Journal of Ethnopharmacology 172 (2015) 30–37

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Immune-stimulating properties of 80% methanolic extract of Ludwigia octovalvis against Shiga toxin-producing E. coli O157:H7 in Balb/c mice following experimental infection Haidar Kadum Yakob a,n, Abd. Manaf Uyub b, Shaida Fariza Sulaiman b a b

Department of Biology, College of Education for Pure Sciences, AL-Anbar University, 00964 Ramadi, Iraq School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia

art ic l e i nf o

a b s t r a c t

Article history: Received 5 February 2015 Received in revised form 18 April 2015 Accepted 6 June 2015 Available online 16 June 2015

Ethnopharmacological relevance: Ludwigia octovalvis is an aquatic plant widely distributed throughout the tropical and sub-tropical regions. It is commonly consumed as a health drink and traditionally used for treating various ailments such as dysentery, diarrhea, diabetes, nephritisn and headache. No information is available on its in vivo antibacterial activity against an important foodborne pathogen, Shiga toxin producing Escherichia coli O157:H7. Materials and methods: Male Balb/c mice were orally administered with the extract at doses of 200 or 400 mg/kg body weight for one week before the infection with E. coli O157:H7 and continued for 14 consecutive days after infection. Serum antibody (IgA, IgG and IgM) levels were quantified at days 7 and 14 post-challenge by an ADVIAs 2400 Clinical Chemistry Auto Analyzer. Nitroblue tetrazolium (NBT) and Ceruloplasmin, as nonspecific immune parameters, were determined enzymatically. Results: A significant increase (po0.05) in IgA serum level was indicated on the 7th day post-challenge with the pathogen in the experimental group received 400 mg/kg of the extract in comparison with other groups. Total IgA serum levels on day 7 post-challenge in groups of PBS negative control, E. coli O157:H7 positive control, E. coli O157:H7 þ200 mg/kg extract group and E. coli O157:H7þ 400 mg/kg extract group were 709.47 149.6, 1655.8 7 139.7, 1728.6 764.3 and 1971.47 135.6 mg/ml, respectively. Serum IgG and IgM did not significantly change among different groups. The extract administered orally to infected Balb/c mice did not affect the NBT as well as ceruloplasmin levels. Conclusions: The extract of L. octovalvis contains biologically active principles which increased systemic immune response to E. coli O157:H7 via potentiating the synthesis of IgA antibodies. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Ludwigia octovalvis in vivo Anti-E. coli O157:H7 Immune response

1. Introduction In recent years, Escherichia coli O157:H7 have caused multiple food and water-borne outbreaks worldwide and considered a serious threat to public health (CDC, 2015). It produces Shiga toxin which causes diarrhea and hemorrhagic colitis which may have adverse effects on the central nervous system, pancreas, lungs and heart with a case fatality rate ranging from 3% to 5% (WHO, 2009; Mohawk and O’Brien, 2011). Infection is often followed by lifethreatening hemolytic uremic syndrome (HUS) and death especially in the elderly and young children (Sukhumungoon et al., 2011; Mendonça et al., 2012).

n

Corresponding author. Tel.: þ964 7806221599. E-mail address: [email protected] (H. Kadum Yakob).

http://dx.doi.org/10.1016/j.jep.2015.06.006 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

Recently, E. coli O157:H7 has been shown to be multi-drug resistant (Sukhumungoon et al., 2011). More investigations on this pathogen have been carried out in recent years than with any other group of food-borne pathogens to find a treatment, yet this group remains the most difficult to treat (Liu et al., 2013; CDPH, 2014; Itelima and Agina, 2014; Paula et al., 2014; Puttalingamma and Niveditha, 2014; IFSAC, 2015). Currently, there is no specific treatment available for E. coli O157:H7 infections and it is limited to supportive care including rehydration and management of anemia and renal failure, and is important with close attention to fluid, electrolyte and metabolic balance, nutrition and blood pressure. Antibiotic treatment is currently not recommended as it may increase the risk of systemic complications such as acute renal failure associated with HUS (CDC, 2015). Antioxidants are believed to be important for the treatment or protection against E. coli O157:H7 infection, since this organism produces oxygen-free radicals causing oxidative cell membrane damage and intestinal

H. Kadum Yakob et al. / Journal of Ethnopharmacology 172 (2015) 30–37

inflammation (Meyers, 1999). Therefore, this fact has made it necessary to search for new sources of anti-E. coli O157:H7 agents, especially natural products from plants. Medicinal plants and their products have been widely used for the treatment of many kinds of acute and chronic diseases. Using of natural products derived from plants as immunostimulant is more useful than antimicrobial drugs that cause adversely side effects for human. It has been reported that over 142,000 emergency department admissions per year were due to antibiotic side effects in the United States (Donnelly et al., 2014). Moreover, there are a number of side effects of different antibiotics that have been reported (Wawruch et al., 2002; Shehab et al., 2008; Barnhill et al., 2012 ). Although antibiotics belong to relatively safe drugs, many of them can be a cause of a serious damage to the human organism. Antibiotic-induced adverse effects may be due to frequent prescriptions (dose-dependent or augmented), as well as to idiosyncratic reactions (not dose-dependent or bizarre). Augmented adverse effects are associated with pharmacodynamic effect of a drug; they are predictable and preventable. Meanwhile, bizarre adverse effects are not depend on pharmacodynamic effect; they are mediated by organism's immune reactions to a particular drug, as well as by genetic divergences of drug metabolism, which leads to the production of a toxic metabolite (Wawruch et al., 2002; Barnhill et al., 2012). Medicinal plants have a potential application as an immunostimulant primarily because they can be easily obtained, are not expensive and act against a broad spectrum of pathogens. Most of the herbal extracts can be given orally, which is the most convenient method of immunostimulation. The use of such plant products as immunostimulants may also be of environmental value because of their biodegradability (Puri et al., 2013). Herbal extracts, as potent antioxidants, may affect different aspects of the innate and adaptive immune system by shifting prooxidant/antioxidant balance toward antioxidant. The pro-oxidant/ antioxidant balance is believed to be an important determinant of the immune cell function as the immune cells particularly sensitive to oxidative stress due to the high percentage of polyunsaturated fatty acids in their cell membranes (Neyestani, 2008). Moreover, several studies have described the modulating effects of different flavonoids on natural immunity components such as neutrophils and NK-cell, and of adaptive immunity such as lymphocytes and immunoglobulin (Kaku et al., 1999; Ma and Kinneer, 2002; Neyestani, 2008). It is reported that the clinical efficacy of many herbal medicines could be partially attributable to their immunoregulatory properties. In recent years, several medicinal plants have been reported to have immunostimulant or immunosuppressive effect on immune system (Kumar et al., 2011). Ludwigia octovalvis, as a traditional Malay herbal medicine, has been used internally in the treatment of a broad spectrum of infections, including gastrointestinal disorders such as diarrhea and dysentery, and urinary infections (Burkill, 1966). Various pharmacological activities of this plant have been reported e.g. antibacterial, antioxidant (Yakob et al., 2012a), antidiarrhoeal, antidiabetic, anti-inflammatory (Murugesan et al., 2002), anticancer (Wu et al., 2010) and hepatoprotective activities (Lin et al., 2006). However, the potential of this plant in the induction or enhancement of immune response against infectious diseases had not been evaluated. After confirming antioxidant and direct antibacterial activities of the 80% methanol extract of L. octovalvis leaf against Shiga toxin-producing E. coli O157:H7 in vitro (Yakob et al., 2012a), another in vivo antibacterial activity investigation of this extract in an animal infection model was needed. In addition, In-vivo acute and subacute toxicity studies involving 28 days daily

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administration of the extract at 200, 400 and 800 mg/kg using Balb/c mice showed that the extract was not lethal or toxic (Yakob et al., 2012b). Therefore, the present study was undertaken, for the first time, to investigate if oral administration of the extract to Balb/c mice capable of inducing or promoting the immune response following an E. coli O157:H7 challenge.

2. Materials and methods 2.1. Plant material The whole plant of L. octovalvis (Jacq.) [syn: Jussiaea suffruticosa L., Jussiaea pubescens L., Jussiaea angustifolia Lamk.; Family-Onagraceae] was collected at flowering stage from the wet area of the Universiti Sains Malaysia main campus, Pulau Pinang. A voucher specimen number (11090) was deposited at the herbarium of the School of Biological Sciences, Universiti Sains Malaysia. 2.2. Plant extraction and extract yield The leaves of the plant were separated and washed. The samples were then dried at 40 1C for 24 h and ground into powder. The powder (30 g) was sequentially extracted (cold extraction) in a flask with 200 ml of 80% (v/v) methanol by continuous shaking for 8 h. The extract was filtered using a filter paper (150 mm, Whatman, U.K.) and concentrated using a rotary evaporator (EYELA N 1000, USA) and stored at 4 1C until further use. This is slightly different than the traditional preparation which is consumed orally as decoction of leaves in a dose of 50 to 100 ml or as dried powder with honey. The yield percentage of the extract was calculated using the following equation (Banso and Adeyemo, 2006): Yieldð%Þ ¼

WeightoftheextractðgÞ  100 WeightofthegroundplantmaterialðgÞ

2.3. Experimental animals All experiments with laboratory animals were carried out using male Balb/C mice (weighing 25–30 g and aged 8–10 weeks) and approved by the Animal Ethical committee of Universiti Sains Malaysia [USM/Animal Ethics Approval/2009/(50) (143)]. Mice were purchased from the Animal House, Universiti Sains Malaysia and housed there in plastic cages under standard laboratory conditions. They were acclimated to the experimental environment for one week prior to the experiments. Mice were fed with standard pellets (standard rodent chow diet) and water ad libitum throughout the experiments, except prior to E. coli O157:H7 infection. Food was taken from the animal cages the night before bacterial challenge. Water bottles were removed from the cages 2 h prior to infection. Following infection, access to food and water was restored. 2.4. Preparation of E. coli O157:H7 for challenge 2.4.1. Source of E. coli O157:H7 E. coli O157:H7 was collected from the culture collection of the Laboratory of Food Safety and Quality (Jalan Bagan Luar, 12000 Butterworth, Pulau Pinang, Malaysia). The pathogen was isolated from contaminated beef. The sequences of Stx1 and Stx2 specific genes of E. coli O157:H7 used in this study were deposited in GenBank with accession number JX161807 and JX161808, respectively. For all pathogenic E. coli O157:H7 infection studies, 10 μl of the stock culture of the organism (stored at 70 1C) was inoculated onto a tryptic soy agar plate and incubated for 18 h at 37 1C. A

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single colony was picked up from the agar plate, inoculated into tryptic soy broth and then incubated with shaking over night at 37 1C. The bacteria were harvested from the broth by centrifugation for 15 min at 5000g and the pellet washed twice and resuspended in sterilized phosphate buffered saline (pH 7.4). The concentration of bacterial cell suspension was adjusted to 0.5 McFarland standard (bioMérieux, France) for a final density of approximately 1  108 CFU/mL and confirmed by counting the CFU of the overnight plating (in triplicate) of serial decimal dilutions of the inoculum and then diluted to an appropriate concentration with phosphate buffered saline. Throughout each experiment mice in all groups were given 5 mg/ml streptomycin in their drinking water one day before oral infection with the bacteria. 2.4.2. Determination of E. coli O157:H7 LD50 in Balb/c mice The 50% lethal dose (LD50) of E. coli O157:H7 was determined using a dose–response method (DePass, 1989). Twenty four male BALB/c mice were randomly divided into six groups. After 1 day of streptomycin treatment, mice were then starved for food (but not for water) for 12 h before inoculation. The next morning, animals in different groups were administrated intragastrically with approximately 1  105, 1.5  105, 2  105, 2.5  105 or 3  105 CFU of E. coli O157:H7 in PBS at a volume of 200 ml/mouse. Control animals were orally administered sterile PBS alone. Food was then reintroduced and provided ad libitum. Animals were observed four times per day for the first 72 h and twice per day thereafter for clinical signs and mortality over a period of 14 days. Mice that were still alive 14 days after bacterial challenge were recorded as survivors. The total number of dead mice at each dose level was recorded and the LD50 was determined from a dose lethality curve using the Prism 3.02 statistical software of GraphPadPrism (San Diego, CA). 2.5. Evaluation of immunomodulatory properties of the extract in BALB/c mice 2.5.1. Experimental design For this study, 66 male Balb/c mice were randomly divided into six groups of 11 animals per group. The assigned groups for this study are presented in Table 1. Male Balb/c mice were orally administered with the extract at the doses of 200 or 400 mg/kg body weight. The treatment with the extract started one week before the infection with E. coli O157: H7 and continued for 14 consecutive days after infection. Doses of the treatment and PBS were administered by oral gavage, using 20-gauge by 38-mm malleable animal feeding needles (Popper & Sons, Inc., USA), at a volume of 10 mL/kg body weight in a single dose. Measurement of antibody levels, nitroblue tetrazolium and

ceruloplasmin activities was done in treatment and control groups at days 7 and 14 post-challenge with E. coli O157:H7. Survival of mice was observed daily and recorded after 14 days postchallenge. 2.5.2. Measurement of total serum IgA, IgG and IgM levels To determine whether 80% methanol extract of L. octovalvis has immuno-modulatory effects in vivo, changes in IgA, IgG and IgM serum antibody level were measured in male BALB/c mice orally infected with E. coli O157:H7 after continuous administration of the test extract over a period of 21 days (excluding the first 7 days of acclamization). Serum antibody levels (IgA, IgG or IgM) were quantified in all experimental groups at day 7 and 14 postinfection. At days 7 and 14 post-challenge with E. coli O157:H7, five mice from each group were fasted overnight (water was not restricted) and then anaesthetized with diethyl ether. Whole blood was collected individually via cardiac puncture using a 23 G needle. Blood samples were then placed in serum collection tubes and allowed to clot on ice for at least 30 min and then centrifuged at 5000g for 10 min. The serum was collected and stored at  20 1C prior to analysis. The measurement of antibody levels in sera was made at the Gribbles Pathology Laboratory, Penang. Total serum IgA, IgG and IgM was determined by an ADVIAs 2400 Clinical Chemistry Auto Analyzer (Siemens Healthcare Diagnostic, Deerfield, USA). 2.5.3. Determination of nitroblue tetrazolium (NBT) activity The Nitroblue tetrazolium (NBT) assay was performed according to Anderson and Siwicki (1995) with slight modification. The blood (100 ml) was transferred into a glass tube containing equal amount (100 ml) of 0.2% (w/v) NBT solution (prepared in PBS). For the control, 100 ml blood was mixed with PBS instead of NBT. The tubes were incubated at room temperature for 30 min. Then 3 mL N, N-dimethyl formamide (DMF) [BDH, UK] was added to each tube. Then the tubes were centrifuged at 3000g for 5 min at room temperature. The supernatant was collected and the sample absorbance at 540 nm was determined on a spectrophotometer (Thermo Multiskan Ex, Finland). 2.5.4. Determination of ceruloplasmin activity Ceruloplasmin was determined enzymatically by the method of Sunderman and Nomoto (1970) with slight modification. The blood sample (50 ml) was mixed with 500 ml acetate buffer (pH 5.2) containing 0.1% (w/v) p-phenylene diamine (PPD, Sigma, USA) as substrate. The mixtures were incubated at 37 1C for 15 min. Then 2.5 mL of 0.25% (w/v) sodium azide solution was added to stop the reaction. For the control, 50 ml blood was mixed with acetate buffer instead of PPD. The absorbance of sample was

Table 1 The assigned groups of Balb/c mice. Measurement of antibody levels, nitroblue tetrazolium and ceruloplasmin activities was done in all experimental animals at days 7 and 14 post-challenge with E. coli O157:H7. Survival of mice was observed daily and recorded after 14 days post-challenge. Group

Treatment

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Control mice; mice in this group were orally administered PBS alone. Infected mice; mice in this group were infected orally with one dose of LD50 of E. coli O157:H7. Mice in this group were orally administered 10 mL/kg body weight of 200 mg/mL of the extract and infected, on day 8, with one dose of LD50 of E. coli O157:H7. Mice in this group were orally administered 10 mL/kg body weight of 400 mg/mL of the extract and infected on day 8, with one dose of LD50 of E. coli O157:H7. Mice in this group were orally administered 10 mL/kg body weight of 200 mg/mL of the extract alone. Mice in this group were orally administered with 10 mL/kg body weight of 400 mg/mL of the extract alone.

H. Kadum Yakob et al. / Journal of Ethnopharmacology 172 (2015) 30–37

determined at 540 nm using a spectrophotometer (Thermo Multiskan Ex, Finland).

b

b

1500 1000

d

d

cd

cd

cd

c

c cd

d

500

Ex ) t2 00 ( Ex 7d t2 ) 00 (1 4d Ex ) t4 00 (7 Ex d) t4 00 (1 4d EC ) (7 d) EC Ex (1 4d t2 ) 00 +E Ex C t2 ( 7d 00 ) +E C Ex (1 t4 4d 00 ) +E Ex C t4 (7 00 d) +E C (1 4d )

4d (1

S

S

(7

d)

0

PB

Relative percentage survival Number of surviving mice after challenge  100 ¼ Number of mice injected with bacteria

a 2000

PB

Survival of mice was observed daily through all the experiment and percentage of live animals was recorded. Any mice, which became moribund during the experiments, were considered as dead. The Relative percentage survival at each dose level was determined using the following formula:

2500

Total serum IgA (µg/mL)

2.6. Survival of L. octovalvis-treated mice after challenge with LD50 of E. coli O157:H7

33

Treatment (days post-challenge)

2.7. Statistical analysis Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS for Windows, version 17, SPSS Inc., Chicago, IL). Data summary was done with descriptive statistics such as the mean and standard deviation of the mean. Statistical significance was determined by One-Way Analysis of Variance (ANOVA). A p value of less than 0.05 was considered statistically significant.

3. Results 3.1. Plant extract yield The percentage yield of the 80% methanol crude extract which was obtained by cold extraction was 16.67%. 3.2. Determination of E. coli O157:H7 LD50 in Balb/c mice Over the course of infections, it is noticed that the experimental group that was infected with E. coli O157:H7 exhibited symptoms of illness on days 3–7 post E. coli O157:H7 challenge that included slowing of activity, ruffled fur, lethargy, ataxia, tremor and convulsions before death. Conversely, all control mice survived. It was found that the E. coli O157:H7 at the highest dose (3  105 CFU) caused death within 10 days, with two of them died five days post-infection. The LD50 of the E. coli O157:H7 was determined to be 2.25  105 CFU in male Balb/c mice (Fig. 1). Therefore, for all subsequent experiments, mice were orally infected intragastrically by gavage with LD50 of the pathogen.

Fig. 2. Total serum IgA antibody concentration in different groups of mice at 7 and 14 days post-challenge with 2.25  105 CFU E. coli O157:H7. All values are the means 7standard deviation. Values followed by different letters (a, b, c, d) are significantly different (p o 0.05) based on one way analysis of variance (ANOVA), followed by Tukey HSD test. PBS: mice treated with PBS. Ext 200: mice treated with 200 mg/kg body weight of the extract. Ext 400: mice treated with 400 mg/kg body weight of the extract. EC: E. coli O 157:H7. d: days.

3.3. Total serum IgA, IgG and IgM levels 3.3.1. Serum IgA antibody levels The changes in total IgA levels in different groups are shown in Fig. 2. Mice challenged with the E. coli O157:H7 yielded significantly higher levels of antibody IgA at the seventh day postinfection, than the levels found in the PBS control group (po0.05). However, the IgA serum levels in the experimental group receiving 400 mg/kg of the extract and infecting with the pathogen were significantly increased in day 7 post-infection in comparison with other groups. No significant change was detectable in IgA concentration among mice in 14 day post-challenge. Interestingly, IgA levels in mice treated with the extract alone at doses of 200 or 400 mg/kg were not significantly different (p 40.05; Fig. 2). 3.3.2. Serum IgG antibody levels The changes of total IgG concentration of the mice in different groups are shown in Fig. 3. The serum IgG levels in the group of mice infected with E. coli O157:H7 were significantly higher (p o0.05) than in other mice after 14 days of infection. Moreover, the results demonstrated no statistically significant difference in the IgG antibody levels among all treated groups when compared to the PBS control group, except the mice in group 3 which were treated with 200 mg/kg of the extract for 14 days post-infection. 3.3.3. Serum IgM antibody levels The changes in total IgM levels in the mice are shown in Fig. 4. The serum IgM concentrations were not significantly changed in all experimental groups compared with the PBS control mice. Moreover, the IgM level in the serum of mice treated with the extract alone did not significantly change. 3.4. Nitroblue tetrazolium (NBT) activity

Fig. 1. Dose lethality curve of E. coli O 157:H7 in Balb/c mice.

Nitroblue tetrazolium (NBT) assay was used to determine neutrophil activity as a nonspecific immune parameter. Table 2 shows that OD540 of all the groups was not significantly different (p 40.05), while a slight increase in absorbance was observed in the infected mice on the 7th day post-infection, but it was not significantly different (p 40.05) compared to control.

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H. Kadum Yakob et al. / Journal of Ethnopharmacology 172 (2015) 30–37

2500

Total serum IgG (µg/mL)

a 2000

bc c

c

c

bc

bc bc

Table 2 Effect of 80% methanolic extract of L. octovalvis leaves on NBT activity (OD540) in different experimental groups after infected with E. coli O 157:H7.

ab bc

c

c

Group Treatment

1500

1000

1 2

500

3 4

4d Ex ) t2 00 (7 Ex d) t2 00 (1 4 Ex d) t4 00 ( Ex 7d ) t4 00 (1 4d ) EC (7 d) EC Ex (1 4d t2 ) 00 + Ex EC t2 (7 00 d) +E C Ex (1 t4 4 d) 00 +E Ex C t4 ( 7d 00 ) +E C (1 4d )

(1 S

PB

PB

S

(7

d)

0

Treatment (days post-challenge) Fig. 3. Total serum IgG antibody concentration in different groups of mice at 7 and 14 days post-challenge with 2.25  105 CFU E. coli O157:H7. All values are the means 7 standard deviation. Values followed by different letters (a, b, c, d) are significantly different (po 0.05) based on one way analysis of variance (ANOVA), followed by Tukey HSD test. PBS: mice treated with PBS. Ext 200: mice treated with 200 mg/kg body weight of the extract. Ext 400: mice treated with 400 mg/kg body weight of the extract. EC: E. coli O 157:H7. d: days.

5 6

Non-infected control (PBS) Infected control (E. coli O157: H7) 200 mg/kg þinfected with E. coli O157:H7 400 mg/kg þinfected with E. coli O157:H7 200 mg/kg extract uninfected 400 mg/kg uninfected

Total srum IgM (µg/mL )

After 14 days of challenge

0.56 7 0.01 0.53 7 0.02

0.49 70.02 0.45 70.02

0.497 0.02

0.49 70.01

0.52 7 0.01

0.50 70.01

0.50 7 0.02 0.53 7 0.01

0.50 70.01 0.47 70.02

Table 3 Effect of 80% methanolic extract of L. octovalvis leaves on ceruloplasmin activity (OD540) in different experimental groups after challenged with E. coli O157:H7. Group Treatment

1 2 3

200

After 7 days of challenge

Values are mean 7 S.D. Values are not significantly different (p 40.05) based on Dunnett's test.

400

300

NBT activity (OD540)

4 5 6

Non-infected control (PBS) infected control (E. coli O157: H7) 200 mg/kg þinfected with E. coli O157:H7 400 mg/kg þinfected with E. coli O 157:H7 200 mg/kg extract uninfected 400 mg/kg uninfected

Ceruloplasmin activity (OD540) After 7 days of challenge

After 14 days of challenge

0.357 0.02 0.36 7 0.01

0.37 70.01 0.36 70.02

0.34 7 0.01

0.36 70.01

0.32 7 0.02

0.34 70.02

0.357 0.02 0.377 0.01

0.3570.01 0.3570.02

100

d) Ex t2 00 (7 d) Ex t2 00 (1 4d ) Ex t4 00 (7 d) Ex t4 00 (1 4d ) EC (7 d) EC (1 Ex 4d t2 ) 00 +E Ex C (7 t2 d 00 ) +E C (1 Ex 4 t4 d) 00 +E Ex C (7 t4 d) 00 +E C (1 4d )

14 S(

PB

PB

S(

7d

)

0

Table 4 Percentage of mouse survival in different groups, following challenge with E. coli O157:H7. Group Treatment

No. of mouse survivors

Survival rate (%)

1 2 3

6 2 3

100 33.3 50

5

83.3

Treatment (days post-challenge) Fig. 4. Total serum IgM antibody concentration in different group of mice at 7 and 14 days post-challenge with 2.25  105 CFU E. coli O157:H7. All values are the means 7 standard deviation. Values are not significantly different (p 40.05) based on one way analysis of variance (ANOVA) followed by Tukey HSD test. PBS: mice treated with PBS. Ext 200: mice treated with 200 mg/kg body weight of the extract. Ext 400: mice treated with 400 mg/kg body weight of the extract. EC: E. coli O 157: H7. d: days.

4 5 6

Non-infected control (PBS) infected control (E. coli O157:H7) 200 mg/kg þinfected with E. coli O157:H7 400 mg/kg þinfected with E. coli O157:H7 200 mg/kg extract uninfected 400 mg/kg uninfected

6 6

100 100

3.5. Ceruloplasmin activity Based on ceruloplasmin assay (Table 3), the extract at the doses of 200 and 400 mg/kg did not affect ceruloplasmin levels as no significant difference (p 40.05) in OD540 was observed between all treatment groups and control.

from the group representing treatment with the extract at dose of 200 mg/kg body weight and five mice (83.3%) from 400 mg/kg body weight treatment group survived after being challenged by the oral route with the pathogen. Two (33.3%) of the infected but untreated control mice remained alive. All mice in other groups (PBS and L. octovalvis extract alone) survived.

3.6. Survival of the extract-treated mice after challenge with the LD50 of E. coli O157:H7 4. Discussion Survival of mice was recorded daily for 2 weeks post-challenge and is shown as percentage of animal's survival (Table 4). The rate of survival of infected mice improved with increasing doses/kg body weight of extract when compared with untreated infected controls. On 14th day post-infection, three mice (50%)

In patients infected with E. coli O157:H7, treatment with antimicrobial agents is not recommended because of a risk that the disease will progress from a gastro enteric disease to HUS (CDC, 2015). Therefore, alternative treatment strategies of this

H. Kadum Yakob et al. / Journal of Ethnopharmacology 172 (2015) 30–37

pathogen are urgently needed and the control of E. coli O157:H7 at the level of immune system via the use of medicinal plants could be a promising strategy through enhanced protective immunity. As a first step in the in vivo study of E. coli O157:H7 infection, dose response experiments were conducted to determine the LD50 of the pathogen in Balb/c mice. The LD50 was 2.25  105 CFU in male Balb/c mice. Therefore, for all subsequent experiments, mice were orally infected intragastrically by gavage with LD50 of the pathogen. Humoral immunity, especially the antibody secretion from different compartments in body, plays an important part in defending against infectious diseases including E. coli O157:H7 infection. Therefore, the effects of humoral immune activation by the 80% methanolic extract of the plant were investigated in Balb/c mice challenged with E. coli O157:H7 by measuring total IgA, IgG and IgM antibodies in sera samples at days 7 and 14 postchallenge. The results showed that the immunoglobulin levels were not significantly affected by oral administration of the extract among all treated groups, except the group of mice administered 400 mg/ kg body weight of the extract and challenged with the pathogen as a significant increase in the IgA serum level was indicated on the 7th day post-challenge. This interesting result demonstrated that the administration of the extract at this dose significantly increased the systemic immune response to E. coli O157:H7 via potentiating the synthesis of IgA antibodies. IgA is thought to be important in the primary immunological defense against local gastrointestinal infection. Protective activities of IgA against enteropathogenic bacteria have been reported by several researchers (Ogawa et al., 2001; Shu and Gill, 2002). It is known that E. coli O157:H7 binds itself to M cells in Peyer's patches (Fitzhenry et al., 2002), thus, an increase of IgA might be sufficient to prevent or attenuate the infection. Conlan and Perry (1998) reported that an enhanced resistance to E. coli O157:H7 in Balb/c mice correlated to the presence of serum and fecal IgA. The

35

protective action of IgA might prevent the invading organism from attaching to and penetrating the epithelial surface (Coico et al., 2003). The increase of IgA concentration could be due to the specific immunologic responses to the pathogen. These responses are mediated by the gut-associated lymphoid tissue (McBurney, 1994). Gut-associated lymphoid tissue consists of lymphoid follicles (Peyer's patches) located in the small intestine, appendix and large intestine, isolated follicles, the mesenteric lymph nodes, and a diffuse tissue comprising the intraepithelial lymphocytes and lamina propria (Shanahan, 1994). Peyer's patches consist mainly of lymphocytes of type B, T, macrophage and accessory cells. The peyer's patches are the central focus for the induction of T and B cell responses following an oral immunization. The mucosal immune response is initiated by contact between the antigen and the lymphocytes and antigen presenting cells in the dome of the peyer's patch (Ogra et al., 1994). IgA, which is secreted from conventional B cells or from B-1 cells, plays a prominent role in the gut mucosal immune system, which serves to protect the body from the continuous threat of infection from intestinal bacteria or exogenous pathogenic bacteria (Kroese et al., 1996). Nevertheless, the high level of the IgA in serum may be attributable to the ability of the immunoactive constitutes from the leaf of L. octivalvis, such as flavonoids, terpenoids and saponins which have shown immunomodulatory activity (Ríos, 2010; Licciardi and Underwood, 2011). According to Licciardi and Underwood (2011), the extract passes through gastrointestinal tract which might bring it in intimate contact with gut associated lymphoid tissue and mesenteric lymph node. These lymphoid tissues function as immunological component of gastrointestinal tract. Nitroblue tetrazolium (NBT) and ceruloplasmin assays were used to assess the ability of L. octovalvis extract to enhance the nonspecific immune response against E. coli O157:H7 in mice. The results of this study showed that the extract administered orally to

Fig. 5. Paradigm for the immunomodulatory activity of plant-derived medicines. Adapted from Licciardi and Underwood (2011).

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infect Balb/c mice did not affect the NBT as well as ceruloplasmin levels. This result suggested that the extract was not able to stimulate the microbicidal activity of polymorphonuclear leukocytes and leucocytosis, suggesting that different non-specific immunomodulating mechanisms may be involved. The results obtained from survival study revealed that oral administration of L. octovalvis extract to mice, at doses of 200 or 400 mg/kg body weight, substantially increased the protective efficacy on mice against E. coli O157:H7 infection as 50% or 83.3% mice survived from the challenge infection after 14 days postchallenge, respectively. The reason for the survival of mice could be due to: 1) direct anti-bacterial activity of the extract, 2) increasing of IgA levels, or 3) anti-adhesion effect of the extract. Phenolics have anti-adhesion effect on the pathogenic bacteria, such as E. coli, Helicobacter pylori and Streptococcus mutans (Ofek et al., 2003; Reed and Howell, 2008; Abreu and Barreto, 2012). Phenolics block the attachment of harmful bacteria to the receptor site present on mucosal surfaces making bacteria not capable to bind any more to the mucosal surfaces. The adherence of E. coli O157:H7 to intestinal epithelial cells is considered to be the first, critical, important and indispensable step during the pathogenesis of E. coli O157:H7 for attaching and effacing lesions and then developing gut colonization and infection (Wales et al., 2002). Bacteria resistant to antiadhesion agents may also be expected to emerge, but because these agents do not act by killing or inhibiting growth of the pathogen, as, antibiotics do, it is reasonable to assume that strains resistant to anti-adhesion agents will be diluted with the sensitive bacteria whose adhesion is inhibited and are shed out of the host (Ofek et al., 2003). Intimate adherence of E. coli O157:H7 to the intestinal mucosa is mediated through a type III secretion system, resulting in histological changes of the epithelial cells, which has been termed an attaching-and-effacing (A/E) lesion that includes F-actin aggregation (Garmendia et al., 2005). We suggest that when the extract passage through gastrointestinal tract might bring it in intimate contact with gut associated lymphoid tissue and mesenteric lymph node. These lymphoid tissues function as immunological component of gastrointestinal tract. Fig. 5 shows a paradigm for the immunomodulatory activity of plant-derived medicines. The 80% methanolic extract of L. octovalvis leaf was rich in phenolics that have shown potent antioxidant and antibacterial activities (Yakob et al., 2012a). Therefore, its immunostimulant activity may be due to the high phenolic content of this extract. Antioxidants, especially flavonoids, are believed to have an immunostimulating and the immune cell functions are specially linked to reactive oxygen species generation, such as that involved in the microbicidal activity of phagocytes, cytotoxic activity or the lymphoproliferative response to mitogens (Neyestani, 2008). Several studies have described the modulating effects of different flavonoids on natural immunity components such as neutrophils and NK-cell, and of adaptive immunity such as lymphocytes and immunoglobulin (Kaku et al., 1999; Ma and Kinneer, 2002; Neyestani, 2008). Overall, the present study indicated that the extract had immunostimulatory activity which was influenced by the extract concentration. The results provided evidence that the 80% methanol extract of L. octovalvis offered good protection against E. coli O157:H7 infection in Balb/c mice via either enhancing the humoral and systemic immune response by increasing IgA level, or probably directly killing the pathogen or preventing its adherence to epithelial surfaces in-vivo. Our results support the ethnopharmacological uses of this plant in folk medicine as antibacterial agent and may scientifically explain the rationale of the traditional use of L. octovalvis in the

treatment of bacterial infections. This finding contributes towards the development of inexpensive, safer and alternative strategies for the treatment of bacterial infections requiring use of immunomodulatory agents from plants. The quantification of a specific anti-E. coli O157:H7 IgA responses in relation to treatment by L. octovalvis extract will be needed. Future works are required to elucidate the exact mechanism responsible for immunomodulatory activity of the plant and to explain their suitability for prevention or attenuation of lethal bacterial infections.

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