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Research Gbedema et al., 2010: Herbal extracts modulate Tetracycline activity.ISSN 0976 4852

2010, IJCRR, All Rights Reserved

1 September, 2010|Volume 01|Issue 04|

International Journal of Contemporary Research and ReviewModulation effect of Herbal extracts on the antibacterial activity of Tetracycline

Gbedema SY*, Adu F, Bayor MT, Annan K1

Departments of Pharmaceutics and1Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences,

Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. W. Africa.

Abstract

As part of our ongoing study to screen local herbs for their possible usefulness in chemotherapy of infectious diseases, we assessed

extracts from sixteen herbs for their antimicrobial properties and their influence on the activity of tetracycline. The minimum inhibito

ry concentrations (MIC) of tetracycline against Staph. aureus, B. subtilis, P. aeruginosa, E. coli and S. typhi were determined aloneand in the presence of sub-inhibitory concentrations of the extracts by the KirbyBauer agar diffusion method of antibacterial assay.

Twelve of the extracts potentiated the antibacterial activity of tetracycline against at least one of the test bacteria. Extracts ofB. arun

dinacea, B. pilosa, C. membranaceus, E. guineensis, H. sabdariffa, J. curcas root, M. oppositifolius, M. lucida and S. campanulata

significantly (p


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Science and Technology, where herba-

rium specimens have been kept. The plant

materials were separately air-dried for 15

days and milled into coarse powders using

a Laboratory Mill Machine (Type 8,

Christy & Norris, UK).

Extraction and Phytochemical screening

Five hundred grams each of the powdered

plant materials were separately cold-

macerated with methanol (Sigma-Aldrich,

St. Louis, MO, USA) over 48 h. The ex-

tracts were filtered through Whatman

filter paper number 1 and concentrated

under reduced pressure in a rotary evapo-

rator. They were then dried in an oven

(Gallenkamp, Leicestershire, UK) to con-

stant weights at 40oC. Portions of the

dried extracts were phytochemically

screened for the presence of tannins, sa-

ponins, anthraquinones, cardiac and cya-nogenetic glycosides, flavonoids, terpeno-

ids and alkaloids using the procedures

outlined by Wall et al. (1952), Harbon

(1973) and Sofowora (1993).

Antibacterial assay

The antibacterial activities of the extracts

were assessed against; Staphylococcus

aureus (ATCC 25923), Bacilus subtilis

(NCTC 10073), Pseudomonas aeruginosa

(ATCC 27853), Escherichia coli (NCTC

25922) and Salmonella typhi (NCTC8385). These test organisms were from

the stock kept at the Microbiology Section

of the Department of Pharmaceutics,

KNUST, Kumasi, Ghana. Minimum inhi-

bitory concentration (MIC) values of the

extracts and tetracycline (Sigma-Aldrich,

St. Louis, MO, USA) were determined

using the Kirby-Bauer agar diffusion me-

thod of antibacterial susceptibility testing

(Jones et al., 2001). A suspension of 24 h

culture equivalent to a 0.5 McFarland

standard was prepared in saline and

spread onto pre-dried Mueller-Hintonagar (MHA) plates. Four wells of 8 mm

diameter were equidistantly bored in the

agar and filled separately with 10 l of 2,4, 8 and 16 mg/ml of the extracts which

were reconstituted in 50% methanol. The

50% methanol was also tested as a con-

trol. Zones of growth-inhibition were read

after 24 h incubation at 37oC. The MICs

were then calculated from semi-log plo

of the values of concentration and mean

zones of inhibition.

Tetracycline modulation assay

In the antibiotic activity modulation as-

say, the minimum inhibitory concentra-

tions of tetracycline in the presence of

sub-inhibitory concentration of the ex-

tracts were determined. 10 g/ml solu

tions of the various extracts were prepared

and used as vehicle for reconstituting the

tetracycline for use as detailed under anti-

bacterial activity assay.

Results and discussionOut of the 18 plant extracts tested 11

(61.1%) exhibited significant antimicrobial activity against at least one of the five

test bacteria with MICs below 20 mg/m

(Table 1). Extracts ofT. cacao

Table 1. Minimum inhibitory concentrations of the plant extracts

Key: bk = bark, lf = leaf, wh = whole herb, rt = root, cx = calyx, sd = seed, Sta = Staphylococcus aureus, Pa = Pseudomonas aeruginosa, Bs =Bacillus subtilis , Ec =

Escherichia coli, Sal = Salmonella typhi, - = no activity observed and a= g/ml.

Plants Extracts

Minimum inhibitory concentration (mg/ml)

Part Sta Pa Bs Ec Sal

Anthoclesta nobilis bk 0.40.01 0.40.02 0.30.02 0.40.01 2.00.04

Bambusa aurambincea lf >20 - >20 >20 >20

Bidens pilosa wh - - - - -

Croton membranaceus rt >20 160.3 >20 >20 130.04

Cryptolepis sanguinolenta rt 800.1a 400.3a 100.01a 600.05a 0.30.02

Elaeis guineensis lf >20 - >20 - -

Hibiscus sabdariffa cx 20.03 - 20.1 40.06 20.04

Jatropha curcas lf - - - - -

rt >20 - >20 20.01 80.04

Mallotus oppositifolius lf 110.03 6.50.01 110.04 - -

Momordica charantia wh >20 - 110.01 30.02 >20

Morinda lucida bk 120.06 100.04 120.01 90.02 80.04

Moringa oleifera rt >20 - >20 - -

Plumbaga zeylanica rt 40.01 160.03 120.02 160.01 20.01

Psidium guajava lf 20.02 90.02 100.03 40.01 100.02

Spathodia campanulata bk >20 150.04 >20 >20 190.04

Theobroma cacao lf - - - - -

sd - - - - -


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leaf and root, J. curcas leafand B. pilosa

were not active at the concentrations

tested in this study. Extracts ofC. sangui-

nolenta,A. nobilis,H. Sabdariffa, P. gua-

java and P. zeylanica showed broad spec-

trum antibacterial activity and the highest

activities were exhibited by C. sanguino-

lenta root, A. nobilis stem bark and H.

sabdariffa calyx. The presence of phyto-

chemical compounds such as tannin, fla-

vonoids, alkaloids, glycosides, anthraqui-

nones and terpenoids in the extracts (Ta-

ble 2) could account for their observed

antibacterial activities. These compounds

are largely untapped reservoir of mole-

cules with diverse chemical structures for

potentially new antimicrobial drug leads.

Additionally, plants have a long history of

use in traditional medicine and have been

the source of several promising novel

antimicrobial agents (Gordien, et al.

2009; Liu et al., 2009 Murphy 1999)

These compounds therefore, when devel

oped into new antimicrobials may with-

stand the problem of bacterial cross-

resistance development which is often

encountered among many of the currently

employed antibiotics such as the penicil

lins and cephalosporins.

Table 2. Phytoconstituents of the plant extracts.

Plants Extracts

Phytoconstituents

Parts Sap Card Cyan Flav Alk Terp Tan Anth

Anthoclesta nobilis bk + - - + + + + -

Bambusa arundinacea lf - - + + + - - -

Bidens pilosa wh + + - + + - + -

Croton membranaceus rt + - - + + + + -

Cryptolepis sanguinolenta rt + - - + + + + -

Elaeis guineensis lf - - - + + + + -

Hibiscus sabdariffa cx + - - + + + + +

Jatropha curcas lf + - + + + + + -

rt + - + + + + + -

Mallotus oppositifolius lf + + - + - + + -

Momordica charantia wh + - - - + + - -

Morinda lucida bk + - - + + + + +

Moringa oleifera rt + - - + + + + +

Plumbaga zeylanica rt + - - + - + + -

Psidium guajava lf + + - + - + + -

Spathodia campanulata bk + - - + + + + -

Theobroma cacao lf + - - + + + + -

sd + - - + + + + -

Key: + = present, - = absent, bk = bark, lf = leaf, wh = whole herb, rt = root, cx = calyx and sd = seed. Sap = saponins, card = cardiac glycosides, Cyan =

cyanogenetic glycosides, fla v= falvonoids, Alk = alkaloids, Ter = terpenoids, Tan = tannins, Anth = anthraquinones.

Tetracycline exhibited far smaller MICs

attesting to its superiority over the plant

extracts. Tetracycline inhibits bacterial

growth by binding to the 16S part of the

30S ribosomal subunit and thus prevents

the binding of amino-acyl tRNA to the

mRNA-ribosome complex. Bacteria can

become resistant to tetracycline by adding

an acetyl group to the tetracycline mole-

cule (enzymatic inactivation), developing

efflux pumps to actively pump the drug

out of the cytoplasm or producing ribo-

somal protection proteins that interfere

with tetracycline activity (Sanford-Chee,

et al., 2001; McMurry, et al., 1980). Te-

tracycline-resistant bacteria survive, tole-

rate and replicate in the presence of the

usual dose of the antibiotic and may only

be inhibited when the dose of the antibiot-

ic is significantly increased. Any agen

that reduces the MIC of tetracycline can

be said to have modified these mechan-

isms to allow the drug to act efficiently

against the pathogen.

.The presence of sub-inhibitory concentra-

tion (10 g/mI) of 9 extracts ( B. arundi-

nacea, B. pilosa, C. membranaceus, E.

guineensis, H. sabdariffa, J. curcas root,

M. oppositifolius, M. lucida and S. cam

panulata) significantly (p


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the MIC of tetracycline against Staph au-

reus. Tetracycline activity against B. sub-

tilis and P aeruginosa were enhanced by 3

(H. sabdariffa, J. curcas and M. oleifera)

and 4 ( J. curcas root , M. oppositifolius,

M. charantia and S. campanulata,) ex-

tracts respectively. B. arundinacea, C.

membranaceus, M. charantia, M. lucida

and S. campanulata extracts also signifi-

cantly reduced the MIC of tetracycline

against E. coli while the susceptibility of

S. typhi was enhanced by the presence of

5 extracts (Fig 1).

Fig.1. MICs of tetracycline only and tetracycline in the presence of

sub-inhibitory concentrations of plant extracts.

Interestingly, J. curcas root extract did

not exhibit any antimicrobial activity

against Staph. aureus, B. subtilis and P.

aeruginosa but its presence potentiated

tetracycline activity against these organ-

isms in addition to S. typhi. Staph. aureus,

P. aeruginosa and S. typhi were respec-

tively not susceptible to B. pilosa, M. cha-

rantia and M. oleifera extracts but they

potentiated tetracycline. These findings

are in line with earlier reports of plant

constituents including alkaloids (Bren-

wald et al., 1997), flavonoids and couma-

rins (Tsutomu et al., 2005), tannins and

saponins (Lee et al., 2000), terpenoids and

steroids (Silvia et al., 1999) exhibiting

bacterial resistance modulation properties

These plants therefore appear to be a rich

source of agents for chemotherapeutic

application. Combination chemotherapy

is preferred in the management of resis

tant bacterial infections. A typical exam-

ple is seen with Augmentin (from Glax-

oSmithKline) where clavulanic acid inhi-


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bits the function of -lactamases produced

by resistant bacteria and thus prevents

destruction of the amoxicillin. We also

recently reported the antibiotic-modifying

properties of friedelin (isolated from

Paullinia pinnata) and extracts of Cory-

nanthe pachyceras (Annan et al., 2009;Adu et al., 2009). The antibiotic modula-

tion compounds of these plants may be

useful as adjuvants in tetracycline formu-

lations for treatment of bacterial resistant

infectious diseases encountered especially

among HIV/AIDS patients.

Conclusion

It has been demonstrated that plant ex-

tracts even in the absence of antimicrobial

activity can potentiate the activity of some

antibiotics. Hence concomitant use ofherbal products and antibiotics without

prior investigation may lead to toxic side

effects and should be discouraged.

Acknowledgement

We will like to thank the government of

Ghana for providing funds for this study.

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