Burtt" is the result of experiments conducted at the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, India, under the guidance of Prof. Burtta” by Ishani Chakrabartty (No. an authentic record of the results obtained from the research work carried out under my supervision in the Department of Biosciences and Bioengineering, IITG. I am also grateful to her for her exemplary guidance, supervision and encouragement during the course of my Ph.D.
I am grateful to the Department of Biological Sciences and Bioengineering (BSBE), IIT Guwahati and Central Instrumentation Facility (CIF) of the Institute for infrastructural support. I would also like to take this opportunity to sincerely apologize to anyone I may have hurt over the years; nothing was intentional - it was just the times!.
Evaluation of the biopharmaceutical potential of the isolated compound
Studying the hemato - compatibility of the isolated compound
Screening the antimicrobial potential of the compound and deciphering its possible mode of
- Bacteria
Screening the antimicrobial potential of the compound and deciphering its possible mode of
Metallic nanoparticles
Microemulsions
List of abbreviations
List of NMR units
List of units
Comparison of experimental Raman spectra with calculated DFT spectra of pure labdane diterpene at (A) 488nm (B) 514nm. Comparison of SERS spectra of labdane diterpene with and without CuNPs with FT-Raman of labdane diterpene at (A) 488 nm and (B) 514 nm. Specific growth rate (A) as a function of labdane concentration; (B) in the presence of fixed concentration of labdanes and (C) in the absence of labdanes diterpene as a function of substrate concentration; (R2 > 0.96).
Different concentrations of labdanum – ME used; C+ indicates positive control, C- indicates negative control, and C-ME indicates blank ME without labdan. A and E represent bacteria after negative control treatment; B and F represent bacteria after treatment with positive control; C and G represent bacteria after treatment with blank ME, and D and H represent bacteria after treatment with labdanum-loaded ME.
List of tables
Experimental wavenumbers of IR spectra of labdane diterpene (with and without CuNPs) and their corresponding vibrational modes. 68 4.8 Lipinski parameters for isolated labdane-diterpene 74 4.9 Bioavailability and toxicity parameters of labdane-diterpene 75 5B 2.1 Inhibitory efficacy of different concentrations of labdane. 6B.3 Composition of pre-titration mixture at Smix: CoS of 4:1 139 6B.4 Water titration volumes for pre-titration mixture at Smix: CoS of.
6B.5 Composition of titration mixture at Smix : CoS at 1:1 141 6B.6 Water titration volumes for titration mixture at Smix : CoS at. 6B.7 Composition of titration mixture at Smix : CoS at 1:4 143 6B.8 Water titration volumes for titration mixture at Smix : CoS at.
Abstract
CHAPTERS
Introduction
- Objectives
She sought old documents of traditional Chinese medicine for her groundbreaking work and thus, further strengthening the importance of traditional medicine. From early times, India was and is still believed to be the botanical garden of the world as it is the largest producer of medicinal plants. The country not only houses thousands of herbal medicines but also serves them to the rest of the world (Seth and Sharma 2004).
As such, it is no wonder that the region has a rich knowledge of traditional medicine, which developed independently and in parallel with established practices such as Ayurveda, Unani and Siddha in the rest of the country. Finally, the problem of poor solubility of the compound was addressed and some formulation strategies were devised for future therapeutic application.
Review of literature
- Introduction
- Plants as traditional medicine
- Plant derived products as drugs
- Medicinal plants and Zingiberaceae
- Alpinia nigra: The plant under study
- Botanical description
- Phytochemistry
- Bio-pharmaceutical potential
- Labdane diterpene: The compound under investigation
- Antimicrobial potential of plant-derived compounds: The inspiration for development of modern antimicrobials
- Solubility of natural bioactive compounds: The issue that is usually ignored Solubility is the property of a solid, liquid or gaseous substance (called solute) dissolvable
- Importance of solubility and solubility limitations
- Strategies to determine and improve solubility
It has the richest diversity of plants and constitutes about 50% of India's biodiversity (Mao et al. 2009). Oil content estimated from different parts of Alpinia nigra (Ghosh et al. 2014; Islam et al. 2014). In addition, the aqueous extracts of flowers and seeds also show moderate antibacterial activity (Ghosh et al. 2014).
This complex biological response involves a large number of immune cells to eliminate noxious stimuli (Ferrero-Miliani et al. 2007). Most of the prescribed antidepressants have a high risk-to-benefit ratio with prominent long-term side effects (Newman et al. 2004).
Oil extraction and physico – rheological characterization of organic extracts obtained from the seeds of A. nigra
- Introduction
- Materials and methods
- Collection of fruits of A. nigra
- Processing of dried fruits
- Preparation of organic extracts
- Physico – rheological characterization .1 Physical properties
- Statistical analysis
- Results and discussion
- Preparation of organic extracts
- Conclusion
However, no reports are available on the optimization of the yield of extracts from the seeds of A. Most organic plant extracts (except oil-producing plants such as Jatropa curcas or Pongamia pinnata) are usually non-flowing, sticky gums or resins. Furthermore, the physicochemical and rheological characteristics of the organic seed extracts were carefully determined and analyzed.
Colour, odor and condition of the extracts were noted at room temperature by visual inspection (Onyeike and Acheru 2002). An Ubbelohde-type capillary viscometer with a temperature-controlled system was used to measure the viscosity of the two plant extracts. The average yield of the hexane and ethyl acetate seed extracts at different seed-to-solvent ratios using room temperature extraction is reported in Table 3.5.
The observed density of the samples is within the range of seed oil as previously reported (Onyeike and Acheru 2002). Both extracts showed some degree of optical activity, but the activity of the hexane extract is much higher than that of the ethyl acetate extract. The reduced and intrinsic viscosity of the extracts as a function of concentration and temperature, respectively, is shown in Figure 3.5.
The reduced viscosity appeared to increase with increasing sample concentration at all temperatures tested. It is evident that both extracts, especially the hexane extract of the seeds of A. This work also covers the physico-rheological characterization of the extracts obtained from the seeds of A.
Isolation, purification and characterization of bioactive compounds from the seeds of A. nigra
- Introduction
- Materials and methods .1 Isolation of the compound
- Purification of the compound
- Characterization of the compound .1 Spectroscopic characterization
- In silico studies
- Results and Discussion .1 Isolation of the compound
- Purification of the compound .1 pTLC
- Characterization of the compound
- In silico studies
- Conclusion
For mass spectral analysis, 1 mg of the pure isolated compound was dissolved in 2 mL of methanol (HPLC grade). ISOL = Intensity of the normal Raman spectra of the compound (without CuNP) CSERS = Concentration of the compound in the SERS mixture. The likelihood of the isolated compound to be a drug was verified using Advanced Chemistry Development (ACD/I-lab).
The optimum yield of the compound from hexane extract w.r.t seed-to-solvent ratio was found to be 1:4 as shown in Fig 4.2. The yield of the compound from ethyl acetate extract was found to be negligible in all the ratios (Fig 4.3). The structure of the compound (Fig 4.4) was deduced using a number of analytical techniques given below.
The mass of the isolated compound was confirmed by HRMS and LC-MS (Figures 4.5 and 4.6). The IR spectrum of CuNP conjugated with labdan diterpene was consistent with the spectrum of the individual compound (Figure 4.14). All peaks of the mixture sample confirmed the excellent match of CuNPs with the compound.
The reduced and intrinsic viscosity of the compound as a function of concentration and temperature, respectively, are shown in figure. However, the log P value of labdane diterpene met the claims of the 'modified Lipinski's rule of five'. The research with vibrational spectroscopy (FT-IR and Raman) shows that the experimental spectra of the compound correspond well with the theoretical wave numbers calculated with DFT.
Evaluation of the biopharmaceutical potential of the isolated compound
- Studying its hemato - compatibility
- Screening the antimicrobial potential of the compound and deciphering its possible mode of action against
- Candida albicans
From Figure 5A.4 B (inset), it became clear that pores were indeed formed on the surface of erythrocytes after treatment with the compound. Cell morphology was slightly distorted and no such deviations were observed in healthy erythrocytes. FESEM was used to visualize the changes in bacterial morphology after treatment with the compound (0.006 mg/ml and 0.4 mg/ml).
Labdan inhibition at higher concentrations was comparable to that of the positive control. However, low concentrations of the compound (0.012 mg/ml and 0.006 mg/ml) could not inhibit the growth of E. The viability assessment of 4 bacteria after treatment with labdanum diterpene is shown in Figure 5B 1.2.
Whereas, membrane disintegration, cell shrinkage and cell lysis were observed in Gram-negative bacteria treated with 0.4 mg/ml compound (Figure 5B 1.3 D and E). The culture was then freshly inoculated into YPD broth and treated with different concentrations of the compound. Dissolved oxygen (DO) in the media is a direct measurement of bacterial respiration and reflects the growth state of the microorganism (Krishnaraj et al. 2010).
However, the minimum effective concentration of the compound by the broth micro-dilution method was found to be 0.000625. The concentration of the substrate decreased as the organism multiplied in the presence of different concentrations of the compound (Fig 5B 2.8 A). The substrate concentration in the medium was comparable to the growth stage of the organism.
Formulating a strategic approach for the delivery of the isolated compound as a potential antibacterial agent using
A plot of labdane diterpene concentration vs % transmission was made to determine the solubility of the compound in the aqueous and NB culture media used. The initial concentration of the CuNPs and AgNPs was 0.34 mg/ml and 0.2 mg/ml respectively. The growth curve analysis of the 4 bacteria was performed after treatment with the NPs according to the protocol given in the previous chapter (section 5B 1.2.2.1).
It was very necessary to determine the viability of the bacterial cells after treatment with the NPs in order to determine the suitable concentration of NPs to be used in combination with labdane diterpene. Viability determination of the bacterial cells after treatment with the NPs and a combination of NPs and labdans was done according to the protocol given in the previous chapter (section 5B 1.2.2.2). FESEM was used to visualize the changes in the morphology of the Gram-negative bacteria after treatment with the NPs and the combination of compound and NPs.
FESEM was performed to determine the size and morphology of CuNPs and AgNPs and to visualize their interaction with the compound. However, both CuNPs and AgNPs could not show any growth inhibition of Gram-positive bacteria. The viability assessment of 2 Gram-negative bacteria after NP and NP + labdan treatment is shown below in Figure 6A.11.
The concentration of the NPs used in this study was in the hemato-compatible range (< 0.1 mg/ml). The effect of the combination of the compound with CuNPs and AgNPs on erythrocytes was also investigated. The increase in the efficiency of the compound at lower concentrations was achieved by using CuNPs and AgNPs.
The antibacterial potential of the synthesized ME (loaded with labdanes) was tested on the Gram-negative bacteria, E. The physiological state of the bacterial populations after treatment with the labdanes-loaded ME was analyzed and determined using flow cytometry.
