Pharmaceutical

Communication

Biosci. Biotech. Res. Comm. 10(1): 44-50 (2017)

Evaluation of antimicrobial and synergistic effects of

selected medicinal plants of Hail area with antibiotics

Abdel Moneim E. Sulieman, Sherif M. Shaarawy, Ahmed A. Alghamdi, Vajid N. Veettil*,

Mohanad Abdelgadir and Nasir A. Ibrahim

Department of Biology, Faculty of Science, University of Hail, Hail, Kingdom of Saudi Arabia

ABSTRACT

In this study, the antibacterial activity of selected medicinal plants and their synergistic effect with antibiotics were

investigated. Different antibiotic resistant bacterial species were employed including: Escherichia coli (E. coli), Kleb-

siella pneumoniae (K. pneumonia), Acinetobacter baumannii (A.baumannii), Pseudomonas aeruginosa (P. aeruginosa),

Staphylococcus aureus (S. aureus) and Salmonella spp. The results indicated that methanol extracts of most of the

tested plants inhibited growth of the tested bacteria, however, the highest inhibition was by Senna followed by Har-

mal, saf ower and Guaada, where the inhibition zone diameter was 8 mm, 4 mm, 3 mm, 2 mm and 2 mm, respec-

tively. The synergism between plants extract and antibiotics resulted in inhibition of the tested organisms; generally,

the synergism with Harmal extract was more effective in inhibition of antibiotic-resistant bacteria, followed by Senna

and  nally Saf ower. The outcomes additionally demonstrated that methanolic concentrate of the plant has more

inhibitory impact than that of the water extract. Further investigation of the plants extract to seclude and recognize

the dynamic  xings is prescribed.

KEY WORDS: ANTIBIOTIC-RESISTANT BACTERIA, INHIBITION ZONE, SAFFLOWER, METHANOL.

ARTICLE INFORMATION:

*Corresponding Author: vajidnv@gmail.com

Received 12

Jan, 2017

Accepted after revision 22

March, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

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Online Contents Available at: http//www.bbrc.in/

INTRODUCTION

Plants as a source of medicinal compounds have kept on

assuming to play a dominant role in the maintenance

of human health since ancient times. As indicated by

the World Health Organization plant extracts or their

dynamic constituents are utilized as people prescription

in customary treatments of 80% of the total population.

More than half of all modern clinical drugs are of regu-

lar item source (Kirbag et al., 2009). Medicinal plants

possess immunomodulatory and antioxidant properties,

prompting to antibacterial activities. They are known

to have adaptable immunomodulatory activity by

stimulating both non-particular and particular immu-

nity (Pandey and Chowdhry, 2006). In the most recent

couple of years, various reviews have been directed in

Abdel Moneim E. Sulieman et al.

various countries to demonstrate such effectiveness.

Many plants have been utilized in light of their antimi-

crobial traits, which are due to compounds synthesized

in the secondary metabolism of the plants, (Nascimento

et al., 2000 Amenu 2014, Wink 2015 and Egamberdieva

et al., 2017).

Antimicrobial screening of plant extracts and phy-

tochemicals speaks to a beginning stage for antimicro-

bial medication disclosure. Phytochemical studies have

pulled in the consideration of plant researchers because

of the advancement of new and complex strategies.

These systems assumed a critical part in the scan for

extra assets of crude material for pharmaceutical indus-

try. (Shakeri et. al., 2012).

Microbiologists differentiate two groups of antimicro-

bial agents used in the treatment of infectious disease:

antibiotics, which are natural substances produced by

certain groups of microorganisms, and chemotherapeu-

tic agents, which are chemically synthesized. A hybrid

substance is a semisynthetic antibiotic, wherein a molec-

ular version produced by the organism is in this way

adjusted by the scienti c expert to accomplish desired

properties. Furthermore, some antimicrobial substances,

originally discovered as microbial products, can be man-

ufactured entirely by chemical methods. In the medi-

cal and pharmaceutical worlds, all these antimicrobial

agents used in the treatment of disease are referred to

as antibiotics. Different antibiotics practice their inhibi-

tory action on various pathogenic organisms (Chanda

and Rakholiya, 2011). Multiple drug resistance in human

pathogenic microorganisms has been developed due to

unpredictable utilization of commercial antimicrobial

ordinarily utilized as a part of the treatment of infectious

diseases.

In general, bacteria have the genetic ability to trans-

mit and acquire resistance to drugs used as therapeutic

agents (Nascimento et. al. 2000). The improvement of

antimicrobial resistance is multifactorial, including the

particular way of the relationship of bacteria to antibi-

otics, the utilization of antibacterial agent, host charac-

teristics and environmental factors. This situation has

constrained researchers to scan for new antimicrobial

substances from different sources as novel antimicro-

bial chemotherapeutic agents, yet the cost generation

of manufactured medications is high and they create

antagonistic impacts contrasted with plant determined

medications (Abiramasundari et. al., 2011).

Due to development of bacterial resistance to many

of the extensively utilized antibiotics has necessitated

the scan for new antibacterial agents or a mix of drugs

to have the capacity to combat new resistant patho-

genic bacteria. Therefore, in this study, we evaluated

the antimicrobial activity of selected medicinal plants

in Hail area and the possible synergism between water

and methanol extracts of these plants and certain known

antibiotic drugs.

MATERIAL AND METHODS

COLLECTION OF THE SELECTED PLANT

SAMPLES

A total of 7  owering plants growing wildly were col-

lected from various geographical sites in Hail area, in

addition to commercial samples. The plant materials

included: Pergularia tomentosa L. (Umlebena), Peganum

harmala L. (Harmal), Senna italica Mill (Senna), Aspho-

delus  stulosus (Bargog), Teucrium polium (Guaada)

and Carthamus tinctorius (Saf ower) were collected

from various sites in Hail area. The plants were identi-

 ed through consultation of the  ora of Saudi Arabia

(Chaudhary, 2008), The plants samples were representa-

tive of medicinal plants as a whole. Repeated sampling

was implemented. The collected plant materials were

rinsed thoroughly with distilled water to remove extra-

neous contaminants and then cut into small pieces,

oven-dried at 50°C until the dry weight stabilized, and

ground into a powder with an electric-grinder.

Tested microorganisms

The tested bacteria used in the study which were anti-

biotic resistance, included: Escherichia coli (E. coli),

Klebsiella pneumoniae (K. pneumonia), Acinetobacter

baumannii (A.baumannii), Pseudomonas aeruginosa (P.

aeruginosa), Staphylococcus aureus (S. aureus) and Sal-

monella spp. These microorganisms were obtained from

King Khalid Hospital, Hail. The microorganism were

maintained on Brain Heart Infusion (BHI) agar bauman-

nii medium (HiMedia) at 4 ºC for further experiments.

Preparation of plant extracts standard concentrations

For aqueous extraction, 20 g of air-dried plant powder

were added to150 ml of distilled water and boiled on

slow heat for 2 hours. Then it was  ltered through 8

layers of muslin cloth and centrifuged at 5000g for 10

min and the supernatant was collected. This procedure

was repeated twice; after 6 hours, the supernatant was

collected at an interval of 2 hours, pooled together and

concentrated to make the  nal volume one-fourth of the

original volume

One g of each aqueous extract and alcohol pre-

prepared (each separately) were taken and the aqueous

extract was dissolved in 5 grams sterile distilled water,

while alcoholic extracts was dissolved in 5 ml of DiMe-

thyl Sulphoxide (DMSO). Thus 200 mg / ml of stock

were obtained as a standard concentration of aqueous

and alcoholic extracts. Aqueous extracts were sterilized

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EVALUATION OF ANTIMICROBIAL AND SYNERGISTIC EFFECTS 45

Abdel Moneim E. Sulieman et al.

using 0.22 μm membrane  lters and alcoholic extracts

was pasteurized for 15 minutes at temperature 62 ºC.

Preparation of culture media, inoculums, reagents and

antibiotics

Types of media which was used include: Brain Heart

Infusion broth, Nutrient agar (biolife) and Mueller-

Hinton agar (HiMedia). Also methanol and water were

used for extraction process. These media and the solvent

were purchased from some company in Jeddah city.The

Antibiotics mixture used in the study included: Van-

comycin, Cefotaxime, Tetracyclines, Chloramphenicol

and Ampicillin, these were purchased from pharmacies

in Hail city. For preparation of inocula, stock cultures

were maintained at 4°C on nutrient agar slants for bac-

teria. Active cultures for experiments will be prepared by

transferring a loopful of culture to 5 ml of Brain Heart

Infusion broth and incubated at 37 °C for 24 hours.

Plant extracts activity assay

A suspension of testing microorganisms was spread on

Muller Hinton Agar (MHA) medium. The  lter paper

discs (5mm in diameter) will be placed on the agar plates

which were inoculated with the tested microorganisms

and then impregnating with 20μl of plant extract (con-

centration 200 mg/ml). The plates were subsequently

incubated at 37°C for 24 Hrs. After incubation the

growth inhibition zone was quanti ed by measuring the

diameter of the zone of inhibition in mm (Kumar et al.,

2009).

Synergism between plant extract, antibiotics and Non-

antibiotics

The bacterial cultures were grown in BHI broth at 37º C.

After 4 h of growth, each bacterium was inoculated on

the surface of Mueller-Hinton agar plates. Subsequently,

the antibiotic disk (diameter =5mm) were placed on the

surface of each inoculated plate and then added 20 μl

of plant extract, to identify synergies effect between the

plant extract at a concentration of 200mg/ml) and anti-

biotics. The plates were incubated at 37º C for 24 h. The

diameters of clearing zones will be measured.

REULTS AND DISCUSSION

Medicinal plants used in the study

Plants are the greatest medication stores ever known on

Earth, interminable bioactive invention blends which

in uence animal and human health (Abdallah et. al.,

2012). They are rich in secondary - metabolites and are

potential source of drugs which can serve to increase the

income of the producers. Detailed information regard-

ing the plant families, the scienti c and common names,

the families of tested plant species, the plant parts used,

the solvents used for extraction, have been tabulated in

Table (1).

The antibacterial properties

The present study investigated the effect of water and

methanol extracts of the tested plants which included:

Pergularia tomentosa (Umlubena), Peganum harmala

(Harmal), Senna italica (Senna), Carthamus tinctorius

(Saf ower) and Teucrium polium (Guuda) on growth of

the tested bacteria which included: P. aeruginosa, E. coli,

A. baumannii and K. pneumonia. From the results it is

clear that water extracts of most of the tested plants did

not inhibited growth of most of the tested bacteria with

exception to P. aeruginosa which was inhibited by Senna,

Umlubena and Harmal where the inhibition zone diame-

ter was 2 mm, 6 mm and 3 mm, respectively. On the other

hand, E. coli bacterial growth was inhibited by all metha-

nolic extract of the tested medicinal plants, however, the

highest inhibition was by Senna followed by Harmal, saf-

 ower and Guaada , where the inhibition zone diameter

was 8 mm, 4 mm, 3 mm, 2 mm and 2 mm, respectively.

Table 1. Medicinal plants used in the study

Pl ant scienti c name Plant common name Family Plant part Solvent used

Pergularia tomentosa L Umlubena Asclepiadaceae Leaves 1- Water (W)

2- Methanol (M)

Peganum harmala L Harmal Zygophyllaceae Leaves 1- Water (W)

2- Methanol (M)

Cassis italic Mill Senna (Senamaka) Fabaceae Leaves 1- Water (W)

2- Methanol (M)

Asphodelus  stulosus . Bargog Asphodelaceae Leaves 1- Water (W)

2- Methanol (M)

Carthamus tinctorius Saf ower Asteraceae Leaves 21- Water (W)

2- Methanol (M

Teucrium polium Guaada Lamiaceae Leaves 1- Water (W)

2- Methanol (M

46 EVALUATION OF ANTIMICROBIAL AND SYNERGISTIC EFFECTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Abdel Moneim E. Sulieman et al.

FIGURE 1. Inhibition zone diameters of the tested plants against

the tested bacteria

FIGURE 2. Inhibition of S.aureus by the tested plants , antibiotic

and synergetic effect between plant extract and antibiotic

A. baumannii was highly inhibited by Harmal fol-

lowed by Saf ower, then Senna, however, and the inhi-

bition zone diameter was, 13 mm, 5 mm and 3 mm,

respectively. However, the microbe was not inhibited

by either Bargog or Guaada plant extract. Methanolic

extract of all tested medicinal plants did not inhibit

growth of P. aeruginosa although the water extract of

some of these plants inhibited its growth.K. pneumo-

nia bacteria was only inhibited by methanolic extract of

Harmal and Senna where the inhibition zone diameter

was 3 mm and 2 mm, respectively.

In general, the mathanolic extract of the tested medic-

inal plants has more inhibitory effect against the tested

bacteria compared to the water extract of these plants.

Moreover, both Harmal and Senna plants exhibited the

highest antibacterial effects compared to the other tested

plants.

Synergism between plant extract and antibiotics

The synergism between plant extract and antibiotics is

presented in Fig. (2-5). It is clearly seen that all tested

plants inhibited the tested microorganism by using anti-

biotics and the inhibition zones increased upon adding

the antibiotics to the plant extracts. This means the syn-

ergism between plant extracts and antibiotics resulted

in inhibition of tested microorganisms with varying

degrees. However, Harmal plant exhibited the highest

inhibition, followed by Senna and  nally Saf ower.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EVALUATION OF ANTIMICROBIAL AND SYNERGISTIC EFFECTS 47

Abdel Moneim E. Sulieman et al.

FIGURE 3. Inhibition of K. pneumonia by the tested plants , antibiotic

and synergetic effect between plant extract and antibiotic

FIGURE 4. Inhibition of E.coli by the tested plants , antibiotic

and synergetic effect between plant extract and antibiotic

FIGURE 5. Inhibition of A. baumannii by the tested plants, anti-

biotic and synergetic effect between plant extract and antibiotic

48 EVALUATION OF ANTIMICROBIAL AND SYNERGISTIC EFFECTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Abdel Moneim E. Sulieman et al.

Moreover, the methanol extract was more effective than

water extracts for all tested plants.

Generally, the synergism between Harmal extract

was more effective in inhibition of antibiotic-resistant

bacteria, followed by Senna and  nally Saf ower. The

most inhibited microorganism was Staphylococci aureus

where the inhibition zone diameter was 16 mm and 9

mm for the combination of Harmal extract plus antibi-

otic and for antibiotic only, respectively.

Eschrichia coli bacteria were highly inhibited by Har-

mal, Senna and Saf ower water and methanolic extract

combined with antibiotics. However, Harmal-antibiotic

combination has the highest inhibitory effect followed

by Senna-antibiotic and saf ower-antibiotic, where the

inhibition zone diameter was 14 mm, 13 mm and 12 mm

, respectively. Moreover, the methanolic extracts of the

tested plants with added antibiotic was more effective

compared to the water extracts of the tested plants with

added antibiotic. Acinetobacter baumannii was inhibited

by Harmal-antibiotic and Senna-antibiotics combinations

at the same extent and the inhibition zone diameter of the

methanolic and water extract combined with antibiotics

was 13 mm and 10 mm, respectively. Saf ower water and

methanolic extract also inhibited the Acinetobacter bau-

mannii, however, combination of the methanolic plant

extract with antibiotic was more effective.

In the present study, the plant extracts had different

synergistic ability to inhibit the growth of microorgan-

isms. However the zones of inhibition of all tested micro-

organisms resulted from plant extract-antibiotic combi-

nations were greater than those resulted from antibiotics

merely. Therefore, increased plants antimicrobials have

been found to be synergistic enhancers in that though

they may not have any antimicrobial properties alone,

but when they are taken concurrently with antibiotics

they enhance the effect of these drugs.

Drug synergism between antimicrobial agents and

plant extracts has been reported (Nascimento et. al.,

2000; Aburjai et. al., 2001; Aqil et. al., 2005; Esimon et.

al., 2006; Ibezim et. al., 2006).

Staphylococcus aureus is recognized as one of the

major causes of infections in humans occurring in both

the community and the hospital. Methicillin-resistant and

multidrug resistant staphylococci have become a major

nosocomial pathogen (NNIS System, 2004). Therefore, it

is of vital importance to identify new effective antimicro-

bial agents. Among the potential sources of new agents,

medicinal plants have long been investigated.

CONCLUSION

In c onclusion, the medicinal plants which included: Per-

gularia tomentosa L. (Umlabina), Peganum harmala L.

(Harmal), Cassis italic Mill (Senna), Asphodelus  stulo-

sus (Bargog), Teucrium polium (Guaada) and Carthamus

tinctorius (Saf ower) collected from various sites in Hail

area have antimicrobial properties against the tested

antibiotic-resistant bacteria with various degrees which

may re ect the antibacterial activity of plant active

ingredients that inhibit bacterial growth. However, both

Harmal and Senna plants exhibited the highest anti-

bacterial effects against the antibiotic-resistant bacteria

compared to other tested plants. The results will also

reveal the importance of plant extracts when associated

with antibiotic drugs in control of pathogenic and spoil-

age bacteria. Future investigation of the plants extract

to isolate and recognize the active ingredients is highly

recommended.

ACKNOWLEDGEMENTS

The authors express their gratitude to the Deanship of

Scienti c Research of University of Hail, Saudi Arabia

for  nancing this research (Project No. 01150009).

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