Screening of Antibacterial activity in Vitro of Cyclamen hederifolium tubers Extracts

 

Oussama Mansour^1* , Rim Salamma², Lubna Abbas³

¹Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al Andalus University, Tartous-Syria

²Department of Pharmacognosy, Faculty of Pharmacy, Tishreen University, Lattakia –Syria

³Department of    Pharmacognosy, Faculty of Pharmacy, Al Andalus University, Tartous -Syria

 

ABSTRACT:

This study was conducted to evaluate the antimicrobial activity of four extracts of Cyclamen hederifolium tubers plant belonging to the Primulaceae (Myrisinaceae) family collected from the coastal surrounding, Lattakia, Syria. Many efforts have been made to discover new antimicrobial compounds from a variety of sources such as micro-organisms, animals and plants. One such source is folk medicine.  Systematic screening of them may result in the discovery of novel effective compounds. These extracts were prepared with increasing polarity solvents: petroleum ether, chloroform, ethyl acetate and methanol solvents by steeping extraction method. The antimicrobial activity of the extracts was then assessed using the disk diffusion method against gram-positive (Staphylococcus aureus), and gram-negative (Escherichia coli) bacteria. The methanolic extracts of Cyclamen Hederifolium tubers have shown greater antimicrobial activities than the other extracts against tested bacteria, followed by ethyl acetate and chloroform extracts. The activity was compared with potent broad spectrum antibiotics such as Imepinem.

 

 

 

INTRODUCTION:

Anti microbial agents is undeniably one of the most important therapeutic discoveries of the 20th century. However, mankind is now faced with the global problem of emerging resistance in virtually pathogens.¹ Concern has been expressed about the rising prevalence of pathogenic microorganisms, which are resistant to the newer or modern antibiotics that have been produced in the last three decades.² The toxic side effects and high cost of new generation antibiotics with limited effective span have resulted in an increase in morbidity and mortality.

 

Therefore, there is a need to look for substances from other sources with proven antimicrobial activity.³ For over thousands of years, natural plants have been considered as a valuable source of medicinal agents with proven potential of treating infectious diseases and with lesser side effects compared with the synthetic drug agents. The World Health Organization estimates that 80% of people in developing countries (65% of the world’s population) still rely on traditional medicine.⁴

 

Plants extracts have been rich sources of medicines because they produce a host of bioactive molecules, which probably evolved as chemical defenses against infection. ⁵Most active compounds in these extracts remain unidentified, and their presence is only detected by biological tests. The identified compounds are products of plant secondary metabolism and belong to the classeso f alkaloids, polyphenols, triterpenes, and steroid glycosides.⁶ Cyclamen hederifolium (ivy-leaved cyclamen) is a species of flowering plant in the genus  Cyclamen, of the family Myrsinaceae. It is the most widespread cyclamen species, the most widely cultivated after the florist's cyclamen (Cyclamen persicum), and the most hardy and vigorous in oceanic climates.⁷It is native to woodland, shrubland, and rocky areas in the Mediterranean region.⁸ C. hederifolium is known by abundance of common names including Ivy-leaved Cyclamen, Sowbread, Cyclamen europaeum and Cyclamen neapolitanum.⁹It widely grows throughout the coastal surrounding, Syria. The tubers of the plant are used commonly to treat menstrual complaints, emotional disorders/nervous states and digestive problems. It is also used in Homeopathic therapy for migraine and its accompanying autonomic symptoms, and for the treatment of premenstrual syndrome.¹⁰ Many researchers have been conducted with the aim of studying the biological activities of medicinal plants and using them for the treatment of microbial infections as possible alternatives to chemically synthetic drugs to which many infectious microorganisms have become resistant¹¹. This study was aimed at evaluating the antimicrobial property in vitro of four extracts of C. hederifolium tubers.

 

MATERIALS AND METHODS:

Collection and Identification of Plant Materials

Cyclamen hederifoluim was collected from the coastal Surrounding, Lattakia, Syria during the hibernate stage (winter 2014). The study was carried out at the Departments of Pharmacognosy and Biology, Faculty of Pharmacy, Tishreen University, Lattakia. Syria.

 

Plant Extracts

The tubers of the plant material were washed and dried in shade at room temperature (25ºc) and then grounded to a fine powder in a mechanic grinder. The powdered plant materials (30g) were then extracted with 300mL of each solvent (petroleum ether, chloroform, ethyl acetate and 70% methanol) for 72 hours by Steepingmethod.^12,6

Following filtration with Whatman filter paper (No 1), all extracts were concentrated and evaporated to dryness at room temperature. The yields from the different extracts were weighed and dissolved in dimethyl sulphoxide (DMSO) to form a mother solution (500 mg/1ml) for each extract, then three dilutions (20,50,100 mg/ml) were made from each mother solution. All extracts were maintained at +4°C until being used for disk diffusion assay.^12,11

 

Test Microorganisms

The extracts inhibitory effects were tested against two microbial species including Escherichia coli, Staphylococcus aureus obtained from Al-Assad Hospital, Lattakia. The bacterial cultures of test organisms were maintained on Muller Hinton Agar at 4°C, and were subcultured in petri plates (90mm) prior to use.¹³ DMSO was used as a negative control under the same condition for the tested microorganisms. Imepinem (Primaxin®, 10μg/disc) was used as a positive control. The tests were carried out in duplicate. Antimicrobial activity was evaluated by measuring the zone of inhibition (mm) against the tested microorganisms.¹⁴

 

Determination of Antimicrobial Activity

The antimicrobial activities of the petroleum ether, chloroform, ethyl acetate and methanol extracts by the different concentrations as well as the positive control Imepinem were tested by means of the disk diffusion assay against two human pathogenic bacterial strains, including Gram positive (S aureus) and Gram negative (E coli).¹⁵ Approximately three cultures from each bacterial stain were inoculated over the surface of petri plates containing Muller Hinton Agar using sterile swab sticks. Whatman paper disk injected by 20 μl of each concentration and Imepinem disk were placed on the surface of petri plates. The plates were incubated at 37°C for 24 hours.¹⁶ The inhibition zones were calculated by measuring the diameters of inhibition in mm (including disk). Experiments were performed in duplicate and the inhibition zones were compared with the positivecontrol.¹⁷

 

RESULTS:

The anti-bacterial activities of tubers extracts were evaluated in vitro against E.coli, S.aureus, which are known to cause common infectious diseases. The results of antibacterial activity were recorded as zone of inhibition in mm around the active extract against the tested microorganisms compared with the standard antibiotic Imepinem used as a positive control.¹⁸ Some of C. hederifolium extracts have shown antibacterial activity against the test microorganisms. The diameters of zone inhibition of extracts against S. aureus were (16-25mm) compared with the diameters of zone inhibition of Imepinem (35-45 mm). The diameters of zone inhibition of extracts against E. coli were (23-28 mm) compared with the diameters of zone inhibition of Imepinem (28-30 mm).

 

As shown in table1, all concentrations of methanolic extract have shown good inhibitory effects against the tested bacteria. While all concentrations of ether petroleum extract showed no inhibitory effects against the tested bacteria. The chloroformic and ethyl acetate extracts have shown no inhibitory effects against the test bacteria except at 100mg/ml concentration which have shown inhibitory effects against the test bacteria. In conclusion, the methanolic extracts of C.hederifolium tubers have shown greater antimicrobial activity than the other extracts, followed by ethyl acetate and chloroformic extracts.

Table 1: Antibacterial activity of four Cyclamen hederifolium tubers extracts

(-) refers to no inhibition (AB) refers to Imepinem

 

DISCUSSION:

C.hederifolium is known to contain stratch, organic acids and Triterpene saponins: including cyclamine, deglu-cocyrlamineI, deglucocyclamine II, ardisicrenoside D, cyclaminorin, hederifoliosides A-E. ¹⁹Saponins are a major family of secondary metabolites that occur in wide range of plant species. Saponins are steroid and triterpen glycosides so named because of their soap like properties. ²⁰Most of Terpenes and saponins have potent against microorganisms as fungi, bacteria, viruses and protozoa.²¹

 

The antibacterial properties of saponins are generally ascribed to the ability of these molecules to complex with sterols in bacterial membranes, thus causing pore formation and loss of membrane integrity.²² Therefore, the presence of triterpene saponins in the tuber C. hederifolium extracts is the main reason for the antibacterial activity demonstrated by some tuber extracts, because that constituents are dissolved in polar solvents as methanol (they are present as glycosides within the plant) and have weak dissolution in non-polarsolvents²³. This may explain that polar extracts have shown the best antibacterial activity against the tested bacteria.

 

CONCLUSION:

The extracts of C.hederifolium tubers were investigated individually for antimicrobial activity by disc diffusion method against selected species of Escherichia coli and Staphylococcus aureus to find the inhibitory activities of the microbes. The methanolic extracts followed by ethyl acetate and chloroformic extracts have showed considerably good activity. These results were compared with standard antibiotic Imepinem. In conclusion, Cyclamen hederifolium can be supplemented in the treatment of infectious diseases and leaves extracts of the plant may have the potential to be used as antibacterial agents for searching new medicines.

 

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Accepted on 02.07.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(11): 1837-1839