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Summer Research Fellowship Programme of India's Science Academies

Caging plant hormone with weak intermolecular interactions

Shyamlee Tiwari

M.Sc. I year, Guru Ghasidas Vishwavidalaya, Koni, Bilaspur, Chhattisgarh 495009

Dr. Apurba Lal Koner

Assistant Professor, Chemistry, Indian Institute of Science Education and Research, Bhopal Bhopal Bypass road, Bhauri, Bhopal 462066

Abstract

Titration of Cucurbit[7]uril with thiochrome and hormones has been performed to solubilize and stabilize the water insoluble hormones and used them in bio-medical applications. Fluorescence and NMR spectroscopic techniques were employed to check the binding of hormone with synthetic macrocyclic host cucurbit[7]uril and the purity of synthesized compounds respectively. The concept behind the research was based on the reversible and competitive binding of a fluorescent dye and the substrate to a macrocyclic host. Therein, binding of the substrate or product to the macrocyclic receptor has been coupled to the displacement of a fluorescent dye. Frequently, the inclusion of a fluorescent guest into the inner cavity of a macrocyclic host is accompanied by changes of the fluorescence properties of a dye. For example, fluorescence quenching or fluorescence enhancement by complex formation with the macrocycle may be observed, which allows for the determination of the binding constant of the fluorescent dye. Addition of another guest, which binds more strongly to the macrocycle, will compete with the fluorescent dye for the macrocyclic cavity and displace the fluorescent dye, whereupon the fluorescence properties of the free dye in solution are regenerated. The resulting “supramolecular tandem assays” exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate.

Keywords: cucurbit[7]uril, glycolurils, thiochrome, auxin, melatonin, encapsulation,

Abbreviations

Abbreviations
CB7 Cucurbit[7]uril
NMR Nuclear magnetic resonance
Angstrom
IAA Indole-3-acetic acid
Trp Tryptophan
UV Ultraviolet
nm nanometer
NaOH Sodium hydroxide
mL milliliter
g gram
HCl Hydrochloric acid
Celsius
rpm Rotation per minute

INTRODUCTION

In 1905, Behrend and co-workers characterized the condensation products of glycoluril and formaldehyde under strongly acidic conditions as “white, amorphous compounds, which are weakly soluble in dilute acid and base and absorb large quantities of water without loosing their dusty powdery character”. One of those products were found to contain “at least three molecules of glycolurils”, condensed with twice as many formaldehyde units, thereby corresponding to the formula C18H18N12O6. More than a century later, this characterization of what was likely a mixture of cucurbit[n]uril. In 1981, Mock and co-workers revisited Behrend’s experiments and upon complexation with calcium sulfate, successfully crystallised a hydrated macrocyclic bearing six glycolurils units linked by twelve methylene bridges and interacting with the calcium cations via its two carbonylated rims. The authors named the structure “cucurbituril” for its resemblance to “a gourd or a pumpkin” (which belong to the Cucurbitaceae family). Because of their exceptional recognition properties in aqueous medium, these pumpkin-shaped macrocycles have been generating some tremendous interest in the supramolecular community.[1] They have also become key units in various self-organizing and stimulus controlled assemblies, as well as in advanced materials and drug carriers. CB[n]s bear two hydrophilic carbonylated rims and a hydrophobic cavity. Their total depth is 9.1 Å, if one includes the van der waals radii of the oxygen atoms, and the depth of the cavity is 7.4 – 7.8 Å, if one considers the separation between two planes of local electrostatic potential minima at both portals.[2]

When guest molecules are noncovalently encapsulated inside water-soluble macrocyclic container compounds such as cucurbiturils, a modification of their chemical and physio-chemical properties always occurs owing to the altered microenvironment as well as the confinement and isolation of the guest.[3] Frequently, the complexation of the guest has beneficial effects on its aqueous solubility, because the encapsulation reduces the tendency of the guest to undergo aggregation or unspecific adsorption. Further, the complexation may enhance the thermal, as well as photochemical, stability of a guest by isolating it from the aqueous bulk and thereby preventing bimolecular reactions – in the simplest case, oxidation by molecular oxygen. [4]

    Melatonin is a hormone that regulates the sleep-wake cycle. It is primarily released by the pineal gland. In animals (including humans), melatonin is involved in synchronizing the circadian rhythm including sleep-wake timing, blood pressure regulation and seasonal reproduction. Many of its effects are through activation of the melatonin receptor, while others are due to its role as an antioxidant. In plants, it functions to defend against oxidative stress.

      Melatonin

      Auxins were the first of the major plant hormones and is present in all parts of a plant, although in very different concentrations. The pattern of auxin distribution within the plant is a key factor for plant growth, its reaction to its environment, and specifically for development of plant organs (such as leaves and flowers). It is achieved through very complex and well coordinated active transport of auxin molecules from cell to cell throughout the plant body- by the so called polar auxin transport.

        Auxin (IAA)

        Thus, a plant can (as a whole) react to external conditions and adjust to them, without requiring a nervous system. On the molecular level, all auxins are compounds with an aromatic ring and a carboxylic acid group. The most important member of the auxin family is indole-3-acetic acid (IAA), which generates the majority of auxin effects and is the most potent auxin. Auxins promote cell elongation, inhibit growth of lateral buds (maintains apical dominance). They are produced in the stem, buds and root tips. Auxin also promotes cell elongation. Auxin moves to the darker side of the plant, causing the cells there to grow larger than corresponding cells on the lighter side of the plant. This produces curving of the plant stem tip toward the light, a plant movement known as phototropism.

        CB7 HORMONE representation.jpg
          Fluorescent indicator displacement method for determining binding constants of analytes to macrocyclic hosts and its transfer to supramolecular tandem assays. Upon formation of the macrocyclic dye complex either fluorescence quenching or fluorescence enhancement and the addition of a more strongly binding competitor will restore the fluorescence properties of the free dye. Similarly, thiochrome is fluorescent but addition of water-soluble CB7 enhances the fluorescence intensity. The rotation of thiochrome inside CB7 is restricted and addition of hormones causes displacement assays.

          Background

          Fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample. A variety of molecular interactions can result in quenching. These include excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching. Fluorescence quenching has been widely studied both as a fundamental phenomenon and as a source of information about biochemical systems. These biochemical applications of quenching are due to the molecular interactions that results in quenching. Both static and dynamic quenching require molecular contact between the fluorophore and quencher. In case of collisional quenching, the quencher must diffuse to the fluorophore during the lifetime of a excited state. Upon contact, the fluorophore returns to the ground state, without emission of a photon. In general, quenching may occur without any permanent change in the molecules, that is, without a photochemical reaction. In static quenching, a complex is formed between the fluorophore and quencher and this complex is non-fluorescent. For either static or dynamic quenching to occur the fluorophore and quencher must be in contact. The requirement of molecular contact for quenching results in the numerous applications of quenching. For example, quenching measurements can reveal the accessibility of fluorophores to quenchers.[5]

          Quenching of fluorescence is, when a fluorophore (F) and a quencher (Q) are creating a non-fluorescent complex (FQ) before excitation of F. The chemical equation therefore is:

          F  +  Q  FQF\;+\;Q\;\leftrightarrow FQ

          The chemical equilibrium (KS) between the fluorophore, the quencher and the complex FQ is formed by the law of mass action and equal to the Stern-Volmer constant (KSV). There [FQ] stands for the concentration of the complex FQ, [F] for the concentration of the loose fluorophore and [Q] for the loose quencher.

          KSV = Ks = [FQ]/[F][Q]

          The Stern-Volmer equation describes the dependence of the fluorescent intensity of a fluorescent dye on the concentration of an quencher. It was created by two physical chemists Otto Stern and Max Volmer in 1919.

          F0/F = 1 + KSV . [Q]

          where, F0 is the fluorescent intensity of a fluorescent dye without the quencher

          F is the fluorescent intensity of a fluorescent dye with the quencher

          KSV stands for the Stern-Volmer constant

          [Q] is the concentration of the quencher.

            The Stern Volmer plot

            Melatonin

            Synonyms N-acetyl-5-methoxytryptamine
            Bioavailability 30-50%
            Metabolism Liver via CYP1A2 mediated6-hydroxylation
            Metabolites 6-Hydroxymelatonin, N-acetyl-5-hydroxytryptmine, 5-methoxytrptamine
            Elimination half-life 30-50 minutes
            Excretion kidneys
            Formula C13H16N2O2
            Molar mass 232.283 g/mol
            Melting point 117℃

             The pineal organ of all vertebrates synthesizes and secretes melatonin in a rhythmic manner due to the circadian rhythm in the activity of arylalkylamineN-aceytltransferase (AANAT)- the rate limiting enzyme in melatonin synthesis pathway. Night-time increase in AANAT activity and melatonin synthesis depends on increased expression of AANAT gene. In mammals, only one AANAT gene is expressed. The activity of AANAT enzyme is regulated by the degree of expression of AANAT gene which exhibits diurnal rhythm in its expression in the pineal gland of majority of mammals resulting in more than 150 fold increase in AANAT gene expression during night time.[6]

               When eyes receive light from the sun, the pineal gland’s production of melatonin is inhibited and the hormones produced keep the human awake. When the eyes do not receive the light, melatonin is produced in the pineal gland and the human becomes tired.  (https://en.wikipedia.org/w/index.php?title=Melatonin&oldid=914796755)

              Biosynthesis of Melatonin

              Biosynthesis of melatonin jpg.jpg
                Biosynthesis of Melatonin

                Auxin

                Indole-3-acetic acid (IAA), the most important natural auxin in plants, is mainly synthesized from the amino acid tryptophan (Trp). Auxin has long been recognized as a hormone essential for almost every aspect of plant growth and development. It is present in all parts of a plant, although in very different concentrations. The concentrations in each position is crucial developmental information, so it is subject to tight regulation through both metabolism and transport. The result is the auxin creates “patterns” of auxin concentration maxima and minima in the plant body, which in turn guide further development of respective cells, and ultimately of a plant as a whole. The dynamic and environment responsive pattern of auxin distribution within the plants is a key factor for plant growth. It is achieved through very complex and well -coordinated active transport of auxin molecules from cell to cell throughout the plant body by the polar auxin transport. Thus, a plant can (as a whole) react to external conditions and adjust to them, without requiring a nervous system.

                Five naturally occurring auxins in plants include:-

                Five different types of auxin.jpg
                  Naturally occurring auxins

                  Biosynthesis of Auxin:-

                  biosynthesis of auxin jpg.jpg
                    Biosynthesis of Auxin

                    Fluorescence Spectroscopy

                    Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyses fluorescence from a sample. Device that measures fluorescence are called fluorometers.

                    Molecules have various states referred to as energy levels. Fluorescence spectroscopy is primarily concerned with electronic and vibrational states. Generally, the species being analysed has a ground electronic state (a low energy state) of interest, and an excited electronic state of higher energy. Within each of these electronic states there are vibrational states. In fluorescence, the species is first excited by absorbing a photon, from its ground electronic state to one of the various vibrational states in the excited electronic state. Collisions with other molecules cause the excited molecule to lose vibrational energy until it reaches the lowest vibrational state from the excited electronic state. This process is often visualized with a Jablonski diagram.[7]

                      The Jablonski diagram features the energy levels within a molecule where valence electrons could be excited. Light of specific wavelength interacts with an electron and causes its excitation to a higher energy level. Excited states are short-lived, so there are two modes for the release of excess energy. It could either occur through non-radiative transitions or through emission of light quanta. The naming of the electronic states is based on the spin angular momentum configuration of each state. S0 is the singlet ground state of the molecule; S1 is the first excited singlet state; S2 is the second excited singlet state; T1 is the first excited triplet state. The radiative transitions are absorption, fluorescence, phosphorescence and the non-radiative transitions include vibrational relaxation, internal conversion and intersystem crossing Adapted from Principle and application on pharmaceutical analysis. ISRN Spectroscopy. 2013. 1-12.10.1155/2013/230858

                      The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the process. As molecules may drop down into any of several vibrational levels in the ground state, the emitted photons will have different energies and thus frequencies. Therefore, by analysing the different frequencies of light emitted in fluorescent spectroscopy, along with their relative intensities, the structure of the different vibrational levels can be determined.[8] For atomic species, the process is similar; however, since atomic species do not have vibrational energy levels, the emitted photons are often at the same wavelength as the incident radiation. This process of re-emitting the absorbed photon is “resonance fluorescence” and while it is characteristic of atomic fluorescence, is seen in molecular fluorescence as well.

                      What is fluorescence?

                      If an atom or molecule first absorbs energy – for instance a photon, this is called excitation. Very shortly (in the order of nanoseconds) after excitation it emits photon of a longer wavelength. We call that fluorescence. (We call it phosphorescence if the emission occurs from same spin state to a longer wavelength).

                      In a typical fluorescence (emission) measurement, the excitation wavelenth is fixed and the detection wavelength varies, while in a fluorescence excitation measurement the detection wavelength is fixed and the excitation wavelength is varied across a region of interest. In fluorescence spectroscopy, a beam with a wavelength varying between 180-800 nm passes through a solution in a cuvette. We then measure from an angle the light that is emitted by the sample. The concentration of the analyte is directly proportional with the intensity of the emission. There are several parameters influencing the intensity and shape of the spectra. When recording an emission spectrum the intensity is dependent on the:

                      •   Excitation wavelength
                      •   Concentration of the analyte solvent
                      •   Path length of the cuvette
                      •   Self-absorption of the sample

                      When can we use spectrofluorometry?

                      •   Fluorescence analysis is suitable for analytes that can be dissolved in solvents like water, ethanol and hexane.
                      •   The analytes need to absorb UV or visible light.
                      •   The analytes need to emit visible or near infrared radiation.
                      • With fluorescence analysis we can do quantitative measurements of a single analyte in solution (or more than one analytes in solution provided they do not interfere with each other).

                      INSTRUMENTATION

                      Two types of instruments exist: filter fluorometers that use filters to isolate the incident light and fluorescent light and spectrofluorometers that use a diffraction grating monochromators to isolate the incident light and fluorescent light.

                      Both uses the following scheme: the light from an excitation source passes through a filter or monochromator and strikes the sample. A proportion of the incident light is absorbed by the sample and some of the molecules in the sample fluoresce. The fluorescent light is emitted in all directions. Some of this fluorescent light passes through a second filter or monochromator and reaches a detector, which is usually placed at 90ᵒ to the incident beam to minimize the risk of transmitted or reflected incident light reaching the detector. Various light sources may be used as excitation sources, including lasers, LED and lamp; xenon arcs and mercury-vapor lamps in particular. A xenon arc has a continuous emission spectrum with nearly constant intensity in the range from 300-800 nm and a sufficient irradiance for measurements down to just above 200 nm.

                      A monochromator transmits light of an adjustable wavelength with an adjustable tolerance. The monochromator is adjusted to select which wavelengths to transmit. As mentioned before, the fluorescence is most often measured at a 90ᵒ angle relative to the excitation light. This geometry is used instead of placing the sensor at the line of the excitation light at a 180ᵒ angle in order to avoid interference of the transmitted excitation light. No monochromator is perfect and it will transmit some stray light, that is. Light with other wavelengths than targeted. An ideal monochromator would only transmit light in the specified range and have a high wavelength independent transmission. When measuring at a 90ᵒ angle, only the light scattered by the sample causes stray light. This results in a better signal to noise ratio and lowers the detection limit by approximately a factor 10000, when compared to the 180ᵒ geometry.

                      The detector can be single-channelled or multi-channelled. The single-channelled detector can only detect the intensity of one wavelength at a time while the multi-channelled detects intensity of all wavelengths simultaneously.

                         A general layout of fluorescence spectrophotometer

                        Objectives of the Research

                        Displacement assay of thiochrome from CB7 and addition of hormones (Auxin and Melatonin) followed by determination of the binding constant.

                        Formation of thiochrome:- Thiamine is visible in the UV range but its relatively common wavelength for absorbance (λmax = 242 nm) and low extiction coefficient do not lend to specific or sensitive analysis. Hence, most commonly, thiamine is converted to thiochrome prior to measurement. This product of oxidation is highly fluorescent with excitation and emission wavelengths of 360 nm and 450 nm respectively.

                        thiochrome jpg.jpg
                          Oxidation of thiamine to thiochrome

                          Thiochrome was synthesized by oxidation of anuerin or thiamine (Vitamin B1) with potassium ferricyanide in the presence of sodium hydroxide.

                          The reaction mixture (thiochrome) contained:-

                          •   2 mL, 1mM potassium ferricyanide
                          •   2 mL, 3 mM thiamine
                          •   1 mL NaOH

                           Synthesis of Glycouril:-

                          1)      To water (225 mL), 21.75 mL of glyoxal was added, after this 21.3 g of P4O10 was added slowly over a period of 5 minutes, while constantly stirring.

                          2)      After this time, 217 g of urea was added and mixture was heated up to 70℃.

                          3)      After 10 to 15 minutes the solution became cloudy and the product precipitates.

                          4)      The product was collected by filtration on a Buckner funnel and the solid was washed with cold water and dried.

                          5)      The product were characterized by 1H NMR spectroscopy.

                          glycoluril jpg.jpg
                            Synthesis of Glycourils from urea and 1,2- dicarbonyl (glyoxal) compounds.

                              Reaction Mechanism

                               Synthesis of Cucurbit[7]uril:-

                              Chemicals required:- Glycouril, paraformaldehyde, 37% HCl

                              Detailed procedure:-

                              1.      Take 10 g (70.35 mmol) glycouril in a 250 mL round bottomed flask and add 14.2 mL HCl to it.

                              2.      Set the reaction upon a magnetic stirrer taking a magnetic bead inside and stir for 5 minutes.

                              3.      Add 4.22 g paraformaldehyde and attach condenser. Set the temperature 97℃ and keep it for 18 h, after stabilizing the temperature.

                              4.      Bring the reaction mixture at room temperature and add 200 mL methanol. Leave it to settle for one day.

                              5.      Dissolve the dried solid in 250 mL 20% aq. Glycerol (v/v) and heat the mixture at 60-80℃ in open atmosphere.

                              6.      Filter it and to the filtrate add 200 mL methanol. Leave it undisturbed overnight.

                              7.      Centrifuge it at 4℃ 10000 rpm for 30 minutes (take the mixture in 50 mL falcon tubes).

                              8.      Characterize using NMR spectroscopy.

                              METHODOLOGY

                              All fluorescence measurements were performed using Horiba Jobin Yvon Fluorolog fluorimeter, using 1 cm quartz cuvettes. Thiochrome, glycoluril and CB-7 were synthesized in the laboratory as mentioned above. Methanol were purchased from Fluka. Milli-Q water, using Millipore water purification set up from Merck was used.

                              Results and Discussions

                                NMR data of synthesized glycoluril

                                1H NMR (DMSO-d6) ⸹ 7.16 (4H, NH), 5.24 (2H, CH).

                                  NMR data of synthesized CB7

                                  The NMR data of glycoluril was pure while the NMR data of CB7 had slight impurity of aliphatic compound and the circles indicate the protons corresponding to CB7.

                                   Fluorescence measurements:- The fluorescence spectra of displacement assays is given as below:-

                                  thiocb7mela.pnj.png
                                    The emission spectra of Thio-CB7- Melatonin

                                    Solvent- 480 uL (Milli Q water)

                                    Excitation wavelength- 360 nm

                                    Melatonin concentration- 5uM

                                    The emission spectra shows the displacement of thiochrome from [CB-7- thiochrome] host-guest complex followed by binding of competitor hormone, Melatonin. Further, increase in intensity confirms the caging of thiochrome inside CB-7. Quenching shows that the thiochrome has been displaced by Melatonin and free thiochrome has been regenerated.

                                    thioauxin.png
                                      Emission spectra of Thio-CB7-Auxin

                                       Solvent- 480 uL (Milli Q water)

                                      Excitation wavelength- 360 nm

                                      4 mM CB7 in 5uM thiochrome

                                      The titration shows the binding of CB-7 and thiochrome complex while the quenching shows the displacement of thiochrome and binding of Auxin.

                                      thio auxin reverse.png
                                        Reverse titration of Auxin-CB7-Thiochrome

                                        Solvent- 480 uL (Milli Q water)

                                        Excitation wavelength- 360 nm

                                        Auxin concentration- 5uM

                                        To check whether Auxin has been properly caged inside the CB-7, we had performed the reverse titration. Since the excitation wavelength was set at 360 nm (at this wavelength excitation of thiochrome occurs) and while performing the reverse experiment, initially Auxin showed very less fluorescence. But on further addition of thiochrome and CB7, the fluorescence intensity was matching with the actual experiment confirming the binding of Auxin inside CB-7 cavity.

                                        graph.png
                                          Host-guest binding titration plots of thiochrome with CB7. It should be noted that the host-guest titrations is fitted to a 1:1 binding model.

                                           Applications of the Research

                                          • Cucurbiturils, have been demonstrated to show a low, if not negligible, cellular and acute toxicity, possessing potential for drug delivery and stabilization. They stand out, in particular, with respect to their high binding constants with hydrophobic as well as cationic guest molecules. Moreover, they have been shown to effectively shift the pKa values of encapsulated guests.
                                          • An important application for the use of macrocycles in hormone encapsulation is the reversibility of the host-guest complexation, which allows full or partial retention of the biological activity of the encapsulated hormone.
                                          •  Hormones are mostly water-insoluble but on complexation with CB7, the solubility as well as biocompatibility is enhanced.
                                          •  Another application is the optimum release of the hormone from host-guest complex. Slow release of hormones leads to long lasting effect on body.
                                          •  The host-guest complex can be used for treating sleep disorders caused due to low levels of melatonin.
                                          •  The improper growth of plants due to deficiency of hormone Auxin can be reduced by injecting the host-guest complex.

                                          CONCLUSIONS

                                          As one of the most water-soluble members in the macrocyclic cucurbit[n]uril, CB7 has attracted increasing attention in pharmaceutical and biomedical fields. Due to its appropriate size to accommodate a variety of guest molecules of biomedical interest, CB7 has been extensively investigated as a potential carrier for cationic guests as the molecular encapsulation by CB7 often improved the chemical stability and solubility of the encapsulated guests, controlled their release and modulated their toxicity. Till date, there is no literature report of complexation of Auxin and Melatonin with CB7. Hence, this host-guest complex can be very useful in the area of research as well as biomedical applications.

                                           REFERENCES

                                          1. Masson, E.; Ling, X.; Joseph, R.; Kyeremeh, L.; Lu, X.; 2012, 2, 1213-1247.

                                          2. Zhang, X.; Xu, X.; Li, S.; Wang, L. H.; Zhang, J.; Wang, R.; (2018) 8:8819. doi :10.1038/s41598-018-27206-6.

                                          3.  Koner, A. L.; Ghosh, I.; Saleh, N.; Nau, W. N.; Can. J. Chem. 89: 139-147 (2011). doi:10.1139/V 10-079.

                                          4. Saleh, N.; Koner, A. L.; Nau, W. N.; Angew. Chem. Int. Ed. 2008, 47, 5398-5401.

                                          5. Principles of Instrumental Analysis F. James Holler, Douglas A. Skoog, Stanley R. Crouch 2006.

                                          6. Saha, S.; Singh, K. M.; Gupta, B. P.; General and comparative Endocrinology 2019, 279, 27-35.

                                          7. J. R. Lakowicz, Introduction to fluorescence, Principles of fluorescence spectroscopy, 3rd Ed. Springer US (2006) pp 3-6.

                                          8. Bernard Valeur, Molecular Fluorescence: Principles and applications.

                                          ACKNOWLEDGEMENTS

                                          I am using this opportunity to thank IASc-INSA-NASI for giving me the golden opportunity to carry out this project and granting me fellowship for the period as a part of Science Academies’ Summer Research Fellowship Programme 2019.

                                          I am deeply indebted to Dr. Apurba Lal Koner, Assistant Professor, Department of Chemistry, Indian Institute of Science Education and Research, Bhopal, for his guidance, valuable suggestions and constant supervision throughout this work.

                                          It is my privilege to express my profound regards to Prof. Goutam Patra, Department of Chemistry, Guru Ghasidas Central University, Bilaspur (C.G.) for providing my letter of recommendation to Indian Academy of Sciences.

                                          I want to thank my beloved parents for their moral support throughout the research work. Above all, I thank Almighty God for blessing me with health and vitality to complete this project.

                                          I am also grateful for having a chance to meet so many wonderful people who led me through this internship period.

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                            

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                           

                                          Source

                                          • : https://en.wikipedia.org/w/index.php?title=Melatonin&oldid=914796755 
                                          • : Principle and application on pharmaceutical analysis. ISRN Spectroscopy. 2013. 1-12.10.1155/2013/230858
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