In vitro antimicrobial activities of Glycyrrhiza glabra and Fagonia arabica

The aim of this study was to test the antimicrobial activities of crude chloroform, hexane, ethyl acetate and ethanol extracts of the leaves Glycyrrhiza glabra (GG) and Fagonia arabica (FA) against bacteria (Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus and Bacillus subtilis). Antimicrobial properties of G. glabra and F. arabica were tested using Agar well diffusion method and Agar disc diffusion method. Streptomycine was used as standard drug with significant activity values, that is, 23 mm against E. coli, 36 mm against S. epidermidis, 34 mm against S. aureus and 26 mm against B. subtilis. Analysis of data showed that the crude extract of G. glabra and F. arabica in dichloromethane exhibited superior activity against E. coli and S. epidermidis. Results were compared concomitantly to standard drugs; streptomycine. Phytochemical screening of G. glabra and F. arabica showed the presence of terpenoids, saponins, flavonoids, alkaloids, tannins, glycosides and reducing sugar components. Based on the current conclusion, it can be accomplished that these plants has antimicrobial activity, which is as potent as standard antimicrobial drugs against definite microorganisms.


INTRODUCTION
Life and diseases go together where there is life, diseases are bound to exist.Dependency and sustainability of man and animal life has been revolving around plants through their uses as food, clothing and shelter, but also plants have been used to control diseases; therefore, the use of plants as medicines is an ancient and reliable practice (Arshad and Rao, 2001).Recently, it is reported by WHO that more than 80% of the world's population depends on traditional medicine for the treatment of their illnesses (Norman et al., 1985).
The phytochemical research, in which ethno pharmacological properties of plants are evaluated, leads to the discovery of new anti-infective agents from higher plants.Owing to the development of drug resistive strains in human pathogens against commonly used antibiotics, it is necessary to discover new antimicrobial substances from plants and other sources.Glycyrrhiza glabra is one of the oldest used herbs, both as a medicine and a flavoring herb to disguise the unpleasant flavor of other medication (Biondi, 2005).The sweet taste of licorice is due to the active constituent called glycyrrhizina glycolside in the combined potassium and calcium salt form of glycyrrhizic acid (GA).The constituent is 50 times sweeter than sugar, and thus widely used as a sweetening agent in food industry (Acharya, 1993).G. glabra has been used for centuries in the treatment of sore throats, coughs, gastric ulcers, allergic reactions, eczema, rheumatism, arthritis and hepatitis (Aly, 2005;Ates, 2003;Rossum, 1998).Nowadays, licorice is widely used for its antimicrobial, antioxidant, anti-inflammatory, anticoagulative, antiulcer, expectorant, antiallergic, antiviral and anxiolytic activities and a number of other pharmacological actions (Shin, 2007;Statti, 2004;Vaya, 1997;Fukai, 2002).
Fagonia arabica is a widely used medicinal plant in Pakistan.F. arabica is an important Ayurvedic herb that grows throughout arid regions of India, and has been widely used as a folk remedy by the indigenous people for its anti-inflammatory, analgesic and antipyretic effects (Satpute, 2009).F. arabica/cretica has been used by various village dwellers in Indo-Pakistan region, for curing fever, dysentery, urinary discharges, erysipelas, typhoid, alexipharmic, reducing tumors and purifying the blood (Sheikh, 2007).It is also used for skin diseases, small pox and for endothermic reaction in the body (Watt, 1972).The twigs of the plant are used as remedy for snake bite and also applied externally as paste on tumors and for the swellings of neck (Hooker, 1982;Rizvi, 1996).Fagonia species were extensively studied by many workers regarding their medicinal uses, since these plants were antitumor, antioxidant, analgesic, astringent, febrifuge and prophylactic against small-pox agents.Thus, the purpose of this current investigation is to evaluate the chloroform, hexane ethyl acetate and ethanol fractions of G. glabra and F. arabica against several bacteria in vitro.

Plant
G. glabra were collected from Afghanistan and F. arabica was picked from District Malakand, Khyber Pakhtunkhwa, Pakistan.The taxonomic identification of both plants was carried out by Dr. Farrukh Hussain at the Department of Botany (DOB), University of Peshawar, Pakistan.A voucher specimen under the scientific name of the plant (bot. 20011 (pup) and bot. 20010 (pup)) was deposited at the herbarium of Department of Botany, University of Peshawar, Pakistan.

Preparation of crude extracts
Plants materials were dried under shade and powered.From these powered materials, weighed amount was taken in separate thimbles.This was suspended above the flask containing the solvent n-Hexane fitted with reflux condenser.The flask was heated (60 to 65°C for 5 to 6 h) on "Heating mantle" the evaporated solvent upon condensation trickled into the extraction chamber containing the plant material.
The extraction chamber was designed so that when the solvent surrounding the sample exceed a certain level, it overflowed and trickled back down into the boiling flask.At the end of the extraction process, the flask containing the n-Hexane extract was removed.After the removal of n-Hexane extract, the plant material was left in the thimble and next solvent of high polarity was passed like dichloromethane and extract was obtained and then ethyl acetate and at the end highly polar solvent, ethanol was passed and thus different extract were obtained according to increasing polarities of solvents.The solvents obtained were evaporated under vacuum using Rotary evaporator.The plants extracts were collected in labeled vials.The plant materials taken for extraction were G. glabra (65 g) and F. arabica (60 g).The crude extracts were subjected for antimicrobial activity and their phytochemical screening.

Qualitative analysis of chemical constituents
Chemical analysis for the presence of major classes of secondary metabolites (terpenoids, saponins, flavonoids, alkaloids, tannins, glycosides and reducing sugar) in the crude extracts was carried out according to the method described by Pearson (1976).The results are shown in Tables 1 and 2.

Alkaloids
About 0.2 g of the extracts was warmed with 2% H2SO4 for 2 min.It was filtered and few drops of Dragendroff reagent were added.Orange red precipitate indicates the presence of alkaloids.

Tannins
Small quantity of the extract was mixed with water and heated on water bath.The mixture was filtered and ferric chloride was added to the filtrate.A dark green solution indicates the presence of tannins.

Anthraquinones
About 0.5 g of the extract was boiled with 10% HCl for few minutes in a water bath.It was filtered and allowed to cool.Equal volume of chloroform was added to the filtrate.Few drops of 10% NH3 were added to the mixture and heated.Formation of rose-pink color indicates the presence of anthraquinones.

Glycosides
The extract was hydrolyzed with HCl solution and neutralized with NaOH solution.A few drops of Fehling's solution A and B were added.Red precipitate indicates the presence of glycosides.

Reducing sugars
The extract was shaken with distilled water and was filtered.The filtrate was boiled with drops of Fehling's solution A and B for minutes.An orange red precipitate indicates the presence of reducing sugars.

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 miss of small bubbles) shows the presence of saponins.

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.

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.

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

Preparation of medium
Solid and liquid media were used.Nutrient agar 28 g was dissolved in distilled water and volume was made up to 1 L. It was autoclaved at 121°C for 15 min.Media was cooled and allowed to solidify in Petri dishes.Nutrient broth (1.9 g) was dissolved in distilled water and volume was made up to 150 ml. 8 ml of this broth was added to screw capped test tubes, which were placed in autoclave at 121°C for 15 min, then refrigerated at 25°C.

of tested materials
The test sample was prepared by dissolving 22 mg of crude extract in 1 ml of dimethyl sulfoxide (DMSO) in Eppendorff tube and was kept for 1 h.To another Eppendorff tube, 1 mg of streptomycin was dissolved in 1 ml DMSO and was kept for 1 h.

Cup-plate diffusion method
Antibacterial activity of plant extracts was carried using cup-plate agar diffusion method (Murray et al., 2009) with some small modifications.One milliliter from each standard bacterial stock suspension was mixed thoroughly with 40 to 45 ml of sterile Molten Mueller Hinton agar at 40°C, poured into sterile Petri-dishes and left to solidify.Then, four cup-shape wells (120 mm diameter) were made in each plate using sterile cork-borer.The agar disks were removed and four alternate cups were filled with extract using sterile adjustable pipettes.Four Petri-dishes with two alternate cups were used with the respective solvent instead of the extracts as control.The plates were then incubated in upright position for 22 to 24 h at room temperature.Two replicates were carried out for each extract.After incubation period, the inhibition zones diameters were measured.

Disc diffusion method
The antibacterial assay for plant extract was also conducted using disc diffusion method as illustrated by Abdel-Wahab (2009).The nutrient agar solution (16 ml) was poured and kept overnight in a refrigerator.Whatman filter paper (No.1) discs of 6 mm diameter were impregnated with 10 μl of the solution of crude extract (at 4 mg/ml) dissolved in DMSO.Standard disc of streptomycin sulphate (10 μg/disc) was used as positive control, while DMSO was used as a negative control.The Petri-dishes were inverted and incubated for 24 h at 37°C.Clear inhibition zones around the discs indicated the presence of antimicrobial activity.

Antimicrobial screening
Table 3 summarizes the results.However, results were interpreted in terms of commonly used terms: sensitive, intermediate and resistant.Findings of cup-plate diffusion method for ethanolic extracts of G. glabra and F. arabica are shown in Figures 1 and 2.

Antibacterial activities of G. glabra and F. arabica
The antibacterial activities of crude extract of the selected plants are n-Hexane, dichloromethane, ethylacetate and ethanol.Traditionally, G. glabra and F. arabica were used to treat bacterial infections.The results from the current study were screened for their microbial activity against four standard bacteria, namely, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis and Escherichia coli.The pattern of inhibition varied with the plant's extract; the solvent was used for extraction and the organism was tested.DMSO was used as negative control and no inhibition was shown by it against all the bacteria (Bacterial growth inhibition=0 mm The dichloromethane extract of G. glabra were found significant against E. coli and S. epidermidis and its zone of inhibition were 20 and 24 mm, respectively, good against B. subtilis with zone of inhibition (16 mm), and non-significant against S. aureus (10 mm).Similarly, the dichloromethane extract of F. arabica showed low activity against E. coli and its zone of inhibition was 14 mm and exhibited no appreciable activity against S. epidermidis, B. subtilis and S. aureus.
The ethylacetate extract of G. glabra was examined and was discovered to exhibit low activity against S. epidermidis and E. coli as its zone of inhibition were 15 and 14 mm, respectively, was good against B. subtilis (18 mm) and non-significant against S. aureus.No appreciable activity was found by the ethylacetate extract of F. arabica against S. epidermidis, B. subtilis and S. aureus, and was non-significant against E. coli and its zone of inhibition was 12 mm.Low activity was exhibited by the ethanolic extract of G. glabra against E. coli with zone of inhibition (14 mm), non-significant against S. epidermidis and B. subtilis and its zone of inhibition were 12 and 10 mm, respectively and did not show activity against S. aureus.Similarly, the ethanolic extract of F. arabica was examined and it was found not to have activity against E. coli, low activity against S. epidermidis and non-significant activities against B. subtilis and S. aureus.Table 3 summarizes the percentage inhibition of each extract (Streptomycin) activity against different bacteria.

Conclusion
The antimicrobial effects of G. glabra and F. arabica extracts against the studied bacteria suggest that, different parts of G. glabra and F. arabica possess remarkable therapeutic action that can support the traditional usage of this plant in the treatment of bacterial diseases such as gastrointestinal infection, diarrhea, respiratory and skin diseases.These antimicrobial activities are likely due to the presence of secondary metabolites like tannins and flavonoids, alkaloids, saponins, terpenes and glycosides in G. glabra and F. arabica.The high potency of G. glabra and F. arabica against these microbes could provide an example of prospecting for new compounds.

Table 1 .
Secondary metabolites detected in different extracts of G. glabra.

Table 2 .
Secondary metabolites detected in different extracts of F. arabica.

Table 3 .
Antibacterial activity of G. glabra and F. Arabica.
). Streptomycin, an antibiotic was used as a positive control.Streptomycin was used as standard drug with significant activity values, that is, 34 against S. aureus, 36 against S. epidermidis, 26 against B. subtilis and 23 against E. coli.The greater activity was shown by G. glabra and dichloromethane extract of the plant showed the maximum activity among all the extracts of both plants.Antibacterial activities of F. arabica different solvents.E.C: Escherichia coli; S.E: Staphylococcus epidermidis; S.A: Staphylococcus aureus; B.S: Bacillus subtilis.
S. aureus showed more susceptibility towards extracts of all studied parts.The n-Hexane extract of G. glabra were found to be good against E. coli and S. epidermidis and zone of inhibition were 16 and 17 mm, respectively; low activity against B. subtilis (14 mm) and non-significant against S. aureus.n-Hexane extract of F. arabica showed no appreciable activity against S. aureus and nonsignificant against E. coli, S. epidermidis and B. subtilis.