Ajay Kalamdhad for their practical advice, suggestions and timely guidance in completing the thesis. Chapter eight describes the significance and salient features of this study with the scope of further in-depth studies in the future.
LIST OF TABLES
Objectives
4] Studies on molecular docking and inhibition kinetics of labdane diterpenes on α-amylase and α-glucosidase enzymes to combat type 2 diabetes. 5] Studies of labdane diterpenes against matrix metalloproteinase inhibitory activity and cytotoxicity on HT1080 fibrosarcoma cells.
Introduction
Increases in pollution, unhealthy lifestyles, stress, loss of traditional medicinal practices and declines in plant biodiversity have increased in recent times, leading to an alarming disruption in the structure and function of nature (Dubey et al. 2004; Alves and Rosa 2007). The demand for various drugs, health products and drugs from plant sources is increasing around the world due to their healthy interaction with physiological flora, relatively less side effects and cheaper (Dubey et al. 2004; Sharma et al. 2008).
History of herbal medicine
Two analogues (first generation) of camptothecin are in use for the treatment of ovarian cancer and many new analogues (second generation) are included in clinical trials. Taxol is clinically approved for the treatment of ovarian cancer and is currently used against various other cancers including breast cancer.
Natural products in drug discovery
This represents a major breakthrough in natural product-based drug discovery research and addresses the limitations of conventional natural product screening. Nevertheless, these examples demonstrate the importance of natural products in the discovery of antimicrobial agents.
Herbal medicine: An Indian scenario
Due to the diverse collection of medicinal plants in India, it is often called the "Medicinal Garden of the World". Due to lack of proper scientific management and inputs in herbal drug development, India was not able to perform well in international herbal drug trade.
Plant secondary metabolites: Source of herbal medicine
- Chemistry of terpenes
- Therapeutic potential of diterpenes
- Labdane type diterpenes: A promising source of bioactivity
- Antibacterial activity
- Anticancerous activity
- Anti-inflammatory and analgesic activity
- Neuroprotective and antioxidant activity
- Antidiabetic activity
- Other activities
Future perspectives
Introduction
Chemical profiling has been performed using various analytical techniques and is also strongly recommended for quality control of herbal medicines (Chitlange et al. 2008). In the plant kingdom, members of the Zingiberaceae family are known for their medicinal properties and diverse pharmaceutical uses (Li et al. Many species belonging to the genus Alpinia have been reported to have medicinal properties and are widely used in folk medicine.
Burtt is a member in the genus Alpinia distributed mainly in China, Thailand and other Southeast Asian countries, including NEI (Wu 1981).
Materials and methods
- Sample collection and maintenance
- Preparation of the organic extracts
- Determination of total soluble phenolics (TSP)
- Phytochemical screening of organic extracts
- Chemical profiling of organic extracts
- Fourier transform infrared (FTIR) spectroscopy analysis
- Nuclear magnetic resonance (NMR) analysis
- Gas chromatography mass spectrometry (GC-MS) analysis
- Determination of DPPH radical scavenging activity
The extraction for the same fixed amount of the sample (200 g) was performed according to the polar strength of the solvent (300 ml each) starting from non-polar (n-hexane) to polar (ethyl acetate and methanol), respectively. Few drops of Mayer's reagent were added to the filtrate next to the test tube. The formation of a foam layer on top of the test tube indicated the presence of saponins.
The absorption spectrum of the pellets (samples) was obtained with a Perkin-Elmer FTIR spectrophotometer (Norwalk, Connecticut) in the range 450-4000 cm−1.
Results and discussion
- Plant material and herbarium
- Preparation of the organic extracts
- Estimation of phenolic content
- Phytochemical screening of organic extracts
- FTIR spectral analysis
- NMR spectral analysis
- GC-MS analysis
- DPPH assay
Moreover, the extraction yield and biological activity of the extracts have a strong relationship with the solvent used, mainly due to the different polarity of the chemical compounds (Moure et al. 2001). Many phenolic bioactive compounds have been identified from the different members of the genus Alpinia (Ghosh and Rangan 2013). From the present results it could be stated that the methanolic extract (S-Met) is rich in most of the phytochemicals tested viz.
It appears to be based on the reduction of DPPH in alcoholic solution in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical form DPPH-H in the reaction.
Conclusion
The chapter describes the isolation and identification of two diterpene compounds of the labdane type and a triacylglyceride from A.
Introduction
Members of the genus Alpinia have a complex chemical profile and possess diverse types of bioactive compounds. The presence of two flavone glycosides (astragalin and kaempferol-3-O-glucuronide) in the seed clusters of A. nigra was previously described by Qiao et al. They also reported kaempferol-3-O-glucuronide as the major compound in the fruit pulp of A. Previous study also described the presence of two volatile oils, β-Pinene and α-Pinene, in the fruit and rhizome of A.
Furthermore, the purified compounds were investigated for their drug probabilistic properties and subsequently the biocompatibility of these bioactive compounds was determined using human erythrocytes.
Materials and methods
- Isolation of compounds
- Thin-layer chromatography (TLC)
- Column chromatography (CC)
- Purification of the selected fractions
- High-performance liquid chromatography
- Characterization of purified compounds
- Nuclear magnetic resonance (NMR) analysis of compounds
- Fourier Transform Infrared (FTIR) analysis of compounds
- High Resolution Mass Spectrometry (HRMS) of compounds
- Physico-chemical properties
- RBC hemolysis assay
- Statistical analysis
The absorption spectra of the samples were obtained with a Perkin-Elmer Fourier Transform Infrared (FTIR) spectrophotometer (Norwalk, Connecticut) in the 450-4000 cm−1 range. Mass spectrum of the isolated compounds was recorded on instrument Water Q-TOF premier mass spectrometer (USA) by electro-spray ionization (ESI) technique with a flow rate of 0.2 ml/min) on the C-18 column and at a total run time of 30 minutes To understand the suitability of the characterized compounds as a drug, analysis of the Lipinski descriptors (Lipinski et al. 2001) for estimating bioavailability using the SciFinder® program.
The software takes into account the parameters describing the molecular properties of the compounds that are important for the pharmacokinetics of drugs in the human body.
Results and discussion 1. Identification of compounds
- Identification of compound I
- Spectroscopic fingerprint of compound I
- Identification of compound II
- Spectroscopic fingerprint of compound II
- Identification of compound III
- Spectroscopic fingerprint of compound III
- Physico-chemical properties
- Hemolytic assay
The 1H and 13C NMR data of compound I agree with previously published data (Sirat et al. 1994). The 1H and 13C NMR fingerprints of compound II were in agreement with a previous report (Haraguchi et al. 1996). Often, compounds isolated from natural sources were cited as an exception to Lipinski's "rule of five."
They also revealed that the majority of the compounds mentioned in The Dictionary of Natural Products showed no violations of Lipinski's rule and moreover 85%.
Conclusion
Introduction
Among various types of phytoconstituents, diterpenes are known to exhibit a wide spectrum of biological activities, including antibacterial activity (Kuzma et al. Previous studies have shown that various classes of diterpenoids, such as pimarans, clerodanes, kauranes, isopimaranes, labdanes and others have been used as a potential source of antimicrobial agents (Kalpoutzakis et al. In recent years, extensive work has been carried out using various plant extracts and isolated bioactive molecules for their antibacterial properties, but the effect of crude extracts or any isolated compounds of A.
Materials and methods
- Study materials
- Bacterial strains
- Determination of minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
- Determination of antibacterial activity using flow cytometry (FC)
- Field emission scanning electron microscopy (FESEM) study
- Effect of extracts on bacterial cell membrane
All the tested bacteria were grown and maintained on Nutrient Agar (NA) as described earlier by Kesari et al. The minimal inhibitory concentration (MIC) was determined using the microdilution method in 96-well microtiter plates (Camporese et al. 2003). The antibacterial effects of extracts were determined according to the fluorescence intensity of PI which correlates with the damage of bacterial cell membrane (Paparella et al. 2008).
Morphological examinations of the bacterial cells before and after exposure of all extracts and compounds were performed using a field emission scanning electron microscope.
Results and discussion
- Antibacterial activities of extracts
- Flow cytometric investigation
- FESEM study
- Effect of extracts on bacterial cell membrane
Conversely, the positive control (HK-heat-killed bacterial population) showed significant increases in relative fluorescence in all bacteria tested (Fig. 5.1A-G) and confirms that the main cell populations are damaged or dead. Like the FC assay with extracts, the vehicle control (ethanol-treated cells) showed minimal changes in relative fluorescence intensity relative to control cell populations (Fig. 5.2A-G). Whereas, the positive control (heat-killed bacteria) showed a significant increase in relative fluorescence intensity for all tested bacteria (Fig. 5.2A-G) and confirmed cell damage or death.
FESEM study of untreated bacteria revealed characteristic morphological features (Fig. 5.3A, E), however, shrinkage and degradation of the cell walls were observed in bacterial cells treated with seed extracts (Fig. 5.3B. nigra seed extracts possesses antibacterial activity and it causes lysis of bacteria by breaking down bacterial cell walls and damaging cytoplasmic membrane proteins.
Conclusion
Studies on molecular coupling and inhibition kinetics of labdane diterpenes on α-amylase and α-glucosidase enzymes for the control of Type 2 diabetes. The chapter describes inhibitory activity and inhibition kinetics for two carbohydrate hydrolyzing enzymes, α-amylase and α-glucosidase using the purified labdane diterpene compounds. Furthermore, molecular docking simulation on human pancreatic α-amylase and maltase glucoamylase illustrates the possible mode of inhibition of isolated diterpene molecules.
Introduction
Therefore, the screening of natural products using bioassay-guided isolation and ethnopharmacological information has provided a logical direction towards the identification of potential α-amylase and α-glucosidase inhibitors from plant sources (Sales et al. Bioactive molecules from medicinal plants are more sought after sources and also alternative means adopted to bypass the side effects of synthetic drugs, and it has been recommended for diabetic treatments (Matsui et al. However, the labdane diterpenes in the present study have not been investigated for its antidiabetic potential, although they are previously well documented as a potential bioactive agent against various diseases (Abe et al.
Burtt seed extracts and purified labdana diterpene derivatives against various pathogenic bacteria towards its antibacterial potential.
Materials and methods
- Study material
- Inhibitory assay
- Kinetics of enzyme inhibition
- Statistical analysis
- Molecular docking
The α-glucosidase inhibitory assay was performed according to the chromogenic method described by Matsui et al. Kinetic parameter values (Km, Vmax and Ki) were determined according to the type of inhibition for both enzyme-catalyzed reactions for each compound. The best docking poses for each docking experiment were subjected to LigPlot analysis ( Wallace et al. 1995 ).
The best anchored poses for each anchoring trial were subjected to LigPlot analysis (Wallace et al. 1995).
Results and discussion
- The inhibitory effect on α-amylase
- The inhibitory effect on α-glucosidase
- Inhibition kinetic studies for α-amylase
- Inhibition kinetic studies for α-glucosidase
- Molecular docking of α-amylase
- Molecular docking of α-glucosidase
The predicted interactions between both the compounds and HPA active site residues are shown in Fig. The best-coupled poses obtained for I and II with 3L4Z and interacting residues in the active site were highlighted accordingly (Fig. 6.15). Here, blue dotted lines represent the H-bonding interactions between the ligand molecules and 3L4Z. C) Predicted non-bonded electrostatic interaction between acarbose and the residues in the active site region.
In the case of acarbose, the interacting residues in the active site of ctMGAM are shown in Fig.
Conclusions
Studies on labdane diterpenes towards matrix metalloproteinase inhibitory activity and cytotoxicity in HT1080 fibrosarcoma cells. The chapter describes the apoptotic activity of these labdane diterpenes in human fibrosarcoma HT 1080 cells. The effect of labdane diterpenes on matrix metalloproteinase (MMPs) activity, cell cycle arrest, nuclear integrity and apoptosis were also studied.
Introduction
