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BS7200-21-22 EXPERIMENTAL TECHNIQUES AND LABORATORY PRACTICE

solution:

  1. Technique introduction:

Western blotting was done to visualize the effect on COX-1, COX-2, Mpges-1 protein expression without or with the treatment of different concentrations of LPS and resveratrol. In this technique for immunoblotting, anti-COX-1, anti-COX-2, and anti-mPGES-1 antibodies were used to visualize COX-1, COX-2, and mPGES-1 proteins, respectively, and β-actin was used as the loading control. The microglial cells were seeded in a 24 well plate at a density of 3 x 10^6 cells/well. The cells were treated with LPS (10ng/mL) and variable concentration of resveratrol (0,1,5,10,25,50 µM) for 24 hrs and incubated in optimal conditions inside a CO2 humidified chamber at 37o C. After 24hrs the plate was washed gently with PBS (Phosphate buffer saline) 2-3 times to remove cell debris. The cells were lysed with lysis buffer using 1.3X sodium dodecyl sulfate and orthovanadate (100 µM). The homogenization of lysate was carried out using a 26-gauge syringe needle. The homogenization was carried out to release the cytoplasmic content of the cell which suppose to contain proteins and other cell components for better results. The sample was then centrifuged for 10 mins at 10,000 rpm at 4o C. This centrifugation of homogenized cells removed the unlysed cells and the cellular debris from the lysate (Bi et al., 2005).

Furthermore, the protein estimation of lysate was carried out using the BCA protein estimation method using the kit supplied from the Pierce company. The Cu ions from the BCA reagent react with amino acids in proteins to give a coloured product (Candelario-Jalil et al., 2007). The intensity of colour formation is directly proportional to protein concentration in the sample. For the protein estimation standards were prepared using Bovine serum albumin (BSA) and was used for the unknown protein concertation determination for the different lysates prepared from the standard graph. The concentration of protein was calculated using the slope from the standards and was used for further experiment. All the protein lysates were then used for SDS-PAGE, followed by western blotting. The DTT (final concentration 10mM) and bromophenol blue were added to all the cell lysate samples, and the lysate was heat-inactivated at 95o C for 10 min. The SDS-PAGE gel was prepared using the electrophoretic assembly. The gel cassette was then subjected to electrophoresis assembly, and a running buffer was added to the buffer tank. Each protein lysate was loaded at a concentration of 60 µg in each well and followed by reducing SDS-PAGE (Poly-acryl amide gel electrophoresis) for the protein separation. The different protein lysate samples were loaded as follows:

Lane 1: Untreated microglial cells

Lane 2: LPS treated cells (10 ng/mL)

Lane 3: LPS (10 ng/mL) and resveratrol 1 µM treated cells

Lane 4: LPS (10 ng/mL) and resveratrol 5 µM treated cells

Lane 5: LPS (10 ng/mL) and resveratrol 10 µM treated cells

Lane 6: LPS (10 ng/mL) and resveratrol 25 µM treated cells

Lane 7: LPS (10 ng/mL) and resveratrol 50 µM treated cells

Once the bromophenol blue dye ran for 3/4th of gel, the electrophoresis was stopped, and the gel was removed carefully in the buffer. The protein was then transferred using semi-dry blotting on to polyvinylidene fluoride (PVDF) membrane. Once protein was transferred on to PVDF medium, the membrane was kept in buffer and blocking was done using 5% BSA for 1 h. The PVDF membrane was then washed with Twin-TBS buffer 3 times to remove unwanted interactions. The PVDF membrane was then carefully incubated with primary 1o antibodies against each protein as mentioned above; the membrane was then developed using goat-anti-COX-2, goat-anti-COX-1, and rabbit-anti-mPGES-1 primary antibody in 1:500 dilution in Tris-buffer saline (containing Twin and BSA) and incubated at 4o C for overnight. Blots were then washed with twin-TBS buffer 3 times using a rocker. Then blots were incubated using HRP coupled rabbit anti-goat IgG and goat anti-rabbit IgG for 1h at RT and developed using chemiluminescent reagents. As soon as the chemiluminescent reagents were added to the blot, the image was captured using ScanPack 3.0 software, and the blots were also quantified using the same software.

The use of western blotting for the protein product is not much suitable when the treatment is given to analyze the RNA expression as all RNA produced do not convert into protein. However, in the detection of protein, western blotting is a highly sensitive technique that can detect the presence of protein even femtomoles or picomoles quantity.

  1. Figure interpretation: In the present study, to analyze the effect of resveratrol on the expression of 3 genes, COX-1, COX-2, and mPGES-1 in microglial cells. Therefore, they treated microglial cells with LPS (10ng/mL) and variable concentration of resveratrol (0,1,5,10,25,50 µM) to study its effect on the mRNA expression of COX-1, COX-2 and mPGES-1 gene. The microglial cells were plated in a 24-well cell culture dish and then followed by pre-incubation for 24 h with LPS (10 ng/mL). Then after the incubation, the media from the plate was removed gently and washed with serum-free media, and then the treatment of resveratrol (0-50 µM) was carried out for 15 min. The protein product of mRNA of all these genes was visualized using western blotting.

The western blot from the figure clearly shows that in the absence of LPS and resveratrol, the expression of COX-2 mRNA and mPGES-1 mRNA was negligible in control microglial cells. It means that microglial cells do not express COX-2 and mPGES-1 in normal physiological conditions. The expression of COX-1 and mPGES-1 is generally linked with the activation of neuroinflammatory pathways in rat brain cells (Tanabe et al., 2002). However, several studies have reported that bioactive compounds show anti-inflammatory activity in brain cells which helped in the reduction of gene expression of PGE-2 and COX-2 protein (Choi et al., 2008). Moreover, the addition of LPS to microglial cells increases the inflammatory protein expression such as COX-2, mPGES-1 and PGE-2 (Wendeburg et al., 2009). This study also observed that the treatment of LPS in microglial cells had increased reactive oxygen species intracellularly in a dose-dependent manner. Previous studies have shown that LPS induces signal transduction in rat microglial cells which is responsible for PGE2 expression (Kis et al., 2004). The western blot showed the expression pattern of COX-1, COX-2, and mPGES-1 in microglial cells while β-actin served as the loading control. The induction of microglial cells also showed a marked increase in the level of PGE-2, which is involved in the inflammatory pathway (Schwab et al., 2002). To check whether resveratrol shows anti-inflammatory activity in microglial cells. So, it was observed that the addition of LPS alone enhanced the basal level expression of COX-2 and mPGES-1 (Block et al., 2005) in microglial cells. It means that LPS induces inflammatory response and thus increases reactive oxygen species and eventually increases the protein expression of COX-2 and mPGES-1. On the basis of this data, when the microglial cells were treated with resveratrol at increased concentrations from 0-50 µM, it was observed that the increased concentration of resveratrol decreases the mPGES-1 mRNA expression near to basal level in western blot. The anti-inflammatory activity of resveratrol can be established with the decrease in the protein expression of mPGES-1. Interestingly, the increased resveratrol concentration from 0-50 µM also enhanced the mRNA levels of COX-2. Moreover, the treatment of LPS, as well as resveratrol, did not show any effect on the mRNA expression of COX-1. This indicates that the LPS and resveratrol did not play any role in COX-1 expression and thus, the basal level of COX-1 was the same throughout the experiment. COX-1 gene is also reported to be linked with inflammation in microglia/macrophages (Candelario-Jalil et al., 2006) In the present study, the COX-1 protein expression was not affected with the treatment of resveratrol in microglial cells, which indicates that the specificity of resveratrol in the reduction of specific proteins involved in inflammation such as COX-2 and mPGES-1 but not COX-1. However, the quantification of the blots was not done, which could have given a more detailed picture of the gene expression of all three genes.

Question 2

Figure 2 displays the H1NMR spectra of distinct peaks in d6-DMSO, revealing that there were a total of 13 peaks. All of the hydroxyl atoms in the molecule were exchanged with the deuterium oxide solvent in H1NMR spectra, resulting in deuteroxyls. As a result, the hydroxyl atoms produce signals that do not appear in the NMR spectrum and hence do not couple with the other signals, simplifying and making the spectrum easier to interpret. The number of magnetically varied protons is approximated by the sum of signals recorded in an H1NMR spectrum. Protons that are magnetically varied are frequently chemically diverse as well. As a result, the number of signals seen in H1NMR spectra corresponds to the number of chemically different protons or groups of protons present in the substance. Twelve of the 13 peaks correspond to the hydrogen atoms in the molecule. The peak that showed up the most in the sample was d6-DMSO. The proton ratio was used to assign peaks. Peak assignment in the below H1NMR spectra was done based on observed ratios. In the spectra, the ratio of 1 represents the signal hydrogen atom connected to a neighbouring atom, while the ratio of 2 reflects CH2, but the ratio of 3 reflects CH3. Peak assignment was done using this information, and it was discovered that there were three CH3 peaks. In addition, two CH2 peaks and seven separate CH1 spectra were found in the spectra.

Figure 2: Peaks assigned to 1HNMR spectra d6-DMSO for an unknown compound

However, during H1NMR, each signal from the proton NMR spectrum can divide into one or more peaks or cannot split. This sort of numerous peaks in the H1NMR spectrum is known as signal multiplicity; various terminology for these types of multiple peaks includes a singlet, doublet, triplet, quartet, and multiplet. The n+1 rule is a regularly used notion for signal multiplicity. The rule states that protons implicated in neighbouring carbons from the compound would fragment the signal recorded in HNMR spectra for the proton under investigation into n+1 peaks, with n denoting the number of such protons in the compound. The presence of several proton signals is highly beneficial in predicting the fate of hydrogen atoms in neighbouring carbon structures.

The peak integration in the H1NMR spectrum from the integrals shows that in the spectra between 6.8 and 8.2 ppm, one multiple of four peaks, also known as a quadruplet, and three multiples of two peaks, also known as duplets, were seen. Furthermore, two peaks in the spectrum were tall, sharp signals, which were most likely CH signals from the compound. It means that the single peak seen between 6.8 and 7.0 ppm in the chemical has no hydrogen atoms nearby. However, the quadruplet signal cloud of peaks at 7.2 ppm indicates that it could be a CH atom from the compound’s pyridinium molecule. Furthermore, two duplets of multiple signals at 7.4 ppm could be due to the compound’s 1,4-dimethyl benzene structure.

The lone appearance in the structure in H1NMR spectra is indicated by the solitary strong peak found at 7.6-7.8 ppm. At 8.2 ppm, two distinct peaks of CH were identified. Both of these peaks were duplets, indicating that both hydrogen atoms in the molecule were encircled by one extra hydrogen atom.

Figure 3 shows six distinct peaks in a cluster, with the peak between 6.8 – 7.0 ppm likely resulting from either the amine unit hydrogen occurring in the architecture. Furthermore, the quadruplet and duplet peaks observed between 7.2-7.6 ppm may be due to aromatic hydrogens present in the unclear chemical from H1NMR. The single sharp peak observed at 7.6-7.8 ppm can be attributed to the compound’s phenolic bunch, while the duplet peak observed at 82 ppm can be attributed to the presence of carboxylic corrosive gathering.

Figure 3: Expanded region for 1HNMR spectra d6-DMSO for the unknown compound to understand the multiplicity

Question 1b

Appearance of spot (pH1.0)Appearance of spot (pH8.0)
IndomethacinVisible spotNo spot
EphedrineNo spotVisible spot
MorphineVisible spotVisible spot

In this experiment, a rudimentary model of the gastrointestinal tract is created by exposing ethyl acetate to water at pH 1 and pH 8 for three different medicines. The lipid lining of the intestinal system is resembled by ethyl acetate, and aqueous solutions of two distinct pH correspond to the acidic stomach contents (pH 1) and the alkaline intestine contents (pH 2). (pH 8). To understand the effect of pH on the absorption of different types of medications, the molecules indomethacin, ephedrine, and morphine are used as examples of acid, basic, and neutral pharmaceuticals, respectively. The hydrophobicity [lipid solubility] of medicine, its formulation, and the manner of delivery all influence its absorption. In most cases, a medicine must pass through the lipid bilayer of cell membranes in order to enter cells, hence it must be lipid-soluble unless it requires active transport or polar channels to do so. The drug absorption process for weakly acidic and weakly basic medications is very pH dependant, and the drug is rendered lipid-soluble only when it is in an unionised state.

The aqueous solutions of three different medicines, Indomethacin, Ephedrin, and Morphin, were produced and then extracted with ethyl acetate and spotted on a Thin Layer Chromatography [TLC] plate and studied under UV light at two different pH 1.0 and pH 8.0. The pH of the various portions of the gastrointestinal system is maintained. The stomach contains an acidic component, while the intestine contains an alkaline component. The varying pH water solutions of each of the three medications represent distinct regions of the gastrointestinal system, whereas the organic solvent ethyl acetate represents the lipidious nature of tissue linings where absorption occurs.

The hydrophobicity [lipid solubility] of medicine, its formulation, and the manner of delivery all influence its absorption. The pKa of indomethacin is somewhere equal to the acidic pH of around 1 and thus is maintained in an unionized state in this pH 1.0. During this state, it resembles a lipid-soluble state and can be easily absorbed by the intestinal linings. Unlike in the pH8.0 state, the pKa of the indomethacin is such that it remains in an ionized state and therefore cannot be absorbed by the hydrophobic lipid linings of the intestines. The pKa of ephedrin is somewhere equal to the basic pH of around 8 or so and thus is maintained in an unionized state in this pH 8.0. During this state it resembles lipid soluble state and can be easily absorbed by the intestinal linings. Unlike in the pH1.0 state, the pKa of the ephedrine is such that it remains in an ionized state and therefore cannot be absorbed by the hydrophobic lipid linings of the intestines. On the other hand, there are two pKa of morphine and is somewhere equal to both the acidic pH of around 1 and the basic pH8.0. It is thus maintained in an unionized state in both the pH values. During both the pH, its state resembles lipid soluble state and can be easily absorbed by the intestinal linings. In this pH 1.0, the pKa of indomethacin is about equal to the acidic pH of around 1 and hence remains unionised. It mimics a lipid-soluble form in this state and can be easily absorbed by the gut linings. In contrast to the pH8.0 state, the pKa of indomethacin stays ionised and hence cannot be absorbed by the hydrophobic lipid linings of the intestines. The pKa of ephedrin is almost equivalent to the basic pH of roughly 8 and so remains unionised in this pH 8.0.

It mimics a lipid soluble form in this state and can be easily absorbed by the gut linings.

Indomethacin has a pka value of 4.5, which means that at pH 4.5, it has a net zero charge. However, when an indomethacin spot was found at pH 1, it can be attributed to the drug’s slightly acidic pka. As a result, under strongly acidic pH, indomethacin acts stable and does not move, making it available for absorption by parietal cells of the stomach. The chemical structure of indomethacin may exhibit a net neutral charge at pH 1, as shown below. However, at pH 8, which is also similar to the pH of the intestine, no spot was found, indicating that indomethacin has a charge on it and hence does not stabilise at that pH. Due to indomethacin’s instability, it can be absorbed in the intestine.

Furthermore, the structural changes in indomethacin at pH 8 can be deduced from its structure, leading to the conclusion that it may lose OH- ions from the carboxylic group in the compound, resulting in a net negative charge.

The detectable spot of ephedrine at pH 8.0 indicates that the pKa value of ephedrine is 9.65, and the pH 8.0 makes it a suitable solvent, and therefore the charge on ephedrine remains neutral, stabilising the medication in the intestine. However, the absence of a spot at pH 1.0 indicated that the medicine was not stable at that pH and so could not be absorbed in the stomach. The structure of ephedrine demonstrates that at pH 1.0, the NH atom in the chain can be destabilised, resulting in additional hydrogens at N. This acidic environment results in more protons at the N atom, making the ephedrine molecule positively charged and thereby destabilising the atom at that pH.

Whereas in neutral pH, ephedrine is close to the pKa value; nevertheless, the lack of electron accessibility at neutral pH makes ephedrine negatively charged and destabilises the structure. Nevertheless, at a pH that allows ephedrine to pair with OH- ions from the solvent, it becomes stable and neutralises the charge on it, making it acceptable for absorption in the gut.

Question 2

LC-MS/MS is among the most effective techniques for analysing various chemical compounds or active medicinal components utilised in biological analysis. Excellent throughput procedures, such as UPLC, give high accuracy, specificity, and sensitivity without compromising sample and result quality. Importantly, UPLC requires a relatively small amount of material for analysis, therefore thus is well suited to the examination of clinical or biological samples. Aspirin is an acetyl derivative of salicylic acid that is widely used to treat moderate to mild discomfort, fever, and, in certain cases, inflammation. Aspirin is a nonsteroidal anti-inflammatory medication. Salicylic acid is created by hydrolyzing acetylsalicylic acid or aspirin.

Figure 4 shows the UPLC spectrum of a standard molecule, which was aspirin or acetylsalicylic acid. The retention period for both chemicals was investigated, and literature revealed that aspirin elutes faster than salicylic acid. Based on these findings, we determined that the UPLC standards were of salicylic acid and aspirin. In UPLC, aspirin elutes faster than salicylic acid, as seen in figure 5, which shows the peak for the salicylic acid standard. Figures 4 and 5 clearly show that the first peak eluted in figure 6 was of aspirin according to the standard from figure 4, and the second peak eluted was of salicylic acid according to the standard from figure 5.

Figure 4: UPLC spectrum of standard Aspirin

Figure 5: UPLC spectrum of standard salicylic acid

Based on the retention time in the chromatogram (Johnson et al., 2003) from figure 5 it can be seen that aspirin moves faster than salicylic acid. The reason behind the faster elution of aspirin than salicylic acid is due to the acetyl group attached to the salicylic acid structure. This leads to lesser interaction of aspirin molecules with stationary phase in the UPLC column. Moreover, the OH group from the salicylic acid leads to higher retention time in UPLC and which interacts with the stationary phase in UPLC. The OH group from the salicylic acid can interact due to the weak H ion as compared to the acetyl group in the aspirin which makes it more stable and less interactive with the UPLC column and thus aspirin elutes faster than salicylic acid during UPLC.

Furthermore, Figure 5 depicts the aspirin and salicylic acid peaks. The area under the curve for aspirin was 3699.67, while the area under the curve for salicylic acid was 1628.71. Figure 5 also showed only two peaks in the chromatogram, which were aspirin and salicylic acid in the combination, respectively. This demonstrated that there are no contaminants in the combination and that the mixture is only composed of the two compounds of interest. Which showed that there are no impurities in the mixture and the mixture is only made up of both the interested compound only.

Alternatively, the level of impurities may be calculated using the area under the curve for each peak, as shown in the formula below.

% impurity= peak area of impurity/ sum of peak area observed *100

Calculate the area under the curve for all of the peaks in the chromatogram to find the impurity in the compound. Because the purpose of this experiment was to determine the retention of both aspirin and salicylic acid, the chromatogram only included two peaks. Apart from the solvent system, this indicated that only aspirin and salicylic acid were present in the solution. Figure 5 shows the peak of the solvent system between 0.3 and 0.5 minutes. This indicates that the solution was pure and that there were no signs of contamination in it.

As can be observed from the retention time in the chromatogram in figure 5, aspirin moves faster than salicylic acid. Because of the acetyl group connected to the salicylic acid molecule, aspirin elutes faster than salicylic acid. As a result, the aspirin molecule has less interaction with the stationary phase in the UPLC column. Because of the presence of an acetic group in its structure, aspirin is more lipophilic than salicylic acid. Aspirin’s lipophilic nature aids in decreased column retention when compared to salicylic acid. Furthermore, the temperature employed in UPLC is substantially higher, resulting in a decrease in the polarity of water, making it a more appropriate solvent for aspirin than salicylic acid.

Aspirin may elute faster than salicylic acid during UPLC as a result of this. In addition, as the temperature rises, the viscosity decreases. Reduced viscosity allows compounds like aspirin to travel faster (Fekete et al., 2015) through the column, resulting in a shorter retention time. Furthermore, the OH group from salicylic acid causes a longer retention time in UPLC and interacts with the stationary phase. Due to the weak H ion, the OH group from salicylic acid can interact with the UPLC column, whereas the acetyl group in aspirin is more stable and less interactive, and so aspirin elutes faster than salicylic acid during UPLC.

Figure 5: UPLC spectrum of the unknown sample

Bibliography:

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Block, M. L., & Hong, J. S. (2005). Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Progress in neurobiology, 76(2), 77-98.

Candelario-Jalil, E., de Oliveira, A. C. P., Gräf, S., Bhatia, H. S., Hüll, M., Muñoz, E., & Fiebich, B. L. (2007). Resveratrol potently reduces prostaglandin E 2 production and free radical formation in lipopolysaccharide-activated primary rat microglia. Journal of neuroinflammation, 4(1), 1-12. https://doi.org/10.1186/1742-2094-4-25

 Candelario-Jalil, E., Akundi, R. S., Bhatia, H. S., Lieb, K., Appel, K., Muñoz, E., … & Fiebich, B. L. (2006). Ascorbic acid enhances the inhibitory effect of aspirin on neuronal cyclooxygenase-2-mediated prostaglandin E2 production. Journal of neuroimmunology, 174(1-2), 39-51.

Choi, S. H., Langenbach, R., & Bosetti, F. (2008). Genetic deletion or pharmacological inhibition of cyclooxygenase‐1 attenuate lipopolysaccharide‐induced inflammatory response and brain injury. The FASEB Journal, 22(5), 1491-1501.

Fekete, S., Veuthey, J. L., & Guillarme, D. (2015). Comparison of the most recent chromatographic approaches applied for fast and high resolution separations: theory and practice. Journal of Chromatogra

Johnson, K. J., Wright, B. W., Jarman, K. H., & Synovec, R. E. (2003). High-speed peak matching algorithm for retention time alignment of gas chromatographic data for chemometric analysis. Journal of Chromatography A, 996(1-2), 141-155.

Kis, B., Snipes, A., Bari, F., & Busija, D. W. (2004). Regional distribution of cyclooxygenase-3 mRNA in the rat central nervous system. Molecular brain research, 126(1), 78-80.

Schwab, J. M., Beschorner, R., Meyermann, R., Gözalan, F., & Schluesener, H. J. (2002). Persistent accumulation of cyclooxygenase-1—expressing microglial cells and macrophages and transient upregulation by endothelium in human brain injury. Journal of neurosurgery, 96(5), 892-899.

Tanabe, T., & Tohnai, N. (2002). Cyclooxygenase isozymes and their gene structures and expression. Prostaglandins & other lipid mediators, 68, 95-114.

Wendeburg, L., de Oliveira, A. C. P., Bhatia, H. S., Candelario-Jalil, E., & Fiebich, B. L. (2009). Resveratrol inhibits prostaglandin formation in IL-1β-stimulated SK-N-SH neuronal cells. Journal of Neuroinflammation, 6(1), 1-8.

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