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irispublishersagriculture · 3 years ago

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Iris Publishers - World Journal of Agriculture and Soil Science (WJASS)

Antimicrobial Effects of Herb Extracts Against Foodborne Pathogen Listeria monocytogenes in Vitro

Authored by Hua Yang

Listeria monocytogenes is a gram-positive foodborne pathogen that is widely distributed during food preparation, storage, and distribution. A variety of ready-to-eat (RTE) foods such as milk, cheeses, ice cream, raw meat, fresh vegetable and fruits may be contaminated with Listeria monocytogenes [1,2]. Consumption of foods contaminated with L. monocytogenes is linked to an increased risk of listeriosis. To control L. monocytogenes in food products, meat industry uses chemical preservatives such as sodium acetate, sodium lactate and various nitrites. However, it is acknowledged that uses of chemical antimicrobials have increased the consumer concerns and created a demand for “natural” and “minimally processed” food. As a result, there has been a great interest in natural antimicrobial agents.

Plant-derived extracts have been used since ancient times, especially in China [3] and India [4,5]. In addition to the uses as flavoring material, plant extracts and essential oils represent a natural alternative in the nutritional, pharmaceutical, and agricultural fields. Due to their antimicrobial properties, plant extracts have been suggested to be used as antioxidant and preservatives in food products, to incorporate into food packaging materials, plant and crop protectants against insect pests, and medicinal products for human and livestock [6]. In recent times, plant extracts have gained great interests especially in food industry. Most plant extracts are classified as generally recognized as safe by U.S Food and Drug Administration, and are easily degradable in human body [7,8]. Previous studies have been proven that many spices and plant essential oils exhibited inhibition and/or bactericidal effects against L. monocytogenes in food products. For example, cinnamon essential oil and oregano reduced the growth rate of L. monocytogenes by 10% and 19% respectively in ham at 4 °C [9]. Thyme and clove essential oils reduced populations of L. monocytogenes in zero-fat beef hotdogs by 1.3 log CFU/g and 1.0 log CFU/g respectively with 5 min treatment at room temperature (21 °C) [10]. The objective of this study is to evaluate potential inhibitory and bactericidal effects of nine herb extracts (HEs) against foodborne pathogenic L. monocytogenes in vitro in order to select natural antimicrobial agents for the control of foodborne L. monocytogenes in food products.

Introduction

Experimental design

HEs can be used to inhibit the growth of foodborne pathogen and/or reduce pathogen populations. In this study, we conducted three experiments to evaluate their potential uses as antimicrobial agents in food products. In experiment 1 (Exp. 1), a minimum inhibitory concentration (MIC) study was conducted to compare inhibitory effects of each of nine HEs against each of five L. monocytogenes strains individually in Mueller-Hinton broth (MHB). In experiment 2 (Exp. 2), the HE with the lowest MIC was used to determine its reductions of each of five L. monocytogenes strains individually at 37 °C for 30 min in MHB. In experiment 3 (Exp. 3), those HEs which could inhibit the L. monocytogenes growth in Exp. 1 were evaluated for their inhibitory effects against a five-strain L. monocytogenes cocktail in MHB up to 11 days at 12 °C.

Five strains of L. monocytogenes which isolated from epidemics were used in this study and are listed in Table 1. According to [11], these five L. monocytogenes strains were selected from a total of 46 strains which represented a genetic diversity of ribotypes, pulsedfield gel electrophoresis types, serotypes, and lineages. In addition, these five strains are believed to cover the genetic diversity of human disease- associated L. monocytogenes and to provide a valuable tool for evaluating the effectiveness of antimicrobials to inactivate or inhibit L. monocytogenes. Therefore, we used these five genetically distinct strains of L. monocytogenes to evaluate inhibitory and bactericidal efficacies of nine HEs. All strains were activated from 20% glycerol frozen stocks (-80 °C) by two transfers in tryptic soy broth (TSB) (Difco, Spark, MD) at 37 °C for 24 h and were subsequently subculture on tryptic soy agar (TSA) (Difco, Spark, MD) at 37 °C for 24h. Each activated strain was kept on TSA plates at 4 °C.

Herb extracts preparation

A total of nine types of herbs were obtained in the form of powder. Each of nine herbs was extracted with sterile deionized water followed by the procedure of [12] with modification. The HEs were prepared before the day of experiment. Each of the HEs was made by combining 10g of each herb powder with 90 mL of sterile deionized water, incubating in a water bath at 45 °C for 30 minutes, and then boiling for 15 minutes. Each of the nine HEs was then cooled to room temperature and was centrifuged at 6000 x g for 15 minutes at room temperature (Thermo Scientific Sorvall Legend X1R Centrifuge, Am Kalkberg, Germany). The supernatant of each HE was transferred into a 50 mL polypropylene tube and stored at 4 °C until use next day.

Exp. 1: Determining MICs of the HEs

Each strain of the L. monocytogenes listed in Table 1 was inoculated in TSB individually and was incubated at 37 °C for 24h. After the incubation, each strain was serially diluted in MHB (Difco, Spark, MD) to approximately 106 CFU/mL. Nine HEs were diluted with the sterile deionized water to six concentration levels: 100, 60, 30, 15, 10, 5 mg/mL. Five mL of each diluted strain was mixed with 5 mL of each diluted HEs in glass sterile test tubes, to make the final concentrations to be 50, 30, 15, 7.5, 5, 2.5 mg/mL for each HEs and approximately 5 x 105 CFU/mL for each strain. Negative control samples were prepared by combining 5mL of each of nine diluted HEs with 5 mL of MHB separately to make the same final herb concentrations for each HE listed above but without inoculum. Positive control samples were prepared by combining 5mL of each diluted strain with 5mL of MHB separately to make same final bacteria concentrations for each diluted strain listed above but without any HE. All tubes were subsequently incubated at 37 °C for 24h. After 24h incubation, all treatment and control samples were visually examined. The lowest herb concentration at which each treatment sample did not show turbidity were designated as the MIC. All tests were performed in two independent replication trails with three samples on each trail (n=6).

Exp. 2: Reduction of L. monocytogenes cells treated with the HE 4

The HE 4 exhibited inhibitory effect against L. monocytogenes with the lowest MIC in Exp. 1. In this experiment, the HE 4 was determined for its reduction of L. monocytogenes cells. After the 24h incubation, each strain was serially diluted in MHB to approximate concentration of 106 CFU/mL. The HE 4 was diluted in sterile deionized water to the concentration of 50 mg/mL. Two mL of each of diluted L. monocytogenes strains was combined with 2mL of the diluted the HE 4 separately, to make a final concentration of 25 mg/mL of the HE and approximate 5 x 105 CFU/mL of each strain. The positive control samples were prepared by combining 2mL of sterile deionized water and 2mL of each of the five diluted strains separately, to make the same concentrations of each strain as the treatment samples but without HE 4. All treatment and control samples were incubated for 30 min at 37 °C. Our preliminary data showed that the HE 4 exhibited the best reductions against each of five L. monocytogenes strains at 37 °C (data not shown). In a previous published study, thyme and clover have been reported to reduce populations of L. monocytogenes after 5 min treatment in peptone water at room temperature (21 °C) [10]. In our study, each of five strains were treated 30 min with HE 4, which was six times longer than [10]. After 30 min treatment, all treatment samples were immediately diluted with sterile deionized water to a concentration of 0.25 mg/mL for the HE 4, in order to terminate its further antimicrobial activity. Our preliminary study has shown at the concentration of 0.25 mg/mL, the HE 4 could not inhibit the growth of each of five L. monocytogenes strains (data now shown). Each of treatment and positive control samples was subsequently serially diluted in 0.1% buffered peptone water (BPW) and each diluted sample were then plated onto tryptic soy agar (TSA) with two duplications. The TSA plates were then incubated for 48 h at 37oC to enumerate the numbers of surviving L. monocytogenes cells. All tests were performed in two independent replication trails with two samples on each trail (n=4).

Exp. 3: Antimicrobial effects of HEs against L. monocytogenes cocktail at abused refrigerated temperature

In Exp. 1, HEs 2, 4, 5 and 8 which inhibited L. monocytogenes growth at or below 50 mg/mL concentrations. In this experiment, those four HEs were evaluated for their inhibitory effects at 12 °C, which represents the abused refrigeration temperature. Each of the five L. monocytogenes strains listed in Table 1 was cultured in TSB separately for 24h at 37 °C. A five-strain L. monocytogenes cocktail was prepared prior to the study. A 10-mL volume of each 24h grown culture was pooled and mixed in a 50 mL sterile falcon tube. After centrifugation at 6000 x g for 15 min at 4 °C, the supernatant was removed. The cell pellet was washed once with a 10-mL volume of phosphate-buffered saline (PBS), and subsequently resuspended in 50 mL PBS. The L. monocytogenes cocktail was serially diluted in MHB to an approximate 5 x 102 CFU/mL concentration.

The HEs 2, 4, 5, and 8 were diluted in sterile deionized water to three levels of concentrations, 6.25, 3.13 and 1.56 mg/mL. Concentrations of HEs were determined based on the preliminary data (data not shown). The treatment samples were prepared by combining 2 mL of each of four diluted HEs and 2mL of the diluted L. monocytogenes cocktail separately in glass test tubes, to make the final concentrations of each of the four HEs at three levels, 3.13, 1.56 and 0.78 mg/mL, and approximately 2.5 x 102 CFU/mL of the L. monocytogenes cocktail. The positive control samples were prepared by combining 2 mL of diluted L. monocytogenes cocktail and 2 mL of sterile deionized water separately but without any HE. Surviving cells from control samples were enumerated immediately after inoculation (day 0). All treatment and control samples were incubated for up to 11 days at 12 °C. All samples were serially diluted in 0.1% BPW and subsequently plated onto two duplicate TSA plates daily from day 1 to day 5, and every two days from day 7 to day 11. TSA plates were incubated for 48h at 37 °C to enumerate the numbers of surviving L. monocytogenes cells. Each treatment sample and control sample were performed in three independent replication trails with two samples on each trail (n=6).

Statistical Analysis

Microbiological data were converted to log CFU/mL prior to the statistical analysis. Statistical analyses were conducted using analysis of variance via the glimmix procedure of SAS (SAS Studio Basic Edition 3.8, SAS Institute, Inc., Cary, N.C.). Least square means were calculated and significant differences between means were detected at the P < 0.05 in the Exp. 2 and at P < 0.001 in the Exp. 3.

Results and Discussion

MICs of nine herb extracts

MIC is defined as the lowest concentration of an antimicrobial agent which prevents visible microbial growth under designed conditions [13]. In this study, the visible microbial growth was determined by comparing the turbidity between treatment samples and control samples after 24h incubation at 37 °C. The MIC value for each of the nine HEs against each strain are shown in Table 2. Four HEs 2, 4, 5 and 8 inhibited the growth of the five L. monocytogenes strains at MIC values ranging from 5 to 50 mg/mL. The other five HEs 1, 3, 6, 7, 9 did not exhibited inhibition effects at up to 50 mg/ mL. Based on the MIC values, the inhibitory effects of those four HEs were ranked from the strongest to weakest as follows: HE 4 (5 mg/mL) > HE 5 (15 mg/mL) > HE 2 (15-30 mg/mL) > HE 8 (50 mg/mL).

The sensitivity to different natural antimicrobials varies in some Gram-positive and Gram-negative bacteria. For example, studies have shown that Gram-positive L. monocytogenes were more sensitive to some essential oils and HEs than Gram-negative E. coli and Salmonella enterica Enteritidis [14-16]; The Ocimum sanctum extract was found to be equally effective against Gramnegative bacteria (E. coli, S. typhimurium and P. aeruginosa) and Gram-positive bacteria (Staphylococcus aureus) [17]; however, Gram-negative pathogens, V. parahaemolyticus and S. typhimurium, were more sensitive to eugenol than Gram-positive S. aureus [18]. As a result of Exp. 1, four out of nine HEs inhibited the growth of L. monocytogenes. Further studies can be conducted to evaluate and compare the antimicrobial effects of those nine HEs against other foodborne Gram- positive and Gram-negative pathogens.

Reduction of L. monocytogenes cells treated with HE 4

In Exp. 2, the HE 4 was chosen to evaluate its reductions of five L. monocytogenes strains individually at 37 °C for 30 min treatment since HE 4 exhibited the strongest inhibition effect with the lowest MIC (5 mg/mL) in Exp. 1. After 30 min incubation with HE 4 at a concentration of 25 mg/mL, differences (P < 0.05) of surviving cells between treatment samples and control samples were observed for each of five L. monocytogenes strains (Figure 1). Cell reductions of HE 4 against five L. monocytogenes strains were calculated: N1-227 (0.91 log CFU/mL), C1-056 (0.87 log CFU/mL), R2-499 (0.85 log CFU/mL), J1-177 (0.59 log CFU/mL), N3-013 (0.38 log CFU/mL).

In a previous published study, at the concentrations of 0.5 mL/L, essential oils of thyme and clover have been reported to reduce populations of L. monocytogenes from 7.2 to 1.8 log CFU/mL and from 7.1 to 1.2 log CFU/mL respectively after 5 min treatment in peptone water at room temperature (21 °C) [10]. In addition, another study indicated that essential oil of origanum reduced populations of each of five L. monocytogenes strains in a range of 1-2 log CFU/mL after 30 min treatment in 0.9% saline solution at room temperature [19]. In our study, Although HE 4 reduced less than 1 log CFU/mL for each strain, populations of surviving cells of each strain were significant (P < 0.05) after HE4 treatment compared with control samples. The result indicated that using HE 4 solely against L. monocytogenes might be less effective than essential oils of thyme, clover and organum. However, there has been increased interests to the use natural antimicrobial agents in their combinations for controlling foodborne pathogens. The effects of the combined substances were observed to be greater than the sum of individual effects against L. monocytogenes in combinations of carvacrol/linalool [20] and oregano/rosemary [21]. HE 4 was expected to be used in combination with other compounds to increase antimicrobial effects.

Inhibitory effects and reductions of four herb extracts against L. monocytogenes cocktail at abused refrigerated temperature

Since HE 2, 4, 5 and 8 exhibited inhibitory effects against L. monocytogenes at 37 °C in Exp. 1, we expected that those four HEs could inhibit L. monocytogenes growth at 12 °C, which represented to the abused refrigerator temperature. We investigated the antimicrobial effects of HEs 2, 4, 5 and 8 at three concentration levels (3.13, 1.56, 0.78 mg/mL) against a five-strain L. monocytogenes cocktail. The initial populations of L. monocytogenes cocktail in control and all treatment samples were 2.3 log CFU/mL. For control samples without any HE, bacteria population rapidly increased from 2.3 log CFU/mL (day 0) to 8.4 log CFU/mL by 4 days, and then increased to 8.8 log CFU/mL by day 7. After 7 days, bacteria population did not have further increase in number. For treatment samples, the growth of L. monocytogenes during refrigerated storage was dependent on the type of herb and HE concentration. In general, compared with control samples, lower bacteria populations (P < 0.001) were observed in all treatments except for the HE 8 at the concentration of 0.78 mg/mL (Tables 3-5).

At a concentration of 3.13 mg/mL (Table 3), HEs 2, 4, 5 and 8 reduced inoculated L. monocytogenes populations from 2.3 log CFU/mL to 0.2, 0.1, 0.7 and 0.5 log CFU/mL at day 11, respectively. Compared with positive control samples without any HE, each of four HEs had lower bacterial population (P < 0.001) on each day from day 1 to day 11. This result indicated that at the concentration of 3.13 mg/mL, all four HEs effectively reduced bacteria populations of L. monocytogenes cocktail at 12 °C.

Table 3: Least square means ± standard deviation of Listeria monocytogenes cocktail populations in inoculated Mueller-Hinton broth with each of four herb extracts at concentration of 3.13 mg/mL or deionized water (control), stored at 12 °C for 11 days (n=6).

Inhibitory effects and reductions of four herb extracts against L. monocytogenes cocktail at abused refrigerated temperature

At the concentration of 1.56 mg/mL (Table 4), HE 2 reduced L. monocytogenes populations from 2.3 log CFU/mL to 0.2 log CFU/ mL at day 11, which was 8.6 log CFU/mL lower (P < 0.001) than the control. Although counts of L. monocytogenes in the sample with HE 5 increased from 2.3 log CFU/mL to 4.0 log CFU/mL at day 11, it was still 4.8 log CFU/mL lower (P < 0.001) than the control. However, compared with the control, HE 4 and 8 were lower (P < 0.001) in bacteria populations only up to 5 days. Therefore, antimicrobial effects of those four HE at concentration of 1.56 mg/ mL were ranked from the strongest to weakest as follows: HE 2 > HE 5 > HE 4 = HE 8.

Table 5 shows the inhibitory effects of each of the four HEs in MHB at the concentration of 0.78 mg/mL. Counts of the L. monocytogenes cocktail in the sample with HE 8 were not different (P > 0.001) from the control sample on each day from day 1 to day 11, indicating that HE 8 at a concentration of 0.78 mg/mL could not inhibit bacterial growth. Counts of samples with HE 4 or 5 increased from 2.3 log CFU/ mL to 6.7 and 6.4 log CFU/mL by day 4 respectively, which were lower (P < 0.001) than the control by about 2 log CFU/mL. After 4 days of incubation, the bacterial population of the sample with HE 4 were not different (P > 0.001) with the positive control sample on each day from day 5 to day 11. Although the sample with HE 5 did not show different (P > 0.001) in bacteria population with the positive control sample at day 5 and day 7, the population of HE 5 was 0.5 log CFU/mL lower (P < 0.001) than the control at day 9 and day 11. In addition, comparing with positive control samples, HE 2 slowed the microbial growth and reached to 5.6 log CFU/mL by day 5, which was lower than the controls for 3 log CFU/mL (P < 0.001). After 7 days of incubation, the bacterial population of the sample with HE 2 were not different (P > 0.001) with the control sample on each day from day 7 to day 11. In summary, at the concentrations of 0.78 mg/mL, HE 2 inhibited the microbial growth up to 5 days; HE 4 and 5 inhibited L. monocytogenes growth up to 4 days; HE 8 could not inhibit the microbial growth.

The demand for convenience foods such as RTE foods has increased in recent years. The majority of listeriosis cases are foodborne [22] and linked to the consumption of RTE foods which are contaminated with L. monocytogenes. Due to the high mortality rate of listeriosis, the U.S. Department of Agriculture and the FDA labels L. monocytogenes as an adulterant of RTE foods. Examples of RTE foods that support the growth of L. monocytogenes are milk, high fat dairy products, soft unripened cheese, cooked and raw seafood, deli-type salads, sandwiches, fresh-cut vegetable and fruits [23] and the processed meat which is under refrigerator conditions [24]. Although L. monocytogenes will continue to thrive at low temperature as 1.1 °C [25] the storage temperature and duration of refrigerated storage before consumption are important factors which reduce the risks of foodborne listeriosis [26]. The recommended refrigerator temperature is 40 °F (4.4 °C); however, abuse home refrigerator temperature can rise to more than 12 °C [26,27].

Previous published studies indicated that the inhibitory efficacies of plant-derived antimicrobials may be affected by temperature [28,29]. The results from Exp. 1 showed that HEs 2, 4, 5 and 8 exhibited inhibitory effects against each of five L. monocytogenes strains at 37 °C. However, in order to use those four HEs as food preservatives, they must be effective against L. monocytogenes under food storage conditions. In this experiment, inhibition efficacies of those four HEs were evaluated at 12 °C which represented the abused refrigerator temperature. As discussed above, at concentrations of 1.56 and 0.78 mg/mL, HEs 2, 4, 5 and 8 inhibited growth of a five-strain L. monocytogenes cocktail at abuse refrigeration temperature of 12 °C, except herb extract 8 at the concentration of 0.78 mg/mL. At a concentration of 3.13 mg/mL, these four HEs reduced cell populations in a range of 2.2 to 1.6 log CFU/mL at 11 days. In a previous study, thyme essential oil showed the inhibitory effect against L. monocytogenes cocktails at 10 °C up to 12 days in minced beef [30]. HEs 2, 4, 5 and 8 were also expected to be developed into food preservatives for inhibiting and/or reducing foodborne L. monocytogenes. For example, those four HEs could be added to RTE foods as supplements or incorporated into food packaging materials to control L. monocytogenes growth. Further experiments should be conducted to determine the inhibitory effects and reductions of those four HEs in food products. In addition, since HEs carry specific odor, palatability of the food applied with HEs should be evaluated by sensory panel.

Conclusion

In summary, HEs 2, 4, 5 and 8 exhibited inhibitory effects against L. monocytogenes at 37 °C in a range of MIC between 5 - 50 mg/mL. HE 4 reduced cell populations of each selected strain ranged between 0.38 -

0.91 log CFU/mL after 30 min treatment at 37 °C. In addition, at concentrations of 1.56 and 0.78 mg/mL, HEs 2, 4, 5 and 8 inhibited growth of a five-strain L. monocytogenes cocktail at 12 °C, except the HE 8 at the concentration of 0.78 mg/mL. At a concentration of 3.13 mg/mL, these four HEs reduced cell populations in a range of 2.2 to 1.6 log CFU/mL at 11 days. For their practical application, further experiments should be conducted to determine the inhibitory effects and reductions of those HEs in a variety of food products. In addition, palatability of the foods which applied with HEs should be evaluated by sensory panel.

#agriculture #Agriculture and Soil Science #journal of agriculture #Inter National Agriculture Science #soil science #Food science

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reporterpiner · 2 years ago

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Lzip pgc1a

Membrane Blocking and Antibody Incubations Electrotransfer to nitrocellulose membrane ( #12369).Ĭ.NOTE: Loading of prestained molecular weight markers ( #59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder ( #7727, 10 µl/lane) to determine molecular weights are recommended. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm). Heat a 20 µl sample to 95–100☌ for 5 min cool on ice.Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate).Aspirate media from cultures wash cells with 1X PBS aspirate.Treat cells by adding fresh media containing regulator for desired time.Protein Blotting A general protocol for sample preparation. Detection Reagent: SignalFire™ ECL Reagent ( #6883).ī.Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody ( #7074).Pore size 0.2 µm is generally recommended. Blotting Membrane and Paper: ( #12369) This protocol has been optimized for nitrocellulose membranes.Blue Prestained Protein Marker, Broad Range (11-250 kDa): ( #59329).Biotinylated Protein Ladder Detection Pack: ( #7727).Primary Antibody Dilution Buffer: 1X TBST with 5% BSA for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.10X Tris Buffered Saline with Tween ® 20 (TBST): ( #9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH 2O, mix.10X Tris-Glycine Transfer Buffer: ( #12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH 2O, mix.10X Tris-Glycine SDS Running Buffer: ( #4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH 2O, mix.1X SDS Sample Buffer: Blue Loading Pack ( #7722) or Red Loading Pack ( #7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.10X Tris Buffered Saline (TBS): ( #12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH 2O, mix.20X Phosphate Buffered Saline (PBS): ( #9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2O, mix.NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water. Solutions and Reagentsįrom sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit NOTE: Please refer to primary antibody product webpage for recommended antibody dilution. For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween ® 20 at 4☌ with gentle shaking, overnight.

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moleculardepot · 3 years ago

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Phosphate Buffered Saline with Tween (10X)

Phosphate Buffered Saline with Tween (10X) Catalog number: B2010773 Lot number: Batch Dependent Expiration Date: Batch dependent Amount: 50 mL Molecular Weight or Concentration: 10X Supplied as: Solution Applications: PBS formulation with Tween 20 supplemented with Tween 20, useful for various immunoassays and cell cultures procedures Storage: RT Keywords: PBST, PBS Tween 20 Grade: Biotechnology…

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purmabiologic · 3 days ago

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D-PBS For Efficient Maintenance of Cell Cultures

PurMa Biologics LLC's D-PBS provides a flexible option for cell culture and washing processes. During experimental operations, this product enhances cell survival by maintaining osmotic pressure and pH balance. For dependable use in a range of biological research applications, PurMa Biologics LLC offers premium D-PBS. For more information visit:- https://www.purmabiologics.com/product/dulbeccos-phosphate-buffered-saline-dpbs/

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annieboltonworld · 3 years ago

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Juniper Publishers-Open Access Journal of Environmental Sciences & Natural Resources

Evaluation of Nutrient Stress (Nitrogen, Phosphorus Regimes) on Physio-Biochemical Parameters of Oleaginous Micro algal Strains and SEM Study under Nutrient Stress

Authored by Kulvinder Bajwa

Abstract

The objective of this study was to investigate the accumulation of lipid, biomass, photosynthetic pigment, protein and carbohydrates from various micro algal strains. Algal cultures were grown in BG-11 medium inoculated into nitrogen (N) and phosphorus (P) en rich and stress medium. Three different treatments were set up: N+P+ (control group); (N-P+); (N+P-) respectively for algal growth evaluation in the form of lipid accumulation, biomass yield, protein, carbohydrate and total chlorophyll contents. When the cells were grown in BG-medium under nutrient stress (N,P) for 12 days, in (N+P+) nutrient regime, significant (P≤0.05) higher biomass yield 1.129±0.036 gL-1 and 1.115±0.021gL- 1 have been reported in Nannochloropsis oculata, Chlorella pyrenoidosa respectively. Interestingly, nitrogen deficiency condition promoted (P≤0.05) significant higher lipid accumulation 25.75%, 23.78% and 20.26% in Nannochloropsis oculata, Chlorella pyrenoidosa and Scenedesmus obliquus respectively as comparison was made with other nutrient stress. On the other hand, (nitrogen+ phosphorus) condition promoted higher chlorophyll, carbohydrates and protein content in almost all algal species.SEM study also conducted under normal and stress condition for these micro algal strains (Chlorella, Scenedesmus, Nannochloropsis and Chlorococcum). Results showed, cells wall of algal species smooth under normal condition, while under stress condition distorted cell morphology. Thus, this native micro alga strain could be a potent candidate for feed, food or bio fuel production.

Keywords: Microalgae; Nutrient Stress; Biomass; Lipid; Sem; Oleaginous Algae; Nitrogen; Phosphorus Deficiency; Biomass; Lipid; Scanning Electron Microscopy

Introduction

As global population and consequently energy demand increase over time the introduction and commercialization of renewable sources of energy becomes a critical issue. Microalgal biomass as feedstock for bio-energy production is an attractive alternative to bio-energy derived from terrestrial plant utilization [1,2]. Micro algal biotechnology has gained increasing attention over the last few decades as a next- generation driver for obtaining food, feed and bio fuels and to carry out bioremediation of effluents and CO2 mitigation [3]. Oleaginous microalgae are well known as promising candidates for renewable energy production mainly because of high biomass productivity and lipid content Chisti [4,5]. Microalgae, cultivated under specific stress conditions, can accumulate, along with the lipids and carbohydrates, considerable amount of secondary metabolites, whose industrial exploitation strongly enhances a bio-based economy [6].

Nitrogen and phosphorus, as the two main nutrients, are hypothesized to influence the attachment efficiency and growth of microalgae [7,8]. Similarly, phosphorus is an essential nutrient for the growth of microalgae as it plays a significant role in cellular metabolic processes related to energy transfer, signal transduction, photosynthesis and respiration. Studies have shown that the phosphorous deprived conditions responsible for significant lipid accumulation in Chlorella spp. Chaetoceros spp. Phaeodactylum tricornutum, Isochrysis galbana and Pavlovalutheri [9-13]. In addition to these factors, nitrogen deficiency severely affects protein synthesis and reduces photosynthetic rates which result in metabolic flux towards lipid biosynthesis [14,15]. High lipid accumulation was reported under nitrogen deprived conditions in microalgal species viz., Neochloris oleoabundans, Nannochloris sp., Chlorella muelleri and Scenedesmus sp. [16-19]. According to Li, et al. [20] phosphorus strongly influenced Chlorella vulgaris growth but has little influence on lipid accumulation. Anand, et al. [21] revealed that the 2.27-fold increase in lipid yield (226 mg/L) was observed in nitrogen-depleted condition when compared to nitrogen rich condition (99.33 mgl-1). The present study aimed at to evaluate the nutrient stress of Nitrogen and phosphorus, simultaneously scanning electron microscopy study of various micro algal species viz. Chlorella, Nannochloropsis, Chlorococcum, Scenedesmus sp. Under nutrient stress.

Materials and Methods

Collection of Water Samples Having Algal Growth

The water samples having algal growth were collected in pre cleaned sterilized plastic containers from different fresh water bodies located in Haryana, Punjab, Rajasthan and Uttarakhand. Marine water samples were collected from Mumbai, Maharashtra.

Isolation and Molecular Characterization

The freshwater micro algal species was isolated from the freshwater pond at Shahidawaali village, Dist. Sirsa (Haryana) India. Genomic DNA from micro algal sample was extracted by using cetyl tri methyl ammonium bromide (CTAB) method Scott & Bendich [22]. Polymerase chain reaction (PCR) was analyzed to amplify 18S rRNA gene of microalgae using forward (5”GGGAC C C GTTAC C GTAGGTGAAC CTGC-3”) and reverse primers (5”-GGGATCCATATGCTTACGTTCCGCGGAT-3”). The purified PCR products were sequenced by Amnion Biosciences Pvt. Ltd. (Bangalore, India). Comparisons of nucleotide sequences and statistical significance of matches were carried out with the National Centre for Biotechnology Information (NCBI) nucleotide BLAST program.

Analytical Methods for Physio-Biochemical Parameters

Bligh and Dyer Lipid Extraction Method: The extraction total lipid were carried out by mixing methanol-chloroform (2:1.5 v/v) with the algal samples using slightly modified version of Bligh and Dyer's method Bligh & Dyer [23]. According to Suganya and Renganatha [24] (the oil extraction yield (%w/w) was determined by the following formula:

Dry Biomass estimation: Dry cell biomass was measured as the cell density (dcw) at OD625 of an 11 day old culture at dilutions ranging from 0.2 to 1.0. The dry biomass was calculated using the regression equation as the linear relationship [25].

y = 0.137x + 0.1766, R2 = 0.9859

Extraction and Determination of Photosynthetic Pigment: Chlorophyll content of the algae was estimated spectrophotometrically at 650 and 665 nm. The concentration of chlorophyll was calculated using the formula:

Total chlorophyll (mgmU1) = 2.55 x 10-2E650 + 0.4 x 10-2E665 x 103

Extraction and Determination of Total Soluble Carbohydrate by Anthrone Reagent: Glucose was determined at 625 nm using Anthrone reagent method by Dubois et al. [26]. The sugar content was calibrated against standard curve prepared by using graded conc. of glucose dilution ranging from 0.2 to 1 and expressed in terms of mg ml-1

y = 0.636x + 0.0592, R2 = 0.9595

where y, concentration of glucose, x optical density.

Total Protein Estimation by Lowry Method: The protein content was estimated using Lowry's method. Protein concentration was calculated from the standard curve prepared with bovine serum albumin (BSA) [27].

y = 0.1097x - 0.0005, R2 = 0.9989

Effect of nitrogen and phosphorus stress on physio- biochemical parameters of screened algal strains

To evaluate the ability of screened algal strains to accumulate lipid under phototrophic conditions, screened algal cultures were grown in BG-11 medium inoculated into nitrogen (N) and phosphorus (P) enrich and stress medium. Three different treatments were set up: N+P+ (control group); (N-P+); (N+P- ) respectively for algal growth evaluation in the form of lipid accumulation, biomass yield, protein, carbohydrate and total chlorophyll contents. All the experiments were conducted in triplicate over a cultivation period of 12 days.

Scanning Electron Microscopy (SEM)

In the present study, morphological features and other cellular details of screened algal under nutrient stress (nitrate and phosphate deficient condition) were studied with the help of Scanning Electron Microscope (Carl Zeiss, Model no. SMT EVO 50SEM) as method described by Fowke et al. [28]. Bacterial and algal broths were centrifuged and washed the pellets with phosphate buffer saline for three times and collected the pellet by centrifugation. The fundamental steps for SEM sample preparation are fixing of samples in 0.25% buffered glutaraldehyde (in Sodium phosphate having pH 7.2) and incubated at room temperature for 30 minutes, then freeze dried for 24 hrs, after that fixing is done using tetra oxide of osmium, samples dehydration by different ethanol grading starting; 30%, 50%, 70%, 80%, 90% and 100% and for each ethanol volume incubate for 10 minutes then incubation in 100% ethanol for 1 hour, drying with air dryer, placed in desiccators until constant weight attain, mounting it on stubs using double sided sticky tape coated with carbon. Preparation of SEM stub by applying the adhesive tape and then adding the dried bacterial and algal samples on the tap. The exposed surface was coated with gold with the help of sputter coater device and then the inner surface was scanned at 20 kV potential and various magnifications.

Result and Discussion

Morphological and Molecular Identification of Micro Algal Isolates

Purified algal species were preliminary identified with the help of algal identification guide on the basis of morphological features by using Olympus (CX41) light microscope equipped with digital camera. Microscopic images of these algal strains under (100 x) magnification are depicted in (Figure 1) 18S rRNA sequences of screened algal strains were aligned with global sequence available in Gen bank (NCBI) using the standard nucleotide -nucleotide basic local alignment search tool (BLAST) programme. Sequences alignment outcomes revealed that screened algal strains were exhibiting 100% homology with Chlorococcum aquaticum (Accession No. KT961379), Scenedesmus obliquus (Accession No. KT983434), Nannochloropsis oculata (Accession No. KU160538), Chlorella pyrenoidosa (Accession No. KU236002).

Effects of Nutrient Stress (Nitrogen, Phosphorus Regimes) on Physio-Biochemical Parameters of Algal Strains

To evaluate the ability of screened algal strains to accumulate lipids under nutrient stress conditions, algal cultures were grown in full BG-11 medium were inoculated into nitrogen (N) and phosphorus (P) enrich medium, respectively, with significant (P≤0.05) higher biomass, protein, carbohydrate and total chlorophyll as given in (Figures 2A-2E). Three different treatments were set up: N+P+ (control group); N-P+ (nitrogen deficiency); N+P- (phosphate deficiency). Anova table suggested that significant (P≤0.05) higher lipid content was observed in nitrogen deficiency condition in four algal strains as shown in (Figure 2A). In (N+P+) nutrient regime, significant (P≤0.05) higher biomass yield 1.129±0.036 gL-1 and 1.115±0.021 gL-1 have been reported in Nannochloropsis oculata, Chlorella pyrenoidosa respectively as compared to Scenedesmus obliquus, Chlorococcum aquaticum (Figure 2 B).

The lipid percentage also slightly increases in (N+P+) condition in comparison to other nutrient stress condition in four algal strains. Interestingly, nitrogen deficiency condition promoted (P≤0.05) significant higher lipid accumulation 25.75%, 23.78% and 20.26% in Nannochloropsis oculata, Chlorella pyrenoidosa, and Scenedesmus obliquus respectively as comparison was made with other nutrient stress. Similar to biomass yield, (N+P+) regimes condition also responsible for significant (P≤0.05) higher protein content in Nannochloropsis oculata (0.062±0.005 mgmL-1) and Chlorella pyrenoidosa (0.068±0.003 mgmL-1) respectively (Figure 2D) illustrated that Scenedesmus obliquus, Chlorella pyrenoidosa showed significant (P≤0.05) higher carbohydrate content in (N+P+) condition as compared to nitrogen and phosphorus deficient media. In case of total chlorophyll content, nitrogen + phosphorus condition promoted higher chlorophyll content in Chlorococcum aqauticum (13.02±0.037 ngmL-1) Chlorella pyrenoidosa (13.68±0.029 ngmL' 1) as compared to Nannochloropsis oculata, Scenedesmus obliquus as shown in (Figure 2E).

Anand, et al. [21] revealed that the 2.27-fold increase in lipid yield (226 mg/L) was observed in nitrogen-depleted condition when compared to nitrogen rich condition (99.33mgL-1). In this study, it was validated that four algal strains were able to accumulate large quantity of lipid and reached the highest lipid content (25.75%) in Nannochloropsis oculata under N deficiency, which was in agreement with previous report that N-deficiency was an efficient prompted to induce lipid accumulation (particularly triacylglycerols) in many microalgae [29]. It is known that the different nitrogen sources and levels were effective on the growth of microalgae and biochemical composition [30-34]. Our study also showed that P-deficiency was a suitable condition for lipid accumulation in screened experimental cultures as well. This observation is similar to prior reports [35] who proposed that lipid storage in Monodus subterraneus can be increased by P deficiency. Similarly Feng, et al. [36] found that the lipid contents of Chlorella zofingiensis grown in media deficient of nitrogen (65.1%) or phosphate (44.7%) were both higher than that obtained from cells grown in full medium (33.5%).

Usually, the nitrogen deficiency would result in more metabolic flux and to lipid accumulation in algae cells as the synthetic rate of essential cell structures including proteins and nucleic acids lowered Li et al. [37] Kirrolia et al. [38]. According to Li et al., phosphorus strongly influenced Chlorella vulgaris growth but has little influence on lipid accumulation as we found in our study. Higher protein content reported in our study with (N+P+) condition, similar finding has been revealed by Mutlu et al. [39] and found significantly higher protein content in Chlorella vulgaris in nitrogen and phosphorus rich condition. Dortch et al. [40] also observed that the proteins associated with the chlorophyll-protein complex decreased in nitrogen starved cultures. Smit et al. [41] reported a positive relationship between protein and chlorophyll a. Chlorophyll a is one of the most important nitrogen pools in algae: the pigment may reduce nitrogen limitation. Similar to our study, observed that chl a content of Chlorella vulgaris decreased and also, a yellowish colour was recorded under N-starvation condition.

Scanning Electron Microscopy

Scanning electron micrographs of four algal species of Chlorellapyrenoidosa, Chlorococcum aquaticum, Nannochloropsis oculata and Scenedesmus obliquus under normal as well as stress conditions were taken at potential of 20 kV and under various magnifications. Scanning electron micrographs of Chlorella pyrenoidosa having cell size 2μm revealed that cells of Chlorella pyrenoidosa in normal stage was smooth and compacted as well as covered with irregular network of subtle ribs (Figure 3 A,B) whereas in nutrient stress conditions cells become dispersed with rough cell wall (Figures 3C & 3D). Under nutrient stress no longer smooth surface of algal cells walls and outer region was irregular and cell wall roughly folded. Similar to our present work Kirrolia also found striking changes in cell morphology in Chlorella sp. under nutrient stress condition. Similar to our findings, [42] observed smooth cell wall in scanning electron micrographs of Chlorella sp. in normal condition but cell wall of Chlorella species no longer remained smooth after absorption of metal ions Cu+2 and Ni+2 . Similarly Scanning electron micrographs of Scenedesmus obliquus under normal and stress condition showed characteristics colonies of cells, usually round in shape with prominent nucleus. Under normal conditions, Scenedesmus obliquus cells are compactly arranged in two or four cells and are non-fragmented wheareas, under stress conditions there is fragmentation and separation of Scenedesmus cells (Figure 4 A-D). Kirrolia [43] also observed distorted morphology under stress condition in Scenedesmus quadricauda. Chlorococcum aquaticum is green microalgae having cell size 2 μm round elongated shape with smooth lines over cells walls in normal condition, whereas in stress conditions cell walls distorted with no smooth coverage of fine lines on cell wall (Figures 5A-5D).

Nannochloropsis oculata showed intact structure with no cell lyses. Whereas in stress condition in normal condition, disrupted morphology of micro algal structure and appeared completely broken cells under Scanning Electron Microscope (Figures 6A- 6D). Similar results have been found in Nannochloropsis oculata in normal condition. In addition, acid treatment 1M HCl totally disrupted the morphology of micro algal structure appearing completely broken cells under Scanning Electron Microscope Surendhiran and Vijay [44-46].

Conclusion

Nutrient stress variables for enhancement of micro algal performance towards sustainable biodiesel synthesis could be effectively optimized in (N+P+) nutrient regime; significant (P≤0.05) higher biomass yield 1.129±0.036 gL-1 and 1.115±0.021gL-1 have been reported in Nannochloropsis oculata, Chlorella pyrenoidosa respectively. Interestingly, nitrogen deficiency condition promoted (P≤0.05) significant higher lipid accumulation 25.75%, 23.78% and 20.26% in Nannochloropsis oculata, Chlorella pyrenoidosa and Scenedesmus obliquus respectively as comparison was made with other nutrient stress. On the other hand, (nitrogen+phosphorus) condition promoted higher chlorophyll, carbohydrates and protein content in almost all algal species. It is meaningful to examine the cellular morphology to further understand the cell disruption under nutrient stress. Scanning electron micrographs (SEM) was found to be efficient tool for characterization of change in cell morphology under normal and stress condition in selected indigenous algal strains. The cellular morphology of micro algal strains was investigated by scanning electron microscope (SEM) which certified that the cells damage was caused by both nitrogen and phosphorus stress.Nutrient stress variables for enhancement of micro algal performance towards sustainable biodiesel synthesis could be effectively optimized in (N+P+) nutrient regime; significant (P≤0.05) higher biomass yield 1.129±0.036 gL-1 and 1.115±0.021gL-1 have been reported in Nannochloropsis oculata, Chlorella pyrenoidosa respectively. Interestingly, nitrogen deficiency condition promoted (P<0.05) significant higher lipid accumulation 25.75%, 23.78% and 20.26% in Nannochloropsis oculata, Chlorella pyrenoidosa and Scenedesmus obliquus respectively as comparison was made with other nutrient stress. On the other hand, (nitrogen+phosphorus) condition promoted higher chlorophyll, carbohydrates and protein content in almost all algal species. It is meaningful to examine the cellular morphology to further understand the cell disruption under nutrient stress. Scanning electron micrographs (SEM) was found to be efficient tool for characterization of change in cell morphology under normal and stress condition in selected indigenous algal strains. The cellular morphology of micro algal strains was investigated by scanning electron microscope (SEM) which certified that the cells damage was caused by both nitrogen and phosphorus stress.Nutrient stress variables for enhancement of micro algal performance towards sustainable biodiesel synthesis could be effectively optimized in (N+P+) nutrient regime; significant (P<0.05) higher biomass yield 1.129±0.036 gL-1 and 1.115±0.021gL-1 have been reported in Nannochloropsis oculata, Chlorella pyrenoidosa respectively. Interestingly, nitrogen deficiency condition promoted (P<0.05) significant higher lipid accumulation 25.75%, 23.78% and 20.26% in Nannochloropsis oculata, Chlorella pyrenoidosa and Scenedesmus obliquus respectively as comparison was made with other nutrient stress. On the other hand, (nitrogen+phosphorus) condition promoted higher chlorophyll, carbohydrates and protein content in almost all algal species. It is meaningful to examine the cellular morphology to further understand the cell disruption under nutrient stress. Scanning electron micrographs (SEM) was found to be efficient tool for characterization of change in cell morphology under normal and stress condition in selected indigenous algal strains. The cellular morphology of micro algal strains was investigated by scanning electron microscope (SEM) which certified that the cells damage was caused by both nitrogen and phosphorus stress.Nutrient stress variables for enhancement of micro algal performance towards sustainable biodiesel synthesis could be effectively optimized in (N+P+) nutrient regime; significant (P≤0.05) higher biomass yield 1.129±0.036 gL-1 and 1.115±0.021gL-1 have been reported in Nannochloropsis oculata, Chlorella pyrenoidosa respectively. Interestingly, nitrogen deficiency condition promoted (P≤0.05) significant higher lipid accumulation 25.75%, 23.78% and 20.26% in Nannochloropsis oculata, Chlorella pyrenoidosa and Scenedesmus obliquus respectively as comparison was made with other nutrient stress. On the other hand, (nitrogen+phosphorus) condition promoted higher chlorophyll, carbohydrates and protein content in almost all algal species. It is meaningful to examine the cellular morphology to further understand the cell disruption under nutrient stress. Scanning electron micrographs (SEM) was found to be efficient tool for characterization of change in cell morphology under normal and stress condition in selected indigenous algal strains. The cellular morphology of micro algal strains was investigated by scanning electron microscope (SEM) which certified that the cells damage was caused by both nitrogen and phosphorus stress.

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Biomed Grid | Cell Proliferative and Cell Cycle Effects of Atrazine Using Human Breast Cell Lines

Abstract

Breast cancer is one of the leading causes of cancer related deaths in women. Several risk factors can increase breast cancer occurrences and strong evidence has shown that exposure to endocrine disrupting chemicals including atrazine can lead to breast cancer etiology. This study examined the effect of low level/environmentally relevant concentrations of atrazine on two breast cell lines (MCF-7, an estrogen responsive breast cancer cell line and MCF-10A, a non-cancerous human breast epithelial cell line) by studying cytotoxicity, proliferation and cell cycle events. To study cytotoxicity, cells were exposed to atrazine within a concentration range of 0.315 μg/L to 100,000 μg/L for 96 h and cell proliferation as well as the LC50 were calculated at 24, 48,72 and 96 h using the RT-CES. Cell cycle was studied by exposing the cell to 3 μg/L atrazine, and nuclei was isolated and analyzed using a BD FACSCalibur. The percentage nuclei in the cell cycle was calculated using Modfit LT 3.0. Results showed that there was an overall decreased in LC50 over time in both cell lines. Exposure of MCF-7 and MCF-10A to 3 μg/L of atrazine for 96 h, stimulated an increase in cell proliferation compared to control. Lower concentrations of atrazine showed even stronger impact on cell proliferation. However, ATR induced more cell proliferation in MCF-7 than in MCF-10A. Significant increases were recorded in the percent nuclei in G1 and G2 phases of MCF-7 cells exposed to ATR. Increase in the percent nuclei was only recorded in the G1 phase in MCF- 10A cells exposed to ATR. This adds to the body of evidence that ATR may indeed play a significant role in the onset and progression of cancer.

Keywords: MCF-7; MCF-10A; Atrazine; Breast Cancer

Introduction

Breast cancer is the most frequent cancer among women, and it impacts 2.1 million women each year [1]. It is estimated that 627,000 women died from breast cancer (approximately 15% of all cancer deaths in women) in 2018 [1]. Although breast cancer occurrence is rare among men, it is estimated that 2,670 will be diagnosed with breast cancer in 2019 [2]. The etiology of breast cancer has been linked to several factors including family history/ hereditary of BRCA1 and BRCA2 genes, hormone replacement therapy, obesity and alcohol [3-5]. In addition, studies have shown that ovarian hormones, including estrogen and progesterone, may increase breast cancer risk by affecting rates of cell proliferation in the breast or by supporting enhanced cellular growth induced by estrogen dependent tumors [6]. Furthermore, exposure to environmental pollutant including endocrine disruptors chemicals (EDC) have been implicated as potential risk factor in developing breast tumors [7].

Environmental pollutants including herbicides have been shown to affect cell proliferation and therefore lead to the onset of diseases [8,9]. Low level herbicide such as atrazine (ATR) exposure can result in many adverse effects causing DNA damage, chromosomal aberration, cell cycle perturbation, as well as reproductive and developmental alteration [8,10,11]. Atrazine (2-chloro-4- ethylamino-6-isopropyl-amino-s-triazine) is a ubiquitous broadspectrum herbicide frequently used in corn and soy fields. ATR contaminates water sources via agricultural runoffs (Solomon et al., 1996). More than thirty-four million kg of ATR are applied each year in the United States. Even though the U.S. Environmental Protection Agency (EPA) enforces a maximum contaminant level (MCL) of ATR at 3.0 μg/L (3 ppb), studies have revealed that ATR concentrations in some areas have exceeded the MCL in public drinking water sources, and the concentration in groundwater could range from below3 μg/L to as high as 700 μg/L [12-14]. In 2003 the European Union banned the use of ATR because of its prevalence in drinking water contamination; however, the EPA permitted its continued use in the US that same year [15].

ATR is believed to be a potent endocrine disruptor [16,17] that can cause demasculinization and feminization [18] and can also induce adverse effects on different biological systems. For example, ATR can affect the reproductive system [19,20] the central nervous system [21], and the immune system [22] Since ATR exposure has been associated with cancer development, increased cell proliferation is an expected outcome after exposure as demonstrated in human intestinal epithelial cells [23]. In fact, a study by [24] showed that exposure to ATR induced cell proliferation in human BG-1 and 2008 ovarian cancer cells in a concentration dependent manner. Epidemiological studies have also related increased risk of ovarian cancer in female farm workers in Italy [25] and increased risk of breast cancer in the population of Kentucky to ATR exposures [26]. ATR does not bind or activate the classical estrogen receptor (ER) [24], but scientists have speculated other estrogen related pathways. For example, ATR may induce aromatase (CYP19) activity, converting testosterone and related hormones to estrogens, thus it increases estrogen levels [27]. Other studies have contradicted the endocrine disrupting effects of ATR. Studies funded by Syngenta concluded that there was no causal relationship between exposures to ATR and development of breast cancer [28]. Contradictory results were also found in another study in which higher levels of mixed pesticides, including ATR, were associated with increased breast cancer in one rural county in the UK, but not in a nearby county [29]. ATR is also known to induce alterations in normal cell cycle progression. Studies by Freeman and Rayburn [30] reported that there was a decrease in cells in the G2 phase when Chinese Hamster Ovary (CHO) cells were exposed to ATR [31]. also saw similar results when Hep2G cells were exposed to ATR.

Even though a few researchers have studied the Proliferative effects of ATR using different cell lines, studies of the effects of ATR on cell cycle in human breast cell lines are scarce. Since endocrine disruptors like ATR act like natural hormones which are available in low doses, this study explored the effect of low level/ environmentally relevant concentrations of ATR on two human breast cell lines (MCF-7, an estrogen responsive breast cancer cell line and MCF-10A, a non-cancerous human breast epithelial cell line) by studying cytotoxicity and cell cycle events. The study will help scientists understand whether ATR will increase the risk of women who have already been diagnosed with breast cancer and those who have not.

Material and Methods

Reagents

MCF-7 and MCF-10A cell lines were purchased from American Type Culture Collection (ATTC, Manassas, VA, USA). Minimum Essential Medium (MEM) alpha 1x, Dulbecco’s Phosphate Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), MEM without phenol, and penicillin streptomycin were purchased from GIBCO Invitrogen (Grand Island, NY, USA). Trypsin-EDTA and fetal bovine serum (FBS) were purchased from ATTC. All other supplements were purchased from Cambrex Bio Science. Walkersville, Inc., USA.

Cell Culture

Both MCF-7 and MCF-10A cell lines were incubated at 37°C in 5% CO2 and humidified atmosphere. When MCF-7 cells reached 75-80 % confluency, they were washed with phosphate buffer saline (PBS), trypsinized with 3 ml of 0.25% w/v trypsin, 0.53 mM EDTA and incubated at 37C for 5 minutes. For MCF-10A cells, 3.0 ml of a 0.05% trypsin, 0.53 mM EDTA solution was added and cells were incubated at 37C for 15 minutes. At the end of the incubation period, 3 ml of a solution of 0.1% soybean trypsin inhibitor was added to the MCF10 A to neutralize the trypsin. Both cells types were centrifuged at 3000 rpm for 2 minutes at 4°C. Both cell lines were diluted with MEM, washed with saline and centrifuged at 5000 rpm for 5minutes at 4°C. Cells were counted with Beckman counter and used for exposure assays.

Description of Exposure Studies

Real-Time Cell Electronic Sensing (RT-CES): Cell viability and cytotoxicity effects of ATR on MCF-7 and MCF-10A cells were determined by RT-CES assay (ACEA Biosciences Inc., San Diego, CA, USA). Cell viability was monitored every 10 min for 96 h by the detection of cell impedance as a measure of cell number morphology and adherence. Continuous recording of impedance in cells was reflected by cell index (CI) value which corresponds to cell growth [31].

Cytotoxicity of ATR on MCF-7 and MCF-10A cell lines Using RT-CES: To determine the cytotoxicity of the ATR, MCF-7 and MCF-10A cells were seeded in a 16x E-plate device and grown in the incubator. After 24 h, the cells were treated with ATR. Serial dilution as described by [9] were followed. To create a negative control, the last row of cell culture plate contained the media and cells but was not exposed to ATR. Cells were exposed to ATR within a concentration range of 0.315 μg/L to 100,000μg/L for 96 h and the lethal concentration at which 50% of the cells died (LC50) was calculated at 24, 48,72 and 96 h. In addition, CI was derived to represent cell status based on measured electrical impedance. A rise in CI (rise in electrical impedance) indicated cell proliferation and a drop in CI (drop in electrical impedance) indicated cell death. The experiment was conducted three separate times. ANOVA followed by mean separation using LSD at α = 0.05 was also calculated [32].

Cell Cycle Analysis

Both cell lines were exposed to 3 μg/L environmentally realistic concentrations of ATR. Eighty pico-gram/ml of estrogen was used as a positive control for measuring estrogenic activity of the cell lines. After 96 h exposure period, nuclei were isolated from exposed and control cells then stained with propidium iodide (PI) using a hypotonic lysis solution [33]. Briefly, cells were washed with sterile 1% PBS, followed by the addition of 1.5 ml of the PI solution (0.05mg/ml, 0.1 Triton X 100, 0.1 % sodium citrate, 7 unites /ml RNAse). Plates were refrigerated and tilted every 3-5 minutes to release the nuclei from the lysed cells. The samples were filtered through a 53-um mesh filter and kept on ice until analysis. The refrigeration time differed for each cell line. MCF-7 cells were refrigerated for one hour and MCF-10A cells were refrigerated for 6 hours as nuclei from MCF10Atakes longer time to be collected than does MCF-7 cells. The isolated nuclei were then analyzed using a BD FACSCalibur equipped with 4-Color filters; 530 nm (FITC), 585 nm (PE/PI), 670nm (PerCP) and 661 nm (APC), (San Diego, LA, USA). The excitation wavelength (488 nm) was provided by a 5 W argon ion laser. Approximately 20,000 nuclei per sample were analyzed. The percentage of nuclei of the cell cycle was calculated using Modfit LT 3.0 (Verity Software House Inc., Topsham, ME, USA). ANOVA followed by mean separation using LSD at α = 0.05 was performed on the percentage of nuclei in the G1, S (synthesis), and G2 phases of the cell cycle.

Results

Cytotoxicity of ATR on MCF-7 and MCF-10A cell lines

Median lethal concentration (LC50) is the most widely used criterion for acute toxicity testing. LC50 is described as the concentration of a substance that kills 50% of the test organisms after a specific length of exposure, usually 96 h. The LC50 for MCF-7 cells exposed to ATR were 14,960, 22,100, 14,700 and 14,100 μg/L for 24 h, 48 h,72 h and 96 h respectively. The LC50 for MCF- 10A were 36,660, 9,870, 9,940 and 1,200 μg/L for 24 h, 48 h,72 h and 96 h respectively (Table 1).

Table 1: LC50 (ug/L) of ATR on MCF-7 and MCF-10A cell lines.

*Means within a column lacking a common letter are significantly difference (p≤0.001)

Cell Proliferation After ATR Exposure

Cell lines were exposed to ATR concentrations ranging from 0.315 μg/L to 100,000μg/L. Results showed that exposure of MCF- 7 breast cancer cells to 3μg/L ATR (EPA MCL) for 96 h stimulated an increase of 20% cell proliferation compared to control (Figure 1). Lower concentrations than the EPA MCL for ATR showed even stronger impact on cell proliferation, for example, 1.5 μg/L increased cell proliferation by 31% as compared to control. ATR concentration of 0.75 μg/L also caused growth to increase by 11%. ATR concentrations higher than 25,000 μg/L induced cell death in MCF-7.

Figure 1: Breast Cancer Cell Line (MCF-7) Exposed to ATR for 96 h.

Figure 2: Normal Breast Cell Line (MCF-10A) Exposed to ATR for 96 h.

After 96 h of exposure, ATR concentrations of 3 μg/L stimulated 25% increase in cell proliferation in MCF-10A breast cells compared to control (Figure 2). Lower concentrations as 1.5 μg/L and 0.75 μg/L showed almost same stimulatory effect. Higher concentrations of ATR: 390 μg/L and 780 μg/L induced the highest increase in cell growth; 37% and 32% more than control respectively. Concentrations higher than 12500 μg/L induced cell death.

Impact of ATR on cell cycle

To determine the effect of ATR on cell cycle, both Cell lines were exposed to the EPA MCL concentration of ATR for 96 h. The result indicated that there was a significant increase in the % nuclei in the G1 and G2 phases of MCF-7 cell lines; however, there was no increase in the S phase. MCF-10 A cells showed a significant increase in the % nuclei in the G1 phase but there was not increase in % nuclei in the G2 or S Phase (Table 2, Table 3 & Table 4).

Table 2: G1 Phase Analysis of both cell lines after 96 h of exposure.

*Means within a column lacking a common letter are significantly difference (p≤0.001)

Table 3: S Phase Analysis of both cell lines after 96 h of exposure.

*Means within a column lacking a common letter are significantly difference (p≤0.001)

Table 4: G2 Phase Analysis of both cell lines after 96 h of exposure.

*Means within a column lacking a common letter are significantly difference (p≤0.001)

Discussion

Cytotoxic effects of environmentally relevant concentrations of ATR on breast cancer cells (MCF-7) and normal breast cells (MCF- 10A) were assessed using RT-CES. The E-plates in the instrument are equipped with microelectrodes which causes changes in electrical impedance. Thus, higher cell index indicate that more cells are bound to the microelectrodes. ATR action was recorded online after cell attachment and continuous information about growth, morphological changes and cell death were collected in real time.

LC50 is the concentration of a substance that kills 50% of its test subjects when administered in a single dose. Acute toxicity data are very important because they are used to set guidelines for regulatory measures (PHAGM, 2005). The current study showed that the effect of ATR was time dependent; in general, toxicity increased as exposure time increased. Although, we found a significant decrease in ATR toxicity on MCF 7 after 48 h. However, a significant increase of ATR on MCF 7 cells was recorded after 72 h. This is in accordance with the findings that showed that the toxicity of ATR in C. Puctatus is both time and concentration dependent [34] Although, their study found the LC50 of a 96 h ATR exposure for C. puctatus to be 42,381 μg/L which was quite high compared to our findings. The LC50 found in this current study (14, 100 and 1,200 μg/L for MCF-7 and MCF-10A respectively) is closer to results obtained from previous studies [35-37] that reported the LC50 for ATR after 96 h exposure to be 16,000, 18800 and 9370 μg/L for Lepomis macrochirus, Cyprinus carpio and Oreochromis niloticus, respectively. Different cytotoxicity studies have shown that ATR exposure caused changes in erythrocytes membranes, DNA damage, mitochondrial dysfunction, cell autophagy and apoptosis [38,39]. Ultimately, the cellular changes may lead to severe adverse health effects including cancer.

It has since been purported that ATR, a potent EDC, could induce proliferative effect on breast cancer cells through aromatase activation [27] as well as GPR30 binding [40]. The current study indicates that both cell lines exposed to low environmentally relevant concentrations of 3 ATR for 96 h stimulated cell proliferation. However, exposure of cell lines to the concentration of ATR higher that 25,000 μg/L (MCF-7) and 12,500 μg/L (MCF- 10A) caused cell death. It is important to note however, that the proliferative effect of ATR in the current study was greater in MCF- 7 than in MCF-10A. The increased proliferation may be attributed to the fact that MCF-7 is estrogen responsive and it yielded more response to ATR’s endocrine disrupting effects. Our findings are in line with the study that showed that ATR enhanced transcription of the aromatase gene as well as estrogen production in estrogen responsive breast cell [41,42]. Similarly, studies by [43] found that there was an increase in cell proliferation when MCF-7 cell were exposed to environmentally relevant concentrations. However, they did not record any increase in cell proliferation in MCF 10A. Even though the EPA has a set MCL of 3 ppb, it is very worrisome to know that lower levels can induce cell growth and therefore cancer cell proliferation. ATR has been classified as non-carcinogenic by the EPA, however, strong evidence suggests otherwise. Scientist continue to record more environmental and human adverse effects due to exposure [43] including increased risk of ovarian cancer in female farm workers in Italy [25] and increased risk of breast cancer in the population of Kentucky to ATR exposures [26].

To our knowledge, our team is the first to investigate the cell cycle effects of ATR on MCF-7 and MCF 10A cells. The cell cycle investigation in the present study buttresses the body of evidence that implicates ATR in the etiology of breast cancer as cell proliferation was confirmed by a significant increase in the % nuclei in the G1, and G2 phases in MCF-7. However, significant increase in % nuclei was only recorded in G1 phase for MCF-10A. Increase of nuclei in cell cycle confirms cell duplication and proliferation. This finding is in agreement with a study conducted in HepG2 cell line, where 100, 300, and 500 ppb ATR exposure for 48 h caused nuclei to accumulate in S phase compared to control [31]. Similarly, studies using CHO cells exposed to 43ppm ATR, showed a significant accumulation of nuclei in S phase [30]. Although an increase in the % nuclei in the G phase was recorded in the current study, unlike previous studies, we did not record any increase in the % nuclei in cells in the S phase. This variation may be attributed to the difference in the type of cell lines used in the study. Interestingly, estrogen did not elicit an increase the % nuclei in any of the cell cycle phases in both cell lines. This suggests that estrogen may not exerts its endocrine disrupting effects through cell cycle activities. It is important to also note that even though ATR and estrogen elicit endocrine disrupting effects, ATR does not bind to or activate the classical estrogen receptor, but it up-regulates the aromatase activity in estrogen-sensitive tumor cells [24].

The current study examined the effects of ATR exposure on human breast cell. Specifically, ATR toxicity. Cell proliferation activity and cell cycle events were studied by exposing cells to different concentrations of ATR including very low concentrations that can be present in drinking water. In addition, two human breast cell lines were studied rather than non-human cell lines that are frequently published. The results obtained from studying human breast cell lines may prove more relevant in understanding the etiology of breast cancer in humans. It is imperative that more studies that elucidates the possible mechanistic pathways by which ATR induces cancer in human cells- specifically breast cells, be conducted.

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genefish · 5 years ago

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New in Pubmed: Identification of the autophagy pathway in a mollusk bivalve, Crassostrea gigas.

Identification of the autophagy pathway in a mollusk bivalve, Crassostrea gigas.

Autophagy. 2020 Jan 22;:1-19

Authors: Picot S, Faury N, Arzul I, Chollet B, Renault T, Morga B

Abstract The Pacific oyster, Crassostrea gigas, is a mollusk bivalve commercially important as a food source. Pacific oysters are subjected to stress and diseases during culture. The autophagy pathway is involved in numerous cellular processes, including responses to starvation, cell death, and microorganism elimination. Autophagy also exists in C. gigas, and plays a role in the immune response against infections. Although this process is well-documented and conserved in most animals, it is still poorly understood in mollusks. To date, no study has provided a complete overview of the molecular mechanism of autophagy in mollusk bivalves. In this study, human and yeast ATG protein sequences and public databases (Uniprot and NCBI) were used to identify protein members of the C. gigas autophagy pathway. A total of 35 autophagy related proteins were found in the Pacific oyster. RACE-PCR was performed on several genes. Using molecular (real-time PCR) and protein-based (western blot and immunohistochemistry) approaches, the expression and localization of ATG12, ATG9, BECN1, MAP1LC3, MTOR, and SQSTM1, was investigated in different tissues of the Pacific oyster. Comparison with human and yeast counterparts demonstrated a high homology with the human autophagy pathway. The results also demonstrated that the key autophagy genes and their protein products were expressed in all the analyzed tissues of C. gigas. This study allows the characterization of the complete C. gigas autophagy pathway for the first time.Abbreviations: ATG: autophagy related; Atg1/ULK: unc-51 like autophagy activating kinase; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG12: autophagy related 12; BECN1: beclin 1; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; DNA: deoxyribonucleic acid; GABARAP: GABA type A receptor-associated protein; IHC: immunohistochemistry; MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NCBI: national center for biotechnology information; ORF: open reading frame; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PtdIns3K: class III phosphatidylinositol 3-kinase; RACE-PCR: rapid amplification of cDNA-ends by polymerase chain reaction; RNA: ribonucleic acid; SQSTM1: sequestosome 1; Uniprot: universal protein resource; WIPI: WD repeat domain, phosphoinositide interacting.

PMID: 31965890 [PubMed - as supplied by publisher]

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juniperpublishersmicrobiology · 5 years ago

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Blood Serum Affects Polysaccharide Production and Surface Protein Expression in S. Aureus

Authored by Nazrul Islam

Abstract

Background: S. aureus biofilm serves a major role in pathogenesis. Two of the major components of bacterial biofilm are Polysaccharides intercellular adhesions (PIA) and surface proteins. It is not known how PIA and surface proteins expressions are affected in presence of blood serum. Analyses of surface proteins expressions will provide more effective biomarker discovery that might lead to development of antimicrobial therapeutics to meet the challenges of biofilm-related infections.

Method: Secondary cultures of S. aureus Philips, a biofilm-forming bacterium, were generated by inoculating 1 ml of overnight culture into 50 ml of TSB. Bacteria were cultured at several concentrations of blood serum and found that 12.5% supplemented blood serum provide s similar growth curve as normal TSB (100%). One and 2 D SASPAGE were used to separate proteins and the differentially expressed proteins were identified by nano-LC/MS.

Results: Polysaccharide intercellular adhesions production was significantly increased due to the addition of blood serum in the media. We also identified two serum proteins, apolipoprotein and globulin (Fc and Fab), that remained attached with the membrane fraction of bacterial proteins.

Conclusion: These results have strongly demonstrated that blood serum influences the exopolysaccharide expression in S. aureus.

Keywords: Biofilm; Serum; Staphylococcus aureus; Proteome

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Background

A biofilms are micro Colonies of bacteria adhere to each other and to biotic or biotic surfaces, embedded in an extracellular matrix produced by the sessile bacterial cells [1]. Extracellular matrix (ECM) and ECM proteins in bacterial biofilm play crucial roles in contaminating the agricultural produce starting from the field to the packing. In addition, ECM mediates adhesions protect bacteria from external threats and other stressors of adverse environment. Some of the ECM enzymes hydrolyze macro biomolecules into smaller biomolecules which subsequently is taken up by bacteria [2,3].

Both polysaccharide and protein embedded in extracellular matrix of biofilm play critical roles in biofilm stability Martm- Cereceda et al. (2001); Tsuneda et al. (2003). The polysaccharide intercellular adhesns are the major components (90%) of biofilm. Gutberlet et al. (1997); Gross et al. (2001); Weidenmaier & Peschel (2008); Rupp et al. (1995) [4]. Two types of PIA have been reported based on structure,. PIA type I (typically>80%) is a unique linear beta-1, 6 glucosaminoglycan which is predominantly positively charged. PIA type II (typically<20%) is structurally similar to type I, but contains phosphate and ester-linked succinate, and thus carries a mild negative charge Rupp et al. (1995); Mack et al. (1996). The biofilms are stabilized by the linear structure of these PIAs electrostatic interaction between positively and negatively charged residues Mack et al. (1996). In addition, surface proteins appear to play a critical role in contributing to biofilm stability. For example, nearly all S. aureus clinical isolates possess and express the genes necessary for PIA production (ica-operon, described below), yet many do not form biofilms Fitzpatrick et al. (2005, 2006). This implies that surface proteins may act as additional biofilm stabilizers, possibly cooperating with PIA to mediate intercellular adhesion O'Gara (2007).

In antibiotic therapy, biofilm has been found in 65-80% of the bacterial infections, and is considered refractory to host defenses [4]. Staphylococcus s. aureus, a biofilm forming bacteria, is responsible for severe skin infections to such major diseases as bacteremia, endocarditis and osteomyelitis. Under favorable conditions, S. aureus causes serious complications in devices like implants and catheters by producing biofilms on them [5]. Treatment of such infections becomes even more challenging given that several S. aureus strains show resistance to multiple antibiotics (e.g., methicilin and vancomycin). Extracellular matrix (ECM) proteins in bacterial biofilm play crucial roles in biofilm stability. In addition, ECM mediates adhesins to protect bacteria from external threats and also other stressors under adverse environment.

The mechanisms that how bacteria survive in their diverse natural habitats by using ECM and ECM proteins are yet to be fully understood. In a recent study, Floyd et al. [6] studied spatial proteome of surface-associated single-species biofilms formed by uropathogenic Escherichia coli and concluded the presence of at least two regulatory mechanisms controlling type 1 pili expression in response to oxygen availability. Similarly, a recent study on ECM proteome of Bacteroides fragilis, a widely distributed member of the human gut micro biome, identified several lipoproteins, TonB-dependent transporters and auto transporters [7]. Similar to theses investigations, several studies on ECM proteome in E coli were also performed [8-10]. Although these investigations have provided in-depth information about the certain ECM proteins, it is not known how surface proteins are affected in presence of serum. We, therefore, investigated how blood serum affects ECM and polysaccharide production and surface protein expression in S. aureus using proteomic techniques.

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Materials and Methods

Bacterial strain

S. aureus Philips, a biofilm-forming bacterium, was used in this study. In previous studies, we successfully used this strain, which was originally isolated from a patient diagnosed with osteomyelitis Patti et al. (1994); George et al. (2006); George et al. (2007). Secondary cultures was generated by inoculating 1ml of overnight culture into 50ml of TSB and growing at 37 °C with constant rotation in shake flasks for 16 hours. We grew the bacteria at several concentrations of blood serum and found that 12.5% supplemented blood serum similar growth curve as normal TSB. The growth of the bacterial strains was monitored by measuring the absorbance of the broth at 600nm on a spectrophotometer. The cells were then harvested and resuspended in phosphate-buffered saline (D-PBS; 138mm NaCl, 2.7mM KCl, pH 7.4). Cell concentrations was be determined using a Coulter Multisizer.

Measuring PIA

The cell plate was created from one ml of the culture, transferred to a micro tube and centrifuged at 10,000xg for 10 min at 4 °C. One ml of PBS buffer was used to wash the cell plates. Cells were then resuspended in 100|il of 0.5M EDTA, pH 8.0 and boiled in hot water for 10 min at 100 °C. The sample was then centrifuged at 10,000xg for 10 min at 4 °C. The clear supernatant was transferred to a new micro tube. Boiling cells with 0.5M EDTA is the best method known to date for the isolation of crude PIA from staphylococcal cell surface [11]. The crude PIA quantification was performed by a colorimetric method as described elsewhere [12]. Briefly, 50 |il of the crude PIA was transferred to a micro tube and mixed with 25|il of 80% w/v Phenol solution (Sigma-Aldrich) and 1 ml of concentrated sulphuric acid was added. The solution was kept at room temperature for 10 min, and absorbance was read at 490nm. Normalization of the amount of PIA was performed by dividing by the number of cells used for extraction.

Protein extraction

Cells were washed with PBS containing 0.1% sodium azide and then with PBS without azide, followed by a brief wash with digestion buffer containingm10 mm Tris HCl, 1 mm EDTA, 5 mm MgCl2. Approximately 5x109 bacterial cells were resuspended in 1ml of digestion mixture containing 35% raffinose, protease inhibitor cocktail (1 tablet/ml of digestion buffer), lysostaphin (5units/ml) and then incubated at 37 °C for 30 min. Cell debris were removed by centrifugation at 8,000g for 20 minutes and the supernatant was collected. After digestion and centrifugation, the digest was kept at -20 °C overnight and then centrifuged at 8,000g for 20min precipitated raffinose was discarded. After digestion and centrifugation, the protein solution was subjected to ultrafiltration using the Millipore ultrafiltration tube and centrifuged as per manufacturer's instructions. Protein concentration in the solution was determined using 2 D Quant (GE) and the resulting solution will be stored at -80 °C for 2-DE.

Two dimensional gel electrophoresis

In preparation for 2-DE, 150 ng proteins was resolubilized by adding standard sample solubilization buffers containing urea (8M), thiourea (2M), ASB 14 (1%), DTT (1%), and Carrier ampholytes (0.08%).The resulting solution was diluted to the desired volume with destreak rehydration solutions. Rehydration of IPG strips with the sample was carried out in the Immobiline Dry Strip Re-swelling Tray (GE Healthcare) according to the manufacturer's instructions. IPG strips of pH 3-11 (NL 24 cm) were used. The rehydrated strips were subjected to isoelectric focusing (IEF), performed using IPGphor operated at 20°C in gradient mode (97 kVhr). After focusing, the strips were stored at -80°C for later use. Prior to the second dimension SDS-PAGE, IPG strips were equilibrated for 15 minutes in equilibration solution (15 ml) containing 50mm Tris-HCl, pH 8.8, 6 M urea, 30% w/v glycerol, 2% w/v SDS and traces of bromophenol blue with 100 mg/10 ml (w/v) of DTT.

A second equilibration was carried out for 15 minutes by adding iodoacetamide (250mg/10 ml) instead of DTT in equilibration solution. Second dimension vertical SDSPAGE was performed using large format (26.8x20.5 cm) gels (12.5% T/ 2.6% C) according to the manufacturer's instructions. Electrophoresis was carried out with an initial constant voltage of 10 mA/gel applied for 30 minutes followed by 20 mA/gel for overnight until the bromophenol band exits the gel. The gels was stained with Colloidal Coomassie brilliant blue (BioRad). Gels were scanned as 12-bit TIFF images using Biorad GS-800 densitometer and analyzed by Nonlinear Dynamics Same Spots (v.3.2). Spot volumes were normalized by the software to a reference gel. At least three gels (biological replicates) for each treatment was used for analyses.

Protein identification

For mass spectrometric identification, gel spots were excised, destained, and digested with sequencing grade trypsin (Promega). Peptide samples were analyzed by Nano ESI-MS/ MS using LTQ (Finnigan, Thermo, USA). Nano LC was performed at reversed phase conditions using an Ultimate 3000 (Dionex corporation, USA) C18 column with a flow rate of 1-5 microliter/ min in 70-90% acetontrile containing 0.1% formic acid. MS and MS/MS data was collected and interrogated using SEQUEST against the NCBI non-redundant protein database for S. aureus providing peptide tolerance of 1.4 amu. Searched results were filtered using three criteria: distinct peptides, Xcorr vs Charge state (1.50, 2.00, 2.50, 3.00) and peptide probability (0.001). The confirmation of the protein identification was based on the Xcorr value of more than 50 and Sf score for individual peptide of more than 0.8.

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Results and Discussion

Blood serum affects polysaccharides intercellular adhesins

We have developed an experimental protocol for isolation and quantification of polysaccharides intercellular adhesins of S. aureus by boiling cells with 0.5M EDTA, digesting the PIA with concentrated sulphuric acid and phenol, and then measuring absorbance at 490nm. Although isolation of crude PIA by 0.5M EDTA is a routine procedure for PIA purification [13], to our knowledge it has not been reported for crude PIA quantification. We combined the EDTA extraction [13] with determination of sugars and their derivatives by colorimetry [11]. Using this procedure, we were able to reproducibly quantify PIA from S. aureus. As evident from the Figure 1, significantly higher amounts of PIA were observed in presence of blood serum. Similar to these findings, we also observed increased level of PIA in elevated level of NaCl [14].

PIA biosynthesis is mediated by ica operon-encoded enzymes [15,16]. The icaA, D and C gene products are involved in translocation of the growing polysaccharide to the cell surface [17], while IcaB is responsible for deacetylation of the PIA I molecule (providing its positive charge) which is essential for biofilm formation [18]. In contrast, the icaR gene, located upstream of the ica ADBC operon, encodes a transcriptional repressor which plays a central role in the environmental regulation of the ica operon [19]. For example, exposure to NaCl activates the ica operon in an icaR-dependant manner [18-20]. We anticipate that blood serum might have similar effect on the ica operon in an icaR-dependant manner, which is yet to be explored.

Blood serum and fibronectin binding and collagen binding proteins

In the SDSPAGE (Figure 2) (Table 1), several virulence- associated surface proteins were identified such as fibronectin- binding protein (b2), collagen-adhesins precursor (b4, b7), trigger factor (b8). However, serum supplement significantly reduced the abundance of fibronectin binding protein, although the abundance of collagen binding protein was not affected. In a recent report, Shinji H et al. (2011) studied, Fibronectin-binding protein A (FnBPA) and FnBPB, by constructing constructed fnbA and/or fnbB mutant strains and reported that the serum levels of interleukin-6 and nuclear factor kB (NF-kB) activation have no significant reduction in fnbB mutant infection(18)s. It is probable that the NF-kB of serum we used might have reduced the fibronectin-binding protein.

Bacterial strain

Measuring PIA

Protein extraction

Two dimensional gel electrophoresis

Protein identification

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Results and Discussion

Blood serum affects polysaccharides intercellular adhesins

Blood serum and fibronectin binding and collagen binding proteins

Serum proteins in bacterial surface

We identified two serum proteins, apolipoprotein and globulin (Fc and Fab), in the membrane fraction of bacterial proteins. These results were confirmed from both 1D and 2-DE SDS PAGE. The presence of serum proteins in membrane fraction of bacterial protein has raised several questions. If we consider these proteins as a contaminant from the serum, why were we unable to wash out these proteins while we successfully washed out the most abundant serum protein such as albumin? If not a contaminant, what is causing these proteins to remain attached to the bacterial surface? It is known that Fc and Fab motifs of globulin interact with Spa C and Spa D domains of protein A. But the bacterial strain we used was a mutant of proteins A. In addition, by using a deletion mutant of Newman, we confirmed the presence of Fc and Fab with bacterial membrane associated protein (Figure 3). This raise another question of what components of bacteria are causing this Fc and Fab to remain attached with bacterial proteins.

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Conclusion

Polysaccharide intercellular adhesins production was significantly increased due to the addition of blood serum in the media. We identified two serum proteins, apolipoprotein and globulin (Fc and Fab), remained attached with the membrane fraction of bacterial proteins even after several washing procedures, indicating that these proteins might play a critical role in bacterial processes of biofilm formation.

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Author's contributions

Nazrul Islam designed and conducted the experiment, and corresponding author for this manuscript. Julia M. Ross, Khwaja G. Hossain and Mark R. Marten developed the concept.

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Acknowledgement

This research was supported by Grant no. R01AI059369 from the NIH and also an Institutional Development Award (IDeA) under NIH grant no. P20GM103442.

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juniperpublishersjcmah · 5 years ago

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Exploration of Antibacterial and Anti-inflammatory Activities of Premna integrifolia Plant Extracts in Bubaline Mastitis- Juniper Publishers

Abstract

Mastitis, which affects the milk production of dairy animals, is usually due to mammary gland invasion by bacterial pathogens. Emergence of antimicrobial resistance in bacteria and side effects associated with the use of anti-inflammatory cortisones in mastitis prompted for use of alternate/complementary therapeutics. As the plant Premna integrifolia was reported to exhibit antibacterial, anti-inflammatory/immunomodulatory properties, its leaf and root aqueous extracts were tested for their antibacterial activity against Staphylococcus aureus and Escherichia coli, either individually or in combination with the antibiotics. The anti-inflammatory properties of the extracts were also tested against the bubaline mammary epithelial cells (MEC) infected with S. aureus and E. coli. In microbroth dilution assays for assessing minimum inhibitory concentration (MIC) in vitro, the leaf and root extracts of Premna integrifolia didn’t exhibit any antimicrobial activity against S. aureus but showed significant antimicrobial activity on E. coli. In combination with the plant extract, the sensitivity of S. aureus to amoxicillin is not only increased but also the S. aureus isolates that were resistant to amoxicillin also became sensitive. The Premna integrifolia leaf and root extracts, however, showed antagonism on antimicrobial activity of enrofloxacin in combination. In addition the aqueous root extract of Premna integrifolia exhibited anti-inflammatory activity through down regulation of cytokines IL-6, IL-8 and TNF-α in S. aureus and IL-6 and IL-8 in E. coli infected MEC. These studies reveal antimicrobial activity of leaf and root extracts of Premna integrifolia on E. coli. In combination with amoxicillin these plant extracts increased the sensitivity of S. aureus to amoxicillin. The anti-inflammatory activity of root extract of Premna integrifolia on MEC infected with S. aureus and E. coli is also demonstrated in these studies.

Keywords:Mastitis; S. aureus; E. coli; Premna integrifolia; Mammary epithelial cells; cytokines; Amoxicillin; Enrofloxacin

Introduction

Mastitis in dairy animals is inflammatory reaction of the udder tissue against the invading microbial pathogens. Bacterial pathogens are majorly implicated in the mastitis of cows and buffaloes leading to major production losses in dairy animals resulting in huge economic losses to dairy farmers and industry [1]. Staphylococcus aureus and Escherichia coli are the major bacterial pathogens of bovine/bubaline mastitis [2]. However, the emergence of antimicrobial resistance in bacterial pathogens that cause mastitis in dairy animals is a cause of grave concern [3-5]. Also controlling the inflammation in mastitis is very essential as the persistent inflammation of mammary gland tissue may result in permanent unproductivity in dairy animals [6-7]. Mastitis is the most frequent reason for the use of antimicrobial drugs in dairy herds, which eventually has resulted in antimicrobial resistance [8].

Development of new antibiotics will take long time and there is chance of further developing antimicrobial resistance against these molecules in due course. In this context exploration of natural compounds from medicinal plants that exhibit both antibacterial and anti-inflammatory/immunomodulatory properties may offer promising solution for therapeutic approach to mastitis in dairy animals. Medicinal uses of the plant Premna integrifolia that has prominent value in Indian system of medicine Ayurveda was reviewed by different researchers [9-11]. Reports on increased sensitivity of bacterial pathogens to antibiotics, when used in combination with anti-inflammatory compounds like Non-Steroidal Anti-inflammatory Drugs (NSAIDs) [12], also encourages us to take up research work on the natural compounds with anti-inflammatory activity. As the development of resistance to natural products of plant origin is highly remote and the issue of antibiotic residues in milk doesn’t arise with the natural compounds, the present investigation was taken up to study the antibacterial and anti-inflammatory/immunomodulatory activities of aqueous young leaf and root extracts of the plant Premna integrifolia. The study is aimed to test the anti-bacterial activity of the plant extracts on S. aureus and E. coli, either individually or in combination with the antibiotics. It is also aimed to test the anti-inflammatory/ immunomodulatory activity of the plant extracts on Mammary epithelial cells (MEC) cultured from fresh milk of buffaloes and further infected with the selected bacterial pathogens of mastitis.

Materials and Methods

Plant material

Plant materials were collected from Maharastra region of India. The plant was identified as Premna integrifolia L. belonging to Verbenaceae by Dr. S. K. Srivastava, Scientist-E, BSI, Dehradun with accession no. 116123. Sample herbarium sheets deposited with Northern Regional Centre, Botanical Survey of India, Dehradun.

Preparation of Premna integrifolia extracts

The young roots and leaves of Premna integrifolia were sun dried for 15 days, powdered and successively extracted with soxhlet apparatus with petroleum ether, ethyl acetate, methanol and water in the increasing polarity index. These extracts were dried using a rotatory evaporator followed by lyophilization. Similarly, leaves were dried in shade for 10 days and extracted as above. In the present study the aqueous extracts were evaluated for their anti-microbial and anti-inflammatory effects./p>

Bacterial isolates

The bacterial pathogens Staphylococcus aureus and Escherichia coli were isolated from the mastitic milk samples of buffaloes and the bacteria were subjected to characterization by culturing on selective bacteriological media. Mannitol salt agar (MSA) and Eosin methylene blue (EMB) agar (Oxoid, UK) were used for culture of S. aureus and E. coli, respectively. These bacteria were further characterized in polymerase chain reaction (PCR) test by reactivity with species-specific oligonucleotide primers [2].

Microbroth dilution method for measuring the minimum inhibitory concentration (MIC) of antibiotic/ minimum inhibitory concentration (MIC) of antibiotic/

The antimicrobial activity of the plant extracts was evaluated by microbroth dilution method in serial wells of microtitre plate (Axygen, USA) [13], with suitable modifications. Briefly, two-fold dilution of antibiotic/plant extract (10mg/ml) is made with cation adjusted Mulleur Hinton broth, in their respective wells of 96-well microtiter plate. The antimicrobial activity of the plant extracts was tested individually, also in combination with antibiotic. In the combination studies a fixed volume of 50μl of plant extract (10mg/ml) was added to the wells with serial dilution of respective antibiotic. Separate row(s) of wells with serial dilution of antibiotic alone were also maintained to compare the MIC values of antibiotic with the MIC values of plant extract or antibiotic & plant extract combination. Appropriate controls were also maintained. Amoxicillin and enrofloxacin (SRL, India) antibiotics in powder form were used for S. aureus and E. coli, respectively. To all the wells constant volume of 300μl of 0.5 McFarlands standard bacterial culture (S. aureus/E. coli) was added. The culture plates were incubated for 18hrs. and the absorbance readings were taken at 660 nm (Multiskan plate reader, Thermo). The MIC values of the antibiotic/plant extract or combination of antibiotic & plant extract corresponding to the absorbance readings of respective wells were noted. Then indicator dye p-iodonitrotetrazolium violet (INT) (SRL, India) was added to all the wells to visually appreciate the extent of antimicrobial activity of the compounds tested. The breakpoints of amoxicillin and enrofloxacin/ciprofloxacin in MIC assays were taken as per Clinical and Laboratory Standard Institute (CLSI) guidelines 2012.

In Microbroth dilution method for measuring the MIC a loopful of inoculum was picked up from the wells in microtiter plates where there is inhibition of bacterial growth and streaked on bacteriological medium, further incubated to confirm the absence of live bacteria/bacterial growth in those wells.

Isolation and culture of mammary epithelial cells (MEC) from milch buffaloes

Mammary epithelial cells were isolated form the fresh milk of apparently healthy milch buffaloes maintained at Livestock Farm Complex, NTR College of Veterinary Science, Gannavaram as per the established procedure [14] with suitable modifications. Briefly, the fresh milk samples were centrifuged at 500 x g and the cell pellet was washed with phosphate buffer saline (pH 7.2). Then the cell pellet was cultured in DMEM/F12 (Sigma, USA) medium with 10% Foetal Bovine Serum (Thermo Fisher) for 48 hrs. in 5% CO2 atmosphere. Four groups of the cultured mammary epithelial cells (MEC) were maintained. First group was maintained normal untreated. Second group was maintained as normal & treated (plant extract treated), third group was maintained as infected by infecting with 300μl of 0.5 Mcfarlands standard bacterial culture. The fourth group was maintained as plant extract treated & infected, where in MEC were treated with 300μl of plant extract (10mg/ml). After 6 hrs. of incubation with plant extract the MEC were infected with 300μl of 0.5 Mcfarlands standard bacterial culture and further incubated for 6 hrs. The S. aureus broth culture was used to infect MEC, whereas heat inactivated (65 °C/30 minutes) E. coli was used to treat the MEC.

Detection of cytokines expression in bubaline MEC by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)

Two step qRT-PCR was carried out in this study. In the first step the total RNA from MEC of different groups of cells was extracted, separately, by using Trizol reagent (Invitrogen, USA) as per the manufacturer’s instructions. The quality of RNA was checked in Nanodrop (Thermo, USA). The cDNA from RNA of different groups of cells was synthesized by standard protocol using reagents/chemical/enzymes from Thermo Fisher Scientific, USA. Briefly, the 200 ng of RNA extracted was incubated with Random Hexamers, then treated with RNAase inhibitor RiboLock. The RNA was reverse transcribed to cDNA using M-MuLV Reverse Transcriptase RNaseH+ at 37 °C in a thermal cycler (Eppendorf Master cycler, Germany). Any contamination of genomic DNA was removed by using DNA free TM DNA removal kit. The resultant cDNA was quantified in Nanodrop.

In the second step the qRT-PCR tests were performed in 25μl of reaction volume in Quant Sudio3 Real Time PCR instrument (Applied Biosystems, USA). The levels of gene expression of cytokines Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumour Necrosis Factor - α (TNF-α) in MEC after 6 hrs. of infection with bacterial pathogens in normal and plant extracts treated MEC were studied. The house keeping β-actin gene was kept as endogenous control. The sequence of oligonucleotide primers used in this study (Bioserve Biotechnologies, India) were adopted from the earlier research reports (15). In the qRT-PCR tests KAPA SYBR Fast qPCR master mix based on SYBR Green technology was used under the test conditions of initial denaturation 95 °C/ 3 minutes; then 94 °C / 3 sec, 60 °C / 3 sec & 70 °C / 10 sec for 50 cycles, followed by standard melt curve conditions.

results

A total of 42 isolates of S. aureus and 11 isolates of E. coli were isolated from mastitic milk samples of buffaloes in and around Gannavaram, Krishna District, Andhra Pradesh. Certain mastitic milk samples were positive for mixed infections of S. aureus and E. coli. The S. aureus produced typical mannitol fermentation on MSA and the E. coli produced greenish metallic sheen on EMB agar, during the culture. In PCR test the S. aureus produced a specific PCR product of 1250 bp (Figure 1a) and E. coli produced a specific PCR product of 662 bp (Figure 1b).

In MIC assays, 31% isolates (n=13) of S. aureus were found to be resistant to amoxicillin. The isolates were GV28, GV40, GV42, GV43, GV45, TVCC41, TVCC47, TVCC49, TVCC53, PMNR1, KSP35, KSP36 and KSP39. Both the plant extracts (each extract separately) didn’t exhibit any significant antimicrobial activity against all the isolates (n=42) of S. aureus. However, for 45.2% of isolates (n=19) amoxicillin exhibited antimicrobial activity even at a lower concentration when combined with the plant leaf extract. The isolates were GV29, GV30, GV35, GV38, GV39, GV40, GV41, GV42, GV43, GV44, GV45, TVCC42, TVCC43, TVCC47, TVCC49, TVCC53, KSP35, KSP36 and KSP39. The MIC values of amoxicillin in antibiotic & leaf extract combination wells are found to be lower (to the extent of 0.00006μg/ml of concentration) than the MIC value of amoxicillin alone. Out of 13 isolates of S. aureus that were found to be resistant for amoxicillin, 10 isolates showed sensitivity to amoxicillin, when it is used in combination with the leaf extracts. For 23.8% isolates (n=10) of S. aureus there is no significant variation in MIC values of amoxicillin, when it is used alone or in combination with leaf extract. For 40.5% of isolates (n=17) amoxicillin exhibited antimicrobial activity at a lower concentration when combined with the plant root extract. The isolates were GV30, GV40, GV41, GV42, GV43, GV44, GV45, TVCC46, TVCC48, TVCC49, WG3, WG4, WG5, KSP35, KSP36, KSP39 and KSP43. The MIC values of amoxicillin in antibiotic & root extract combination wells are found to be lower than the MIC values of amoxicillin alone. Out of 13 isolates of S. aureus that were found to be resistant for amoxicillin, 9 isolates showed sensitivity to amoxicillin when it is used in combination with the root extract.

In MIC assays, all the E. coli isolates (n=11) were found to be sensitive to enrofloxacin. The isolates were GV26, GV27, GV28, GV29, WG1, KSP35, KSP38, GV46, GV47, KSP44 and KSP45. The leaf extract exhibited significant antimicrobial activity against 81.81% isolates (n=9) of E. coli. For these 9 isolates of E. coli the MIC values of leaf extract were significantly lower than the MIC values of enrofloxacin. The root aqueous extract exhibited antimicrobial activity against all the 11 isolates of E. coli. The MIC values of plant extracts was in the range of 31.25 to 0.98 μg/ml for different isolates of E. coli, whereas the MIC values of enrofloxacin are in the range of 500 - 62.5 μg/ml. In MIC assays with combination of enrofloxacin & plant extract (each extract separately), the enrofloxacin didn’t exhibit antimicrobial activity at its higher concentration but showed antimicrobial activity at its lower concentration.

After 48 hr. culture the MEC attained full confluence in tissue culture flaks and they were used for infection studies. The cDNA obtained from different groups of MEC was quantified by Nanodrop (Thermo) and same concentration cDNA from all the groups was used in qRT-PCR assays.

In MEC infection studies with S. aureus, the expression of cytokines IL-6, IL-8 and TNF-α genes were upregulated in S. aureus infected MEC (Figure 2a). In plant (young root) extract treated & infected MEC the gene expression of these cytokines was significantly downregulated compared to infected MEC (Figure 2b).

In MEC infection studies with E. coli, the gene expression of cytokines IL-6, IL-8 and TNF-α was upregulated in infected MEC compared to normal MEC (Figure 3a). Figure depicting upregulation of TNF-α gene expression was not shown. The gene expression of the cytokines IL-6 and IL-8 was downregulated in plant (young root) extract treated & infected MEC compared to infected MEC (Figure 3b). However, the gene expression of cytokine TNF-α was found to be upregulated in plant (young root) extract treated & infected MEC compared to infected MEC (Figure 3b).

Discussion

Mastitis in dairy bovines is usually caused by bacterial pathogens leading to inflammation of udder tissue and its further damage [1,2]. As the use of conventional antibiotics and antiinflammatory agents have certain disadvantages like development of antimicrobial resistance in bacteria, presence of antibiotic residues in milk during treatment, immunosuppression associated with cortisone administration etc., it is proposed to explore the antibacterial and anti-inflammatory/immunomodulatory activity of leaf and root aqueous extracts of the plant Premna integrifolia. The antibacterial activity of the plant extracts was tested on clinical isolates of S. aureus and E. coli isolated from mastitic milk samples of buffaloes. The isolated S. aureus and E. coli from different samples in this study were further characterized and the results were in accordance with the earlier reports [2].

Out of 42 characterized isolates of S. aureus 31% showed resistance to amoxicillin. The MIC values for indicating the resistance to amoxicillin in S. aureus were as per the CLSI guidelines, 2012. Due to the emergence of anti-microbial resistance, it is not surprising to find resistance to amoxicillin in S. aureus isolates from mastitic milk samples of dairy bovines [4,5]. Though antibacterial activity was reported with different extracts of Premna integrifolia [11,15-17], in the present study both the leaf and root aqueous extracts of the plant didn’t show any antimicrobial activity against all the isolates of S. aureus. This may be due to use of different solvent in the process of extraction. Also, in the previous studies the antimicrobial activity of the leaf extract was investigated by disc diffusion method [11], whereas in the present study the antimicrobial activity of plant extracts was tested by micro broth dilution method. In addition, all the isolates used in the present study were clinical isolates.

For 45.2% of isolates of S. aureus, amoxicillin exhibited antimicrobial activity at a lower concentration when combined with the plant leaf extract. It was reported that anti-inflammatory drug celecoxib sensitizes S. aureus to antibiotics [12] and the combinatorial effect of celecoxib and ampicillin was further demonstrated [18,19]. Anti-inflammatory activity of Premna integrifolia root was also reported [9]. Therefore, the antimicrobial activity exhibited by amoxicillin at lower concentrations may be due to combinatorial effect of plant extract (with anti-inflammatory activity) and amoxicillin. This may be correlated to the finding that out of 13 isolates of S. aureus that were found to be resistant to amoxicillin, 10 isolates showed sensitivity to amoxicillin when used in combination with the leaf extracts and 9 isolates showed sensitivity to amoxicillin when used in combination with the root extract. However, further research is to be carried out to find out the precise mechanism of this combinatorial effect.

In MIC assays the antibiotic enrofloxacin exhibited antimicrobial activity against all the isolates of E. coli. The sensitivity of E. coli to enrofloxacin in antimicrobial assays was already established [5,20]. Although the leaf extract didn’t exhibit any significant antimicrobial activity against S. aureus isolates, it exhibited significant antimicrobial activity against 9 isolates of E. coli. In fact MIC values of leaf extract were significantly lower than the MIC values of enrofloxacin for these E. coli isolates. The antimicrobial activity exhibited by the leaf extracts against E. coli is in accordance with the earlier reports on antibacterial activity of Premna integrifolia [11,16,17]. The aqueous root extract of the plant also exhibited significant antimicrobial activity against all the 11 isolates of E. coli. Specific research reports on the antimicrobial activity of the root extract are not available. Though the enrofloxacin has an established antimicrobial activity against E. coli when used alone, it is very interesting to observe in the present study that the enrofloxacin in combination with the plant extract (each extract separately) didn’t exhibit antimicrobial activity at higher concentration but exhibited its antimicrobial activity at lower concentrations. So, in two-fold serial dilution wells of enrofloxacin with combination of constant concentration of plant extract (each extract separately), bacterial growth was not inhibited at higher concentrations of enrofloxacin, whereas at lower concentrations of enrofloxacin the bacterial growth was inhibited. However, usually in MIC assays as the dilution of antibiotic progresses in the series of wells its concentration decreases and the bacterial growth is not inhibited in wells of microtiter plates with lower concentration of antibiotic. These findings are also in contrary to the reports on synergism of natural products and antibiotics [21].

From the studies on antimicrobial activity of fluoroquinolone antibiotic ciprofloxacin in combination with antioxidants it was reported that antioxidants exhibited antagonistic activity on ciprofloxacin [22,23]. It was observed that as the fluoroquinolones kill the bacteria by increasing the oxidative stress in bacterial cells, the concurrent/combinatorial use of antioxidants inhibit the oxidative stress induced by the ciprofloxacin. The antioxidant properties of Premna integrifolia were already reported [9- 11]. Therefore, it may be summrised that in the present study the antioxidant properties of the plant extracts antagonized the antimicrobial activity of enrofloxacin, which belongs to fluoroquinolones. This is supported by the observation that with plant extract combination E. coli growth was not inhibited in the microtiter plate wells with higher concentration of antibiotic, whereas in the wells with lower concentration of enrofloxacin the E. coli growth was inhibited. Perhaps there might be optimum levels of enrofloxacin and antioxidant plant extract combination in the microtiter plate wells with higher concentrations of enrofloxacin, leading to antagonistic action of plant extract on enrofloxacin. However, further studies are required for conclusive evidence on this aspect.

The aqueous leaf extract of the plant Premna integrifolia didn’t have any activity on downregulation in the expression of cytokines, IL-6, IL-8 and TNF-α genes in S. aureus and E. coli infection studies in MEC. However, in MEC infection studies with S. aureus the aqueous root extract of the plant Premna integrifolia showed antiinflammatory activity by downregulating the expression of genes of cytokines IL-6, IL-8 and TNF-α. But in MEC infection studies with E. coli the aqueous root extract showed anti-inflammatory activity by downregulating the expression of cytokines IL-6 and IL-8 genes only but not TNF-α. This may be due to the potent action of endotoxin of E. coli on MEC even after heat inactivation. This study thus forms the first report on the pattern of expression of cytokines IL-6, IL-8 and TNF-α genes in Premna integrifolia plant extract treated and infected cells of any system.

Conclusion

In conclusion, although the young leaf and root extracts of the plant Premna integrifolia didn’t exhibit any antimicrobial activity on S. aureus, significant antimicrobial activity was exhibited by these extracts on E. coli in microbroth dilution assays for MIC in vitro. However, in combination with the plant extract, the sensitivity of S. aureus to amoxicillin is not only increased but also the S. aureus isolates that were resistant to amoxicillin also showed sensitivity to the same antibiotic in this combination. The effect of plant extracts on E. coli, however, were in contrast with the findings of S. aureus as the antioxidant natural products showed antagonism on antimicrobial activity of enrofloxacin in its combination with the plant extracts. The aqueous root extract of Premna integrifolia exhibited anti-inflammatory activity through down regulation of genes of cytokines IL-6, IL-8 and TNF-α in S. aureus infected MEC. However, the down regulation of genes of cytokines was limited to only IL-6 and IL-8 only in E. coli infected MEC. Therefore, the plant extracts of Premna integrifolia offer promising solution for therapeutic approach to mastitis in dairy animals with a caution on its antioxidant property as it antagonizes the action of fluoroquinolone antibiotics.

Acknowledgment

The authors acknowledge the funding by National Medicinal Plants Board (NMPB), Ministry of AYUSH, Government of India, New Delhi to carry out this research project (Z. 18017/187/CSS/ R&D/AP-01/2014-15).

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Evaluation of antioxidant and cytotoxic properties of Vernonia amygdalina- Juniper Publishers

Abstract

The present investigation was carried out to evaluate the antioxidant activity and cytotoxic properties of Vernonia amygdalina. The free radical scavenging activity using a stable radical; 2, 2-Diphenyl-1-picryl hydrazyl, lipid peroxidation assay (DPPH), and nitric oxide inhibitory assay gave the highest percentage inhibition as 74.55±1.07%; IC50 = 1.831, 60.42±0.11; IC50 = 3.84 ± 1.03 and 71.26±0.48; IC50 = 0.99mg/ml, respectively. This is comparable to the standards quercetin used (P>0.05). In addition; total phenol, total flavoniods, anthocyanin and proanthocyanidine of the extract were determined using established methods. The results obtained justify the scavenging activity of the extracts. Furthermore, the extracts possessed very low cytotoxicity to brine-shrimp lethality test, when compared with the reference standard (Potassium dichromate, LC50 = 0.003±μg/mL). The results obtained in the study indicate that V. amygdalina can be a safe potential source of natural antioxidant agent; used as a neutralcetical/functional food..

Keywords: 2; 2-Diphenyl-1-picryl hydrazyl; Antioxidant; Cytotoxicity; Veronia amygdalinais

Introduction

Vernonia amygdalina is a shrub that grows predominantly in the tropical Africa. Leaves from this plant serve as food vegetable and culinary herb in soup [1]. Anecdotal evidences suggest the use of V. amygdalina in the treatment of feverish condition, cough, constipation, hypertension and related vascular diseases as well as diabetes. Photochemical screening of this plant leaves extracts showed the presence of Saponins, riboflavin, polyphenols, sesquiterpene and flavonoids [2]. Strong antioxidant activities involving flavonoids extracted from V. amygdalina and its saponins have been reported to elicit anti-tumoral activities in leukemia cells [3]. In addition, peptides from V. amygdalina are known to be potent inhibitor of mitogen activated protein kinase (MAPK) which is involved in the regulation and growth of breast tumour [4].

Previous studies have shown that a good number of plants have antioxidant activities that could be therapeutically beneficial. Consequently, antioxidant agents of natural origin have attracted special interest because of the potential they hold in the maintenance of health and protection of some age related degeneration disorders, such as coronary heart disease and cancer, neurodegenerative disease [5-7].

Although, antioxidants from natural sources are beneficial, it is pertinent to know their bio-safety. In this regard, the brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity of plant extracts; a suggested pharmaco logical screening method in plant extracts. It has been used for the detection of fungal toxins, plant extract toxicity. The shrimp lethality assay was proposed by Michael and co-workers in 1956, and later developed by Vanhaecker and his group in 1981. This is based on the principle, whereby the kill laboratory-culture of an invertebrate, Artemia salina L (the brine shrimp larva) following exposure to a varied concentration of plant extracts, heavy metals, cyan bacteria toxins and pesticides, is assessed for toxicity [8]. The purpose of this study is to evaluate the acute toxicity and antioxidant properties of V. amygdalina in relation to its use as a neutralcetical.

Materials and Methods

V. Amygdalina: Fresh leaves of V. amygdalina were collected from the University Village, Kogi State University, Nigeria. The plant material was identified and authenticated by taxonomist in the Department of Botany, Kogi State University, where the voucher specimen (VA-111) was deposited. Fresh leaves of V. amygdalina were air dried under room temperature until a constant weight was obtained. Thereafter, the leaves were milled to a coarse powder with the use of laboratory Mortar and Pestle. After this, 20g of the plant powder was weighed into a volumetric flask and then extracted using 200mls of distilled water for 72 hours. The crude extract was obtained by concentrating the water soluble extract using rotary evaporator at 45 °C. The working solution of extract was prepared by weighing out 0.02g of crude extract accurately and dissolved it in 20ml of distilled water to give an effective concentration of 1mg /ml.

Radical scavenging activity

In order to determine the antioxidant properties of the plant, radical scavenging activities of the leaves extract, was determined using the stable radical DPPH (2, 2-diphenyl-1 piccrlhydrazyl hydrate) according to the method of Blois (1958) as describe by Babalola and co-workers [9]. The principle is based on the reaction of DPPH, and an antioxidant compound to generate hydrogen, which is reduced (DPPH + RH → DPPH2 + R). The observed colour change from deep violet to light yellow was measured at 517nm. To 1ml of varied concentrations (0.5, 0.25, 0.125, 0.0625, 0.003125mg/ml) of the extract or standard, was added 1ml of 0.3mM DPPH in methanol. The mixture was vortexed, and then incubated in a dark chamber for 30minutes. Thereafter the absorbance was read at 517nm against a DPPH control containing only 1ml of methanol in place of the extract. The antioxidant activity (AA) was then calculated using the formula:

AA = [(Ao – Ac)/Ao] x 100,

Where: Ao = absorbance without extract and Ac = absorbance with extract.

Nitric oxide

Sodium nitroprusside generates nitric oxide in aqueous solution at Physiological pH, which consequently interacts with oxygen to produce nitric ions. This was measured by Griess reaction [10].

Procedure: 3ml of the reaction mixture containing sodium nitroprusside (10mM) in phosphate buffered saline (PBS) together with the varying concentrations of the extract (0.5, 0.25, 0.125, 0.0625, 0.003125mg/ml) were incubated in a water bath at room temperature for 150 minutes. This was followed by the removal of 1.5 ml of the reaction mixture and the addition of 1.5 ml of Griess reagent. After which, the absorbance of the chromophore formed was read using spectrophotometer at 546nm. Percentage inhibition of nitric oxide radical by the extract was calculated using the formula:

NO = [(1-E/C)] x 100,

Where: C= absorbance value of the fully

Ferric reducing antioxidant power assay (FRAP) assay

The FRAP assay used antioxidants as reductant in a redox linked colorimetric method with absorbance measured with a spectrophotometer. A 300mmol/L acetate buffer of pH 3.6 (3.1g of sodium acetate+16ml of glacial acetic acid made up to 1L with distilled water, 10mmol/L 2, 4, 6-tri (2-pyridyl 1, 3, 5-triazine, 98% (sigma-Aldrich) (3.1mg/ml in 40mmol/L HCl) and 20mmol/L of ferric chloride were mixed together in the ratio of 10:1:1, respectively to give the FRAP working reagent.

Procedure: A 50μL aliquot of extract was added to 1.5ml of FRAP reagent in a semi-micro plastic cuvette. Absorbance measurement was taken at 593nm (A593) exactly 10 minutes after mixing using 50μL of water as the reference. Thereafter, to standardize 50μL of the standard, iron (III) sulphate, (1mM) was added to 1.5ml of FRAP reagent. All measurement was taken at room temperature in the absence of light.

Evaluation of total phenolic content

The total phenolic of V.amygdalina extract was determined using the folin ciocalten assay method of Singleton and Rossi (1965) [11]. To 0.1ml of 1mg/ml of extract /standard was added 0.9ml of distilled water. Thereafter, 0.2ml of folic reagent was added. This was vortex-missed. Subsequently, 1ml of 7 % Na2CO3 solution was added to the mixture after 5minutes. The solution was followed by dilution to 2.5ml and then incubated for 90minutes at room temperature. The absorbance was read at 750nm against the reagent blank. Standard preparation was carried out by preparing a stock solution of gallic acid (1mg/ml) aliquots of 0.2,0.4, 0.6,0.8 and 1ml were taken and made up to a total volume of 2ml.

With the equation as shown below, the total phenolic content of the plants was then calculated, and expressed as mg gallic acid equivalent (GAE)/g fresh weight. The analysis was carried out in triplicates.

Equation (1) - - - - -C=c *v/m

Where: C = total content of phenolic compound in gallic acid equivalent (GAE); c = concentration of gallic acid established from the calibration curve, mg/ml; V=volume of extract (ml); m = Weight of the crude methanolic plant obtained

Evaluation of total flavonoids content

Aluminium chloride colorimetric method described by Zhilen was used for the determination of the total flavonoidal content of the plant extract [5]. Water (0.4ml) was added to 0.1ml of extract/ standard, as well as 0.1ml of 5 % sodium nitrite. This was left for 5minutes. Thereafter, 0.1ml of 10 % aluminium chloride and 0.2 ml of sodium hydroxide was added to the solution, and the volume was adjusted to 2.5ml with water. The absorbance at 510nm was measured against the blank.

Standard preparation

A stock solution of quercetin (1mg/ml) was prepared. Aliquots of 0.2, 0.4, 0.6, 0.8, and 1ml were taken and the volume made up to 2ml with distilled water.

The total flavonoid content of the plant extract was then calculated as shown in the equation below and expressed as mg quercetin equivalents per gram of the plant extract. The analysis was conducted duplicates and mean value considered. X = q×V/w: Where X= total content of flavonoid compound in quercetin equivalent; q = concentration of quercetin established from the standard curve; V=volume of extract (ml); w = weight of the crude methanolic extract obtained.

Proanthocyanidin content determination

The proanthocyanidin content of the extract was determined spectrophotometrically [12]. Extracts were diluted to provide a spectrophometric reading between 0.1 and 0.8 absorbance units.

Procedure: A 0.25ml sample aliquot of adequately diluted extract was added to 2.25ml of concentrated hydrochloric acid in n-butanol (10/90, v/v) in a screw top vial. The resulting solution was mixed for 10 to 15 seconds. Extracts were then heated for 90 minutes in an 85 °C water bath then cooled to 15-25 °C in an ice bath. The absorbance at 550nm was measured on a UV visible spectrophotometer. A control solution of each extract was prepared to account for background absorbance due to pigments in the extracts. The control solution consisted of the diluted extract prepared in the hydrochloric acid/n-butanol solvent without heating.

The proanthocyanidin content was expressed as mg cyaniding per Kg of sample.

Where:

ΔA = A550sample – A550control

A550 sample = Sample absorbance at 550nm

A550control = control sample absorbance at 550nm

Є = Molar absorbance co efficient of cyanidin (17,360L-1M- 1cm-1)

L= pathlenght (1cm)

MW= Molecular weight of cyaniding (287g/mol)

DF= dilution factor to express as g/L

1000 is the conversion from grams to milligram

Determination of total anthocyanin content

Total anthocyanin content of the extract was determined by the pH differential method [13].

Procedure: A pH 1.0 buffer solution was prepared by mixing 125ml of 0.2 N KCl with 385 ml of 0.2 N HCl and 490ml of distilled water. The pH of the buffer was adjusted to pH 1.0 with 0.2 N HCl.A pH 4.5 buffer solution was prepared by mixing 440ml 0f 1.0 M sodium acetate with 200ml, 1.0M HCl and 360ml of distilled water. The pH of the solution was measured and adjusted to pH 4.5 with 1.0 MHCl.

0.5ml of the extract was diluted to12.5ml in the pH 1.0 and 4.5 buffers, and allowed to equilibrate in the dark for 2 hours. The absorbance of the samples at 512nm (A512nm) and 700nm (A700nm) was measured on a UV- visible spectrophotometer. The difference in absorbance (ΔA) between the anthocyanin extract diluted in pH 1.0 and pH 4.5 buffers was calculated using the equation below

ΔA= (A512 pH1.0-A700nm pH1.0)-(A512nm pH4.5-A700nm pH 4.5)

The A700nm was employed in the calculation of ΔA to correct for any background absorbance due to turbidity on the extracts. The anthocyanin content was expressed as mg cyaninidin 3-glucoside per 100g berries using a molar absorbance co efficient (Є) of 26900 L-1M-1cm-1(Guisti and Wrolstad, 2001).

TACY = (ΔA×MW) ×DF×1000

Є ×0.1×1

Where:

TACY= Total anthocyanin expressed as mg cyaniding 3-glucoside/ 100g of plant material

MW= molecular weight of cyaniding 3-glucoside (449.2g/L)

DF= dilution factor to expressed the extracts on per gram of plant basis

Є= molar absorption co efficient of cyaniding 3-glucoside (26900 M-1cm-1)

0.1= is the conversion factor for per 1000 grams to 100 grams basis.

Brine shrimp bioassay

Brine shrimp lethality test was carried out using hatched Brine shrimp (Artemia salina L) larvae (nauplii) according to the procedure described by The eggs were hatched in artificial sea water (16g of sea salt in 50ml of distilled water) by adding 100mg of brine shrimp eggs to 50ml of sea water that was partitioned into two compartments. The compartment sprinkled with the cysts was left dark, while the other compartment was supplied with bright white fluorescent light. After 24hours of incubation, the hatched shrimps moved to the illuminated side. Ten brine shrimps larvae were then counted and transferred to each sample vial, using a Pasteur pipette and artificial sea water was added to make 10ml. The sample vials were previously containing solution of the extract prepared by dissolving 0.2g of the extract in 20ml distilled water to give concentration of 1mg/ml. The varied concentrations from the stock solution were transferred to different graduated container with the aid of a micropipette. The survivors were counted after 24 hours. Three independent studies were carried out (n =3).

Statistical Analysis

The results are expressed as mean±SEM using Graph Pad Prism Graphical-Statistical Package version 5. The difference between groups was analyzed by Student t-test followed by Dennett’s test with 5% level of significance (p<0.05).

Results

Antioxidants

The extract was assayed for total content of four major types of antioxidant properties. The antioxidant constituents were: to tal phenol, total flavonoid, proanthocyanidins and anthocyanins. However, the percentage yield of the crude extract used for the assays is given as 10.11±1.08%. The results showed the total phenolic content as 1.588±0.04mgGAE/g, which is considerably high compared to the standard. The total flavonoid content expressed as quercetin equivalent per gram of the plant extract showed that the test material had 0.857±0.15mg QUE/g dry weight for the crude extract (Table 1). These two indices are pointer to an increased antioxidant activity. The concentration of anthocyanin in the sample was 0.099±0.08 cyanidin 3-glucoside/100g for the crude extract, while the concentrations of proanthocyanin was 0.038±0.05 cyanidin 3-glucoside/100g for the crude extract. Tannin was also assayed, and it gave a concentration of 1.188±0.04mg/ml (Table 1).

All values are expressed as mean±SEM (n=3)

Antiradicals

The result of the antiradical assays carried out on the extract is shown in Table 2. Using the DPPH (2, 2-diphenyl-1-piccrlhydrazyl hydrate) assay, a well established antiradical assay, the activity was concentration dependent i.e. activity increases with increase in concentration. The extract gave the highest inhibition of 74.55±1.07% at 0.005mg/ml. The calculated IC50 values for the test extract and standard Quercetin were 1.831±0.15 and 0.00326±0.24mg/ml, respectively Table 2. The extract used showed activity despite the significant difference (P<0.05) between the test and standard.

All values are expressed as mean±SEM (n=3). The level of activity between the crude extract and the standard Quercetin is significantly different (p<0.05)

All values are expressed as mean±SEM (n=3).

The nitric oxide inhibition assay also showed that V.amygdalina is a potent scavenger of nitric oxide as shown by the percentage inhibition and IC50 of 3.84±1.03mg/ml Table 3. The FRAP assay result showed a concentration dependent change when the FRAP values of the test fractions were determined. Results were expressed in mmol Fe2+/L. The concentration of Fe2+ in the reaction mixture at 0.5mg/ml, was given as 1.49±0.18 mmol Fe2+/l for the test extract (Table 4).

Brine shrimp lethality test

As shown in Table 5, the plant extract showed the highest percentage lethality to be 75% with LC50 of 1.49mg/ml, whereas, the LC50 for the positive standard (K2Cr2O7) was found to be 10.91±2.22μg/ml. The plant extract showed concentration at 50% percentage lethality to be a little greater than 1mg/ml compared to the standard. In essence, the test sample at the concentration used could be harmless to the biological system. All values are expressed as mean±SEM. This result is a triplicate of three independent experiments.

Discussion

Studies have shown that consumption of biosafe exogenous and natural antioxidant is beneficial, as regard combating diseas es such as cancer, arthritis, diabetes, among others. These diseases emanates from oxidative stress mostly caused by reactive oxygen species (ROS) [14-16]. Moreover, synthetic antioxidant, including tert-butylhydroquinone (TBHQ), buthylatedhydroxytoluene (BHT) and propylgallate have been found to be beneficial, but toxic, as well as with attendant effects [17,18]. This is shown by comparing the bio-safe syzygium cumini fruit juice, a natural antioxidant to the toxic BHT on serum enzymes such as ALT (alanine transferase), AST (aspartate transferase), alkaline phosphatase and urea in rats [19]. For this reason, it has become imperative to continue to investigate and search for more bio-safe antioxidants that could be relevant in the fight against oxidative stress V. amygdalina is useful in this regard [20-22]. Kahaliw and his group have reported on the biosafety of this plant [23]. Moreover, anecdotal evidence attests to its use in the treatment of different ailments after boiling, as well as its use in the preparation of soup. This informed the aqueous extraction carried out, as opposed to the use of organic solvents, such as methanol and ethanol.

A lot has been reported on V. amygdalina as a functional food. In order to further establish its biosafety, the result in table 5 and the work of Kaali justifies V. amydalina as an anti-malaria agent that is biosafe for all the benefits discoursed above [29]. The study of Patnaik and Bhatnagar is in agreement with this study [30]. Moreover, Thompson showed comparable results [31] Data from alcoholic extract of V.amygdalina [32,33] is statistically indistinguishable compared to this study (Table 5).

Conclusion

On the basis of the data from this current research, V. amygdalina is a potent antioxidant attributable to their flavanoid and phenolic constituent that is biosafe for all the health benefits that is known for.

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