MEDICINAL HERBS AS A SOURCE OF ANTIMICROBIAL SUBSTANCES

Michael R. Gohar. This study aimed to test a variety of naturally occurring some medicinal herbs and their extracts for their antimicrobial potential against a group of some bacterial and fungal pathogens. A total of 6 herbs (Thymus vulgaris, Foeniculum vulgare, Datura stramonium, Matricaria chamomilla, Ammi visnaga linne and Cassia angustifolia) collected from famous Egyptian plant dealers. The antimicrobial activity of aqueous, Methanol and Petroleum ether extracts were assisted by measuring the inhibition zone by agar well diffusion method and minimum inhibitory concentration (MIC) in order to increase the reliability and precision of the study. A number of 5 bacterial, 3 fungal (Molds) and 1(Yeast) species were chosen: G+ Bacteria: Staphylococcus aureus (RCMB 010027), Bacillis subtilis (RCMB 000101(5)). GBacteria: Pseudomonas aeruginosa (RCMB 000102(3)), Escherichia coli (RCMB 000103(9)), Klebsiella pneumonia (RCMB 0010093(12)). Fungi (molds): Aspergillus fumigates (RCMB 02564), Penicillium italicum (RCMB 001003), Syncephalastrum racemosum (RCMB 005003), and (yeast): Candida albicans (RCMB 05035). The three different solvents gave markedly varied abilities to extract the antimicrobial compounds active against versatile groups including G+, Gbacteria and fungi. The active antagonism of crude methanolic extract can be descendingly arranged as thyme>senna>fennel>Datura>chamomil. The extract from Ammi vesnaga gave negative results. The hyphal fungi, Penicellium italicum was less antagonized (11.1 – 21.9 mm IZ) than Aspergillus fumegatus ( 12.3 – 22.6 mm IZ), and Syncephalastrum racemosum (12.6 – 20.9 mm IZ), whereas Candida albicans was not affected by any methanolic extract of 5 herb types and the sixth (thyme) gave relatively moderate activity (20.9 mm IZ). Methanolic extracts gave in MICs of 0.015-7.81 μg/ml (Thyme), 0.49 – 125 μg/ml (Senna) and 31.25 – 500 μg/ml (Fennel) compared to MICs of 3.9 – 500 μg/ml, 31.25 – 500 and 15.63 – 500 μg/ml for those of ether extracts respectively in the same order of herbs. Essential oil and extracts of two herbs (Thymus vulgaris, Foeniculum vulgare), were analysis by Gas chromatography/mass spectrometry analysis revealed the presence of 12 peaks in fennel essential oil which indicating the presence of 12 phytochemical constituents representing 100% of the oil, while GC-MS analysis revealed the presence of 18 peaks in Thyme essential oil which indicating the presence of 14 phytochemical constituents representing 99.98% of the oil. The major constituents of the essential oil in fennel were Anethol (63.37%), D-Limonene (20.18%), L-Fenchone (7.44%) and Estragole (3.40%). The major constituents of the essential oil in thyme were Thymol (62.16%), o-Cymene (9.75%), Caryophyllene (7.30%), δTerpinene (5.66%), Butylated hydroxyl onisole (5.39%).


Introduction:-
The use of plant and its products has a long history that began with folk medicine and through the years has been incorporated into traditional and allopathic medicine (Dubey et al., 2011). Since antiquity, many plants species reported to have pharmacological properties as they are known to posses various secondary metabolites like glycosides, saponins, flavonoids, steroids, tannins, alkaloids, tirpenes which is therefore, should be utilized to combat the disease causing pathogens (Kamali et al., 2010).The use of medicinal plants as a source for relief from illness can be traced back over five millennia to written documents of the early civilization in China, India and the Near east, but it is doubtless an art as old as mankind.
The potential of higher plants as source for new drugs is still largely unexplored. Among the estimated 250,000-500,000 plant species, only a small percentage has been investigated phytochemically and the fraction submitted to biological or pharmacological screening is even smaller. Historically pharmacological screening of compounds of natural or synthetic origin has been the source of innumerable therapeutic agents (Kroschwitz et al., 1992). Antibiotics are undeniably one of the most important therapeutic discoveries of the 20 th Century that had effectiveness against serious bacterial infections.
A wide range of medicinal plant parts is used for extracting raw drugs as they possess varied medicinal properties. The different parts used include root, stem, flower, fruit, twigs exudates and modified plant organs. Although hundreds of plant species have been tested for antimicrobial properties, the vast majority have not been adequately evaluated (Uniyal, 2006). In recent years drug resistance of human pathogenic bacteria has been commonly reported from all over the world. With the continuous use of antibiotics, microorganism have become resistant. In addition to this problem, antibiotics are sometimes associated with adverse effects on host which include hypersensitivity, immunosuppressant and allergic reactions (Lopez., 2001).The antimicrobial effect of plant oils and extracts has formed the basis of many applications, including raw and processed food preservation, pharmaceuticals, alternative medicine and natural therapies.
Thus, innovative scientific methods for the discovery and validation of multicomponent botanical therapeutics are important for the development of medicine and both the standardization of extracts and the identification of the efficient chemical and biological, properties. Since 1990s there has been a growing shift in interest towards plants as significant sources for new pharmaceuticals. Many pharmaceutical companies show interest in plant-derived drugs mainly due to the current widespread belief that 'Green Medicine' is safe and more dependable than the costly synthetic drugs. Essential oil of Thyme was evaluated for its antibacterial activities against six Gram-positive and Gram negative pathogenic bacteria: Staphylococcus aureus, S. epidermidis, Streptococcus sp., Pantoa sp. and Escherichia coli (Jianu et al., 2012).The essential oil extracted from the fruits of F. vulgare exhibited antibacterial effect against food borne pathogens such as Escherichia coli, Bacillus megaterium and Staphylococcus aureus ( Mohsenzadeh., 2007) , Listeria monocytogenes and S. aureus ( Dadalioglu andEvrendilek., 2004 andCantore et al., 2004).
The methanol extracts of D. stromonium and Datura inoxia showed antagonistic activity against Gram positive bacteria in a dose dependent manner. Little or no antimicrobial activity was found against Escherichia coli and Psuedomonas aeruginosa (Takhi and Ouinten., 2011). The essential oil and methanol extract of Matricaria chamomilla L. were tested against bacterial and fungal strains using a broth microdilution method. The results suggest that M. chamomilla, oil and methanol extract have significant antimicrobial activity (Fatouma et al., 2011).

Materials and methods:-
Herbal materials:-All herbal species were identified at the Pharmacognosy Department -Faculty of Pharmacy -Misr University for Science and Technology. The specimens were bought from famous Egyptian plant dealers. The collected fresh herbal materials were washed thoroughly under running tap water to remove all debris contaminants, then cut into small pieces and air-dried in the shade for 15 days and each of the selected parts of each herb was ground in a grinding machine (Pharmacognosy Department laboratory mill) to make the samples smooth and then homogenized to fine powder before being kept in dry and dark place for further work. Among the most widely applied as folk medicine and/or for pharmacological drug industries 6 samples were selected, and the effective portions of the herbs were collected, which include: Thymus vulgaris, Foeniculum vulgare, Datura stramonium, Matricaria chamomilla, Ammi visnaga linne and Cassia angustifolia.  The previous steps were repeated twice, through application of the specific solvent, filtration and evaporation to obtain a pure herb extract. The solvents were evaporated by rotary evaporator of the various extract samples (at 45°C) to make the final volume (10-20%) of the original volume (Parekh et al., 2005)then were autoclaved at 121°C temperature (15 1bs pressure) for 15 min and stored at 4°C in airtight bottles in Refrigerator.
For aqueous extraction:-Thirty g portions of the air-dried powdered herb were taken in 300 ml of distilled water in a 500 ml conical flask plugged with cotton wool and then kept on a rotary shaker at 200 rpm for only 3days. The extract was then treated as previously mentioned in methanol and petroleum ether extracts with some modification, e.g. solvent evaporation occurred at 60°C until (10-20%) of the initial volume. The collected solvents (methanol and petroleum ether and aqueous) extracts were lyophlyzed by using (model Free Zone 6 plus 12L LABCONCO) to ensure the complete dryness. A sample of 1 gm. from each herbal extract was taken and put in screw capped weighed vials then kept in a refrigerator before testing antimicrobial activity and the designed chemical analysis (Sahin et al., 2003).Samples were asceptically taken for testing the various biological and biochemical parameters (Bradly., 1992).
Antimicrobial activity and media used:-Media used:-Nutrient agar (NA) (Shiriling and Gottlieb., 1966) was used to cultivate and maintain the examined bacterial species whereas that of Saboured's Dextrose agar with antibiotic (Midgled et al., 1997) was applied to deal with pathogenic mold and yeast. Both media were prepared and sterilized before being applied as slants in test tubes or in bulk to be poured in Petri dishes.

Antimicrobial activity:-
The screening of antimicrobial activity of the six selected herb extracts was carried out using the agar well diffusion method as described by Holder and Boyce (1994).Negative control was prepared with respective solvents used for extraction. The dry extract was weighed (1gm) and dissolved in sterile 5% dimethylsulfoxide (DMSO) as a carrier and used as a sample extract (Beni et al., 2014).The tested organisms were subcultured in the suitable media i.e. nutrient agar medium for bacteria and Saboured's dextrose agar for molds and yeast. Ampicillin and Gentamycin were used as a standard antibacterial substance to be compared with Gram positive and negative bacteria respectively and Amphotericin B was used as a comparable agent for fungi (mold and yeast) (Agwa et al., 2000).
The bacterial strains were inoculated into nutrient agar medium for bacterial cultures and incubated at 37°C for 24 h, while the fungal cultures (mold and yeast) in Sabouroud´s dextrose agar and were incubated at (25-30°C) for 3-7 days before being assayed for antimicrobial activity.

Preparation of the essential oil sample:-
Three-hundred grams of the air dried powder from each of thyme or fennel were added to 1L distilled water and used the Clavenger distillation apparatus for hydrodistillation to isolate the essential oil (2 ml for each( . It was taken to Cairo University Research Park at the Faculty of Agriculture for the test of GC/MS.

Gas Chromatography / Mass Spectra (GC/MS) analysis:-
The phytochemical investigation of Thyme and fennel essential oils were performed on a GC-MS equipment Experimental conditions of GC

Screening of antimicrobial compounds in herbs:
The three different solvents gave markedly varied abilities to extract the antimicrobial compounds active against versatile groups including G+,G-bacteria (antibacterial) and fungi (molds and yeastantifungal).Collectively under the examined environmental conditions, methanol was the most active solvent for extracting the antimicrobial compounds embedded in tissues of the herbs either as antibacterial or antifungal, followed by Petrolium ether solvent, mean while the aqueous solution extraction was the poorest. The active antagonism of crude methanolic extractas shownin Table(2) can be descendingly arranged as thyme>senna>fennel>Datura>chamomile using molds, G+ and G−bacteria. However, the extract from Ammi vesnaga gave negative results either for antibacterial and antifungal influences, in addition to certain cases e.g. Candida albicans and Pseudomonas aeruginosa were not antagonized by any methanolic extracts, except that of thyme which gave active antagonism against both of them i.e. inhibition zone of 20.9 and 20.3 mm beyond well diameter respectively. Staphylococcus aureus and B. subtilis (G+) were more susceptile to methanolic extracts i.e. 13.6 -29.6 mm and 13.9 -28.2 mm IZ respectively, which are higher than those of E. coli (13.2 -19.9 mm IZ) and Klebsiella pneumonia (13.6 -25.9 mm IZ)(G-). A behavior that was expected P. aeruginosa gave no response to various methanolic extracts except to that of thyme (20.3 mm IZ). Among the hyphal fungi, Penicellium italicum was less antagonized (11.1 -21.9 mm IZ) than Aspergillus fumegatus ( 12.3 -22.6 mm IZ), and Syncephalastrum racemosum (12.6 -20.9 mm IZ), whereas Candida albicans was not affected by any methanolic extract of 5 herbs types and the sixth (thyme) gave relatively moderate activity (20.9 mm IZ). An overall evaluation of the antimicrobial activities against the examined test fungi or bacteria of the various medicinal herbs extracted by methanol, it can be seen that those of thyme were comparably equal to those the concerned standard antibiotics, whereas those of the remainder methanolic extracts were lower or markedly beyond those of the standards which gave inhibition zones of 19.5 -22.9 mm for Amphotericin B against fungi 28.3 -28.9 mm for Ampicillin against G+ bacteria and 20.3 -25.3 mm for Gentamycin against G− bacteria ( see Table 2).     Fig (1) shows representatives of IZ of some herbs and reference antibiotics extracted by the 3 solvents.

Antimicrobial Activity Indices:-
The antimicrobial activity index either for antifungal or antibacterial antagonism of the 6 herbs appear in Graph (1) and (2). It is clear that their values are a reflection of the antimicrobial mechanisms against both fungi and bacteria, since their figures paralleled the increments or declines in the action of the antagonistic compounds. These attributes indicate the entire dependency of the indices on the type of herb, the solvent, applied and the type of selected test organism and reference antibiotic. Some of the herb's extracts showed no activity against certain tested fungi or bacteria therefore have no AI and will be neglected in neither Fig nor description. As for methanolic extracts and irrespective of the examined species, their Activity indices compared to the reference antibiotics, ranged between 0.521 to 1.071 for the examined molds whereas candida albcans was antagonized only with thyme methanol extract with a relatively moderate AI, 0.976 an indicative that this yeast species is not sensitive to the others examined herbs. Again, petroleum ether has relatively lower strength as extraction solvent. Thus almost similar behaviours in AI, shown by methanol extract are repeated with petroleum ether extracts against the examined fungi or bacteria, however with lower levels. As to the aqueous extracts, they have no antifungal against hyphal or unicellular fungi. In case of true bacteria either G-or G+ the calculated AI were either nil or lower or relatively so when compared with those of methanolic or petroleum ether extracts (Graph1, 2).Graph 1: Antibacterial activity index of the 6 medicinal herbs extracted by petroleum ether related to specific standard antibiotic for both G+ and G-bacteria.

Minimum Inhibitory Concentration (MIC):-
The Minimum Inhibition Concentration is known as the highest dilution or the least concentration of the herb extract that inhibits the growth of an organism. MIC is used to determine the potentiality of new antimicrobial substances against a specific microorganism which can be compared with utilizable ones. MIC is widely applicable in microbiological laboratories to evaluate antagonistic activity of antibiotics either produced by pure cultures or embedded in plant tissues.
Three of the herbs containing active antagonistic compounds extracted by two active solvents: Methanol and Petroleum ether were tested by MIC which inhibited the growth of almost all, except in few cases, of the examined organisms and data are shown in Table ( 5).MIC values significantly varied depending upon the type of herb, the solvent applied and the test microorganism. It ranged between nil-500µg/ml compared to 0.49 -7.81 µg/ml for Amphotericin B, 0.015 -0.03µg/ml for Ampicillin and 0.12 -3.9 µg/ml for Gentamycine.
As to the potentiality of the tested solvents ,it can be observed that Methanolic extracts were more active than those of Petroleum ether , therefore and irrespective of the microorganisms, methanolic extracts gave in MICs of 0.015-7.81 µg/ml (Thyme), 0.49 -125 µg/ml (Senna ) and 31.25 -500 µg/ml (Fennel ) compared to MICs of 3.9 -500 µg/ml, 31.25 -500 and 15.63 -500 µg/ml for those of ether extracts respectively in the same order of herbs. These values confirm that Thyme contained more antagonistic compounds against the tested organisms and fennel had the least.
However, as to the specific bacterial groups, it was found that Thyme either methanolic and ether extracts are most active against both Staphylococcus aureus and Bacillus subtilis (0.015-0.03 and 3.9-1.95 mg/ml) and nil or very weak for Fennel and Senna respectively.    Tables (6 and 7) and Graph (3 and 4) for Thyme and Fennel respectively. The result of the chemical composition of Fennel and Thyme essential oils indicate that the overall, GC-MS analysis revealed the presence of 12 peaks in fennel essential oil which indicating the presence of 12 phytochemical constituents representing 100% of the oil. The major constituents of the essential oil tested were Anethol (63.37%), D-Limonene (20.18%), L-Fenchone (7.44%) and Estragole (3.40%). In addition, the tested fennel essential oil also contained considerable amounts of various minor constituents whose contribution was <10% (Table 6). The GC-MS analysis revealed the presence of 18 peaks (4 belonged to Thymol) in Thyme essential oil which indicating the presence of 18 phytochemical constituents representing99.98% of the oil. The major constituents of the essential oil tested were Thymol (62.16%), o-Cymene (9.75%), Caryophyllene (7.30%), δ-Terpinene (5.66%), Butylated hydroxyl onisole (5.39%) Table (7). In addition, the tested Thyme essential oil also contained considerable amounts of various minor constituents whose contribution was < 10%.Generally, the oil was found to be rich in the active monoterpene phenols (thymolandcarvacrol) and their corresponding monoterpenep-cymene and δ-terpinene, which collectively showed synchronized patterns of variation during the different collection periods and in different seasons. Monoterpene phenols (mainly thymol) and their derivatives (mainly carvacrol methyl ether), sesquiterpenes (mainly caryophyllene) and their oxygenated derivatives (e.g. caryophyllene oxide) were found in different levels.  , six chemotypes have also been reported, geraniol, linalool, γ-terpineol, carvacrol, thymol and trans-thujan-4-ol/terpinen-4-ol. Our results differ from those obtained by Ozcan et al,.(2004) who studied the oil composition of thyme of the same species sample in Turkey, in which thymol (46.2%), alpha terpinene(14.1%), pcymene (9.9%) alphapinene (3.0%) were revealed to be dominant. For the Spanish thyme essential oil, the major components quantified were 1,8-cineole, followed by terpenyl acetate, borneol, linalool, beta -pinene, alphaterpineol and camphor (Jordán et al., 2006). On the contrary carvacrol, which was not detected in our sample was found to be the main componenet in the previosereport (Hudaib., 2002).These probably due to the plant varieties or sites, as well as the time of harvesting.