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Antibacterial and Antiinflammatory Properties of Bovine Colostrum Ramesh Yadav1, Trupti Angolkar1, Ginpreet Kaur1 and Harpal S. Buttar2 1
SPP School of Pharmacy & Technology Management, SVKM’s NMIMS, V. L. Mehta Road, Vile Parle (W), Mumbai-400056, Maharashtra, India; 2Department of Pathology & Laboratory Medicine, Faculty of Medicine, University of Ottawa, Ontario, Canada Received: August 10, 2015; Accepted: February 17, 2016; Revised: February 17, 2016
Abstract: Context: Immunity related disorder is one of the leading causes of disease in the world. Oxidative stress and microbial infections play a major role in inflammation-induced diseases. Bovine colostrum (BC) contains immunoglobulins and lactoferrins which help in building the immunity and protect against the bacterial proliferation and growth.
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Aim: This study was designed to investigate the antimicrobial and antiinflammatory activities of BC. Materials and Methods: Antimicrobial activity was determined by the pour-plate method using five different strains of bacteria (Gram -ve and +ve), and carrageenan-induced rat paw edema method was used for the evaluation of antiinflammatory activity in adult Wistar rats. Diclofenac was used as standard antiinflammatory drug, and amoxicillin was used as standard antimicrobial agent. Results: BC showed significant antimicrobial activity against Escherichia. coli, Staphylococcus. aureus, Proteus. vulgaris, Enterobacter. aerogenes and Salmonella. typhi. At 100 g/mL of BC, the inhibition zones were found to be 13 mm, 11 mm, 12 mm, 12 mm, and 11 mm, respectively. The BC zones were comparatively smaller than those of amoxicillin at 10 g/mL, where the inhibition zones were 16 mm, 30 mm, 23 mm, 22 mm and 23 mm, respectively. In the BC treated animals, the percentage edema inhibition was found to be 67.94% at the third hour, suggesting high antiinflammatory activity of BC in rats. Conclusion: BC may be beneficial in reducing the risks of inflammation associated diseases. Further studies are needed before BC can be recommended for therapeutic interventions in humans.
Keywords: Anti-inflammatory, antimicrobial effects, bovine colostrum, carrageenan, oxidative stress peroxidase. INTRODUCTION Bovine colostrum (BC) is the first mammary secretion that all mammals provide for their newborns during the initial 24-48 hours after parturition. It is the foundation of lifelong immunity [1], since it contains various antibodies, immunoglobulin, lactoferrins and has lower fat and higher protein content than ordinary milk [2-4]. colostrum contains greater levels of lactoferrin than BC [5]. Some studies suggested that colostrum is beneficial in eradication of infection and promotion of growth in the neonatal gastrointestinal tract [6]. BC is also found to be beneficial in treating orally mediated infections and helps to build natural immunity [7]. Immunoglobulin and lactoferrin present in BC are found to be beneficial in treating Escherichia coli infection and building immunity [8-10]. Due to the presence of growth hormone and insulin like growth factor-1(IGF-1), BC has cholesterol lowering effect [11, 12]. It also has cardio*Address correspondence to this author at the Department of Pharmacology, SPP School of Pharmacy & Technology Management, SVKM’s NMIMS, V.L. Mehta Road, Vile Parle (W), Mumbai-400056, Maharashtra, India; Tel: (022) 42332000; Extension: 2035; Fax: (022) 26185422; E-mail:
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protective effects and was used to prepare anti-polio vaccine Sabin [3, 13, 14]. Colostrum also provides passive immunity against several diseases in human [15]. It also has an immunomodulatory activity and shows a positive influence on neonatal immunity due to the presence of cytokines [16, 17]. Major classes of Igs found in serum and lacteal secretions of BC are IgG, IgM and IgA antibodies[18]. Ig present in cow colostrum is higher than Ig of human colostrum [3]. Immunoglobulins present in BC form chelating complex with bacterial and viral antigens to show anti-inflammatory activity [5]. Oxidative stress and microbial infections may lead to inflammation response which in turn causes the generation of reactive oxygen species (ROS) and reactive nitrogen intermediate (RNI), which are capable of causing DNA damage[19]. Immunoglobulins, lactoferrin and lactoperoxidase present in colostrum have been shown to reduce inflammation and oxidative stress [5]. High levels of antioxidants present in colostrum are found to be beneficial in athletes by reducing oxidative stress caused due to heavy exercise [20]. The aims of present study were to determine the antimicrobial and anti-inflammatory activities of BC using standardized tests. © 2016 Bentham Science Publishers
2 Recent Patents on Inflammation & Allergy Drug Discovery 2016, Vol. 10, No. 1
MATERIAL AND METHODS The test bacteria used in this study were obtained from N.C.I.M. (National Collection of Industrial Microorganism), Pune, India. Amoxicillin was purchased from sigma Aldrich. Nutrient agar and nutrient broth were supplied by Hi-media, Dimethly sulphoxide from S D Fine-Chem Limited. Carrageenan (AR grade) was purchased from sigma Aldrich. The bacterial strains were cultured on nutrient agar slants. The cultures were maintained by sub culturing periodically and stored at 4ºC prior to use. METHODS USED FOR PROCESSING BOVINE COLOSTRUM POWDER (BCP) Colostrum was obtained from a dairy source, located in Mumbai. Spray drier (Labultima LU 222 Advance) was used, by keeping the inlet temperature at 95ºC± 5ºC and outlet temperature at 35˚C± 5 C. The powdered colostrum (BCP) was then collected from first and the second cyclone separator. To preserve activity of the powder, it was stored at 2-8ºC. Animals Studies were performed using adult albino Wistar rats weighing 150-200gm. The animals were procured from Bharat serums and vaccines limited, Thane, India. For acclimatization, the animals were housed in cages for one week under standard laboratory conditions (25ºC ± 2ºC), relative humidity of 60 ± 5% and 12 hours light and day cycle. The studies were carried out according to the CPCSEA guidelines and the experimental protocol number CPCSEA/ IAEC/SPTM/P-32/2015. Acute Toxicity Studies The acute toxicity studies were performed as per OECD guidelines no.423 [21]. Animal were divided into two groups, each containing six rats, BCP was dissolved in vehicle (water) and administered 2,000mg/kg body weight orally. The daily general overt behavior of treated rats was observed for 14 consecutive days with free access to food and water. DETERMINATION OF ANTIMICROBIAL EFFECTS Antimicrobial activity of BCP was determined using both Gram positive & Gram negative bacterial strains. The strains were Enterobacter aerogenes, Proteus vulgaris, Escherichia coli, Salmonella typhi and Staphylococcus aureus, the pour plate method was adopted for microbial activity [22]. Stock solution of BCP was prepared in Dimethyl sulfoxide (DMSO). Nutrient agar were autoclaved at 120ºC, 15psi (Equtron), and bacterial strain (concentration of 1 10 8 CFU mL -1 ) were added into autoclaved nutrient agar, and poured into the sterile petri dish and allowed to set, all the procedure were performed in laminar air flow (Dynafilters Verticals). Wells (9mm) were made in the pre-solidified nutrient agar plate (19cm) using metal borer. The test sample (20L) were added in to respective wells and kept for incubation (37 °C for 24hrs.) The MIC of colostrum was calculated by serial dilution in triplicate and it was found to be 100g/ml.
Yadav et al.
DETERMINATION OF ANTI-INFLAMMATORY ACTIVITY Antiinflammatory activity was determined by carrageenan-induced paw edema method. Before starting the treatment, the rats were fasted overnight. The average volume of the right paw of each animal was determined, before the treatment using a plethysmometer (Plethysmometer 520 IITC Life Science). Immediately after, the test substances were administered [23]. The rats were divided into four groups, each consisting of six rats. The group I served as control group and received only the vehicle SCMC (Sodium carboxymethylcellulose) (1%). Group II received standard drug diclofenac sodium 30 mg/kg per oral, Group III received test agent BCP 500 mg/kg and Group IV received the combination of BCP 500 mg/kg with diclofenac 10mg/kg per oral (standard drug as an anti-inflammatory agent) [24]. Carrageenan (0.1%) was administered at the dose of 0.1mL sixty minutes later, through sub-plantar route in right hind paw to induce edema. Volume of paw edema of each rat was measured at time points of zero hours immediately after injecting carrageenan, and then after 1, 2, 3 and 4 hr after carrageenan injection using Plethysmometer. The difference of average values between treated animals and control groups was calculated for each time interval. The percentage inhibition of paw edema in the various treated group was calculated as follows: Percentage inhibition= (1- Vt/ Vc ) 100 Where, Vt = edema volume in treated group Vc = edema volume in control group RESULTS BCP showed significant (p > 0.001) (Fig. 1) antimicrobial activity against E. coli, S. aureus, P. vulgaris, E. aerogenes and Salmonella. typhi. It showed minimum zone sizes of inhibition at 100g/mL against Staphylococcus aureus (11mm), followed by Salmonella typhi (11mm), Enterobacter aerogenes (12mm), Proteus vulgaris (12mm) and Escherichia coli (13mm) (Table 1). Anti-inflammatory activity of carrageenan-induced rat paw edema was evaluated by calculating percentage inhibition produced by colostrum alone. Significant (p > 0.001) (Fig. 2) inhibition was observed as, diclofenac showed 77.33% edema reduction at 2 hrs post-treatment. There was 67.94% edema reduction at 3 hrs in the colostrum treated group. Whereas combination of diclofenac with BC showed less potency less than 60% reduction as compared to standard drug 77.33% at 3rd hour, the antiinflammatory results are summarized in Table 2. Statistical Analysis Analysis was done using One-way ANOVA followed by Bonferroni’s test DISCUSSION Immunity related non-communicable diseases are one of the leading cause of long-term morbidity in the world. It is well known that colostrum contains macro- and micro-
Bovine Colostrum: Antibacterial & Antiinflammatory Effects
Recent Patents on Inflammation & Allergy Drug Discovery 2016, Vol. 10, No. 1 3
Fig. (1). Antimicrobial effects of BC and its combination with amoxicillin with BC. All triplicate mean values were compared with positive control (amoxicillin). ***p < 0.001 when compared with positive control (amoxicillin) mean control values. Table 1.
Antibacterial Effects of Amoxicillin Alone, BC Alone, and Combination (BC with Amoxicillin) all the Reading were Taken in Millimeter (mm).
Bacterial Strains
Control
BC with Amoxicillin (mm)
BC Alone 100g/mL (mm)
Amoxicillin Alone 10g/mL (mm)
-ve (mm)
+ve (mm)
Staphylococcus aureus
0.0
30
23
11
30
Salmonella typhi
0.0
23
18
11
23
Enterobacter aerogenes
0.0
22
17
12
22
Proteus vulgaris
0.0
23
19
12
23
Escherichia coli
0.0
16
12
13
16
Table 2.
Antiinflammatory effects of diclofenac, BC and combination (diclofanac with BC) Diclofenac Alone
B C Alone
Diclofenac with BC
(30mg/kg)
(500mg/kg)
(10mg/kg+500mg/kg)
0
46.25 ± 0.07
35.62 ± 0.03***
21.67 ± 0.02***
1
64.56 ± 0.05
50.76 ± 0.02***
37.49 ± 0.02***
2
77.33 ± 0.06
58.80 ± 0.01***
49.38 ± 0.02***
3
76.23 ± 0.11
67.94 ± 0.02***
59.53 ± 0.02***
4
67.38 ± 0.06
56.65 ± 0.02***
48.83 ± 0.01***
Time (hrs)
Diclofenac standard drug was compared with BC at different time interval and inflammation status was calculated by one-way ANOVA. Significant (P < 0.001) reductions in inflammation were found when compared with mean diclofenac values. Diclofenac was used as a positive control group, and the BC alone as well as combination (BC with diclofenac) values were compared with diclofenac alone.
nutrients such as protein, carbohydrate, vitamin, and minerals as well as certain immune growth factors which are responsible for maintaining the immunity and resistance against various microbes among children and adults. At the same time, colostrum helps eliminating infection and stimulating growth of neonatal gastrointestinal tract [25-27]. Colostrum also contain several enzymatic and non-enzymatic antioxidants that have proven to reduce blood glucose level in diabetic rats [28, 29].
BC has the ability to inhibit the endotoxin secreted by the Gram negative bacteria by neutralizing the lipopolysaccharides due to the presence of lactoferrin, thereby causing bacteriostatic and bactericidal effects [30-33]. The results of this study showed that BCP and standard amoxicillin have varied antimicrobial activity against the test organism (Table 1). The study suggests that the BCP and Amoxicillin are broad spectrum in there activity. This correlates with the observation of previous works that, BC contains vitamins, minerals,
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Yadav et al.
Fig. (2). Anti-inflammatory effects diclofenac alone, BC alone, and combination (diclofenac with BC) of All the group were compared with control group. Control did not have any % inhibition. Hence, it is not seen in the graph. ***P < 0001 when compared with control group.
growth factors and other active constituents which are showing antimicrobial activity [34]. The findings of present study have demonstrated the antiinflammatory activity of BCP along with the standard diclofenac in the rats. Inflammation was using carrageenan which triggers various inflammatory mediators such as histamine, bradykinins, prostaglandins responsible for the induction of inflammation [35, 36]. Carrageenan induces inflammation through the inducible COX-2 pathway [37]. The inflammation reduction observed at 3rd hrs. was highest in the BCP alone group (67.94%). The next highest reduction (59.53%) was found with the combination (BCP with diclofenac) at 3rd hrs. as shown in (Fig. 2). The increased antimicrobial effect could be because of synergistic effects of diclofenac with colostrum. Further study is required to confirm the exact mechanism of synergistic effects.
[2]
[3] [4] [5]
[6] [7] [8] [9] [10]
CURRENT AND FUTURE DEVELOPMENTS BCP depicted potent antimicrobial activity in both Gram -ve and +ve strains of bacteria, while it showed moderate anti-inflammatory activity in pathway responsible for oxidative stress. It was found that the combination (BCP with amoxicillin) produce anti-inflammatory and antimicrobial effects greater than that of BCP alone, which suggests that combination treatment may reduce or minimize the side effects of synthetic drugs. Our results suggest that BCP could be beneficial in targeting the microbial infection and inflammation.
[11] [12]
[13] [14] [15] [16]
CONFLICT OF INTEREST
[17]
The authors confirms that the article contents has no conflicts of interest.
[18]
ACKNOWLEDGEMENTS
[19]
We would like to acknowledge SPPSPTM, SVKM’s NMIMS University Mumbai, for technical support.
[20]
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