DESC:FIELD OF THE INVENTION
[0001] The present invention relates to a method of extracting organic compounds from nuts. Specifically, the present invention relates to a method of extracting melatonin and tannins from Pistacia vera nuts. The present invention also relates to a pharmaceutical composition comprising the melatonin and tannins extracted from Pistacia vera nuts.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Members of the Pistacia genus (Anacardiaceae family), include plants like cashew nut, sumac, mango, and poison ivy. Of which, Pistacia vera L. (pistachio) produces edible nuts large enough to be commercially acceptable. Raw pistachios are a source of proanthocyanidins, otherwise known as condensed tannins, oligomeric and polymeric end-products of the flavonoid biosynthetic pathway. The identified tannins compounds include dimers, trimers, tetra-hexamers and hepta-decamers with a total amount of ~258 mg/100 g of raw pistachios.
[0004] Tannins are complex organic compounds present in plants which is known as “a substance that can easily be dissolved in water, and the aqueous solution is highly astringent, and has the tanning property”. Tannins have been industrially used as a tanning agents and as a precipitant for various kinds of metal ions. Furthermore, tannins are used as tannic acid of the Japanese Pharmacopoeia, an adhesive for wood or its filler. In any case, tannins extracted with warm water or hot water as an extraction agent is used as a base agent, and if required, tannins may be further purified with an organic solvent or modified with alkalis or the like.
[0005] Verde et al., (Journal of Food Composition and Analysis 105(3):104180) studied the presence of melatonin in nuts Melatonin was detected in four walnut cultivars with levels similar to those previously reported. A number of other commercial nuts including pistachio were also measured, with melatonin contents varying markedly, and generally being lower than in walnuts. Further, the melatonin levels in non-roasted, non-salted pistachios were higher than those detected in the processed pistachios, although with great differences depending on the origin of the product.
[0006] Mokhtarpour (Annual Research & Review in Biology 4(8):1330-1338) disclosed that 70% aqueous acetone and 50% aqueous ethanol are more convenient solvent for extraction of phenolics and tannins in pistachio by-products (PB). However, the known arts do not disclose about the extraction of tannins and melatonin from pistachio kernels.
[0007] Accordingly, there is a need in the art to simultaneously extract tannins and melatonin from pistachio kernels using an environmentally safe and economical way. The present invention solves the requirements present in the art.
[0008] The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

OBJECTS OF THE INVENTION
[0009] Objects of the present invention are to provide an extract of Pistacia vera nuts.
[0010] An object of the present invention is to provide a method of extracting melatonin and tannins from Pistacia vera nuts.
[0011] Another object of the present invention is to provide a pharmaceutical composition comprising the melatonin and tannins extracted from Pistacia vera nuts.

SUMMARY OF THE INVENTION
[0012] This summary is provided to introduce a selection of concepts in a simplified form that is further described below in the detailed description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0013] Aspects of the present invention relates to a method of extracting organic compounds from nuts. Specifically, the present invention relates to a method of extracting melatonin and tannins from Pistacia vera nuts. The present invention also relates to a pharmaceutical composition comprising the melatonin and tannins extracted from Pistacia vera nuts.
[0014] In an aspect, the present invention provides an economical and environmentally sustainable method of extracting melatonin and tannins from Pistacia vera nuts.
[0015] In an aspect, the present invention provides a method of extracting melatonin and tannins from Pistacia vera nuts comprises the steps of:
a) cleaning and shelling pistachio nuts to obtain the seed kernels;
b) grinding the seed kernels to obtain a coarse powder;
c) extracting the coarse powder using six volumes of an extraction solution comprising ethanol, water, and HCl to obtain first extract, followed by repeating the same three more times to obtain second, third and fourth extracts, respectively;
d) filtering all four extracts with 10-micron filter cloth to obtain a filtrate;
e) adjusting the pH of the filtrate to 4.0 at 20±5°C by adding HCl, followed by distillation in a reactor at 75±5°C under vacuum for a suitable duration to obtain a paste form of the extracts;
f) mixing the paste form of the extracts with four volumes of 50% ethanol and chilling, followed by filtration through a 10-micron filter cloth and vacuum drying at 65±5°C for a suitable duration;
g) recovering melatonin by LC-MS and tannins by titration.
[0016] In an aspect of the present invention, the extraction solution comprises a mixture of 90% ethanol and water, along with 0.2% HCl.
[0017] In an aspect of the present invention, the total dissolved solid content is 2.5-3.0 percent in the first filtrate.
[0018] In an aspect of the present invention, the total dissolved solid content is 1.5-2.0 percent in the second filtrate.
[0019] In an aspect of the present invention, the total dissolved solid content is 1.0-1.5 percent in the third filtrate.
[0020] In an aspect of the present invention, the total dissolved solid content is 0.5-1.0 percent in the fourth filtrate.
[0021] In an aspect of the present invention, the paste form of the extract comprises 3 percent weight of Melatonin; 10 percent weight of tannins; and 2 to 5 percent weight of moisture content.
[0022] In an aspect of the present invention, the molecular formula of Melatonin is C13H16N2O2, and the molecular weight of the Melatonin is 232.278 g/mol.
[0023] In another aspect, the present invention provides pharmaceutical composition comprising the melatonin and tannins extracted from Pistacio vera nuts by the method as disclosed herein; and pharmaceutically excipient Maltodextrin.
[0024] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESSCRIPTION OF THE DRAWINGS
[0025] Figure 1: relates to the flow chart of the method of extracting melatonin and tannins from Pistacia vera nuts.

DETAILED DESCRIPTION OF THE INVENTION
[0026] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention.
[0027] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0028] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0029] In some embodiments, numbers have been used for quantifying weight percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0030] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0031] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0032] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0033] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[0034] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0035] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.
[0036] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present invention. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0037] It should also be appreciated that the present invention can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0038] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0039] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements a, b, and c, and a second embodiment comprises elements b and d, then the inventive subject matter is also considered to include other remaining combinations of a, b, c, or d, even if not explicitly disclosed.
[0040] Embodiments of the present invention relate to a method of extracting organic compounds from nuts. Specifically, the present invention relates to a method of extracting melatonin and tannins from Pistacia vera nuts. The present invention also relates to a pharmaceutical composition comprising the melatonin and tannins extracted from Pistachio vera nuts.
[0041] In an embodiment, the present invention provides an economical and environmentally sustainable method of extracting melatonin and tannins from Pistacia vera nuts.
[0042] In an embodiment, the present invention provides a method of extracting melatonin and tannins from Pistacia vera (Figure 1) nuts comprises the steps of:
a) cleaning and shelling pistachio nuts to obtain the seed kernels;
b) grinding the seed kernels to obtain a coarse powder;
c) extracting the coarse powder using six volumes of an extraction solution comprising ethanol, water, and HCl to obtain first extract, followed by repeating the same three more times to obtain second, third and fourth extracts, respectively;
d) filtering all four extracts to obtain a filtrate;
e) adjusting the pH of the filtrate to 4.0 at 20±5°C by adding HCl, followed by distillation in a reactor at 75±5°C under vacuum for a suitable duration to obtain a paste form of the extracts;
f) mixing the paste form of the extracts with four volumes of 50% ethanol and chilling, followed by filtration through a 10-micron filter cloth and vacuum drying at 65±5°C for a suitable duration;
g) recovering melatonin by LC-MS and tannins by titration.
[0043] In an embodiment of the present invention, the grinding of seed kernels may be conducted by a crusher capable of crushing bark into an appropriate size, for example into particles of 1 mm or less (in particular about 100 to 500 µm) and fed to an extraction process. In some embodiments, the crushing is affected by a method known in the art that does not allow the temperature to rise substantially during crushing.
[0044] In an embodiment of the present invention, the extraction is effected by the extraction solution comprises a mixture of 70-90% ethanol and water (For example, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90%), along with 0.2% HCl. Preferably, 90% ethanol and water along with 0.2% HCl
[0045] In an embodiment of the present invention, the extraction is effected for upto four times, wherein the residue of the first extract is the input for second extraction, while the residue of second extract is the input for third extraction, and so on.
[0046] In an embodiment of the present invention, the nutrient is selected from micronutrient, macronutrient, nitrogen, phosphorous, vitamins, minerals, carbohydrates, fats, protein source, growth factor, and the like.
[0047] In an embodiment of the present invention, the total dissolved solid content is 2.5-3.0 percent in the first filtrate.
[0048] In an embodiment of the present invention, the total dissolved solid content is 1.5-2.0 percent in the second filtrate.
[0049] In an embodiment of the present invention, the total dissolved solid content is 1.0-1.5 percent in the third filtrate.
[0050] In another embodiment of the present invention, the total dissolved solid content is 0.5-1.0 percent in the fourth filtrate.
[0051] In another embodiment of the present invention, the method as disclosed herein further comprises the step of monitoring the parameters (extraction conditions) that may be monitored include, but are not limited to, the temperature, pH, and the presence (or absence) of contamination.
[0052] In another embodiment of the present invention, the method as disclosed herein further comprises the step of modulating the parameters (extraction conditions) including but not limited to, the temperature, pH, and the presence (or absence) of contamination.
[0053] In another embodiment of the present invention, the filtration is affected by a method used in general solid-liquid separation such as pressure, suction, and centrifugation, as well as by filtration under normal atmospheric pressure (e.g. using filter paper). Examples of filtration include centrifugation, sedimentation, reduced-pressure suction and filtration, and pressure filtration. Preferably, the filtration is effected under normal atmospheric pressure with 10-micron filter cloth to obtain a filtrate.
[0054] In another embodiment of the present invention, the wherein the paste form of the extract comprises 3 percent weight of Melatonin; 10 percent weight of tannins; and 2 to 5 percent weight of moisture content.
[0055] In another embodiment of the present invention, the harvested protein-rich biomass is subsequently processed to powders by drying technologies including but not limited to lyophilization, milling or spray drying.
[0056] In another embodiment of the present invention, the molecular formula of Melatonin is C13H16N2O2, and the molecular weight of the Melatonin is 232.278 g/mol.
[0057] In yet another embodiment, the present invention provides a pharmaceutical composition comprising the melatonin and tannins extracted from Pistacia vera nuts by the method as disclosed herein; and one or more pharmaceutically acceptable excipients.
[0058] In some embodiments of the present invention, the pharmaceutically acceptable excipient is selected from but not limited to pharmaceutically acceptable carriers, adjuvants, solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, flavonoids, diluents, stabilizers, binders, flavoring agents, thickeners, coloring agents, humectants, preservatives, olfactory agents, and combinations thereof.
[0059] In an embodiment, the pharmaceutical composition as disclosed herein may be administered in an oral dosage form comprising of tablet, powder, capsule, gums, films, semi-solid, solution, suspension, syrup, decoctions, elixir, granules, microparticles, nano-particles, gel, or concentrates.
[0060] In some embodiments of the present disclosure, the pharmaceutical composition as disclosed herein may be administered as needed, e.g., once, twice, thrice or four or more times daily, or continuously depending on the subject's needs. In a preferred embodiment, the compositions may be administered every other day or every alternative day (i.e. Q.O.D dosage).
[0061] In some embodiments of the present disclosure, the term “subject,” as used herein, means a many-celled vertebrate or invertebrate organism from the animal kingdom. The subject thus may be a person (also termed an individual or a human) or a non-human animal (hereafter, termed only an “animal”). Exemplary animals include but not limited to laboratory animals, farm animals, pets, or sport animals, among others. An animal subject thus may, for example, be a rodent (such as a mouse, rat, hamster, guinea pig), dog, cow, horse, non-human primate, bird, amphibian, reptile, fish, insect, or the like. Non-human subjects may be test species, that is, animals for testing the effect of a composition, chemical compound, or candidate drug, generally prior to human clinical trials and/or use as an approved drug in humans and/or animals. Alternatively, non-human: subjects may be drug recipients after a drug has been tested and/or approved, such as for treatment in a veterinary setting.
[0062] While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
[0063] The present invention is further explained in the form of following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
Material and Methods
[0064] Example 1: The extraction process of melatonin comprises a mixture of ethanol, water and HCl in a ratio of 70:30:0.2 v/v/v at a temperature of 70±5°C for a suitable duration. All washings are consolidated, cooled to room temperature, and filtered through a 10-micron filter cloth. The pH of the filtrate is adjusted to 4.0 at 20±5°C using 6N HCl to prevent the degradation of melatonin and subjected to distillation in a reactor at 75±5°C under vacuum. The paste form of extract is mixed with 6 volumes of 70% ethanol and chilled to 10-15°C for 4-5 hours, followed by filtration through a 10-micron filter cloth and vacuum drying at 65±5°C for a suitable duration. This method ensures the extraction and recovery of melatonin up to 1.5% by LC-MS and tannins to 5% by titration.
[0065] Example 2: The extraction process of melatonin comprises a mixture of ethanol, water and HCl in a ratio of 80:20:0.2 v/v/v at a temperature of 70±5°C for a suitable duration. All washings are consolidated, cooled to room temperature, and filtered through a 10-micron filter cloth. The pH of the filtrate is adjusted to 4.0 at 20±5°C using 6N HCl to prevent the degradation of melatonin and subjected to distillation in a reactor at 75±5°C under vacuum. The paste form of extract is mixed with 6 volumes of 70% ethanol and chilled to 10-15°C for 4-5 hours, followed by filtration through a 10-micron filter cloth and vacuum drying at 65±5°C for a suitable duration. This method ensures the extraction and recovery of melatonin up to 2.0% by LC-MS and tannins to 6% by titration.
[0066] Example 3: The extraction process of melatonin comprises a mixture of ethanol, water and HCl in a ratio of 90:10:0.2 v/v/v at a temperature of 70±5°C for a suitable duration. All washings are consolidated, cooled to room temperature, and filtered through a 10-micron filter cloth. The pH of the filtrate is adjusted to 4.0 at 20±5°C using 6N HCl to prevent the degradation of melatonin and subjected to distillation in a reactor at 75±5°C under vacuum. The paste form of extract is mixed with 6 volumes of 70% ethanol and chilled to 10-15°C for 4-5 hours, followed by filtration through a 10-micron filter cloth and vacuum drying at 65±5°C for a suitable duration. This method ensures the extraction and recovery of melatonin up to 2.5% by LC-MS and tannins to 8.5% by titration.
[0067] Example 4: The extraction process of melatonin comprises a mixture of ethanol, water and HCl in a ratio of 90:10:0.2 v/v/v at a temperature of 70±5°C for a suitable duration. All washings are consolidated, cooled to room temperature, and filtered through a 10-micron filter cloth. The pH of the filtrate is adjusted to 4.0 at 20±5°C using 6N HCl to prevent the degradation of melatonin and subjected to distillation in a reactor at 75±5°C under vacuum. The paste form of extract is mixed with 4 volumes of 50% ethanol and chilled to 10-15°C for 4-5 hours, followed by filtration through a 10-micron filter cloth and vacuum drying at 65±5°C for a suitable duration. This method ensures the efficient extraction and recovery of melatonin up to 3% by LC-MS and tannins to 10% by titration.
Example 5: Assay of melatonin by LC-MS:
Liquid Chromatographic conditions:
Column : C18 (2.1mm x 100 mm, 1.7µm)
Volume of Injection : 1.0 µL
Flow rate : 0.25 mL / min
Run time : 12 min
Temperature : 30?
Diluent : Methanol

Mobile phase:
A: Water with 0.1% formic acid.
B: Acetonitrile with 0.1% formic acid.

Mass spectrometric conditions:
Ionization method: ESI (positive)
Desolvation Temperature: 400?
Desolvation Gas Flow: 600 L/Hr (N2)
Collision Gas: Argon (Ar) or equivalent
Capillary Voltage: 2.8 kV

Test solution:
Accurately 1 g of homogenized sample is weighed precisely and place it in a 50 mL volumetric flask. Add 25 mL of 70% methanol and shake for 30 minutes. Fill up to the mark with 70% methanol and filter through a 0.45 µm membrane filter to use as the test solution. If the concentration of the test solution exceeds the range of the calibration curve, dilute it within the range.
Solvent: For Liquid chromatography or equivalent.
Water: Triple-distilled water or equivalent.
Stock solution: Dissolve the standard substance in methanol to make a concentration of 1,000 mg/L.
Standard solution: Mix and dilute the stock solution to an appropriate concentration.
70% methanol: Mix 300 mL of water with 700 mL of methanol.
Other reagents: Reagent grade or equivalent.
Gradient:
Time (min) A (%) B (%)
0.0 80 20
2.0 80 20
7.0 40 60
8.0 10 90
10.0 10 90
10.1 80 20
12.0 80 20

CALCULATION:
V
Melatonin content (mg/g) = C X ------ X D
S
C: Concentration of melatonin calculated from the calibration curve (mg/L)
V: Final volume of the test solution (L)
S: Amount of sample taken (g)
D: Dilution factor
Example 6: Procedure for Tannins by Titration:
Reagent Preparation:
Indigo sulphonic Acid Preparation: Weigh 500 mg of Indigo carmine and dissolve in 25 mL Sulphuric acid, dilute to 500 mL with distilled water.
Preparation of N/10 Potassium permanganate: Weigh 3.16 g of KMnO4, dissolve in 1000 mL of distilled water, filter & keep for 24 h.
Standardization: Weigh 0.3 g of dry sodium oxalate and dissolve in 150 mL of distilled water and add 4 mL of sulphuric acid, heat the entire solution at 600? for 15 min. Titrate with KMnO4 solution. End point should be in light pink color.
Weigh 1 g of extract sample and dissolve in 100 mL of distilled water, stir for 1h. and filter through Whatman filter paper.
Take 10 mL of the above solution and add 25 mL indigo sulphonic acid, titrate with KMnO4 solution. End point should be in golden yellow color.
Blank titration: Take 10 mL of distilled water and add 25 mL indigo sulphonic acid titrate with KMnO4 solution. End point should be in golden yellow color.
CALCULATION:
(Titer value of Sample-Titer value of Blank) X 0.004157 X 100
% of Tannins =-------------------------------------------------------------------------------x 100
10 X Wt. of the sample
,CLAIMS:1. A method of extracting melatonin and tannins from Pistacia vera nuts comprises the steps of:
a) cleaning and shelling pistachio nuts to obtain the seed kernels;
b) grinding the seed kernels to obtain a coarse powder;
c) extracting the coarse powder using six volumes of an extraction solution comprising ethanol, water, and HCl to obtain first extract, followed by repeating the same three more times to obtain second, third and fourth extracts, respectively;
d) filtering all four extracts with 10-micron filter cloth to obtain a filtrate;
e) adjusting the pH of the filtrate to 4.0 at 20±5°C by adding HCl, followed by distillation in a reactor at 75±5°C under vacuum for a suitable duration to obtain a paste form of the extracts;
f) mixing the paste form of the extracts with four volumes of 50% ethanol and chilling, followed by filtration through a 10-micron filter cloth and vacuum drying at 65±5°C for a suitable duration;
g) recovering melatonin by LC-MS and tannins by titration.
2. The method as claimed in claim 1, wherein the extraction solution comprises a mixture of 90% ethanol and water, along with 0.2% HCl.
3. The method as claimed in claim 1, wherein the total dissolved solid content of 2.5-3.0 percent in the first filtrate.
4. The method as claimed in claim 1, wherein the total dissolved solid content of 1.5-2.0 percent in the second filtrate.
5. The method as claimed in claim 1, wherein the total dissolved solid content of 1.0-1.5 percent in the third filtrate.
6. The method as claimed in claim 1, wherein the total dissolved solid content of 0.5-1.0 percent in the fourth filtrate.
7. The method as claimed in claim 1, wherein the paste form of the extract comprises 3 percent weight of Melatonin; 10 percent weight of tannins; and 2 to 5 percent weight of moisture content.
8. The method as claimed in claim 7, wherein the molecular formula of Melatonin is C13H16N2O2, and the molecular weight of the Melatonin is 232.278 g/mol.
9. A pharmaceutical composition comprising the melatonin and tannins extracted from Pistacia vera nuts by the method as claimed in claim 1; and maltodextrin