ANTIBACTERIAL PLANTS

1
Antibacterial Activity of Selected Edible Medicinal Plants 1

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Mohamad Zainuddin, Mohamad Farhan, Abdul Hamid, Azizah
1,2h*
and Khatib Alfi 3
1
Department of Foood Science, Faculty of Food Science and Technology, Universiti Putra 4
Malaysia, Serdang, Selangor, Malaysia 5
2
Agrobiotechnology Institute, Ministry of Science and Technology, Malaysia. 6
*Corresponding author. Tel:+603-89468374; Fax: +603-89423552 7
Email address: azizah@food.upm.edu.my; azizahhamid@mosti.gov.my 8
9
Abtract 10
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Seventeen edible traditional medicinal plants that are usually being used as salads and 12
traditional medicine in Malaysia were investigated for their antibacterial properties in vitro. 13
Antibacterial activity of methanol extracts of the each plants wereas evaluated by using disc 14
diffusion and agar dilution methods against pathogenic strains of Gram positive (Bacillus 15
cereus, Listeria monocytogenes and Staphylococcus aureus, Bacillus cereus and Listeria 16
monocytogenes) and Gram negative bacteria (Escherichia coli and Salmonella typhimuri and 17
Escherichia coli ). Results from disk diffusion assay showed that nine9 plants had appreciable 18
antibacterial activity including Andrographis paniculata, Borsenbergia rotunda, Cosmos 19
caudatus, Curcuma Xanthorhiza, Kaempheria galanga, Lawsonia inermis, Melicipe lunu, 20
Muraya koenigii and Cosmos caudatus, Piper betle, Melicipe lunu, Lawsonia inermis, 21
Borsenbergia rotunda, Andrographis paniculata, Muraya koenigii and Kaempheria galanga. 22
While ten plantsin agar dilution assay showed 10 plants active by including Pipper longum 23
exhibited antibacterial activity in agar dilution assay. Each of the active plants inhibited at 24
least one strain bacteria with minimum inhibition concentration (MIC) range between 1-32 25
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mg/mL. Interestinglly, C. xXanthorhiza showed the strongest antibacterial activity followed 26
by C.caudatus, P.betle, M.lunu, L. inermis, B. rotunda, A. paniculata, M. koenigii, K. galanga 27
and finally P.longum. While L.inermis and P.betle showed broadest activity against all 28
bacteria tested. The rest of the plantsamples such as Centella asiatica, Gynura procumben, 29
Justicia gendarussa, Morinda cintrifolia, Psophocapus tetragonolobus, Sesbania grandifolia 30
and Talinum triangulare did not revealshow any antibacterial activity. It can be concluded 31
that a number ofcertain edible traditional medicinal plant extracts possesed antibacterial 32
activity and theseit may serve as new sources of antibacterial agents. 33
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Keywords: Malaysian edible medicinal plants; Foodborne pathogen; Antibacterial activity; 36
Disc diffusion method; Agar dilution method 37
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1.Introduction 41
42
Food borne diseases areis still a concern for both consumers and food industry despite 43
the use of various preservation methods. Food safety researchers and regulatory agencies are 44
continuously concerned with the high and growing number of illness and outbreaks caused by 45
some pathogenic and spoilage microorganisms in foods (Shan et al.,2007). It ishad been 46
estimated that 76 million people in United States had suffered from foodborne illness each 47
year (Mead et al., 1999). It WHO (2007) also reported that 30% of people in industrialized 48
countries suffer from food borne diseases each year and in 2005 alone, at least 1.8 million 49
people died form diarrhea related diseases worldwide (WHO, 2007). Many countries losee 50

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billions of dollar due to medical cost, productivity losses and value permature deaths related 51
to food borne diseases (Snowdon et al., 2002). 52
53
Foodborne diseases can be divided into two groups: which are food infection and food 54
intoxication. Foodborne pathogen is the main role cause of disease which many occur as a 55
result of cross contamination, improper handling of food and temperature abuse., it happens 56
via cross contamination, improper handling and temperature abuse. Food intoxication 57
occursn happened when the patient consume food that contains harzadous toxic chemicals 58
produced by bacteria such as Salmonella sp. and Compylobacter (Melzer and Shah, 2009). 59
This can It also can happen even if the toxin producing microorganisms that produced the 60
toxin is are no longer present or unable to cause infections. On the other hande, food infection 61
iwas caused by the presencet of infectious pathogens in the consumption foods. Consumption 62
of contaminated foods will result in multiplication of microorganisms in the intestine, with 63
release toxins which invade and damage the epithelium cells. consumed that will multiply in 64
the intestine and release their toxins which invade and damage the epithelium cells. 65
Salmonella and Compylobacter sp. had been reported to beas one of the common bacteria 66
involved in food infections. Others While thereported microorgaismsthat implicated less 67
frequently in food borne infectionus arewere Listeria monocytogenes, Escherichia coli, 68
Bacillus cereus and Streptococcus sp. (Taylor., 2002). 69
70
71
In an effort toorder to control microbial growth and reduce the incidence of food 72
poisoning and spoilage, many synthetic antimicrobials have been usedproduced (Shan et al., 73
2008). AlEven though synthetic perservatives are effective, there is still concerns regarding 74
consumers are still worried about their potential toxicity (Tang et al., 2008). They have also It 75
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also had been reported to be assosiated with causes many side effects such as sleepinessy, 76
halucination, fever, paranoriaid and amenisia (Willey et al., 2008). In addition, synthetic 77
drugs also haves been implicatedrelated with the evolution of drug resistant microorganism. 78
With the increased awareness of people about these health risks, the consumption of raw 79
vegetables and fruits has increased significantly as individuals have become more heatlh- 80
conscious and aware of the importance of plant-based diets in combating the onset of such 81
diseases (Shuib et al., 2010). 82
83
Due to all these increasing drwabacks, many researchers have attempted to find new and safer 84
sources of antimicrobialagents from natural sourcesthe problems, mmany researcherstend to 85
go back to natural product as an alternative way in finding a new source of antimicrobial 86
agent. Each Pplants possesed their own characteristic of self defence mechanism against 87
viruses, bacteria, fungals, bugs and herbivor animals. From the characteristic of the selected 88
plants, the ancient people had tested those plants until they found which plant is safe and 89
effectively cure specific illnesses. Those knowledges that had been claimed to be safe had 90
been passed down from generation to generation. Today, . Nnumerous traditional medicinal 91
plants have been explored for in more detail to find their bioactivity potential. such 92
Antioxidant activity have been found in Cosmos caudatus, Murraya koenigii (huda et al., 93
2009) as antioxidant, anti diabetic activity in Gynura procumbens (zurina et al.,2010), 94
antimicrobial activity in Morinda elliptica, Borreria latifolia, Sida rhombifolia (ali et al., 95
1995), anticancer activity in Jatropha curcas (Ehsan et al., 2011), anti inflammatory activity 96
in Piper sarmentosum, Psophocarpus tetragonolobus, Sauropus androgynus (Lee et al., 97
2011) and othersmany more. 98
99
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Those plants also were cheap to buy compared to the synthetic drugs and some 100
consumers self planted the plants which seem easy to grow. This fact had been supported 101
from Eloff (1998) where in his report, in 1998, about 80% of the people in developing 102
countries almost exclusively use traditional medicinal plants. 103
104
NumerousSome traditional medicinal plants arecan be consumed as ingredient in 105
cooking or eaten it rawly as salad. There is common belief Most people highly believe that 106
those plants are safe for consumption as they have been consumed from generation to 107
generation without any toxicity reportto consume since there is no complain about the 108
poisoness or toxicity report of the plant from generation to generation. Most of the edible 109
traditional plants have many medicinal effects in our digestion system such as for treating 110
stomach diseases, diarrhea, dysentry and diuretic (Sankaranarayanan et al., 2010; Yasni et al., 111
1994; Chowdhury et al., 2008; Gowril and Vasantha., 2010). Plants have also been used to 112
treat outer body conditions It also had been reported have outer body treatment such as skin 113
diseases, itches, wounds, bruises, irritant, boils and ulcers. (Chaudhary et al., 2010; Shaari et 114
al., 2006; Ridtitid et al., 2008;Manoj et al., 2004; Perry, 1980). All those therapeutic values 115
are related towith the presence of phytochemical compounds phytochemical compounds 116
(flavonoids, isofalavones, lignans, cinnamic acids derivatives, steroids, carotenoids, 117
terpenoids, etc), vitamin, polysaccharides, proteins and minerals presentcontain in the plants. 118
These phytochemicals may also have antibacterial activity. Therfore objectives of this study 119
weare to screen antibacterial activity of seventeen edible medicinal plants by using different 120
method approaches and evaluate the lowest concentration required for the screen and evaluate 121
the potential of antibacterial activity against different strains of food borne bacteria. of 122
selected edible traditional Pplants were seleceted based on their traditional medicinal practice 123
used against different strains of food borne bacteria. 124

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125
126
The sample plants for this research is Andrographis paniculata, Boesenbergia 127
rotunda, centella asiatica, cosmos caudatus, curcuma xanthorhiza, gynura procumbens, 128
justicia gendarussa, kaempferia galangal, Lowsonia inermis, Melicipe lunu, Molinda 129
cintrifolia, Muraya koenigii, Pipper betle, Piper longum, Premna Cordifolia, Psophocapus 130
tetragonolobus, Sesbania grandifolia, Talinum triangulare and Vitex nogundo. These samples 131
were selected based on their used as salad, food ingredient and contribution in medicinal 132
properties. Table 1.1 show the ethnomedicinal uses of selected plants in this study. 133
Since in 1550 BC, Egyptians had been used spices and herbs such as Cinnamon, cumin and 134
thyme as a food perservation and mummification (Webb & tanner, 1994 ; Hirasa & takemasa, 135
1998). Until now numerous studies have been published on the antimicrobial activities of 136
plants extracts against different types of bacteria (Shan et al.,2007; Cos et al., 2002; Kumar et 137
la., 2006; Vivek et al., 2008; Buwa and Van., 2006; Agnihotri et al.,2008; Khan and 138
Omoloso.,2008; Tadhani and Subhash.,2006;Tsai et al.,2008 and Wang et al.,2008). However, 139
only a few studies focused on the potential of consumption vegetables plants as sources of 140
antibacterial compounds that could inhibit foodborne microorganisms. The objective of this 141
study are to determine the antibacterial activity of extracts from salad with traditional 142
medicinal properties against food borne microorgansims 143
144
Table 1.1 Ethnomedicine uses of selected consumption plants salad in Malay communtiy. 145
Species name Common name Part tested Ethnomedicinal uses
Pipper betle

Sireh

Leaves
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Lowsonia
inermis

Inai

leaves
Melicipe lunu

T.Burung

leaves
Borsenbergia
rotunda
T.kunci

tuber
curcuma
xanthorhiza

T.lawak

tuber
Cosmos
caudatus
U.raja
leaves
Andrographis
paniculata
H.bumi

leaves
Muraya
koenigii

Kari

leaves
kaempferia
galangal

Cekur

leaves
Piper longum

Kaduk

leaves
Talinum
triangulare
K.Belanda

leaves
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Sesbania
grandifolia

Turi

leaves (.,
Psophocapus
tetragonolobus
K.botol

pods
Molinda
cintrifolia

Mengkudu

leaves
gynura
procumben

S.nyawa

leaves
justicia
gendarussa

G.rusa

leaves
Centella
asiatica

Pegaga

leaves
146
147
148
2. Materials and methods 149
2.0 materials and chemicals/reagents 150
Traditional Malaysian medicinal plantsplants like Centella asiatica, Cosmos caudatus, 151
Curcuma xanthorhiza, Justica gendarussa, Lawsonia inermis, Kaemperia galangal, Melicipe 152
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lunu, Morinda cintrifolia, Muraya koenigii, Piper betle, Pipper longum, Psophocapus 153
tetragonolobus, Sesbania grandifolia, Talinum triangulare, Gynura procumbens, 154
Borsenbergia rotunda and Andrographis paniculata were selected based on their traditional 155
medicinal uses asare listed in Table 1. The plant parts were collected from the Traditional 156
Medicine Plant Plot, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. Dimethyl 157
sulfoxide from Fisher Scientific (Leicestershire, UK),. Chloramphenicol (30ug) and 158
Tetracycline (10ug), Mueller Hilton Agar (MHA) medium from Oxoid ( (Hampshire, 159
England) and Nunco
TM
Surface (Rosklide, Denmark)., respectively. 160
161
2.3 Microbial strains and culture 162
Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 14579, Listeria 163
monocytogenes ATCC 19115, Campylobacter jejuni ATCC 29428, Salmonella typhimurium 164
ATCC 13311 and Escherichia coli ATCC 25922 were purchased from ATCC (American 165
Type and Collection Centre). Each bacterial strain was cultivated in Nutrient Agar and 166
incubated for 18-20 hours at 37
C in an incubator (Memmert, Germany). Then three or five 167

colonies bateria of the same morphological type form on agar plate were then taken up and 168
suspended in 10mLl sterile saline followed by then vortexingxed thoroughly. The bacterial 169
suspension was prepared according to was followed 0.5 Mcfarland standards. The inocoloums 170
were used within less than 15 minutes ofafter preparation. 171
172
2.2 Preparation of samples 173
plant partscollected from the Traditional Medicine Plant Plot, Universiti Putra 174
Malaysia, Serdang, Selangor, Malaysia. Plant materials obtained were washed underwith 175
running tap water, before being chopped into pieces, followed by drying then dried in 176
conventional oven (Memmert, Germany) at 45
o
C for two days. Dried plants were then and 177

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then grounded into powder form and kept at. The powders were then kept at -20
o
C until 178
further analysis.. 179
180
2.4 Preparation of crude extract 181
100g of sample The sample was mixed with absolute analytical methanol (Merck, 182
Germany) at 1:10 ratio and leftaved in shaking water bath for 24 hours at 45
C. Samples were 183

then filtered using whatman filter paper No.1 and the solvent was removed by using Eyela 184
rotary evaporator (Tokyo Rikikai Co. Ltd, Japan) set at 40
C. 185
186
2.5 Antibacterial screening assay 187
2.5.1 Disk diffusion method 188
The cultures were prepared according to Institute Cclinical Laboratory Standard 189
(2001) standardization and the antibacterial assay was prepared according to Lee et al., (2007) 190
with some modifications. 191
Sterile cotton was dipped into the inoculum and then rotated firmly to remove the excess 192
liquid, followed by streaking into thethen streaked into entire surface of Muller Hilton Agar . 193
This was repeated 3 times at 60 degree rotation in ensuring for three times. Each swab was 194
applied in 60 degree rotation to make sure all inoculums were well distributed and finally 195
swab all around the edges of the agar surface. Twenty microlitter of the extracts 196
(40mg/mL)was impregnated into 6mm diameter paper disc.The extracts were diluted in 197
Dimethyl sulfoxide (DMSO) and adjusted to 40mg/mL concentration. Twenty microlitter of 198
the solution was dispensed into 6mm diameter paper disc. The paper discs were left 30 199
minutes to dry, then placed the disc on the surface of Muller Hilton Agar with inoculum on it. 200
The plates were then incubated in an inverted position at 37
C in an incubator for 18-20 201

hours. Tetracycline was used as a positive control, and the . While for negative controlblank 202

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consisted of was just only Nutrient agar. The antimicrobial activity was assessed based on the 203
measurement of the diameter of the clear zone around the paper discs. In this study, tThree 204
replicates carried outwere prepared for each type of bacteria tested.in this study. 205
2.5.2 Agar Dilution method. 206
The AAgar Dilution method was conducted according to the method proposed by 207
Jennifer, (2001) with some modifications. Crude extract (64mg)Sixteen four mg crude extract 208
was diluted with 10% DMSO and used as stock solutiiton for further experiment. Form Tthe 209
stock solution, (6.25mL) was taken out and mixed with 3.75mL sterile Muller Hilton agar 210
(MHA) in sterile round plate (90 x 15mm) which given a final concentration of 40mg/mL. 211
The temperature of MHA was then brought to 50 C before pouringed to the plate. Then 212
swirled until thoroughly mixed. The plates were then left for 10 minutes and allowed to 213
solidify. Then 1 uL of suspensions from the prepared inoculums were placed on the agar 214
surface. Chloramphenicol was used as a positive control. While for Tthe blank consisted of 215
was MHA only only. The plates were then incubated in an inverted position at 37
C in an 216
incubator (Memmert, Germany) for 18-20 hours. The activity was then documentedwill be 217
noticed based on visualization, whether either the bacteria is ablecapable to growt h or not. 218
Three replicates were carried atprepared for each type of bacteria tested in theis study. 219
220
221
2.6 Determination of Minimum Inhibition Concentration by using Agar Dilution 222
method. 223
The method was theere same aswith the previous agar dilution method with the 224
exception of. Only the concentaration of the sample usedadjusted. The suitable amount of 225
extract was added on to the plate (90 x 15mm) followed by then mixinged with a specific 226
setence volume of sterile Muller Hilton Agar (MHA) to produce final concentration of 0.5, 227

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1.0, 2.0, 4.0, 8.0, 16.0 and 32.0 mg/mL. Chlorampehenicol (30ug) was used as a positive 228
control. Two replicates were prepared for each type of bacteria in this study. 229
230
3. Result 231
In the present study, 17 methanol extracts of selected tropical plant collected from 232
Taman Pertanian Putra (TPU), Universiti Putra Malaysia (UPM), Serdang, Selangor, 233
Malaysia were tested against 3 gGram-negative (Bacillus cereus, Listeria monocytogenes, 234
Staphylococcus aureus) and 2 gram-positive (Salmonella thyphii, Escherichia coli) 235
foodborne bacteria by using on the basis of disc-diffusion and agar dilution assay. Table 3.1 236
showed the screening results of antibacterial activity of plants extracts by using Disc-diffusion 237
method and Agar dilution methods. While mMinimium inhibition concentration (MIC) 238
obtained from were examined by using Agar Dilution assay isas showed in table 3.2. The 239
Rresults revealedshow that only 10 out of 17 plant extracts exhibitedshowed potent 240
antibacterial activity which inhibited at least one selected bacteria strain. 241
quantitaively assesed by the presence or absence of inhibition zones and zone diameters 242
(Table 1), MIC values (Table 2). 243
. 244
Results screening and mic 245
The Sscreening results from disk diffusion method showed that 9 out of 17 246
plants posessed potent antibacterial activity where atleast inhibiting one bacterium tested in 247
this study. Pipper betle and lawsonia inermis extract showed broadest anti-bacterial activity 248
against all tested bacteria as compared to the other plant extracts. where They also showed the 249
highest antibacterial activity in this screening result where the extracts wereit is capable to 250
inhibiting more than 10mm diameter inhibition zone atleast for two selected bacteria. For 251
Melicope lunu, Curcuma xanthorhiza, Cosmos caudatus and Andrographis paniculata assed 252
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in the present study showed moderate antibacterial activity where they it inhibiteds all the 253
gram positive bacteria strain (S. aureus, B. cereus and L.monocytogenes) withhich diameter 254
inhibition zone in the range from 8.0 to 10.0 mm. Low antibacterial activity was possesed by 255
Kaempferia galanga, Boesenbergia rotunda and muraya koenigii showed low antibacterial 256
activity with inhibition of atleast one bacteria and diameter inhibition zone of 6.0 8.0 257
mmwhich capable to inhibit atleast one bacterium strain where diameter inhibition zone show 258
in the range of 6.0 to 8.0 mm. The Oother plant extracts such as Centella asiatica, Justica 259
gendarussa, Morinda cintrifolia, Pipper longum, Psophocapus tetragonolobus, Sesbania 260
grandifolia, Talinum triangulare and Gynura procumbens did not show any antibacterial 261
activity in the present study. 262
263
Mean while screening byResults obtained from the Agar Agar Dilution method 264
revealed that 10 out of 17 plant extracts (40mg/mL) possesed antibacterial activity against at 265
least 1 bacterial strain at the same concentrations as in disk diffusion assay (40mg/mL). As 266
expected, Piper betle, Lawsonia inermis and Melicope lunu showed the broadest spectrum of 267
action against bacteria, inhibiting all the strains tested. Borsenbergia rotunda also showed 268
quite broad activity where it could inhibit 4 out of 5 strains tested. Curcuma xanthorhiza, 269
Cosmos caudatus, Androphanis paniculata, Kaempferia galangal and Muraya koenigii also 270
possesed antibacterial activity against all gram positive strains. The lowest activity was shown 271
by Piper longum where it only inhibited Listeria monocytogenes. While the other plant 272
extracts such as Centella asiatica, Justica gendarussa, Morinda cintrifolia, Psophocapus 273
tetragonolobus, Sesbania grandifolia, Talinum triangulare and Gynura procumbens samples 274
did not show any antibacterial activity. 275
276
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Plant extracts showing potentialBased on Screening activity, only those who have the 277
positive antibacterial activity were then further tested is using the have been proceeded to 278
further study which is evaluating the Minimum Inhibition Concentration (MIC), assay which 279
assesses the of selected sampes. MIC is a minimum concentration requiredneeded to inhibit 280
bacterial growth. The results showed that Piper betle, Cosmos caudatus, Melicipe lunu and 281
Curcuma xanthorhiza showed high antibacterial activity against gram positive bacteria, where 282
it inhibited the bacteria at low concentration (1.0 2.0 mg/mL). Melicipe lunu, Borsenbergia 283
rotunda and Andrographis paniculata showed modest activity where inhibition occurred at a 284
concentration of 4.0 to 8.0 mg/mL respectively. Muraya koenigii, Kaempferia galanga and 285
Piper longum showed low activity in this study where it required high concentrations of 286
extract, in the range of 16.0 to 32.0 mg/mL. The highest activity was shown by Curcuma 287
xantthorhiza where it inhibited all the bacteria tested at a low concnetration of 1 mg/mL. On 288
the other hand, gram-negative bacteria were only affected by Pipper betle, Lawsonia inermis, 289
Melicipe lunu and Borsenbergia rotunda. Piper betle exhibited high antibacterial activity 290
where it inhibited the bacteria tested at 2mg/mL. Followed by Lawsonia inermis which 291
exhibited modest activity ( 4.0 8.0 mg/mL). Lastly, Melicipe lunu and Borsenbergia 292
rotunda showed low antibacteria activity where the MIC value for the bacteria tested were the 293
range of 16 to 32 mg/mL. Piper betle, Lawsonia inermis, Melicope lunu and Borsenbergia 294
rotunda had the broad spectrum of activity where it could inhibit almost all the bacteria tested 295
which MIC value in the range of 32mg/mL to 1mg/mL. While, Curcuma xanthorhiza, 296
Cosmos caudatus, Androphanis paniculata and Kaempferia galangal showed modest range 297
which it only inhibited 3 out of 5 bacteria tested. The lowest antibacterial effect was Piper 298
longum, it only inhibited 1 bacterium which is Listeria monocytogenes . 299
300
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From the bacteria testedThe study revealed that, Esherichia coli showed thewere most 301
resistant and exhibit only slight susceptibility to Piper betle, Lawsonia inermis and Melicope 302
lunu with MIC value ranginge from 2 to 32 mg/mL. The most susceptible bacteria was 303
Listeria monocytogenes, which was inhibited by 10 plant extracts with MIC value range from 304
1 to 32 mg/mL. Negative control was observed that no inhibition of the strain to growth. It 305
can be said that the studied plant extracts were less susceptible to the gram negative bacteria 306
than gram positive bacteria. 307
308
4. Discussion 309
Ten out of 17 plants used From 17 plants that been used as salad and herbs in 310
traditional medicine in Malaysia in the present study were found to , ten of them exhibited 311
potent antibacterial activities against six foodborne bacteria. This study also revealed the 312
antibacterial activity were to same extent assay dependent. Different observation were recrded 313
for disk diffusion and agar dilution assay. This is supported by previous literature (rios et al., 314
1980), who the different screening results between disk diffusion and agar dilution assay. The 315
different had been expected as descibed by Rios et al., (1988), where the author reportedstated 316
that agar diffusion method was only suitable for testingstudying polar compounds since the 317
polar compounds are capable to disolve and diffuse into the agar. On the other hand While 318
non-polar compounds were unable to diffuse into the agar and might end up with false result. 319
However, thisthis method is widelyhad been well used by numerous reseachers as a 320
preliminary study since it iwas affordablecheap, easy and fastless time consuming. On the 321
other hand, agar dilution method is applicable to both polar and non-polar compounds since 322
the microorganisms are can directly in contact with the compound that had been mixed with 323
the agar. However, the Even do this method iswas not very populor due to its being time 324
consuming and utilizing among researchers since it high amount of quite difficult and need a 325
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lots of crude extracts. Agar dilution method has beenwas claimed to be a suitable method in 326
evaluating antimicrobial potential of for complex samplesextract such as plant extracts (Rios 327
et al., 1988). This could explain the different results on antibacterial activity screening of 328
M.lunu, B.rotunda, K.galanga and P.longum extract in this study.. Therefore from the 329
screening results, the samples that have shown antibacterial activity in screening had been 330
carried out to determine their MIC by using agar dilution method. Rios et al., (1988) also 331
reported in his review that agar dilution results were relevant for MIC value. 332
333
Antibacterial activity demostrated by plant extracts is usually attributed to an array of 334
phytochemicals present in the Based on the literature review, bioactive compounds of 335
Melicope .lunu, Kaempheria .galanga and P.iper longum had been reported to contain 336
nemurous of bioactivenon-polar compounds including. For instance, M.lunu possesed 337
limonene and, terpene and -ocimene (Goh et al., 1995), While K.galanga contained 338
consisted ofcavene, cineol, ethyl cinnamate and coumarin (Othman et al., 2006; Ismail 2000). 339
P.longum is richcontained peperine, assarinin, piperidine and sarmintine (Majumdar et al., 340
2002; Sawangjaroen., 2005; Selvendiran et al., 2005; Nalin and Rahim., 2007). All these 341
bioactive compounds have been reported to exhibit potent antibacterial activity (Cowan., 342
1999; Sibel.,2003; Iqbal et al.,2006) In addition,While B.rotunda hasve been reported to 343
contain cChalcones a non which are non polar flavanoid derivatives, as a one of it major 344
compound (Nor AY et al., 2010). P.betle and L.inermis may have high amount of polar and 345
non-polar compounds, which could be the reason that explained the most active antibacterial 346
results in disk diffusion assay. P.betle had been reported to havesimilarily consisted of high 347
phenolic compounds such as terpene , pyrocatechin, cavicol, cavibetol, carvacrol, eugenol and 348
allilpyrocatechol (Farnsworth and Bunyapraphatsara, 1992), alkaloids, saponins and tannins 349
(Anonymous., 1992) thatwhich are known to have very potent antibacterial agent. Similar 350

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bioactive compounds werealso had been found in L. inermis in addition towith some other 351
compounds such as flavanoids, terpenoids, quinones, coumarins, xanthones and lawsone 352
(Mikhael et al., 2004; edrini et al., 2002; Chaudary et al., 2010). In the present study, 353
C.xanthorhiza and C.cosdautus were expected possessed a lot of essential oil compound 354
where Vimala et al. (2003) reportedfound that C.caudatus containing stigmasterol, 355
ssquiterpene and hydroxy eugenols and. Similarly, C.Xanthorhiza has been reported to 356
contain eugenol (Ruslay et al., 2007; Noraida 2005), terpinene and xanthorhizol (Chatterjee et 357
al., 1999) where theose essential oils have been reported to exhibitcointain potent antibacterial 358
activity. Andrographis .paniculata and M.koenigii showed only moderate antibacterial 359
activity, and is probaly due to the lower amount of maybe it is because these plants have less 360
amount of antibacterial bioactive compounds with antibacterial activity. Several studies had 361
found that A.paniculata contained compound such as diterpenoids, eugenol, caffeic acid, 362
tritriacetone and flavonoids (Joganath et al., 2000; Sukrasno et al., 2011).. While, M.koenigii 363
had been found to contain caumarins, terpenoids, murrayazoline and sesquiterpenes (Noraida 364
2005; Yadaz et al., 2002 and Tachibana et al., 2001). 365
366
Discn mode of action 367
The antibacterialmicrobial activities of phytochemicals may involve multiple modes of 368
action as been described by Cowan (1999). For example, Pphenolics are a broad class of 369
compounds that have a variety of antibacterial mechanisms. Each of the subclasses have their 370
own specific mehanisms of action. Simple phenols such as catechol and epicatechin impart 371
their antibacterial activitywork by substrate deprivation and membrane disruption of 372
bacteria`s cell wall. Phenolic acids and quinones act by binding to and form adhesins 373
complexing with cell wall and deactivated the enzymes. Similarly, flavonoids binds to 374
adhesins and form a complex with cell wall. Tannins react by binding to protein and adhesin, 375

18

distrupting membranes, forming complex with the cell wall, inhibiting enzymes and caused 376
subtrate deprivation. Other classes of phytochemical compounds such as terpenoids 377
(capsaicin) and essential oils act by membrane distruption and alkaloid act by intercalating 378
into the cell wall or DNA. However Shan et al., (2007) reported that there are many other 379
compounds and reactions that may had contributed before the final mechanisms of 380
antibacterial activity take it place. 381
Since there are many possibilities of antibacterial mechanisms, all of these mechanism are not 382
separate targets. Some of the mechanism of inhibition are effected as a consequense of 383
another mechanism being targeted (Shan et al., 2007). However the mechanism of 384
antimicrobial agents are also depend on the type of microorganism and mainly related to their 385
cell wall structure and outer membrane arrangement 386
Among the bacteria tested, Escherichia coli and Salmonella thyphii werewas found to 387
be the most resistant strain, that was only inhibited to some extent by . Only P.betle, L.inermis 388
and M.lunu extracts were found able to inhibit this bacterium. This observation was agreed 389
with that of previous studies that gGram-negative bacterial generally were more resistant to 390
traditional herbs extract as compared to that gram positive bacteria (Shan et al., 2007). 391
Numerous In fact, numerous studies have alsobeen reported that gGram-negative bacteria 392
were becoming more resistant towards many available antibiotics current available in the 393
market (Alonso et al.,2000; Sader et al., 2002). This is might be explained due to the 394
differences in morphological constitutions between the microorganisms. Gram-negative 395
bacteria possess an outer membrane and unique periplasmic space which is lacking inere 396
grampositive bacteria was lacking does not ( Nikaido, 1996; Duffy and Power, 2001). The 397
outer membrane of gram-negative bacteria is covered by hydrophilic surface and rich in 398
lipopolysacride molecules that function as a barrier to the penetration of any harmful 399
subtances while the periplasmic space contains several degradative enzymes which are 400

19

capable ofin breaking down the introduced molecules from outside (Russell, 1991; Nikaido, 401
1994;Gao et al., 1999). GThe Gram- positive bacteria on the other hand, does not has an outer 402
membrane, but onlyand the cell wall structure consisting of only having peptidoglycan layer 403
thatwhich is not an effective permeability layer barrier. Thus, antibacterial chemical subtances 404
can easily penetrate into the bacteria cell wall and cyctoplasmic membrane, resultinged in a 405
leakage of the cyctoplasm and it coagulatione (Kalemba and Kunicka, 2003). AlEventhough, 406
the cell walls of gram- negatives are more complex than gram-positive bacteria (Nostro et 407
la.,2000; Hodges, 2002), results from but in the present study, revealed that some of the 408
extracts can have still exert ed some degree of inhibition against selected the gram negative 409
bacteria . 410
It can be said that the studied plant extracts were less susceptible to the gram negative 411
bacteria than gram positive bacteria. 412
413
5. Conclusion 414
In Extracts of Piper betle, Lawsonia Inermis, Melicope lunu, Borsenbergia rotunda, 415
Curcuma xanthorhiza, Cosmos caudatus, Andrographis paniculata, Muraya koenigii , 416
kaempferia galangal and Pipper longum demostrated activity against at least one strain of 417
bacteria was inhibited. The nemurous and different phytochemical compounds in these plants 418
may be responsible for their varying nemurous applicable in due to antibacterial activities. 419
This study has provided on sight on the potential use of certain Malaysian medicinal plants as 420
antibacterial agents. However further studies are required to identify bioactive compound of 421
interest. 422
nalternative and in growth 423
424
425

20

426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450

21

451
452
453
454
4. Discussion. 455
5.Conclusion 456
Table 457
Table 1: Ethnomedicine uses of selected edible traditional medicinal plants in Malaysia 458
communtiy. 459
Species name Common name Part tested Ethnomedicinal uses
Pipper betle Sireh Leaves
Digestive, stimulative, carminative and
aphrodisiac (Sankaranarayanan et al., 2010).
Lowsonia
inermis
Inai Leaves
Alleviating jaundice, skin diseases, venereal
diseases, smallpox and
spermatorrhoea (Chaudhary et al., 2010)
Melicipe lunu Tenggek Burung Leaves
Treatment of itches and wounds (Shaari et al.,
2006),
Borsenbergia
rotunda
Temu Kunci Rhizom
Ailment, illness and confinement. Rhizomes are
also taken as carminatives for relieving
flatulence.(Chan et al., 2008 )
Curcuma
xanthorhiza
Temu Lawak Rhizom
Treatment of stomach diseases, liver disorders,
constipation, bloody diarrhea, dysentery, fever
in children, hemorrhoids, and skin eruptions
(Yasni et al. 1994)
Cosmos
caudatus
Ulam Raja Leaves
Blood cleansing, induction of uterine
contractions and prevention or cure of ailments
such as diabetes, high blood
pressure, cardiovascular disease, arthritis, fever
and coughs (Abas et al., 2006)
Andrographis
paniculata
Hempedu Bumi Leaves
Treatment of upper GI tract and upper
respiratory infections, fever, herpes and other
chronic diseases. (Roy et al., 2010)
Muraya
koenigii
Kari Leaves
Relieve nausea, indigestion, vomiting; treatment
of diarrhea and dysentery (Chowdhury et al.,
2008).
Kaempferia
galangal

Cekur Leaves
Treatment of Tinea versicolor, and eye diseases
and seizures, rheumatism, asthma, headaches,
cough, toothaches, bruises and wounds (Ridtitid
et al., 2008)
Piper longum Kaduk Leaves
Treatment of respirotory tract, cough,
bronchitis, irritant, inflammation. (Manoj et al.,
2004)
Talinum
triangulare
Kerekot Belanda Leaves
Treatment of diuretic, gastro-intestinal
disorder.(Mensah Et al., 2008)
Formatted: Font: 11 pt
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spacing: single, Don't keep with next, Don't
keep lines together

22

Sesbania
grandifolia
Turi Leaves
Aperient, diuretic, and tonic and
disinfect the mouth and throat (gowri1 and
Vasantha., 2010)
Psophocapus
tetragonolobus
Kacang Botol Pods
Treatment of skin sores such as boils and ulcers
(Perry, 1980).
Molinda
citrifolia
Mengkudu Leaves Relief joint pain (Rout et al., 2009)
Gynura
procumben
Sambung Nyawa Leaves
Treatment of malaria, general febrifuge, and
analgesic (Scott., 2006)
Justicia
gendarussa
Ganda Rusa Leaves
Treatment of fever, hemiplegia, rheumatism,
arthritis, muscle pain, lumbago, headache and
earache (Ahmad and Holdworth 2003;
Anonymous 1959)
Centella
asiatica

Pegaga Leaves
Against conjunctivitis and other eye injury,
wound healing but especially for the treatment
of skin diseases such as eczema, leprosy and
psoriasis. Treatment of burns, itching and insect
bites (Gupta et al., 1999; Zainol et al., 2008)
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476

23

477
478
479
480
481
3.1 Disk diffusion method 482
Table 3.1: Antibacterial activity of plant extracts screening of 40mg/mL concetration by using 483
disk diffusion and agar dilution method. 484
SamplesPlant
extract
Disk diffusion method Agar dilution method
Diameter of zone inhibition(mm) Inhibition of bacteria growth
Gram-positve bacteria
Gram-negative
bacteria
Gram-positive
bacteria
Gram-
negative
bacteria
BC SA LM EC ST BC SA LM EC ST
P. betle
7.7
0.3
11.8
0.6
7.3
0.3
15.4
1.7
10.4
0.1
+ + + + +
L. inermis
14.7
0.3
8.4
0.5
14.8
0.5
7.2
0.1
7.0
0.1
+ + + + +
M. lunu
9 .2
0.3
9.0
0.5
9.5
0.4
- - + + + + +
B. rotunda
7. 2
0.2
-
7.1
0.1
- - + + + - +
C.
xanthorhiza
8.0
0.2
7.8
0.3
7.8
0.3
- - + + + - -
C. caudatus
7.9
0.2
8.9
0.4
8.6
0.5
- - + + + - -
A. paniculata
8.0
0.1
8.1
0.2
7.7
0.3
- - + + + - -
M. koenigii
7.1
0.1
7.1
0.2
7.6
0.4
- - + + + - -
K. galangal - -
7.2
0.3
- - + + + - -
P. longum - - - - - - - + - -
T.
triangulare
- - - - - - - - - -
S. grandifolia - - - - - - - - - -
P.
tetragonolob
us
- - - - - - - - - -
M. cintrifolia - - - - - - - - - -
G.
procumben
- - - - - - - - - -
J. gendarussa - - - - - - - - - -

24

C. asiatica - - - - - - - - - -
Tetracyline
(30ug)
17.4
1.4
21.3
3.6
19.9
0.7
19.3
1.6
22.4
2.3
- - - - -
Chloramphen
icol (30ug)
- - - - -
4.0
ppm
8.0
ppm
2.0
ppm
8.0
ppm
8.0
ppm
Concentration used 40mg/mL .(+) positive antibacterial activity (-) no activity; BC= Bacillus 485
cereus ATCC 14579; SA= Staphylococcus aureus ATCC 25923; LM= Listeria 486
monocytogenes ATCC 19115; CJ= Campylobacter jejuni ATCC 29428; EC = Escherichia 487
coli ATCC 25922; ST= Salmonella thyphii ATCC 13311 488
489
3.2 Agar dilution method 490
Table 3.2: Minimum inhibition concentration (MIC) of methanol extract of 17 491
medicinalsalads plants. 492

Methanol extract
Minimum inhibition concentration( mg/mL)

Gram-positive bacteria
Gram-negative
bacteria
No. BC SA LM EC ST
1 Pipper betle 1.0 2.0 2.0 2.0 2.0
2 Lowsonia inermis 4.0 4.0 4.0 8.0 4.0
3 Melicipe lunu 2.0 2.0 1.0 32.0 16.0
4 Borsenbergia rotunda 8.0 16.0 8.0 - 32.0
5 Curcuma xanthorhiza 1.0 1.0 1.0 - -
6 Cosmos caudatus 2.0 2.0 1.0 - -
7 Andrographis paniculata 8.0 8.0 8.0 - -
8 Muraya koenigii 16.0 16.0 16.0 - -
9 kaempferia galangal 32.0 32.0 32.0 - -
10 Piper longum - - 32.0 - -
Formatted: Centered
Formatted: Centered
Formatted: Centered

25

11
Chloramphenicol (ppm)
4.0 x 10
-
3
.0 8.0 x 10
-3
2.0 x 10
-3
8.0 x 10
-3
8.0 x 10
-3

(-) no activity; BC= Bacillus cereus ATCC 14579; SA= Staphylococcus aureus ATCC 25923; 493
LM= Listeria monocytogenes ATCC 19115; CJ= Campylobacter jejuni ATCC 29428; EC = 494
Escherichia coli ATCC 25922; ST= Salmonella thyphii ATCC 13311 495
496
497
498
499
500
501
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Antibacterial Activity of Selected Edible Medicinal Plants 1

C in an incubator (Memmert, Germany). Then three or five 167

C. Samples were 183

C in an incubator for 18-20 201

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