Part B. Isolation of the Odorific Components of Essential Oils. Preparative Gas Chromatography. Experiment 1b. This is to be expected, since the human body distinguishes odor by the interaction of molecules with odor receptors in the nose, which are chiral.
The phenomenon in which a chiral receptor interacts differently with each of the enantiomers of a chiral compound is called chiral recognition. With the exception of their optical rotations, the physical properties of enantiomers are identical.
The only difference in properties that one should observe are the odors and the signs of optical rotation in a polarimeter. Using gas collection tubes, the two major components of the natural oil can be collected separately as they elute from the column. Spearmint oil contains mainly - -carvone, with a smaller amount of limonene and the isomeric terpenes, a - and b -phellandrene.
The gas chromatogram of this oil is also shown in Figure 1. In this case, while it is easy to collect a pure sample of the carvone, limonene cannot be collected in pure form because it will be contaminated by the other terpenes, which have similar boiling points. Your instructor will either assign you spearnint or caraway oil, or have you choose one of the oils. You will also be given instructions on which optional procedures, if any, you are to perform.
Your instructor may have you isolate both the carvone and limonene components or only carvone. In this experiment, you will carry out all aspects of the gas chromatographic separation yourself. The most critical part of the operation is the injection procedure - it is important that once you have pierced the injector with the syringe containing the mixture to be separated, you inject the mixture rapidly. Your T. Inject m L of caraway-seed or spearmint oil onto the gas chromatography column.
Just before a component of the oil limonene or carvone elutes from the column, install a gas collection tube at the exit port. To determine when to connect the gas collection tube, refer to the chromatograms on the wall of the GC room.
These chromatograms have been run on the same instrument you are using under the same conditions. Ideally, you should connect the gas collection tube just before the limonene or carvone elutes from the column and remove the tube as soon as all the component has been collected, but before any other compound begins to elute from the column.
This can be accomplished most easily by watching the recorder as your sample passes through the column. The collection tube is connected if possible just before a peak is produced, or as soon as a deflection in the pen is observed. When the pen has returned to the base line, the gas collection tube is removed. This procedure is relatively easy for collecting the carvone component of both oils and for collecting limonene in caraway-seed oil.
Because of the presence of several terpenes in spearmint oil, it is somewhat more difficult to isolate a pure sample of limonene from spearmint oil see chromatogram in figure. Examination of lung tissues and bronchoalveolar lavage fluid BALF suggested anti-inflammatory effect due to reduced production of pro-inflammatory cytokines. Cyclooxygenase activity is prompted by LPS, cytokines and growth factors.
It also protected against chemically induced dysfunction of macrophages and balanced the pro-inflammatory mediators. Further clinical studies are required to authenticate the use of eugenol and its other active derivatives as anti-inflammatory agents on dendritic cells, along with their modulating effects on cytokines and autoimmune inflammatory diseases. Eventually, results from these clinical trials would significantly improve the immune-protective application of eugenol.
Recent clinical studies of medicinal plants used in numerous traditional medicines have triggered the invention of various chemotherapeutic drugs, like camptothecin, vinblastine, taxol and vincristine. Breast cancer is the most prevalent type of malignant cancer among women and is ranked fourth common reason of cancer related deaths in the world.
Eugenol repressed growth of human breast cancer cells by initiation of cell death both in time and dose dependent manner. Essential oils being extracted by hydro distillation of roots and barks of Uvariodendron angustifolium containing Inducible cyclooxygenase COX-2 inhibitors have been considered as cancer chemo-preventive and anti-inflammatory agents.
According to findings of Kim et al. Remedies for colon cancer through administration of eugenol resulted in decreased concentration intracellular non-protein thiols. Additionally, DNA fragmentation caused by amplification of reactive oxygen species ROS is considered as a core reason for apoptosis in eugenol-subjected colon cancer cells. Eugenol not only transmitted apoptotic signal through reduction of non-protein thiols resulting in decreased potential of mitochondrial membranes, eventually leading to increased production of ROS.
Novel strategies in cancer prevention have initiated the use of eugenol as a potential chemotherapeutic agent. Synergistic effect of eugenol with chemo-preventive drugs has been reported to reduce the drug toxicity on normal cells and augment the cytotoxicity of administrated synthetic agents.
Among all investigated phenyl-propanoids, eugenol explicated maximum cytotoxicity and reduced hemolytic activity validating its use as a chemo-protective agent without any adversative toxicity. Currently, chemo-protective agents being practiced to cure malignant melanoma; typically the most antagonistic skin cancer occurring in melanocytes are unsatisfactory.
They suggested inhibitory mode of action of eugenol as it causes cell cycle arrest and induces apoptosis. In an experimental trial Koh et al. Cytotoxic concentrations of eugenol resulted in reduction of ATP utilization of oxidative stress and an increase in the polyamines and glycolytic metabolites, in normal oral cells and oral squamous cell carcinoma. Another group of researchers have highlighted the inhibitory potential of eugenol against expression of matrix metalloproteinase MMP-9 activity in PMA-stimulated HT cells via inactivation of ERK proteins.
Consequently, they evaluated that eugenol from cloves, explicitly revealed highest inhibitory effect on hydrogen peroxide H 2 O 2 than other ROS reactive oxygen species , and was able to halt lipid peroxidation and DNA oxidation, induced by hydroxyl radical. Therefore, eugenol could be accessible as an outstanding agent for prevention of metastasis associated to oxidative stress.
Several studies have been performed to study the neuroprotective and anti-stress related potential of eugenol. For instance, Prasad and Muralidhara 78 investigated the neuro-protective effectiveness of eugenol and iso-eugenol against acrylamide ACR induced neuropathy model in male albino rats. Momentously, both active ingredients reduced oxidative stress markers i.
Furthermore, they effectually diminished the acetylcholinesterase activity, levels of cytosolic calcium and dopamine in brain regions. Conclusively, they were of a view that eugenol and iso-eugenol have tendency to curtail acrylamide induced neuropathy in rats and therefore possibly could be used as a diet based regime to attenuate various forms of neuropathy in human beings.
Additional studies illuminate the protecting potential of eugenol by modulation of hypothalamic pituitary adrenal cortex HPA axis and brain mono-aminergic pathways against restraint stress irritable bowel syndrome RS-IBS in rats. Alongside, eugenol also deflated RS-induced alterations in levels of norepinephrine and elevated antioxidant immune system in all regions of brain.
Stress is foremost psychopathological reason for numerous psychological and mental impairments. Eugenol is stated to significantly moderate brain functions by regulating the release of neurotransmitters. Garabadu et al. Exposure to stress not only elevated the ulcer index but also increased the plasma norepinephrine and corticosterone levels. However, eugenol administration for 7 days showed significant effect on hypothalamic pituitary adrenal HPA axis by reducing the stress-induced upsurge in ulcer index and plasma corticosterone levels.
Moreover, eugenol also augmented the stress-induced changes in serotonin 5-HT levels in all regions of brain, while decrease in norepinephrine levels was also observed in all portions of brain except for hippocampus. Outcomes of this study suggested anti-stress activity of eugenol is mainly due to modulation of hypothalamic pituitary adrenal HPA and brain monoaminergic systems BMS.
Several research mediations suggested the importance of eugenol as neuro-protective agent which could further be used in stress related pathological conditions. Amongst various strategies to combat diabetes, diet plays a vital part in maintaining blood sugar level, hyper-glycosylation of biologically active molecules linked with various metabolic pathways and inhibition of pathologies. Various epidemiological investigations have illustrated the positive relationship among the utilization of phenolic rich diet and prevention of diabetes.
Various experiments have shown that eugenol is the key bioactive molecule present in spices with anti-diabetic potential. In this context, Srinivasan et al. Furthermore, activities of various enzymes involved in carbohydrate metabolism increased for instance hexokinase Additionally, orally administrated eugenol elevated the levels of hepatic glycogen demonstrating the anti-hyperglycemic capabilities in diabetic rats. The rinsing and transferring operations are best accomplished if the Pasteur pipet is bent slightly at the end.
If you are distilling oil of cloves , continue the steam distillation with frequent rinsing of the Hickman head and collection of the distillate in the 5-mL conical vial until you have obtained 2. If you are distilling cinnamon , continue the steam distillation until you have collected about 1. Raise the assembly and allow the roundbottom vial to cool until you can touch it with your fingers.
Remove the flask and discard the mixture of ground cinnamon and water, but save the stirring bar. It is not necessary to clean the roundbottom vial or the Hickman head at this stage.
Add 0. Reassemble the apparatus and continue the distillation. Rinse the Hickman head as before and transfer the distillate to the same 5-mL conical vial. When you have collected another 1. The essential oil will now be extracted with dichloromethane see Method A of Microscale Extractions. Using a calibrated Pasteur pipet, add 1. Cap the vial securely and shake it vigorously with frequent venting. Allow the layers to separate. Using a filter-tip pipet, transfer the lower dichloromethane layer to a second dry 5-mL conical vial.
Repeat this procedure with two more 1. If there are visible drops of water in the vial, it will be necessary to transfer the dichloromethane solution with a dry pipet into another dry conical vial. Dry the dichloromethane solution with microspatulafuls measured with the V-groove end of granular anhydrous sodium sulfate to the conical vial. Let the mixture stand for minutes with occasional stirring. While the organic solution is drying, clean and dry the first 5-mL conical vial and weigh it accurately.
With a clean, dry filter-tip pipet, transfer the dried organic layer to this tared vial.
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