|
|
|
|
Research Article
|
|
The Effect of Selected Sterilization Methods on Antibacterial Activity of Aqueous Extract of Herbal Plants |
|
S.R. Hashemi,
I. Zulkifli,
Z. Zunita
and
M.N. Somchit
|
|
|
ABSTRACT
|
The aim of this study was to compare selected sterilization
methods to maintain high susceptibility of antibacterial activities of
aqueous extracts of herbal plants. Autoclave-sterilized Impregnated disk
and Impregnated disk sterilized by Acrodisc syringe filter were embedded
on Mueller-Hinton Agar (MHA) plates seeded with the respective test microorganisms.
Among five extracts, Euphorbia hirta exhibited antibacterial activities.
Autoclaving caused less damage to the antibacterial activities of the
tested extract in comparison with syringe filtration.
|
|
|
|
|
|
|
INTRODUCTION
Antimicrobial activities have been exploited intensively in recent years,
mainly in respect to the extensive ban on antibiotics in the animal industries
and antibiotic overuse in human medicine. Earlier studies have shown that
many regional herbs have anti-microbial, anti-pyretic, anti-inflammatory
and immunoreactive properties (Habsah et al., 2000; Schwikkard
and Van Heerden, 2002; Somchit et al., 2005). There are several
reports on the antimicrobial activity of different herbal extracts (in
vitro) in different regions of the world (Polasa and Nirmala, 2003;
Onyeagba et al., 2004; Wiart et al., 2004; Kattak et
al., 2005; Soo Park et al., 2005; Sudhakar et al.,
2006; Yano et al., 2006). Several studies have also reported various
types of contamination of herbal medicines. Contaminants in herbal medicines
that have been reported include other herbs, microorganisms and toxins
produced by microorganisms, pesticides and toxic heavy metals (Talaly
and Talaly, 2001) which question the rightness of the use of the extracts
for different purposes. Food borne pathogens such as diarrheagenic serotypes
of Escherichia coli, Salmonella, Listeria monocytogenes
and Aeromonas hydrophila are widely distributed in nature (Indu
et al., 2006). They may easily contaminate the obtained herbal
extracts.
The main methods for testing the antimicrobial activities of extracts
are disk diffusion method, agar dilution method (Luangtongkum et al.,
2007) and broth dilution method (Kianbakht and Jahaniani, 2003). The most
frustrating problem confronting attempts to antibacterial activity of
herbal plants by disk diffusion method is contamination by other moulds
and fungus which most probably arises when carrying out the aqueous extraction
from fresh materials as aseptic techniques are followed when carrying
out the other stages of the experiments. The methods used to sterile the
extracts may be inappropriate because they seriously change the color
and morphology of the herbal extract. The present study has studied a
few sterilization methods to compare the antibacterial activity of contamination-free
aqueous herbal plants extracts and control (without sterilization).
MATERIALS AND METHODS
Plant materials: Five aqueous extracts obtained from the fruit
of Solanum torvum, whole plant of Euphorbia hirta and rhizomes
of Zingiber officinale, Curcuma longa and Zingiber zerumbet
were tested for antibacterial activities. Fresh Euphorbia hirta
was collected in April 2007 from the agriculture gardens in Universiti
Putra Malaysia (UPM). Zingiber officinale, Curcuma longa,
Zingiber zerumbet rhizomes and Solanum torvum fresh fruits
were obtained from a local market. All plants were authenticated by the
Institute of Bioscience (IBS), Universiti Putra Malaysia.
Preparation of extract: The plants were cleaned and washed with
sterile distilled water and oven-dried at 50 °C.
In order to obtain the plants` extracts, 100 g of each dried plant material
powder was placed in a flask and distilled water was added (1:10 w/v).
The flasks were incubated in a shaking-water bath at 50 °C for 48
h and the obtained extracts were filtered using filter papers (Whatman®
No. 1). The flow-through were stored in a deep freezer at -80 °C overnight
and then subjected to freeze drying (Jouan LP3, France) at -50 °C;
0.2 mbar for 48 h to obtain water-free extracts. Extracts were then weighted
and stored at 4 °C till further use.
Test microorganism: Two Gram-positive (Staphylococcus aureus
and Bacillus subtilis) and two Gram-negative (Escherichia
coli and Salmonella entertidis) were obtained from Bacteriology
Laboratory, Faculty of Veterinary Medicine, UPM, Malaysia.
Antibacterial sensitivity test using filter paper method: Isolated
colonies of the four bacteria were cultured into tubes containing 10 mL
of sterile nutrient broth (OXOID®, CM1, UK) and incubated at room
temperature till the cultures` turbidity reached 0.5 McFarland and were
aseptically swabbed on the surface of sterile Mueller-Hinton Agar (MHA)
plates. Filter paper discs of 6 mm diameter were prepared and sterilized.
Using an ethanol dipped and flamed forceps, these discs were aseptically
placed over Mueller-Hinton Agar (MHA) plates seeded with the respective
test microorganisms. Twenty five microlitres of the various concentration
of each spices extract (50, 100 and 200 mg mL-1) were aseptically
transferred to these discs to give a final concentration of 1.25, 2.5
and 5 mg disc-1, respectively. The plates were incubated in
an upright position at 37 °C for 24 h. The diameter of inhibition
zones were measured in mm and the results were recorded. Inhibition zones
with diameter less than 12 mm were considered as having no antibacterial
activity. Diameters between 12 and 16 mm were considered moderately active
and these with >16 mm were considered highly active (Indu et al.,
2006).
Antibiotic sensitivity testing: The tested microorganisms were
also assayed for their sensitivity against the antibiotics (OXOID®,
UK) tetracycline (30 mcg), chloramphenicol (30 mcg), gentamicin (10 mcg),
kanamycin (30 mcg), erythromycin (10 mcg) and cephalothin (30 mcg) using
the disk diffusion method.
Sterilization methods: After carrying out antibacterial activity
test of five different herbal plants` extracts, Euphorbia hirta
extract was selected to study the effect of the sterilization methods.
Two sterilization methods were applied; autoclaving of impregnated discs
and filtration of the extracts. For autoclaving, impregnated discs were
placed in small glass bottles and then autoclaved at 121 °C for 15
min. Filtration was carried out using Acrodisc syringe filter membrane
of 0.45 μM (Schleicher and Schuell Bioscience Inc., Keene, NH) and
the flow-through was impregnated on blank disks and let stand at room
temperature under fume hood to air-dry. The prepared extract-impregnated
discs were stored at -5 °C before tested (Somchit et al., 2003).
Previously isolated classes of constituents: Zingiber officinale
has two primary extracts: oleoresin and essential oil. The chemistry of
these oils is well documented and includes monoterpenes, sesquiterpenes
and phenols (Mukherjee et al., 1995; Onyeagba et al., 2004)
Steroidal alkaloids, tannins and saponins from Solanum torvum is
isolated (Kattak et al., 2005). Tannins, flavonoids, phenolic acids,
saponin and amino acids are isolated constituents of Euphorbia hirta
(Sudhakar et al., 2006). curcumin. Curcuminoids, a group of
phenolic compounds isolated from Curcuma longa (Somchit et al.,
2003). Sesquiterpene, bicyclic monoterpene and monocyclic sesquiterpene
obtained from rhizomes of Zingiber zerumbet (Polasa and Nimala,
2003).
Statistical analysis: To compare the three methods, a Completely
Randomized Design (CRD) with three replicates was used. Means comparison
was carried out using Duncan`s New Multiple-Range Test (p<0.05). All
statistical analyses were performed using SAS version 9.1 (SAS Institute,
Cary, NC, 2005).
RESULTS AND DISCUSSION
Many studies have been conducted to establish the antimicrobial effect
of the medicinal plants (Habsah et al., 2000; Sudhakar et al.,
2006). The results of different studies provide evidence that some medicinal
plants might indeed be potential sources of new antibacterial agents even
against some antibiotic-resistant strains (Indu et al., 2006).
Among the five herbal plants studied only the aqueous extract of Euphorbia
hirta showed considerable growth-inhibiting activity against Staphylococcus
aureus (Table 1). Significant increase in the inhibitory
zone was observed by increasing the concentration of the extract. The
range of the inhibition zone`s diameter was measured 19 to 27 mm and the
antibacterial activity was a linear function of the concentration (Table
2).
Phytochemicals have been reported to have medicinal uses (Ojo et al.,
2006). Specifically, saponin has been reported to have antimicrobial effects
(Mahato et al., 1992) and could serve as precursors of steroidal
substances with a wide range of physiological activities (Madusolomuo
et al., 1999). The preponderance of saponin in the extracts of
E. hirta that reported by previous researchers (Jonson et al.,
1999; Tona et al., 2004; Sudhakar et al., 2006) could then
justify the use of these plants in the treatment of some microbial infections
mentioned earlier. However, the observation that Zingiberaceae family
contain saponin do not have antibacterial activity but possesses remarkable
antibacterial activities suggested that the activities observed may not
be due to the presence of saponin only, but that other phytochemical components
may be antibacterial active as well. In addition, the biological or therapeutic
activities of medicinal plants are closely related to their chemicals
compounds. These chemicals are classified into major groups such as essential
oils, alkaloids, acids, steroids, tannins, saponins and etc. Each one
of these classes of chemicals may have a preferred effective method of
extraction which facilitates getting the chemicals out of the plant and
into the herbal remedy that is being prepared e.g., some active chemicals
found in plants are not soluble or dissolved in water but are more soluble
in alcohol. Interestingly, this is also the reason why some plants are
prepared in one manner to treat one specific condition, yet are prepared
in a different way to treat a completely different condition e.g. preparing
an infusion/tea of a plant might extract a delicate group of anti-inflammatory
plant steroids to treat arthritis (and leave behind other non-water soluble
chemicals), yet when the same plant is prepared in alcohol as a tincture,
the delicate steroids are degraded or burned-up in the alcohol but different
antibacterial alkaloids (which are only soluble in alcohol) are extracted
instead. It has been reported that plant extracts in organic solvent provided
more consistent antimicrobial activity compared to those extracted in
water (Parekh et al., 2005). It can be rationalized in terms of
the polarity of the compounds being extracted by each solvent and, in
addition to their intrinsic bioactivity, by their ability to dissolve
or diffuse in the different media.
Table 1: |
Antibacterial activity of various concentrations of
aqueous extracts of Zinziber officinale, Curcuma long,
Zinziber zerumbet, Euphorbia hirta and Solanum torvum |
 |
-: Refers to no antibacterial effect of corresponding
medicinal plant to the mentioned bacterial strain at mentioned dose.
± : Refers to antibacterial effect of corresponding medicinal
plant to the mentioned bacterial strain at mentioned dose |
Table 2: |
Mean inhibition zones of Euphorbia hirta aqueous
extracts in comparison with standard antibiotics |
 |
-: Refers to no antibacterial effect of corresponding
medicinal plant to the mentioned bacterial strain at mentioned dose.
Values are Mean ± SE (mm) of triplicate testes |
The effects of the sterilization methods on the antibacterial effect
of the extract are shown in Table 3. It is apparent
that sterilization methods have had negative effects on the properties
of phytochemical compounds and quality of nutrition. It is believed that
high temperature causes serious structural damage and consequently partial
or complete loss of important properties (Gliguem and Birlouez-Aragon,
2005). Nelson et al. (2007) explained that the antimicrobial substance
in the onion extracts, which are mainly phenolic compounds were destroyed
or inactivated by heat. In contrast, present findings indicate that autoclaving
had less negative effects on the antibacterial properties of herbal extract
in comparison with Acrodisc syringe filter. As it was observed in present
study, the use of autoclave-sterilized and syringe-filter sterilized both
reduced the antibacterial activities of the herbal extracts (p<0.05).
However, the reduction was far less for autoclaving. Interestingly the
reduction in antibacterial activities was significantly related to concentration
of the extract. At the concentration of 50, 100 and 200 mg mL-1
the inhibitory zone was reduced to 35, 33 and 30% for autoclaved extract
and 49, 56 and 59% for syringed-sterilized extract respectively in comparison
with the control group (without sterilization). To our knowledge, there
has been no report on the use of autoclaving for sterilization of herbal
extracts so far as it is believed that it may seriously damage their properties.
Indu et al. (2006) studied the antibacterial of some syringe-filter
sterilized herbal extracts and reported effective inhibitory zone against
E. coli. Based on present findings, we believe that syringe-filter
sterilization could have damaged the herbal extracts` ingredients and
if autoclaving had been applied as the means of sterilization, wider inhibitory
zone could be observed. Furthermore, Filtration is a mechanical or physical
operation. Mechanical filtration is typically achieved by passing water
or solvent through materials which act as a sieve, physically trapping
the particulate matter. Contaminants or bacteria are removed by nylon
syringe filter through a membrane having microscopic holes that allow
water or solvent molecules, but not larger compounds, to pass through.
It is also possible that some phytochemical compounds cannot pass through
the filter.
Table 3: |
Comparison of the sterilization methods on the inhibitory
zone of Euphorbia hirta against Staphylococcus aureus |
 |
Values are Mean ± SE (mm) of triplicate testes,
Values with different subscripts are significantly different (p<0.05) |
On the other hand, we experienced difficulty with syringe-filtration
of high concentrated extracts. Interestingly, the results showed a decreasing
trend of the reduction of the inhibitory zone when compared with the control
group by increasing the concentration of the extract, from 49% reduction
at the concentration of 50 mg mL-1 to 59% reduction at the
concentration of 200 mg mL-1.
In conclusion, the results of this study indicate that if it is necessary
to apply sterilization, it is recommended to apply autoclaving rather
than syringe filtration to maintain the antibacterial properties at the
highest possible level.
ACKNOWLEDGMENTS
The authors would like to express their appreciation to Mr. Chong Lin
Check and Miss. Krishnammah Kuppusamy for technical assistance. This research
was funded by a grant (05-01-04-SF0182) from the Ministry of Science,
Technology and Innovation, Malaysia.
|
REFERENCES |
1: Gliguem, H. and I. Birlouez-Aragon, 2005. Effects of sterilization, packaging and storage on vitamin C degradation, protein denaturation and glycation in fortified milks. J. Dairy Sci., 88: 891-899. Direct Link |
2: Habsah, M., M. Amran, M.M. Mackeen, N.H. Lajis and H. Kikuzaki et al., 2000. Screening of Zingiberaceae extracts for antimicrobial and antioxidant activities. J. Ethnopharmacol., 72: 403-410. CrossRef | PubMed | Direct Link |
3: Indu, M.N., A.A.M. Hatha, C. Abirosh, U. Harsha and G. Vivekanandan, 2006. Antimicrobial activity of some of the South-Indian spices against serotypes of Eschrichia coli, Salmonella, Listeria monocytogenes and Aeromonas hydrophila. Braz. J. Microbiol., 37: 153-158. CrossRef | Direct Link |
4: Johnson, P.B., E.M. Abdurahman, E.A. Tiam, I. Abdu-Aguye and I.M. Hussaini, 1999. Euphorbia hirta leaf extracts increase urine output and electrolytes in rats. J. Ethnopharm., 65: 63-69. CrossRef |
5: Kattak, S., Saeed-ur-Rehman, H.U. Shah, W. Ahmad and M. Ahmad, 2005. Biological effect of indigenous medicinal plants Curcuma longa and Alpina galangal. J. Fitoterpia, 76: 254-257. CrossRef |
6: Kianbakht, S. and F. Jahaniani, 2003. Evaluation of antibacterial activity of Tribulus terrestris L. growing in Iran. Iran. J. Pharm. Ther., 2: 22-24. Direct Link |
7: Luangtongkum, T., T.Y. Morishita, A.B. El-Tayeb, A.J. Ison and Q. Zhang, 2007. Comparison of antimicrobial susceptibility testing of campylobacter spp. by the agar dilution and the agar disk diffusion methods. J. Clin. Microbiol., 45: 590-594. CrossRef | Direct Link |
8: Mahato, S.B., A.K. Nandy and G. Roy, 1992. Triterpenoid. Phytochemistry, 31: 2199-2249. PubMed | Direct Link |
9: Madusolomuo, M.A., M.S. Nadro and A.U. Wurocheke, 1999. Antihepatotoxic properties of Cassia sieberiana in acetaminophen–treated rats. Nig. J. Biochem. Mol. Biol., 14: 21-25.
10: Mukherjee, P.K., R. Balsubramanian, K. Saha, M. Pal and B.P. Saha, 1995. Antibacterial efficiency of Nelumbo nucifera (Nymphaeaceae) rhizome extract. J. Indian Drugs, 32: 274-276. Direct Link |
11: Azu, N.C., R.A. Onyeagba, O. Nworie and J. Kalu, 2007. Antibacterial activity of Allium cepa (Onions) and Zingiber officinale (Ginger) On Staphylococcus aureus and Pseudomonas aeruginosa isolated from high vaginal swab. Internet J. Trop. Med., Vol. 3. Direct Link |
12: Ojo, O.O., M.S. Nadro and I.O. Tella, 2006. Protection of rats by extracts of some common Nigerian trees against acetaminophen-induced hepatotoxicity. Afr. J. Biotechnol., 5: 755-760. Direct Link |
13: Onyeagba, R.A., O.C. Ugbogu, C.U. Okeke and O. Iroakasi, 2004. Studies on the antimicrobial effects of garlic (Allium sativum Linn), ginger (Zingiber officinale Roscoe) and lime (Citrus aurantifolia Linn). Afr. J. Biotechnol., 3: 552-554. Direct Link |
14: Parekh, J., D. Jadeja and S. Chanda, 2005. Efficacy of aqueous and methanol extracts of some medicinal plants for potential antibacterial activity. Turk. J. Biol., 29: 203-210. Direct Link |
15: Polasa, K. and K. Nimala, 2003. Ginger: It’s role Xenobiotic metabolism. ICMR Bultin, 33: 58-63. Direct Link |
16: Schwikkard, S. and F.R. van Heerden, 2002. Antimalarial activity of plant metabolites. Nat. Prod. Rep., 19: 675-692. CrossRef | PubMed | Direct Link |
17: Somchit, M.N., I. Reezal, I.E. Nur and A.R. Mutalib, 2003. In vitro antibacterial activity of ethanol and water extract of Cassia alata. J. Ethnopharm., 84: 1-4. CrossRef |
18: Somchit, M.N., M.H.N. Shukriyah, A.A. Bustammam and A. Zuraini, 2005. Anti-pyretic and analgesic activity of Zingiber zerumbet. Int. J. Pharmacol., 277: 277-280. CrossRef | Direct Link |
19: Soo Park, B., J.G. Kim, M.R. Kim, S.E. Lee and G.R. Takeoka (et al)., 2005. Curcuma longa L. Constituents inhibit sortase A and staphylococcus aureus cell adhesion to fibronectin. J. Agric. Food Chem., 53: 9005-9009. Direct Link |
20: Talaly, P. and P. Talaly, 2001. The importance of using scientific principles in the development of medicinal aspects of plants. Acad. Med., 76: 238-247. PubMed | Direct Link |
21: Sudhakar, M., C.V. Rao, P.M. Rao, D.B. Raju and Y. Venkateswarlu, 2006. Antimicrobial activity of Caesalpinia pulcherrima, Euphorbia hirta and Asystasia gangeticum. Fitoterapia, 77: 378-380. CrossRef | Direct Link |
22: Tona, L., R.K. Cimanga, K. Mesia, C.T. Musamba and T. de Bruyne et al., 2004. In vitro antiplasmodial activity of extracts and fractions from seven medicinal plants used in the democratic republic of Congo. J. Ethnopharm., 93: 27-32. CrossRef | Direct Link |
23: Wiart, C., S. Mogana, S. Khalifah, M. Mahan and S. Ismail et al., 2004. Antimicrobial screening of plants used for traditional medicine in the state of Perak, Peninsular Malaysia. J. Fitotera, 75: 68-73. CrossRef | PubMed | Direct Link |
24: Yano, Y., M. Satomi and H. Oikawa, 2006. Antibacterial effects of spices and herbs on Vibrio parahaemolyticus. Internet J. Food Microbiol., 111: 6-11. PubMed | Direct Link |
|
|
|
 |