Traditionally it is used to treat skin ulcers, inflammation, abdominal pain, abdominal worms and muscular weakness³. The leaves extract is also used as immunostimulant in treatment of asthma, jaundice and as antihemorrhagic³. The aqueous and alcoholic extract showed moderate diuretic, tranquilizing, hypoglycermic, antihemorrhagic activity, in addition to antibacterial and antifungal effects., and also act to relieve intestinal and uterine pain³.

Thymus plant species (Family: Lamiaceae) are commonly used as tonic, carminative, digestive, antitussive, expectorant and for the treatment of cold in Iranian traditional medicine. Recent studies imply that these species have strong antibacterial activities ^4,5. Many reports have shown that Thymus capitatus exhibits biological activities viz. antimicrobial activities^6-13; antifungal activity^14-16 and antioxidant activity^11,16,17.

So far no specific study has been carried out for the preliminary phytochemical screening and antibacterial activity of Convolvulus arvensis and T. capitatus grown in Libya. Therefore, in this present study, the preliminary phytochemical testings and antibacterial activity of leaves extracts of both these plants against Gram positive and Gram negative bacteria were investigated and described.

MATERIALS AND METHODS:

Collection of Plants materials:

Convolvulus arvensis and Thymus capitatus plants were collected from various parts of the Al-khoms city of Libya in May/ June months of 2012. The plants were identified by Plant taxonomist of the Department of Biology of Al-Mergheb University, Al-Khoms, Libya. Leaves of each plant were collected, washed under running tap water and then dried under shade and ground into coarse powder in the electronic grinder., The course powder was stored in plastic bags at room temperature and low humidity condition.

Preparation of Extract:

The powdered plants materials were extracted in water and ethanol separately at a 40% (w/v) concentration (200g leaves powder in 500 ml water) by using Soxhlet method for 8 h and filtered. The extracts were stored at 4°C until further use.

Phytochemicals screening:

Chemical tests were carried out on the water and ethanolic leaves extracts of both the plants using standard procedures to identify the constituents as described by Sofowora, 1993 ¹⁸ and Raaman, 2006 ¹⁹.

Tannins: A small quantity of each extracts were mixed with water and heated on water bath and filtered. A few drops of ferric chloride were added to the filtrate. A dark green solution indicates the presence of tannins.

Saponins: About 0.2 g of the extract was shaken with 5 ml of distilled water and then heated to boil. Frothing (appearance of creamy mass of small bubbles) shows the presence of saponins.

Phlobatanins: The extract (0.5 g) was dissolved in distilled water and filtered. The filtrate was boiled with 2% HCl solution. Red precipitate shows the presence of phlobatanins.

Flavonoids: Extract of about 0.2 g was dissolved in diluted NaOH and HCl was added. A yellow solution that turns colorless indicates the presence of flavonoids.

Terpenoids : 0.2 g of the each extract was mixed with 2 ml of chloroform (CHCl₃ ) and concentrated H₂SO₄ (3 ml) was carefully added form a layer. A reddish brown coloration of the interface was formed to indicate positive results for the presence of terpenoids.

Cardiac glycosides: 0.2g of each extract was shaken with 1ml of glacial acetic acid and a drop of ferric chloride and a drop of concentrated sulfuric acid was added. Green blue color to upper layer and reddish brown color at the junction of two layers indicates the presence of cardiac glycosides.

Alkaloids: About 0.2g of the extracts was warmed with 2% H₂SO₄ for two minutes. It was filtered and few drops of Dragondorff reagent were added. Orange red precipitate indicated the presence of alkaloids.

Test organisms:

Two Gram positive strains (Staphylococcus epidermidis ,Staphylococcus saprophyticus) and three Gram negative bacterial strains (Proteus vulgaris, Eschericia coli, Citrobactor freundii ) from standard cultures were used as test strains. Standard bacterial strains were procured from Microbiology Medical Laboratory, Central Hospital, Al-Khoms, Libya.

Determination of Antibacterial activity by disc diffusion method:

Antibacterial activity of ethanolic extracts was investigated against three Gram positive bacterial strains and two Gram negative bacterial strains by the paper disc diffusion method²⁰. From the 50 mg/ml stock solution of ethanolic extracts of both plants, 40 μl aliquots were transferred onto blank sterile paper discs (6 mm diameter)⁴. Dried discs were placed onto nutrient agar medium (UK) previously inoculated with a bacterial suspension and incubated at 37ºC for 24 h. Ethanol solvent was used as control to determine the sensitivity of the tested strains. After incubation, plates were then examined for the presence of zones of growth inhibition, and the diameters of these zones were measured in mm, if any. All tests were performed under sterile conditions in duplicate and repeated two times.

RESULTS:

a) Phytochemicals screening:

Table 1 shows the results of phytochemicals screening of aqueous and ethanolic leaves extracts of Convolvulus arvensis and Thymus capitatus plants.

Phytochemical screening of the aqueous extract of Convolvulus arvensis revealed the presence of tannins, saponins, flavonoids, terpenoids and cardiac glycosides while the ethanolic extract revealed the presence of tannins, saponins, flavonoids, terpenoids, cardiac glycosides and alkaloids. However, the aqueous extract of Thymus capitatus revealed the presence of tannins, saponins, flavonoids and terpenoids, and the ethanolic extract revealed the presence of tannins, saponins, phlobatanins, flavonoids, terpenoids and cardiac glycosides .

Table 1. Phytochemical screening of aqueous and ethanolic leaves extracts of Convolvulus arvensis and Thymus capitatus :

Chemical Components

Convolvulus arvensis

Thymus capitatus

Water Extract

Ethanol Extract

Water Extract

Ethanol Extract

Tannins

Saponins

Phlobatanins

Flavonoids

Terpenoids

Cardiac glycosides

Alkaloids

Key: + = present ; - = absent

b) Antibacterial activities:

Table 2 shows the results of antibacterial activity of both of the plants extracts.

The ethanolic extract of Convolvulus arvensis exhibited antibacterial activity against both tested Gram positive bacterial strains (S. epidermidis, S. saprophyticus) and a Gram negative bacterial strain (Proteus vulgaris) among the three tested Gram negative bacterial strains. While the ethanolic extract of Thymus capitatus revealed antibacterial activity against a Gram positive bacterial strain (S. saprophyticus) among the two tested Gram positive bacterial strains and all tested Gram negative strains (Proteus vulgaris, Eschericia coli, Citrobactor freundii). Convolvulus arvensis and Thymus capitatus exhibited maximum antibacterial activity against Gram negative bacteria Proteus vulgaris (zone of inhibition: 10 mm and 11mm, respectively).

Table 2. Antibacterial activity of leaves ethanolic extracts (50 mg/ml) of Convolvulus arvensis and Thymus capitatus :

Chemical Components

Convolvulus arvensis

Thymus capitatus

Zone of inhibition (mm)

S. epidermidis

S. saprophyticus

Proteus vulgaris

Eschericia coli

Citrobactor freundii

Note : Discs injected with 40ml of ethanol solvent served as controls against each bacterial strain and showed nil zone of inhibition.

DISCUSSION:

The results of the screened phytochemicals (tannins, saponins, phlobatanins, flavonoids, terpenoids, cardiac glycosides and alkaloids) of aqueous and ethanolic extracts of both the plants showed the common presence of. tannins, saponins, flavonoids, terpenoids, however, ethanolic extractions showed the presence of more phytochemicals including cardiac glycosides and alkaloids in Convolvulus arvensis and glycosides and phlobatanins in Thymus capitatus. This may be due to stronger phytochemicals extraction capacity of ethanol than water. Ethanol formulations are relatively safe for human consumption as compared with other organic solvents such as acetone and methanol which are frequently used by the researchers. Further, ethanol extraction is widely used to obtain crude extracts of phytochemicals from plant materials in the herbal medicine industry for therapeutic applications. Most of the previous studies also indicated that ethanolic extracts of the plants leaves contains more biological activity and showed richer in phytochemicals concentrations than the aqueous and other organic solvents extracts of other parts of the plants. Therefore, ethanolic leaves extract of the plants was chosen for the preliminary investigation of both the plants for antibacterial activity.

Previous reports have indicated that the aqueous and alcoholic extract of Convolvulus arvensis and Thymus capitatus are found to have remarkable biological and therapeutic effects including antibacterial action^3,13. The different level of anti-bacterial activity of Convolvulus arvensis of different areas of Pakistan have been reported by Raza et al, 2012.The results on both of the studied plants of Libyan origin lacking in previous reports prompted us for the present study. The results of anti bacterial activities of both of the plants leaves ethanolic extracts have shown good antibacterial effects against standard strains of Gram positive and Gram negative bacterial strains. Zone of inhibition produced by the extracts (50 mg/ml) in paper disc diffusion method against the susceptible bacteria ranged from 8 to 11 mm diameter. It was interesting to note that Proteus vulgaris (Gram negative bacterial strain) indicated maximum sensitivity to the investigated both of the plants leaves extracts. The extract contains many phytochemicals substances including flavonoids. The different components diffusing in different rates may have been responsible for the varying zones of inhibition obtained in our study against the susceptible microorganisms. Both of these plants are assumed to have flavonoids metabolites which have a potential antibacterial activity. Flavonoid’s activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls. Lipophilic flavonoids may also disrupts bacterial membranes. If the components could be separated and tested further we might find use as individual bacterial agent against bacterial infections⁴.

The present study showed that Convolvulus arvensis and Thymus capitatus plants leaves are rich in phytochemicals. Both plants exhibited potent antibacterial activity. This would be helpful to create awareness among people for taking control measures based on, herbal plants against infectious diseases and herbal based medicines can be recommended alternate to antibiotics. This study also suggest for the detailed investigation and chemical characterization of the active principle of leaves of the plants combating resistant bacteria.

ACKNOWLEDGEMENT:

Authors are grateful to the Head of the Microbiology Medical Laboratory, Central Hospital, Al-Khoms, Libya for providing us all bacterial strains used for the present study.

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Received on 18.03.2013

Modified on 03.04.2013

Accepted on 03.05.2013

Research Journal of Pharmacognosy and Phytochemistry. 5(5): September – October 2013, 220-223