BIOACTIVITY OF CRUDE EXTRACT AND PURIFIED COMPOUNDS AGAINST BACTERIA
CONCLUDING REMARK
ABSTRACT
UNITS
(D) Dihydrofolate reductase and (E) DNA gyrase subunit B. 6.5 The predicted docked conformation of Karanjin against regular E. A) Dihydropteroate synthase; (B) β-ketoacyl-acyl carrier protein synthase II (KAS II or FabF);. (D) Dihydrofolate reductase and (E) DNA gyrase subunit B. 6.9 The predicted docked conformation of Glabrin against regular E. A) Dihydropteroate synthase; (B) β-ketoacyl-acyl carrier protein synthase II (KAS II or FabF);.
LIST OF TABLES
Introduction
Furthermore, the efficacy of the crude extracts and purified compounds was studied with regard to antibacterial activity in the perspective of herbal medicines. Furthermore, the biological efficacy of the compounds for the development of herbal medicines was investigated.
Literature Review
Introduction
Historically, most new drugs have been synthesized or inspired by natural products due to their excellent chemical scaffolding. The structural elucidation and characterization of natural products geared to the morphine structure was determined in 1923 (Gulland and Robinson, 1923), the structure of quinine in 1908 (Rabe, 1908) and cocaine in 1898 (Willstatter and Muller, 1898).
The downfall in herbal drug research
Other remarkable achievements were the discovery of „Camptothecin‟ and „Taxol‟ which are considered life-saving drugs in cancer therapy (Oberlies and Kroll, 2004). Many natural products and their derivatives are still used to combat various infectious and non-infectious diseases and are considered to be potent antibacterial, anticancer agents (Butler, 2008; Newman and Cragg, 2012).
Plant secondary metabolites
- Phenolic compounds: Plants produce a variety of secondary compounds that are naturally occurring phenolic compounds, occurring both in free state and as glycosides
The various functions include their role in plant physiology, stress defense and structural integrity (Bhattacharya et al., 2010). Nitrogen-containing alkaloids and sulfur-containing compounds: A large variety of plant secondary metabolites have nitrogen as part of their structure.
Flavonoids and their biological activity
Theobroma cacao (-)-epicatechin and (+)-catechin Immunomodulatory activity Ramiro et al., 2005 Waltheria indica (-)-epicatechin, quercetin and tiliroside Anti-inflammatory activity Rao et al., 2005 Caesalpinia pulcherrima 2´-hydroxy tetramethoxy chalcone Immunomodulatory activity Madagundi et al., 2012. The main analytical methods used by researchers to elucidate and characterize compounds include mainly spectroscopic and chromatographic techniques (Sasidharan et al., 2011).
Leguminaceae (Fabaceae)
Pongamia pinnata
It has also been used against ulcer, chronic fever, rheumatoid arthritis and leprosy (Sangwan et al., 2010) due to its medicinal properties. Stigmasterol-3-O-β-D-galactoside Sterol glycoside Shameel et al., 1996 Methylcarranic acid Aromatic carboxylic acid Baki et al., 2007.
Introduction
All parts of this plant are used as a source of traditional medicine such as Ayurveda and Unani. Seed oil is used for the treatment of various inflammatory and infectious diseases, such as leukoderma, leprosy, lumbago and muscular and articular rheumatism (Mahli et al., 1989; Singh and Pandey, 1996; Khare, 2004).
Materials and methods 1. Sample collection
- Preparation of the organic extract
- Thin layer chromatography (TLC) of crude extract
- Thermal stability of crude extract
- Isolation and purification of the compound from EtOAc crude extract The glass column (3 × 50 cm) was sealed with cotton at the end of the column. The
Therefore, the current chapter focuses on the extraction of crude extracts from the mature seeds of P. After a series of optimizations (using solvents as mobile phase), crude extracts were subjected to column chromatography and solvent extraction.
Results and discussion 1. Plant material
- Isolation and purification of compound(s)
- Isolation and purification of the compound from EtOAc crude extract The major compound isolated from EE by column chromatography was named as PPS-E
Compound Isolation and Purification from Crude MeOH Extract The crude methanolic (ME) extract (50 g) was homogenized in 0.01 N HCl (100 mL) for 8 min. In the present study, the thermal property of the crude organic extract is monitored as a function of temperature.
PPS-E)
Isolation and purification of the compound from MeOH crude extract Compound was also isolated from ME by solvent extraction. After crystallization, brown
In this chapter, crude extracts of seeds were obtained by hot solvent extraction method using Soxhlet extractor. Moreover, the thermoanalytical method was considered as a versatile technique to study the thermal degradation profile of crude extracts, revealing its stability over a temperature range.
Structural characterization of compounds and correlation of its vibrational assignment with density functional theory
Introduction
- High-resolution mass spectrometry (HRMS)
- High-performance liquid chromatography (HPLC)
- Raman spectroscopy and density functional theory (DFT)
- Nuclear magnetic resonance (NMR)
- X-ray diffraction (XRD)
- Thermal gravimetric analysis (TGA)
Ab initio electronic structure calculations of PPS-E and PPS-M were performed using the Gaussian 09 software (Frish et al., 2004). One dimension spectral analysis of PPS-E was performed by dissolving 5 mg of the compound in 600 µL CDCl3 and transferred to 5 mm NMR tube. The detailed structures of PPS-E and PPS-M were elucidated and confirmed by single crystal XRD CrysAlisPro Agilent Technologies, Germany.
Results and discussion 1. Identification of PPS-E
- Fourier transform infrared spectroscopy (FTIR)
- Raman Spectroscopy
- Nuclear magnetic resonance (NMR)
- X-ray Diffraction (XRD)
- Thermoanalytical analysis
- Identification of PPS-M
- Fourier transform infrared spectroscopy (FTIR)
- X-ray diffraction (XRD)
Due to antibond repulsion, the phenyl ring of Karanjin is shifted slightly above the plane (Figure 4.7 D) to minimize its surface energy (Pandey et al., 2014). FESEM image of Karanjin shows that it is needle-shaped crystal with sharp edges (Figure 4.8) Size of the crystal obtained after purification and crystallization is in the range of 0.1-0.5 mm. In the present work, the vibrational spectrum of Glabrin was obtained and compared with the result obtained from DFT calculation (Figure 4.12).
UV-Visible absorption and fluorescence spectra of Karanjin in different solvents and solvent mixture
Introduction
It is calculated using equation 1.2. 1.2) where, Ψa = wave function of the ground state, Ψb = wave function of the excited state, μ. The difference between the maximum wavelengths of absorption and emission of the fluorophore is called The fluorescence of the molecule can be measured by two different techniques, namely: steady-state and time-resolved.
Materials and methods 1. Compound and solvent
- UV-Visible spectroscopy of Karanjin
- Absorption spectra of Karanjin in different solvent
- Critical micellar concentration (CMC) determination
- Time-resolved fluorescence investigation of Karanjin
Fluorescence spectra of Karanjin in different solvents were recorded with Fluoromax 4 fluorescence spectrofluorometer at room temperature (22-25°C). Spectra of Karanjin (1, 5 and 20 μM for water-MeOH; 10 μM for glycerol-water and glycerol-MeOH solvent mixtures) were also recorded. Excitation wavelength (ex) used was according to the excitation maxima (em) of Karanjin in respective solvents.
Results
- UV-Visible spectroscopy of Karanjin
- Absorption spectra of Karanjin in neat solvent
- Molar extinction coefficient of Karanjin
- Absorption study in solvent mixture
- Transition dipole moment and oscillator strength
- CMC determination
- Steady-state fluorescence study of Karanjin in neat solvent
Chapter 5|76 Figure 5.7 Molar extinction coefficients calculation of Karanjin in different solvents at two absorption maxima. The CMC of SDS in water in the presence of Karanjin (20 μM) was determined as a function of surfactant concentration at room temperature (~23°C) (Figure 5.9). Fluorescence spectra of Karanjin (1 μM) in different solvents are obtained by excitation at 260 and 305 nm (Figure 5.11).
320-370 nm Solvent/Excitation
Stokes shift
The fluorescence of Karanjin shows large Stokes shift in water and SDS, and the magnitude of these shifts increases with solvent polarity from MeOH to water. Karanjin in MeOH showed the least Stokes shift of 174 and 152 nm at 260 and 305 nm excitations, respectively. The largest Stokes shift of 230 and 182 nm when Karanjin dissolved in water excited at 260 and 305 nm, respectively.
Steady-state fluorescence study of Karanjin in solvent mixture 1. Glycerol in MeOH
The gradual addition of glycerol increases the viscosity of the medium, and consequently the fluorescence intensity also increases. The fluorescence emission spectra of Karanjin in the binary glycerol-water solvent were excited at 260 and 305 nm, as shown in Figure 5.14. The fluorescence intensity increases with the increase in the percentage of glycerol in aqueous solutions.
The fluorescence intensity, area under the emission curves, and λmax values were plotted against different percentages of water in the methanol solution, as shown in Figure 5.15. The fluorescence intensity of Karanjina showed a bell shape where the intensity increases up to 50% water volume fraction and then gradually decreases until it reaches 100% water volume fraction (Figures 5.15 A and B). A continuous red shift of λmax is observed with an increase in the proportion of water in the mixture at other excitations of 261 and 305 nm, except at the high concentration (20 μM).
Fluorescence spectra of Karanjin (1, 5, and 20 μM) in different MeOH-water mixtures were obtained by excitation at 261 and 309 nm. The emission spectra showed that the gradual addition of water as a cosolvent increases the polarity of the mixtures. Chapter 5|90 Figure 5.15 Steady-state fluorescence study of Karanjin (1, 5 and 20 μm) excited at 261 and 309 nm, showing the fluorescence intensity (A and B), area under the curve (C and D) and λmax (E and F), on the gradual addition of water to MeOH solutions.
Intensity
Area under the curve
Steady-state fluorescence study of Karanjin
Steady-state fluorescence emission spectra of Karanjin in various solvents were obtained after excitation at 295 and 340 nm (Figure 5.16). Fluorescence intensity, area under the curve, λmax and Stokes shift are summarized in Table 5.9 and Table 5.10.
Time-resolved fluorescence study of Karanjin
Chapter 5|93 weighted residual values obtained by fitting the raw decay data from Karanjin in various solvents. Good fit of the fluorescence decay data is observed and judged by reduced χ2 values for respective conditions. A fairly good linear relationship (R2 = 0.98 at ex 295 nm; R2 = 0.93 at ex 340 nm) is found between area under the emission curve obtained from steady-state fluorescence study and mean lifetime obtained from TCSPC data as shown in Figure 5.19.
UV-Visible absorption of Karanjin
- Absorption spectra of Karanjin and its molar extinction coefficient in neat solvent
- Absorption spectra of Karanjin in binary solvent
- Transition dipole moment and oscillator strength of Karanjin
- CMC of SDS in water in the presence of Karanjin
- Stokes shift
- Steady-state fluorescence of Karanjin in solvent mixture 1. Glycerol in MeOH
From the experimental data, the absorbance of Karanjin fluctuates in mixed solvents due to continuous change in the polarity of the environment. Thus, both the fluorescence intensity and the yield of Karanjin can be correlated to the viscosity of the surrounding environment. Karanjin's fluorescence emission spectrum shifts in different percentages of glycerol in water (Figure 5.14) may be due to change in the polarity of the microenvironment.
Time-resolved fluorescence investigation of Karanjin
Chapter 5|108 findings provide interesting insight into the solvent hydrogen bonding effects on the fluorescence emission of Karanjin. This chapter highlights the application of absorption and fluorescence spectroscopic methods in the study of the photophysical properties of Karanjin in various solvents and solvent mixtures. There is a significant improvement in the photophysical properties of Karanjin in micellar solution due to shielding from water inside the micelle and inaccessibility of solvent molecules around the fluorophore molecule in surfactants.
Bioactivity of crude extract and purified compounds against bacteria
Introduction
In vitro study includes sensitivity test (Langfield et al., 2004), Raman scattering (Das et al., 2013), flow cell assay (Oonmetta-aree et al., 2006), flow cytometry (Leonard et al ., 2016) and electron microscopy (Ghosh et al., 2013) which are routinely used for the evaluation of antibacterial properties. Flow cytometry is emerging as a leading bacterial population technology in food biotechnology (Bunthof et al., 2001), industrial biotechnology (Looser et al., 2001) and environmental samples (Hoefel et al., 2003). This methodology uses commercially available dyes (carboxyfluorescein diacetate, cFDA and propidium iodide, PI) that allow the quantification of the different population within the bacterial sample (Antolinos et al., 2014).
Materials and methods
- Study material and reagents
- Bacterial strains
- In silico drug-likeness and toxicity prediction
- Lipinski’s rule
- Molecular docking study 1. Protein and ligand retrieval
- Binding site determination
- Docking study
- Antibacterial property
- Determination of MIC by microbroth dilution assay
- Antibacterial study by Raman spectroscopy
- Effect of compounds on bacterial cell membrane
- Filed emission electron microscopy (FESEM) study
- Determination of antibacterial action by flow cytometry
- Statistical analysis
Characterization of bacterial cells before and after Karanjin exposure was performed at SA and ETEC using Raman spectroscopy (Horiba LabRAM HR spectrometer) in backscattering mode using Argon-ion laser at 514 nm wavelength as. Mean OD values of respective treatment and vehicle control were compared independently at each time point. Field emission scanning electron microscopy (FESEM) was used to visualize changes in SA and ETEC cell morphology before and after treatment with.
Results and discussion
- In silico drug-likeness and toxicity prediction
- Lipinski’s rule of five
- Antibacterial activity of Karanjin
- Antibacterial activity by microbroth dilution assay
- Antibacterial activity by Raman spectroscopy
- Docking study
- FESEM study
- Antibacterial activity of Glabrin 1. Docking study
- FESEM study
- Flow cytometry study
Chapter 6|128 Figure 6.4 Predicted docked conformation of Karanjin against regular S. A) Dihydropteroate synthase; (B) β-ketoacyl-acyl carrier protein synthase III or KAS III or FabH; (C) β-lactamase; (D) Dihydrofolate reductase and (E) DNA gyrase subunit B. Figure 6.5 Predicted coupled conformation of Karanjin against common E. A) Dihydropteroate synthase; (B) β-ketoacyl-acyl carrier protein synthase II (KAS II or FabF); (D) Dihydrofolate reductase and (E) DNA gyrase subunit B. Figure 6.9 Predicted coupled conformation of Glabrin against common E. A) Dihydropteroate synthase; (B) β-ketoacyl-acyl carrier protein synthase II (KAS II or FabF);
Concluding remark
Significance and salient features of the study
All things considered, this is the first report describing the structure and photophysical properties of the substance. The study also highlights the promising antibacterial potential of the isolated compounds, which can certainly help in the development of the new drug in herbal medicine research. It further confirms the use of the purified compound in the antibacterial formulation as the compound effectively kills pathogenic bacteria related to foodborne illness and nosocomial infection.
Future scope
Antioxidant activity of methanolic extract of Pongamia pinnata on lead acetate-induced liver injury in rats. Investigation of the antihyperglycemic activity of aqueous and petroleum ether extract of stem bark of Pongamia pinnata on serum glucose level in diabetic mice. Analgesic and anti-inflammatory effect of the alcoholic extract of the stem bark of Pongamia pinnata (L.) Pierre.
