract of leaves of the plant was suspended in water and successively fractioned with different solvents chloroform (CHCl₃), ethyl acetate (EtOAc) and n-butanol (BuOH) respectively. Each fraction was concentrated under vacuum using rotatory evaporator till dryness and kept in dry conditions.

Total phenolic content

The phenolic content of the defatted methanolic extract of the leaves of M. paniculata and its fractions were determined using spectrophotometric method [13]. The reaction mixture was prepared by mixing 0.5 ml of sample soluble in MeOH (250 mg/ml), 2.5 ml of Folin-Ciocalteus reagent (10 %) dissolved in water and 2.5 ml NaHCO₃ (7.5%). Blank was prepared containing 0.5 ml methanol, 2.5 ml of Folin-Ciocalteus reagent (10 %) and 2.5 ml NaHCO₃ (7.5%). The mixture was shaken and incubated for 45 min at 45°C. The absorbance was measured at 760 nm against the blank sample and gallic acid used as a standard. All determinations were carried out in triplicate. The total content of phenolics was expressed in terms of gallic acid equivalent per gram dry weight extract (mg GAE/g of extract).

Total flavonoid content

The content of flavonoids of the defatted methanolic extract of the leaves of M. paniculata and the fractions were determined using a colorimetric assay [14]. 0.5 ml of extract was mixed with 2 ml distilled water and 150 μl of NaNO₂ (5%) for 6 min, then 150 μl of AlCl₃ (10%) was added and allow to stand 5 min then 2 ml of NaOH (4%) was added and adjusted to 5 ml with 200 μl distilled water. The mixture was incubated at room temperature for 30 min. The absorbance was measured at 510 nm against blank sample and rutin used as a standard. Estimation of flavonoid content was carried out in triplicate. The total flavonoid content was estimated as mg rutin equivalents per gram extract (mg RE/g extract).

In vitro antioxidant assays

DPPH radical scavenging activity assay

DPPH (2, 2'-diphenyl-1-picrylhydrazyl) free radical scavenging method was carried out [15]. 2 ml of extract concentration solution was mixed with 2 ml of DPPH in methanol (0.1 m mol/l). The control takes in 2 ml of MeOH and 2 ml DPPH solution without the extract. The mixtures were shaken well and kept in dark for 30 min at 37°C. The absorbance was measured at 517 nm against the blank. Ascorbic acid was used as standards and the experiment was carried out in triplicate. The DPPH scavenging activity expressed as SC₅₀ (Concentration of sample required to scavenge 50% of DPPH radicals compared with that of ascorbic acid). The lower SC₅₀ value is an indication of higher scavenging activity. The antioxidant activity of plant extracts was calculated from the following equation:

Scavenging activity % =[(control absorbance - sample absorbance)/(control absorbance)] × 100

Total antioxidant capacity (Phosphomolybdate assay)

The total antioxidant capacity assay was carried out [16]. An aliquot of 0.5 ml of sample (200 μg/ml) solution get mixed with 5 ml of reagent solution (0.6 M sulphuric acid, 28 mM disodium hydrogen phosphate and 4 mM ammonium molybdate). Blank was prepared to contain 5 ml of the reagent solution and the appropriate volume of the same solvent used for the samples. The tubes were coverd and incubated at 95°C for 90 min. After the samples had cooled down to room temperature, the absorbance of the mixture was measured against the blank at 695 nm. The experiment was repeated for three times. The antioxidant activity of the extracts was expressed as the number of equivalents of mg ascorbic acid per gram extract (mg AAE/g extract).

Determination of antimicrobial activity

Test microorganisms

The pathogenic microbes used in this study were gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and gram positive bacteria (Bacillis subtilis and streptococcus pneumonia). In addition to four fungal strains which are Aspergillus fumigates, Syncephalastrum, Candida albicans and Geotrichum candida. They were obtained from the regional center for Mycology and Biotechnology Unit, Al-Azahar University, Cairo, Egypt.

Disc diffusion assay

The antimicrobial activity assay is based on the clear zone formed around the disk. Partial inhibition was indicated by a semi-clear zone and complete inhibition by a clear zone. The sterilized media was poured onto sterilized Petri dishes (20 ml, each Petri dish) and be allowed to solubility. Wells of 6 mm diameter were made in the solidified media with the help of sterile borer. A sterile swab was used to evenly distribute microbial suspension over the surface of solidified media and solutions of the tested samples were added to each well with the help of micropipette. The plates were incubated at 37°C for 24 h in case of antibacterial activity and 48 h at 25°C for antifungal activity. Ampicillin and gentamycin were used as antibacterial standards and ampholericin was used as an antifungal standard. This experiment carried out in triplicate and zones of inhibition measured in mm scale. This test was carried out at Antimicrobial activity unit Faculty of Science, Al-Azhar University, Cairo, Egypt.

HPLC-ESI-MS analysis

HPLC-ESI-MS analysis was performed using HPLC instrument (Waters Alliance 2695) and mass spectrometry (Waters 3100). The mobile phase was filtered carefully by using 0.45 μm filter membrane disc and degassed by sonication before analysis. 20 μl of samples volume of MeOH extract and EtOAc fraction of M. paniculata were injected into the HPLC instrument contain reversed phase C-18 column (Phenomenex 250 mm, 5 μm particle size). Mobile phase elution was made with the flow rate of 0.4 ml/min using gradient linear mobile phase containing two solvents: solvent A is H₂O acidified with 0.1% formic acid and eluent B is CH₃CN/MeOH (1: 1, v/v) acidified with 0.1% formic acid. The mobile phase elution was carried out by the following gradient: 5% B, 0-5 min; 5%-10% B, 5-10 min; 10%-50% B, 10-55 min; 50%-95% B, 55-65 min; 5% B, 65-70 min. The parameters for analysis were performed by using negative ion mode as follows: source temperature 150°C, capillary voltage 3 kV, cone voltage 50 eV, cone gas flow 50 L/h, desolvation temperature 350°C, and desolvation gas flow 600 L/h. Mass spectra were detected in the ESI negative ion mode between m/z 50-1000. The peaks and spectra were processed using the Maslynx 4.1 software and tentatively characterized by comparing its mass spectrum and retention time (t_R) with literature.

Statistical analysis

The data analysis was carried out using SPSS (13) software for evaluation of the IC₅₀. The data were presented as mean ± standard deviation (SD).

Conclusion

As a conclusion, EtOAc fraction derived from the defatted MeOH extract of M. paniculata exhibited significant the highest content of antioxidants compared to the tested extracts. The antimicrobial test showed that the ethyl acetate and methanol extract had similar results against S. pneumonia. On the other hand, the results for other bacteria and fungi showed that the ethyl acetate extract has the lead compared to other extracts. HPLC-ESI-MS analysis of MeOH extract and its derived fractions (EtOAc and BuOH) demonstrated the presence of three major bioactive compounds, viz. flavonoids, phenolic acids, and coumarins. These bioactive secondary metabolites are responsible for the different bioactivities of M. paniculata extracts. Therefore, it would suggest that M. paniculata extracts may be used for the treatment of microbial infections.

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