EXTRACTION OF CARDAMOM BY DIFFERENT EXTRACTION METHODS AND ISOLATION AND IN-SILICO ANALYSIS OF THE MOLECULES IN LUNG CANCER

1. EXTRACTION OF CARDAMOM BY DIFFERENT
EXTRACTION METHODS AND
ISOLATION AND IN-SILICO ANALYSIS OF THE
MOLECULES IN LUNG CANCER
SHAKTI SWARUP MOHAPATRA
shaktisworup3@gmail.com
Contact no. -9348110349

2. CONTENTS
Introduction
C𝑶𝟐Extraction
Qualitative analysis of cardamom
Quantitative analysis of cardamom
Chromatography
In-silico analysis
Conclusion

3. INTRODUCTION
➢ Cardamom is popularly known as Queen of species due to its pleasant
spicy taste, commonly used as flavoring agent for preparation of delicious
foods, Cardamom herb belongs to Zingiberaceae family foundly native in
western ghats of southern India and take third position of world-wide
spices.
➢ Being spice cardamom is rich in phytochemicals which have anti-
inflammatory, anti-proliferative , antioxidant , anti cancer properties.
➢ Ligand taken from flavonoids show anti lungs cancer properties when
interaction is made with 4lvt protein.
➢ The interaction between ligand and protein is studied in auto Dock vina.
➢ These interaction give a brief idea about the anti cancer properties of
ligand which qualify for oral drug preparation
cardamom
Anti-michrobia
Anti-Cancer
Gastroprotective
Anti-inflamentory
Cardio
Vascular
protective

4. Lung Cancer
➢ A cancer that begins in the lungs and most often occurs in people who smoke.
➢ Two major types of lung cancer are non-small cell lung cancer and small cell
lung cancer.
➢ Lung cancer happens when cells in the lung change (mutate). They grow
uncontrollably and cluster together to form a tumor. Lungcells most often change
because they are exposed to dangerous chemicals that we breathe.
➢ Lung cancers, on average, double in size in four months to five months.

5. C𝑶𝟐Extraction
➢ This process is known as Super critical fluid extraction.
➢ Super critical fluid extraction is a process of separating one
component from another using super critical fluid as the C𝑂2
act as liquid extracting solvent.
➢ Substance with a pressure and temperature above its critical
point where distinct gas and liquid phase do not exist.
➢ Mostly C𝑂2 are used as supercritical fluid.

6. Procedure
➢ First we have to prepare our raw material by grinding cardamom seeds. Then fill it in the extractor.
➢ In the cooling bath the compressed Liquid C𝑂2 gas gets supercritical under high pressure and
temperature.
➢ Then it flowed into the extraction vessel containing cardamom samples which being controlled by a
digital controller.
➢ The process continue with fixing the temperature and variable the pressure facto.
➢ Lowest temperature is required for cardamom extraction. At temperature 35℃ and 100bar
pressure highest amount i.e. 58 gm of cardamom essential oil is extracted.
Results

7. Qualitative Analysis
Qualitative analysis determine the non numerical information like present of flavonoid and carbohydrate
in the sample of cardamom.
A. Flavonoid Test: (For the estimation of Flavonoid)
2 ml of extract
Appearance of
Yellow colour
Solution became
colourless
Colorless of sample after addition of dil. NaOH and dil HCl indicates the presence of Flavonoid.
Results:

8. Barfoed’s Test
Barfoed’s test is used for detecting the presence of monosaccharides in the sample.
2 ml of cardamom
sample
Boiling at 70℃
And then allow to cool
No change of colour
In sample
Barfoed’s reagent
Development of brick red precipitate indicates presence of
manosaccharide. But no colour change in cardamon sample indicates the
presence of disaccharide in it.
Results:

9. Paper Chromatography
Taking Whatman’s paper in 9.8cm and level in 2 cm and mark in pencil.
Then preparation of buffer solution containing N-butanol, acetic acid and water with ratio 4:1:5.
Prepare the coloring reagent containing 𝐾2C𝑂3(10gm), KMn𝑂4(1.5gm), NaOH(1gm). In the paper drop by
drop add standard solution by micropipette then dry.
After that the buffer is placed in the chamber then prepared paper fit in the chromatography chamber with
clip then salute tape it because for not evaporate.
The solute arises in the paper, will wait until the solvent buffer reach in certain level. After approx. 2 hours
mark the line by pencil and dry it. Then spray the coloring reagent in the paper and yellow color appears in
some areas.

10. The yellow color appears in G and F pores. But in samples yellow color is not found. That concluded
that the sample does not contain monosaccharide. It contained disaccharide.
Fig-1: Paper chromatography chamber Fig-2: showing monosaccharide in G and F

11. Thin layer Chromatography
Plate preparation:
➢ This TLC plates are usually commercially available, with standard particle size ranges to improve
reproducibility.
➢ This mixture is spread as thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or
plastic. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C.
➢ The thickness of the adsorbent layer is typically around 0.1- 0.25 mm for analytical purposes and around 0.5-
2.0 mm for preparative TLC.
Capillary spotters:
➢ Place a melting point capillary and in the dark blue part of the Bunsen burner flame.
➢ Hold it there until it softens and starts to sag. Quickly remove the capillary from the flame and pull on
both ends to about 2-3 times its original length.
➢ Allow the capillary to cool down, and then break it in the middle. Make sure to break off the closed
end on one of them.

12. Spotting the plate:
➢ The thin end of the spotter is placed in the dilute solution; the solution will rise up in the capillary . Allow the
solvent to evaporate and spot at the same place again. This way we will get a concentrated and small spot.
➢ The spots should be far enough away from the edges and from each other as well. spot the compound or
mixture together with the starting materials and possible intermediates on the plate.
Fi- 1: TLC paper after KMnO4 spray Fig 2: TLC plate in UV visible machine

13. In silico analysis of Phytochemicals
➢ After preparation, the compounds were filtered based on the molecular properties for predicting their solubility and
permeability in drug discovery.
➢ The best known of the physical property filters is “Lipinski’s rule-of five and Veberrules”, which focuses on
bioavailability.
➢ The rule states that the compounds have molecular mass less than 500 Daltons, not more than 5 hydrogen bond
donors, not more than 10 hydrogen bond acceptors and an octanol-water partition coefficient XlogPnot greater than 5.
➢ The filtered compounds were then downloaded in ‘.csv’format for toxicity assessment.
Ligand Selection and Filtration:
➢ This is mainly the parameter to check the toxicity of the ligand. (bioavailability, Mutagenic, Tumorigenic, Irritant
and Reproductive)
➢ According to Lipinski’s rule, a ligand molecule should have the following properties to become a good drug
candidate.
➢ The molecular weight of that ligand should be below 500 Daltons, the cLogPvalue should be less than 5, solubility
value should be greater than -4, TPSA value should be less than or equal to 140, drug likeness values must be in
positive range and drug score should be greater than 5.
Drug likeness and toxicity prediction of the ligands:

14. ➢ The ligands selected are of the phytocompounds which have passed the toxicity parameters are
Ethyl linalool1[CID - 526764], 2-(5-Ethyl-5-methyloxolan-2-yl)propan-2-ol, [CID-5317140],
Dehydrosabinaketone [CID-527426],Glabranin [CID-124049], 3,4-flavandione [CID-265703]
➢ Those ligands of the phytocompounds which have passed the toxicity parameters of the Lipinski' rule was
then downloaded from the Corina Online web server(https://www.mn-am.com) in ‘.pdb’ format. Then the
.pdbfiles was converted to. pdbqtfiles using the software Open Babel (http://openbabel.org) which was then
used for Virtual screening studies of those five phytocompounds against Lung cancer ‘ 4lvt ’.
➢ Cooperation of ligandswith 4lvt were examined via AutoDock Vina to portray a potential compliance and
direction for the ligand at its limiting site.
➢ The protein was attracted PyRx programming (https://sourceforge.net) and protein structure that contain
hydrogen in all polar buildup was saved in pdbqt document. In this condition, all obligations of ligands were
set to be rotatable.
➢ All computation for protein-fixed ligand-adaptable docking were broke down by the Lamarckian Genetic
Algorithm (LGA) strategy.
➢ The normal restricting energy for best postures was taken as the last restricting energy esteem. This cycle was
rehashed multiple times.
Molecular Docking Analysis

15. Bcl_2-Navitoclax (ABT-263) Complex
Classification: APOPTOSIS REGULATOR/INHIBITOR
Organism(s): Homo sapiens
Expression System: Escherichia coli
Mutation(s): Yes
4lvt protein :-
➢ The structure of 4lvt is , an anticancer drug that act as a cancer inhibitor..
➢ It binds to a hydrophobic pocket in the major cancer protein and prevents uncoating of the viral genome.
➢ 4lvt protein in anti cancer properties has been downloaded from RCSB site, and this proteins are involved for
causing Lung Cancer.

16. The PubChem CID numbers with their IUPAC names and their molecular formula of the 5ligands selected
after the virtual screening.
Ligand CID IUPAC Name Molecular Formula
124049 5,7-dihydroxy-8-(3-methylbut-2-
enyl)-2-phenyl-2,3-dihydrochromen-
4-one
C20H20O4
265703 2-phenylchromene-3,4-dione C15H10O3
527426 5-propan-2-ylbicyclo[3.1.0]hex-3-en-
2-one
C9H12O
5317140 2-(5-ethyl-5-methyloxolan-2-
yl)propan-2-ol
C10H20O2
526764 3-ethyl-7-methyloct-6-en-3-ol C11H22O

17. Interaction Profile Analysis
➢ The protein molecule was treated as a receptor molecule and plant metabolites was treated as the
ligands. The docked protein and ligand are prepared for docking process.
➢ In molecular docking process the protein-ligand interaction has been done by using BIOVIA Discovery
Studio.
➢ The docking stances of everyone of the five Cardamom ligands uncovered that every ligand has just one
restricting posture.
➢ Out of 20 poses of ligand a single pose file should be created. Then single pose PDB file in ligands and
docked protein open in discovery studio. Then successfully protein-ligand interaction has been shown.
The interactions are visualized in 2D diagram to see the interaction show active site .
124049 265703

18. Summary And Conclusion
➢ It was early studied known that Elettaria cardamomum plant has medicinal action against various disease .
➢ The existence of various phytochemicals such as unstable mixtures, flavones, phenols, unsaturated fats,
including organic molecules can be credited for the observed medical benefits. As a response, using
commercial software and web tools, the Insilco approach used in this study aided in the discovery of ligands
for prostate cancer . This technique saves money and time by decreasing the cost and time.
➢ Elaichi is rich in antioxidants and anti cancer activities , black cardamom helps in combating the symptoms
of cold and cough, while the oil derived from its seeds acts as an anti cancer agent which is help to cure
against lungs cancer.

19. ➢ Using autoDock vina software molecular docking action was performed to identify the phytochemicals (3-ethyl-7-
methyloct-6-en-3-ol,2-(5-ethyl-5-methyloxolan-2-yl)propan-2-ol,2-phenylchromene-3,4-dione,5-propan-2-
ylbicyclohex-3-en-2-one,5,7-dihydroxy-8-(3-methylbut-2-enyl)-2-phenyl-2,3-dihydrochromen-4-one) which can
have a dominant role with the protein 4lvt . This work gives information about the presence of this metabolites are
most functional constituents in cardamom plant that reduces the risk of Lung Cancer.

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