1

Correlation of Bioactive Compounds of Antibacterial Activity in Cosmos caudatus and 1

Melicope lunu by using multivariate analysis. 2
3
Mohamad Zainuddin Mohamad Farhan, Abdul Hamid Azizah* and Khatib Alfi 4
Department of Foood Science, Faculty of Food Science and Technology, Universiti Putra 5
Malaysia, Serdang, Selangor, Malaysia 6
*Corresponding author. Tel:+603-89468374; Fax: +603-89423552 7
Email address: azizah@food.upm.edu.my 8
9
Abtract 10
11
12
13
14
Keywords: Malaysian edible medicinal plants; Foodborne pathogen; Antibacterial activity; 15
Disc diffusion method; Agar dilution method 16
17
18
19
1.Introduction 20
21
Food borne 22
23
24
Food borne diseases is still a concern for both consumers and food industry despite 25
the use of various preservation methods. Food safety researchers and regulatory agencies are 26
continuously concerned with the high and growing number of illness outbreaks caused by 27
some pathogenic and spoilage microorganisms in food (Shan et al.,2007). It had been 28
estimated that 76 million people in United State had suffered from foodborne illness each year 29
(Mead et al., 1999). WHO (2007) also reported that 30% of people in industrialized countries 30
suffered from a food borne diseases each year and in 2005 alone, at least 1.8 million people 31
died form diarrhea diseases worldwide. Many coutries lossing billion of dollar due to medical 32
cost, productivity losses and value permature death (Snowdon et al., 2002). 33
34


2

Foodborne diseases can be devided into two groups which are food infection and food 35
intoxication. Foodborne pathogen is a main role cause of the desease, it happens via cross 36
contamination, improper handling and temperature abuse. Food intoxication happened when 37
the patient consuming foods that contain hazadous toxic chemicals that produced by some 38
bacteria such as Salmonella sp. and Compylobacter (Melzer and Shah, 2009). It also can 39
happen even the microorganism that produced the toxin is no longer present or unable to 40
cause infections. On the other hande, food infection was caused by the present of infectious 41
pathogens in the consumption food that will multiply in the intestine and release their toxins 42
which it will invade and damage the epithelium cells. Salmonella and, Compylobacter sp. had 43
been reported as one of the common bacteria involving in food infections. While the other 44
reported bacteria that implicates less frequently in food borne infectious were Listeria 45
monocytogenes, Escherichia coli, Bacillus cereus and Streptococcus sp. (Taylor., 2002). 46
47
48
In order to control microbial growth and reduce the incidence of food poisoning and 49
spoilage, many synthetic antimicrobial have been produced (Shan et al., 2008). Even though 50
synthetic perservatives are effective, consumers still worried about their potential toxicity 51
(Tang et al., 2008). It also had been reported have many side side effects such as sleepy, 52
halucination, fever, paranoid and amenisia (Willey et al., 2008). In addition, synthetic drug 53
also has been related with the evolution of drug resistant microorganism. With an increased 54
awareness of people about these health risks, the consumption of raw vegetables and fruits 55
has increased significantly as individuals have become more heatlh-conscious and aware of 56
importance of plant-based diets in combating the onset of such diseases (Shuib et al., 2010). 57
58
Due to the problems, mmany researchers tend to go back to natural product as an 59
alternative way in finding a new source of antimicrobial agent. Each plant possesed their own 60
characteristic of self defences mechanism against viruses, bacteria, fungals, bugs and herbivor 61
animals. Based from the characteristic of the selected plants, Our ancient people had been 62
tested those plants until they found which plants is safe and effeciently to cure on the specific 63
illness. Those knowledge that had been claimed to be safe had been pass down from 64
generation to other generation. Today, Numerous of traditional medicinal plant have been 65
explored in more detail in order to find their bioactivity potential such as antioxidant, anti 66
diabetic, antimicrobial, anticancer, anti inflammatory and many more. Those plants also were 67
cheap to buy compare to the synthetic drugs and some consumers self planted the plants 68
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seems it was quite easy to growth. This fact had been supported from Eloff (1998) where in 69
his report, in 1998, about 80% of the people in developing countries almost exclusively use 70
traditional medicinal plants. 71
72
Some traditional medicinal plant can be consumed as ingredient in cooking or eat it 73
rawly as salad. Most of the people are highly believe that those plants are safe to consume 74
since there is no complain about the poisoness or toxicity report of the plant from generation 75
to the other generation. Most of the edible traditional plants have many medicinal effect in our 76
digestion system such as treating stomach diseases, diarrhea, dysentry and diuretic 77
(Sankaranarayanan et al., 2010; Yasni et al., 1994; Chowdhury et al., 2008; Gowril and 78
Vasantha., 2010). It also had been reported have outer body treatment such as skin diseases, 79
itches, wounds, bruises, irritant, boils and ulcers (Chaudhary et al., 2010; Shaari et al., 2006; 80
Ridtitid et al., 2008;Manoj et al., 2004; Perry, 1980). All those therapeutic values are related 81
with the presence of phytochemical compounds ( flavonoids, isofalavones, lignans,cinnamic 82
acids derivatives,steroids, carotenoids, terpenoids, etc), vitamin, polysaccharides, proteins and 83
minerals contain in the plants. The objective of this study is to evaluate the potential of 84
antibacterial activity of selected edible traditional plants based on their traditional medicinal 85
practice against different strains of food borne bacteria. 86
87
88
89
90
91
The sample plants for this research is Andrographis paniculata, Boesenbergia 92
rotunda, centella asiatica, cosmos caudatus, curcuma xanthorhiza, gynura procumbens, 93
justicia gendarussa, kaempferia galangal, Lowsonia inermis, Melicipe lunu, Molinda 94
cintrifolia, Muraya koenigii, Pipper betle, Piper longum, Premna Cordifolia, Psophocapus 95
tetragonolobus, Sesbania grandifolia, Talinum triangulare and Vitex nogundo. These samples 96
were selected based on their used as salad, food ingredient and contribution in medicinal 97
properties. Table 1.1 show the ethnomedicinal uses of selected plants in this study. 98
Since in 1550 BC, Egyptians had been used spices and herbs such as Cinnamon, cumin and 99
thyme as a food perservation and mummification (Webb & tanner, 1994 ; Hirasa & takemasa, 100
1998). Until now numerous studies have been published on the antimicrobial activities of 101
plants extracts against different types of bacteria (Shan et al.,2007; Cos et al., 2002; Kumar et 102
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la., 2006; Vivek et al., 2008; Buwa and Van., 2006; Agnihotri et al.,2008; Khan and 103
Omoloso.,2008; Tadhani and Subhash.,2006;Tsai et al.,2008 and Wang et al.,2008). However, 104
only a few studies focused on the potential of consumption vegetables plants as sources of 105
antibacterial compounds that could inhibit foodborne microorganisms. The objective of this 106
study are to determine the antibacterial activity of extracts from salad with traditional 107
medicinal properties against food borne microorgansims 108
109
Table 1.1 Ethnomedicine uses of selected consumption plants salad in Malay communtiy. 110
Species name Common name Part tested Ethnomedicinal uses
Pipper betle

Sireh


Leaves
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
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Piper longum

Kaduk

leaves
Talinum
triangulare

K.Belanda

leaves
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
111
112
113
2. Material and methodology 114
2.1 Samples 115
Traditional Malaysian plants like Centella asiatica, Cosmos caudatus, Curcuma 116
xanthorhiza, Justica. gendarussa, Lawsonia inermis, Kaemperia galangal, Melicipe lunu, 117
Morinda cintrifolia, Muraya koenigii, Piper betle, Pipper longum, Psophocapus 118
tetragonolobus, Sesbania grandifolia, Talinum triangulare, Gynura procumbens, 119
Borsenbergia rotunda and Andrographis paniculata were selected based on their traditional 120
medicinal uses are listed in Table 1. The plant parts were collected from the Traditional 121
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Medicine Plant Plot, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. Plant materials 122
were washed with running tap water before being chopped into pieces, then dried in 123
conventional oven (Memmert, Germany) at 45
o
C for two days and then grounded to powder. 124
The powders were then kept at -20
o
C. 125
126
2.2 Preparation of crude extract 127
The sample was mixed with absolute analytical methanol (Merck, Germany) at 1:10 128
ratio and leaved in shaking water bath for 24 hours at 45

C. Samples were then filtered using 129

whatman filter paper No.1 and the solvent were removed by using Eyela rotary evaporator 130
(Tokyo Rikikai Co. Ltd, Japan) at 40

C. 131
132
2.3 Preparation of solvent fractionation 133
The methanol extract was diluted in 25 mL Methanol and mixed with 100 mL distilled 134
water then mixed with 125 mL of serial solvent( hexane, Dichloromethane, ethyl acetate, 135
butanol) in 1000 mL seperated funnel. The ratio of the mixer of Water: Methanol: Serial 136
solvent was 4:1:5. Each of the solvent fraction were removed by using Eyela rotary 137
evaporator (Tokyo Rikikai Co. Ltd, Japan) at 40

C. 138
139
2.3 Microbial strains and media 140
Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 14579, Listeria 141
monocytogenes ATCC 19115, Campylobacter jejuni ATCC 29428, Salmonella typhimurium 142
ATCC 13311 and Escherichia coli ATCC 25922 were purchased from ALL EIGHTS (M) 143
SDN BHD (Subang, Selangor, Malaysia). Each bacterial strain was cultivated in Nutrient 144
Agar and incubated for 18-20 hours at 37

C in incubator (Memmert, Germany). Then three or 145

five of the same morphological type form on agar plate were took up and suspended in 10ml 146
sterile saline then vortexed thoroughly. The bacterial suspension was followed 0.5 Mcfarland 147
standards. The inocoloums were used less than 15 minutes after preparation. 148
149
2.4 Inoculums and inoculation 150
The bacteria were reculture from the stock culture. Two or three single well colonies 151
were pick up and streaked on Nutrient Agar by using loop. The cultures were incubated at 37 152

C for 18-20 hours in incubator (Memmert, Germany). Then three or five of the same 153
morphological type form on agar plate culture were took up and suspended in 10 ml sterile 154


7

saline then vortex thoroughly. The bacterial suspension then is compared to 0.5 McFarland 155
standards. The inoculums should be used less than 15 minutes after prepare it. 156
157
158
2.6 Determination of Minimum Inhibition Concentration by using Agar Dilution 159
method. 160
The minimum inhibition concentration (MIC) of the extracts were tested by using 161
Agar Dilution method according to method that proposed by Jennifer, (2001) with some 162
modification. The extracts were diluted using 10 % DMSO and adjusted to 8mg/mL as stock 163
solution then two-fold dilution were made from 4mg/mL up to 0.5mg/mL. The suitable 164
amount of extract was added on to the well of 24 well plate then added with suitable amount 165
of sterile Muller Hilton agar (MHA). The temperature of MHA was cold to 50 C before 166
poured to the well. Then was swirled up until thoroughly mixed. The plates were left for 10 167
minutes and allowed to solidified. Then 1 uL of suspensions from the prepared inoculums 168
were placed on the agar surface. Chloramphenicol was used as a positive control. While for 169
the blank was MHA only. The plates were incubated in an inverted position at 37

C in 170
incubator (Memmert, Germany) for 18-20 hours. The MIC was read as the first concentration 171
that inhibits the organism growth completely. In the present study, five replicates were 172
prepared for each sample. 173
174
2.7 GCMS analysis 175
176
2.8 Statistic analysis 177
178
2.9 Multivariate analysis 179
180
181
182
183
184
185
3. Result 3.2 Agar dilution methodTable 3.2: Minimum inhibition concentration (mg/mL) of 186
methanol extract of 17 salads plants. 187


8

(-) no activity; BC= Bacillus cereus ATCC 14579; SA= Staphylococcus aureus ATCC 25923; 188
LM= Listeria monocytogenes ATCC 19115; CJ= Campylobacter jejuni ATCC 29428; EC = 189
Escherichia coli ATCC 25922; ST= Salmonella thyphii ATCC 13311 190
191
Introduction 192
4. Discussion 193
194
195
5. Conclusion 196
. 197
the mode of action of antimicrobial agents also Conclusion 198
199
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202
203
204
205
206
207
208
209
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212
213
214
215
216
217
218
219
220
221
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222
223
224
4. Discussion. 225
5.Conclusion 226
Table 227
Table 1: Ethnomedicine uses of selected edible traditional medicinal plants salad in 228
Malaysia communtiy. 229
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 Kari Leaves Relieve nausea, indigestion, vomiting;


10

koenigii 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)
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)
230
231
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233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
3.1 Disk diffusion method 256
Table : Results of weight collected from different fraction solvent. 257
Plants
Weight percentage collected %
Hexane Dichloromethane Ethyl acetate Butanol Water
Melicope lunu 11.8 1.42 74.912.65 6.260.3 26.080.51 5.880.42
Cosmos caudatus
258
259
260
Table : minimum inhibition concentration of extract in different fraction layer. 261
Plants Extract of minimum concentration inhibition (mg/mL)


12

fraction layer
Staphylococcus
aureus
Bacillus cereus
Listeria
monocytogenes
Melicope lunu
Hexane <0.5 <0.5 <0.5
Dichloromethane <0.5 <0.5 <0.5
Ethyl acetate <0.5 <0.5 <0.5
Butanol 4 2 2
water 1 <0.5 <0..5
Cosmos caudatus
Hexane 4 4 4
Dichloromethane 4 2 2
Ethyl acetate 2 1 2
Butanol 1 <0.5 <0.5
water 2 1 2
262
263
264
265
266
267
268
269
270
271
272
273
274
3.2 Agar dilution m 275