Synergistic antibacterial effect of Lepidium sativum and Coriandrum sativum against standard and drug resistant clinically isolated pathogenic bacteria

Emerging and increasing resistance to antibiotics has become a threat to public health globally. Spices and herbs are considered as rich source of bio-active antimicrobial compounds. Thus the aim of this study was to assess the antibacterial effect of Lepidium sativum and Coriandrum sativum seed extracts and their synergistic action against some selected pathogenic bacteria; namely: Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumonia and Escherichia coli clinical isolates. Ethanol, methanol and chloroform crude extracts of L. sativum and C. sativum were evaluated against tested pathogenic bacteria using agar well diffusion method; the inhibitory zones were recorded in millimeters. Tetracycline and Vancomycin were used as positive controls, while sterile distilled water was served as negative control. The minimal inhibitory concentration (MIC) of the plant extracts against test bacteria were assessed using agar well dilution and broth dilution method; and then Minimum Bactericidal Concentration (MBC) was evaluated. The inhibition zone of all crude seed extracts of L. sativum ranged from 19.64-25.68 mm against S. pneumonia; and S. aureus were significantly (P value ranges from 0.02 to 0.04) greater than the inhibition zone of other clinical isolates. On the other hand, the inhibition zone (24.86 mm) of ethanol extract of C. sativum seed against K. pneumoniae was significantly (P= 0.01) greater than the rest clinical isolated test pathogenic bacteria. The results of this study also demonstrated that ethanolic extracts used against almost all test bacteria showed antimicrobial and synergistic effect with most antibiotics better than methanolic and chloroform extracts. The inhibition zone of the synergistic antibacterial effect of L. sativum and C. sativum seed extracts against tested pathogenic bacteria was significantly (P value ranges from 0.01 to 0.02) greater than the antibacterial agents L. sativum and C. sativum used separately. Thus, the present finding supports the traditional use of these plants in combination for treating pathogens by the community. And also there is a need for detailed scientific study of traditional knowledge to ensure that valuable therapeutic knowledge of some plants is preserved as well as to provide scientific evidence for their efficacies.


INTRODUCTION
There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action because there has been an alarming increase in the incidence of new a reemerging infectious diseases.Another big concern is the development of resistance to the antibiotics in current clinical use.In line with this, researchers have so far discovered more than 10,000 medicinal plants with biologically active compounds of microbial origin (Awoyemi et al., 2012).
Herbs and Spices; the most important parts of human diet are among the traditional medicinal plants used in the world.They synthesize substances that are useful to the maintenance of health in humans and other animals.In addition to boosting flavor, are also known for their preservative and medicinal value (Sutradhar and Choudhury, 2015).
Except the few mere traditional usage, in Ethiopia majority of these spices and herbs have not yet been studied scientifically for their individual or combined (synergistic) effect against various infections.From herbs: Lepidium sativum; and from spices: Coriandrum sativum, of which this study is particularly focused on, are the most widely used traditional herbs and spices, respectively, for the treatment of various bacterial infections.Several reports are available in literature regarding the antimicrobial activity of plant crude extracts from these plants (Mahajan and Badgujar, 2012).L. sativum, also known as pepper cressor Elrashad, belongs to the family Brassicaceae (cruciferae).The vernacular name of L. sativum is "Fetto" in Amharic and different names are given in different localities of English speakers (Mahajan and Badgujar, 2012).
C. sativum which is sometimes called Chinese parsley has different names in different localities of English speakers.Its vernacular name is "Dinbilal" in Amharic speakers.Coriander refers to the same family as the parsley, but has a more spicy taste and flavor and more tender leaflets.Coriander can be used as a medicine and widely useful as spice.It contains rich vitamins, decanal, nonanal, linalool and fragrant beans fine substances (Rahman et al., 2013).In view of this paper, the focus is on its antibacterial activity in combination with L. sativum against selected pathogenic bacteria and provided certain basis for coriander as a new secure natural food preservative (Mohammad et al, 2014).

Study area and duration
The study was conducted in Gondar, North West Ethiopia.In particular, this work was carried out in University of Gondar, Molecular Biology laboratory of the Department of Biotechnology from November, 2013 to June, 2014.Its climatic condition is very suitable for the people to live healthfully.The area has two seasons; the wet season from June to September and the dry season from October to May.The average rainfall of 800 mm (of which 84% of rain expected), while the annual mean minimum and maximum temperature of the area vary between 12.3-17.7°Crespectively (Central Meteorology Agency of Ethiopia, 2005).

Study design
The study design was cross-sectional experimental based; using appropriate methods such as determination of antibacterial activities, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) that performed from November, 2013 to June, 2014.

Collection and Identification of plant materials
Dried and healthy seed samples were collected from a local market of Gondar town, the so called "Arada" in November, 2013.The plants were selected based on the indigenous knowledge of the local traditional medicinal plant healers.A voucher (NUMERO) specimen of these plants seed were identified and confirmed by a Botanist in Department of Biology, University of Gondar.The cleaned, washed, dried, and mechanically ground plant seeds were brought to the research laboratory, Department of Biotechnology, University of Gondar for further investigation.

Preparation of plant extracts
The collected plant seed materials were thoroughly washed in running tap water to remove debris and dust particles and then rinsed in distilled water.The seed samples were dried in the laboratory in an open air at room temperature for about five days and protected from sunlight.Once completely dry, these were grounded to a fine powder using an electronic blender or mechanical grinder, and the powder was stored in a sterile bottle at room temperature in dark place.The dried and powdered seed of each plant (50 g) was extracted with 300 ml ethanol, chloroform and methanol with a Soxhlet extractor for 48 h at temperature not exceeding the boiling point of the solvent (Shama et al., 2013).The extracts were filtered through a sterile Whatman No.1 filter paper and then were concentrated in a vacuum at 40°C using a rotary evaporator.Each extract was transferred to glass vials and kept it at 4°C until use (Obeidat and Shatnawi, 2012).

Sources and preparation of the tested organisms
The bacteria used in this study included nine different bacterial strains; Staphylococcus aureus (ATCC25923), Streptococcus pneumoniae (ATCC63), S. aureus and S. pneumonia AMBAS Klebsiella pneumoniae, Escherichia coli (ATCC2592), E. coli, K. pneumoniae (ATCC13883), and Shigella flexneri (ATCC12022).Bacterial strains were obtained and collected from Gondar College of Medicine and health Sciences Hospital.The bacterial cultures were maintained in their appropriate agar slants at 4°C until use.

Preparation of inoculums or bacterial suspensions
The tested were separately cultured on nutrient agar at 37°C for 24 h.This was achieved by streaking the inoculating loop containing the bacteria at the top end of the agar plate moving in a zigzag horizontal pattern until 1/3 of the plate was covered.Then, three to five well-isolated overnight cultured colonies of the same morphological type were selected from an agar plate culture.The top of each colony was touched with a sterile bent wire-loop and the growth was transferred into a screw-capped tube containing 5ml of a suitable broth medium, which is Tryptic Soy Broth (TSB).The broth cultures (test tubes) were incubated without agitation for 24 h at 37°C until it achieves or exceeds the turbidity of the 0.5 McFarland standards.The turbidity of the actively growing broth culture was adjusted with sterile saline to obtain turbidity optically comparable to that of the 0.5 McFarland turbidity standard 1.5×10 8 colony-forming units (CFU)/ml.To perform this step properly, there was an adequate light to visually compare the inoculums tube and the 0.5 McFarland turbidity standards (Murray et al., 2015).

Inoculation of test plates
Optimally, within 15 min after adjusting the turbidity of the inoculum suspension, a small volume about 0.1 ml of the bacterial suspension was inoculated onto the dried surface of Mueller-Hinton agar plate and streaked by the sterile cotton swab over the entire sterile agar surface.This procedure was repeated by streaking two more times, rotating the plate approximately 60 rev/min each time to ensure an even distribution of inoculum and finally the rim of the agar was swabbed.The lid was left a jar for 3 to 5 min, but no more than 15 min, to allow for any excess surface moisture to be absorbed before applying the crude extracts on the respective well.

Antibacterial activity (Agar-well diffusion method)
The antibacterial activities of the seed extracts were tested against the selected bacterial strains.Suspensions of the bacterial isolates were made in sterile normal saline and adjusted to the 0.5 McFarland's standard.The 40 ml of sterilized Mueller Hinton Agar (MHA) medium was poured in to each sterile large sized petri-plate and allowed to solidify.The test bacterial cultures were evenly spread over the appropriate media by using sterile cotton, Swab.Then a well was made in the medium by using a sterilized cork borer with 6mm diameter, 4mm deep and about 5.0 cm apart to minimize overlapping of zones.Then 100 µl of each methanol, ethanol, and chloroform extracts of each seed were transferred into separated wells.After this, plates were incubated at 37°C for 24-48 h.After incubation, the results were observed and measured the diameter of inhibition zone around the each well.The tested microorganisms were also tested for their sensitivity against the commonly prescribed antibiotics using similar methods used for the determination of antimicrobial activity of L. sativum and C. sativum extracts.Antibiotic discs (Tetracycline 30 µg and Vancomycin 30 µg) were served as positive controls while sterile, distilled water was served as negative control.

Determination of the minimum inhibitory concentration (MIC)
The minimal inhibitory concentration (MIC) values of seed extracted from L. sativum, C. sativum, and mixture of them were determined based on agar well dilution and broth macro-tube dilution methods.In the determination of MIC, sterile screw-capped test tubes were arranged on a suitable rack in a number of rows and labeled each of them including the negative and positive control test tubes.Each seed extract was diluted to concentrations ranging from 3.125 to 50%.Each test tube contained 10 ml of extract and nutrient broth and 50%, which implies that (5 ml of crude extract and 5 ml of nutrient broth), 25% (2.5 ml of extract and 7.5 ml of nutrient broth), 12.5% (1.25 ml of extract and 8.75 ml of nutrient broth), 6.25% (0.625 ml of extract and 9.375 ml of nutrient broth), and 3.125 (0.3125 ml of extract and 9.6875 ml of nutrient broth) to bring 10 ml.To each dilution of L. sativum, C. sativum, and a mixture of both, 100 µl of the bacteria inoculum was carefully dispensed in sterile screw-capped tubes which consist of crude extracts and nutrient broth.Nutrient broth with bacterial inoculation but no any extract (positive control tubes) and nutrient broth only with no bacterial inoculation (negative control tubes) were included for every test microorganism to demonstrate an adequate microbial growth over the course of the incubation period and media sterility, respectively.Then tubes were incubated aerobically at 37°C for 24 h and examined for bacterial growth.The lowest concentration (highest dilution) of the extract that produced no visible bacterial growth (turbidity) after overnight incubation was recorded as the MIC.

Determination of the minimum bactericidal concentration (MBC)
To determine the MBC, all the agar wells, macro-test tubes used in the MIC, which did not show any visible growth of bacteria after the incubation period were sub-cultured on to the surface of the freshly prepared Mueller Hinton Agar (MHA) plates and incubated at 37°C for 24 h.The MBC was recorded as the lowest concentration (highest dilution) of the extract that did not permit any visible bacterial colony growth on the agar plate after the period of incubation (Mueller and Mechler, 2015).

Data analysis
All data were analyzed using the SPSS software package version 16.0 for windows.Means and standard deviations of the triplicates analysis were calculated (analyzed) by one-way analysis of variance (ANOVA) to determine the significance differences between the means followed by Duncan's multiple range test (P≤0.05).The statistically significant difference was defined as P≤0.05.And the graphs of all MIC and MBC values were sketched using the application of Microsoft Office Excel 2007.

Evaluation of L. sativum and C. sativum seed crude extracts against test bacteria
The diameter of inhibition zone of L. sativum, C. sativum, and the synergistic seed extracts of ethanol, methanol and chloroform solvents were evaluated against standard and clinical isolated pathogenic bacteria and is given in Tables 1, 2 and 3, respectively.
The mean inhibition zone of chloroform extract of L. sativum (9.60 mm) against E. coli (ATCC2592) was significantly (P=0.04)less than the ethanol and methanol extracts; whereas the inhibition zone (11.05 mm) of the   1).
The mean inhibition zones of all extracts of C. sativum ranges from 3.97-5.89mm against both E. coli (ATCC2592) and E.coli (clinical isolate) which is below the expected standard measure of inhibition zone (6 mm) (Biruhalem, et al., 2011).There was no statistically significant difference between the inhibition zone of methanol and chloroform extracts of C. sativum against S. aureus (clinical isolate) but the inhibition zone of ethanol against this test bacterium was significantly (P=0.04)greater than methanol and chloroform extracts.In addition to this, there was no statistically significant difference between the inhibition zone (15.30mm) of Moreover, the mean inhibition zone (24.86 mm) of the ethanol extract against K. pneumoniae (clinical isolate) was significantly (P=0.01)greater than the inhibition zone of other extracts (which are 20.63 and 17.70 mm).But there was no statistically significant difference within inhibition zones (20.18-24.68mm) of all extracts against S. pneumoniae (13883), and also within inhibition zones (18.72-22.08)against S. pneumoniae (clinical isolate) (Table 2).

Evaluation of synergistic effect of L. sativum and C. sativum seed crude extracts against tested bacteria
Whenever the mean inhibition zones of the mixed crude extract was evaluated for antibacterial activity, there was no statistically significant difference among inhibition zones which ranges from (10.41-12.06mm) of all extracts against both E. coli (ATCC2592) and E. coli (clinical isolate).However, the mean inhibition zone (27.68 mm) of ethanol extract of L. sativum and C. sativum mixture against S. aureus (ATCC25923) was significantly (P=0.02)greater than methanol and chloroform extracts.On the other hand, the inhibition zone (15.19 mm) of chloroform crude seed extracts mixture against S. flexineri (ATCC12022) was significantly (P=0.03)less than the inhibition zone of ethanol and methanol extracts  3).

Comparison of ethanol, methanol and chloroform crude seed extracts of L. sativum, C. sativum and commercial antibiotic discs against test bacteria
The inhibition zone of ethanol, methanol and chloroform crude seed extracts of L. sativum, C. sativum and commercial antibiotic discs against gram negative and gram positive pathogenic bacteria is given on Tables 4, 5, 6 and 7, respectively.The inhibition zone of antibiotic discs Tetracycline 30 µg (0.00 mm) and Vancomycin 30 µg (0.00 mm) against the organisms K. pneumoniae and E. coli (clinical isolate) were statistically (P=0.01)less than the mean inhibition zones of ethanol, methanol and chloroform crude seed extracts of L. sativum and C. sativum (Tables 6 and 7, respectively).And also the inhibition zone of Tetracycline 30 µg (0.00 mm) and Vancomycin 30 µg (0.00 mm) against S. pneumoniae  sativum (20.18-24.68 mm).

Determination of minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC)
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of different concentration of crude seed extracts of L. sativum, C. sativum and mixture of the two crude seed extracts (v/v) is given in Figures 1a, b, 2a, b and 3a, b, respectively.As shown in Figure 1a, the minimum inhibitory concentrations of ethanol and methanol crude seed extracts of L. sativum against E. coli (ATCC2592) and E. coli (clinical isolate) were 12.5%.Whereas MIC of L. sativum crude seed extracts with all solvents against S. pneumoniae (ATCC63) and extracts of ethanol and methanol against S. pneumoniae (clinical isolate) were 3.125%.But in the rest test organisms, the MIC of L. sativum crude seed extracts by the help of three solvents was 6.25% (Figure 1a).The minimum bactericidal concentration (MBC) of ethanol, methanol and chloroform crude seed extracts of L. sativum against both E. coli (ATCC2592) and E. coli (clinical isolate), and chloroform extract of this seed against S. aureus (ATCC25923) were 25%.And also the MBC of all solvents of L sativum seed extracts against K. pneumoniae (ATCC13883), K. pneumoniae (clinical isolate), and S. aureus (clinical isolate), ethanol and methanol extracts against S. aureus (ATCC25923), methanol and chloroform extracts against S. flexineri (ATCC12022), and chloroform extract of this seed against  The MBC of the chloroform extract in the combination of  et al., 2012).Similarly, the active compounds aliphatic 2Ealkenals alkenals and alkanals, isolated from the fresh leaves of C .sativum were found to possess bactericidal activity against various frequently emerging human pathogenic bacteria (Isao et al., 2014).In support of these three ideas, the results of the present study are similar to those reported by Ejoba et al. (2011) andRathabai et al. (2012) and other constituents of C. sativum have been found to be active against a range of bacteria including S.
The seeds of L. sativum showed remarkable antibacterial activity against a number of pathogenic bacteria responsible for severe infections; this might be due to the presence of phyto-constituents like flavonoids, saponins, tannin and phenols in the crude extracts of L. sativum (Shama et al., 2013).Infections caused by gram positive and gram negative bacteria, especially those with multidrug resistances, are among the most difficult ones to treat with conventional antibiotics; therefore, it seems likely that the antibacterial compounds extracted from L. sativum might inhibit bacteria by different mechanisms than that of currently used antibiotics and have therapeutic values as antibacterial agents (Chandra et al, 2012).
The present study also revealed that L. sativum alcoholic extract had maximum antibacterial activity, which is identical with results obtained from other researchers (Parekh and Chanda, 2011).Therefore, comparisons of the current finding with former literatures are showing similar result.The inhibition zone of crude seed extracts of L. sativum against most tested pathogenic bacteria were significantly (p≤0.05)greater than the inhibition zone of C. sativum, but less than the inhibition zone of their synergism.On the other hand, the inhibition zone of the synergistic antibacterial effect of mixture of L. sativum and C. sativum crude seed extracts against all tested pathogenic bacteria was significantly (p≤0.05)far greater than the mean inhibition zone of the antibacterial agents of L. sativum and C. sativum alone.This might be the basic reason why the local community used the mixture of the two to treat different pathogenic bacterial infections even though very few people are familiar with this art.As a result, mixture of L. sativum and C. sativum crude seed extracts may be effective to treat both gram-positive and gram-negative bacterial infections at relatively low concentrations.
However, it is necessary to underline that the crude seed extracts of L. sativum and C. sativum separately have also good potential of a broad spectrum of activity against both gram-positive and gram negative bacteria.But for those gram negative bacteria, the crude extracts of the two plants in combination or individually showed relatively less antibacterial activity than gram positive bacteria.This was due to the presence of an extra outer membrane in gram negative bacteria which consists of lipo-polysaccharide and makes them their cell wall impermeable to lipophilic extracts; whereas the gram positive bacteria were more susceptible because of having only an outer peptide-glycan layer which is not an effective permeability barrier.So, this was a valuable reason and agreed with the previous report (Parekh and Chanda, 2011).In addition to this, the results of crude seed extracts of L. sativum and C. sativum were compared with the common commercial antibiotic discs (Tetracycline 30 µg and Vancomycin 30 µg).The mean inhibition zones of L. sativum crude seed extracts against all tested pathogenic bacteria were significantly (p≤0.05)greater than the inhibition zones of the commercial antibiotic discs (Tetracycline 30µg and Vancomycin 30 µg); while the mean inhibition zones of the antibiotic disc (Vancomycin 30 µg) against the tested organisms S. aureus (ATCC25923), S. pneumoniae (ATCC63), E. coli (clinical isolate), and E.coli (ATCC2592) were significantly (p≤0.05)greater than the inhibition zones of C. sativum crude seed extracts.
The zone of inhibition varied among suggesting that the varying degree of efficacy and different phytoconstituents of the extracts on the target organism.This difference in antibacterial effects may be due bacterial strains differences used in the study.From this study it was found that the ethanolic extract of L. sativum and C. sativum possessed significant antibacterial activity when compared with the other extracts and standard antibiotics/ drugs used in the study; so that while screening of various extracts of L. sativum and C. sativum against various Gram positive and Gram negative bacteria.The results of statistical analysis also showed a significant difference (p≤ 0.05).According to this study, MIC and MBC of L. sativum, C. sativum and mixture of them against most clinical isolated pathogenic bacteria were 12.5 and 6.25% but for those standard pathogenic bacteria, the MIC and the MBC were reduced by half (6.25 and 3.125%).This was because of the clinical drug resistant pathogenic bacteria have the ability to resist and were not easily killed at lowest concentration (highest dilution) by using the mixture or separate extracts of L. sativum and C. sativum when compared with standard pathogenic bacteria with the exception of C. sativum against K. pneumoniae (clinical isolate) which has greater inhibition zone than S. pneumoniae (ATCC13883).But the standard pathogenic bacteria were easily killed at the lowest concentration (highest dilution) due to their sensitivity for any antibiotic agents.This result was agreed with the earlier reports (Shama et al., 2013).
Traditionally, L. sativum is known to act synergistically with C. sativum in very rare localities of north and south Gondar district rural villages for the treatment of many different clinical isolated pathogenic bacteria that are difficult to eradicate.The traditional medical practitioners using their indigenous knowledge open a way to use the medicinal plant, L. sativum and C. sativum in combination for the treatment of different bacterial ailments.Thus, this finding was largely favor the claim of the local society or community to use the combination of L. sativum and C. sativum rather than using L. sativum and C. sativum separately for the treatment of different pathogenic bacteria infections and it opens a door to consider and acknowledge the traditional medical practices for the treatment of different infectious ailments using natural resources such as L. sativum and C. sativum.

CONCLUSION AND RECOMMENDATION
In the present study, it can be concluded that the synergistic antibacterial are preferred, as microbial tolerance is less likely to develop against substances having more than one type of modes of action.This emphasizes as the mixture of substances under investigation has potential application against drug resistant pathogenic bacteria.
The antibacterial activity of L. sativum and C. sativum crude seed extracts were greater than the activity of currently used antibiotics (Tetracycline and Vancomycin) against the selected organisms.As a result, the growth of all pathogenic bacteria was completely inhibited with the exception of C. sativum for E. coli.All extracts of C. sativum were poorly potent against E. coli strains.It is also possible to say Gram negative bacteria were relatively resistant than gram positive bacteria.
Moreover, from this study it was found that the ethanolic extract of L. sativum and C. sativum exhibited relatively very satisfactory inhibitory activity.All clinical isolated pathogenic bacteria used in this study did not show any inhibition zones against the commonly prescribed commercial antibiotic discs used in this study, with the exception of Vancomycin for S. aureus (clinical isolate).This was believed to be due to their resistance for these commercial antibiotic discs.
The antibacterial potency of these medicinal plants is generally believed to be due to bioactive compounds found in the seeds.Therefore, with the ever increasing resistant strains of microorganisms to the already available and synthesized antibiotics, the naturally available L. sativum and C. sativum could be a potential alternative, especially, when they are used in combination though the habit of using them as such is very rare.

Figure 1 .Figure 2 .Figure 3 .
Figure 1.(a) MIC and (b) MBC determination of ethanol, methanol and chloroform crude seed extracts of L. sativum against standard and drug resistant clinical isolated pathogenic bacteria.

Table 1 .
Comparison of the efficiency of the solvents on L. sativum seed extracts.

Table 2 .
Comparison of the efficiency of the solvents on C. sativum seed extracts.
bc K. pneumoniae (clinical isolate) e *Values were means of triplicate determinations.Values of the same column followed by different letters are significantly different at (p≤0.05).Et, Ethanol; Met, Methanol; Chl, Chloroform.(25.68 mm) of ethanol seed extract of L. sativum against S. pneumonia (clinical isolate) was significantly (P=0.04)greater than the inhibition zone of methanol and chloroform extracts (Table

Table 3 .
Comparison of antibacterial activity of the extracts alone and in combination.

Table 4 .
Comparison of inhibition zone of ethanol, methanol, and chloroform crude seed extracts of L. sativum and commercial antibiotic discs against gram-negative test bacteria.
Et, Ethanol; Met, Methanol; Chl, Chloroform.against this organism.The inhibition zone (25.18 mm) of methanol seed extract of L. sativum and C. sativum mixture against K. pneumonia (clinical isolate) was significantly (P=0.03)greater than the inhibition zone of ethanol and chloroform extracts.But the inhibition zone (25.74 mm) of methanol seed extract of L. sativum and C. sativum mixture against S. pneumonia (clinical isolate) was significantly (P=0.04)less than the inhibition zone of ethanol and chloroform extracts against this test bacteria (Table

Table 5 .
Comparison of inhibition zone of ethanol, methanol, and chloroform crude seed extracts of L. sativum and commercial antibiotic discs against gram-positive test bacteria.

Table 6 .
Comparison of inhibition zone of ethanol, methanol, and chloroform crude seed extracts of Coriandrum sativum and commercial antibiotic discs against gram-negative test bacteria.

Table 7 .
Comparison of inhibition zone of ethanol, methanol, and chloroform crude seed extracts of Coriandrum sativum and commercial antibiotic discs against gram-positive test bacteria.