Invention of an Anticancer Moiety from Indian River Spinach using Pharmaceutical Analytical Techniques

 

Sasikala M*1,2, Sundaraganapathy R2, Mohan S1

1Karpagam College of Pharmacy, Coimbatore – 641032, Tamil Nadu, India

2Faculty of Pharmacy, Karpagam Academy of Higher Education, Coimbatore – 641021, Tamil Nadu, India

*Corresponding Author E-mail: sasikalampharm@gmail.com

 

ABSTRACT:

Introduction: Since to meet the worlds challenging diseases, there is a huge requirement for finding remedies. The remedy might be derived from any source like synthetic, mineral, biological, microbiological, marine and herbal. Among which, herbal, the natural remedy stands first to treat an illness without side effects. The active secondary metabolites have been spread widely across the world. The herb that acts both as nutraceutical and as pharmaceutical majorly is various species of spinach. Hence, the water spinach is selected for getting an excellent moiety by means of different analytical procedures. Aim and Objectives: The aim is to characterize isolated herbal compound from Ipomoea aquatica Forsskal with the use of spectroscopic and chromatographic techniques. Method: The authenticated herb was extracted for its active metabolites. The screened phytochemicals by preliminary identification tests and TLC were applied for isolation. The high yield isolate from water fraction was evaluated for characterizing its chemical and biological properties. The structural elucidation was done with FTIR, 1H NMR and LCMS. The biological property was analyzed by colorimetric method using MCF-7 cell lines. Results and Discussion: The yield of isolate was 13.9 %w/w. The compound was characterized as halo aromatic alcoholamide with molecular weight of 1677.60 g/mol. IC50 value of active molecule by MTT assay was detected as 173.1 μg/ml. Conclusion: The invented anticancer natural drug might be isolated in bulk to prepare herbal formulations. The medicine could be utilized for treating various life threatening diseases because of its functional groups and pharmacological actions.

 

KEYWORDS: Isolation; MCF-7; Water Spinach; FTIR; LCMS; NMR.

 

 


INTRODUCTION:

The plant is lying under spinach category to build with great number of phytoconstituents having nutrient and therapeutic effects. The antioxidant activity has been reported on this herb. This leads the molecules to exist with anticancer property1-3.

 

The process to extrude out phytochemicals from the plant in the form of coarse powder is extraction. The methods are applied based on thermolabile and thermostable metabolites of an herb using either cold, ambient or heat conditions to recover them1-6.

 

 

 

The various identification tests are performed on the extract to identify what class of metabolite being existed in the plant. Thin layer chromatographic analysis meant to separate similar group of metabolites with different set of mobile phase. The separated molecules would be detected by means UV light or by means of visualizing reagents. The Rf, qualitative parameter in TLC to detect the compound comparing it with reference standard7-10.

 

The group of separated metabolites are picked into its individual moieties. The process is achieved efficiently with a support of column chromatograph. It is one of the best purification techniques of mixture of samples11-13.

 

When the infrared radiation is passed on to a chemical, it will cause vibrational energy changes in it. This is associated with atomic mass and bond energy. Hence, the spectral analysis helps to reveal the functional group present in the molecule. The mid IR region composed of Functional group and fingerprint region which will support structural elucidation11-13.

 

Chemical shift values of different alkyl, aromatic and functional groups linked with proton nucleus could be analyzed clearly with the assistance of an NMR spectrum. Any atomic nucleus with odd mass number can be characterized by NMR spectroscopic analysis12.

 

Separation and characterization is achieved simultaneously with minute amount of sample by LCMS analysis. This determines molecular weight of parent ions, fragment ions, metastable ions and isotopes of a molecule along with the molecular formula also using fragmentation patterns and rules12.

 

It is an invitro or exvivo analytical method to reveal the anticancer activity of a drug molecule derived from natural or from synthetic source. The technique is carried to detect half maximal inhibitory concentration of drug samples using different cancer cell lines13-17.

 

The aim is to characterize isolated herbal compound from Ipomoea aquatica Forsskal with the use of spectroscopic/chromatographic techniques chemically and biologically.

 

MATERIALS AND METHODS:

Materials:

The aimed plant contents are listed in the table 1. The instruments, chemicals/reagents and glass wares/apparatus effective for the research are described in the table 2, table 3 and table 4 respectively. 

 

Table 1: Plant details

S. No.

Parameters

Subject

1.

Plant Name

Water Spinach

2.

Botanical Name

Ipomoea aquatica Forssk.

3.

Family

Convolvulaceae

4.

Location

Parambikulam – Aliyar Riverine, Pollachi

5.

Part of the plant

Whole plant

6.

Authentication No.

BSI/SRC/5/23/2017/Tech./3269

7.

Place of Authentication

BSI, Coimbatore-641003, Tamil Nadu, India

 

Table 2: Instruments used

S. No.

Name of the Instrument

Model Name

1.

Precision Balance

Wensar

2.

Hot plate

Cintex

3.

Ultra Sonicator

Labman

4.

Electrical Water bath

Technico

5.

UV cabinet

CAMAG and Deep Vision

6.

FTIR

Shimadzu

7.

LCMS

PE Sciex API3000

8.

NMR

Spect

9.

Double beam UV/Visible spectrophotometer

Shimadzu 1800

10.

CO2 incubator

11.

Tecan Plate reader

 

Methods:

Preparation of coarse powder of collected plant:

The authenticated plant was collected in large numbers. They were washed many times with distilled water to remove soil and other foreign materials. Then, they were dried under shade for nearly two weeks. The dried plant was powdered by means of electrical blender and sieved to get uniform particles.

 

Extraction:

The weighed uniform particles were admitted for maceration technique.  The amount of powder taken was 350.0 g. The volume of menstrum utilized for the process was 50.0 %v/v ethanol in distilled water in the ratio of 1:10. The amount of hydroalcoholic extract obtained was 101.2 g.

 

Preliminary phytochemical Screening:

The identification tests for alkaloids, flavonoids, glycosides, triterpenoids, saponins and tannins. The group of constituents was separated using thin layer chromatographic analysis using fluorescence detection methods.

 

Isolation by Column Chromatography:

Isolation of active phytoconstituents were done taking 10.0 g of dark-brown colored extract with the eluents of petroleum ether, chloroform, ethyl acetate, methanol and distilled water (from non polar to polar). The highest yield of water fractions allowed its characterization studies by further techniques.

 

Table 3: Chemicals/Reagents used

S. No.

Name of the Reagent

Company

Location

1

Petroleum Benzine boiling range 60.00C-80.00C GR (Petroleum ether)

Merck Specialties Private Limited

Mumbai –

400 018

2

Silica gel G 60 – 120 mesh for column chromatography

3

Pyridine GR

4

Ethanol AR 99.9%

Jiangsu Huaxi International Trade Co., Ltd.

China

5

Distilled water

 

 

6

Toluene (Sulphur free)

Reachem Laboratory Chemicals Private Limited

Chennai –

600 098

7

Formic acid LR

8

Acetic acid Glacial LR

9

Acetone LR

10

Methanol LR

S d Fine Chemicals Limited

Mumbai –

400 030

11

Ethyl acetate LR

12

Silica gel G for TCL

Loba Chemie Private Limited

Mumbai – 400 005

13

Potassium bromide for IR

14

MTT Powder

15

DMSO

16

Cell lines

ATCC

17

DMEM/F12

Invitrogen

18

Fetal Bovine Serum

19

Penicillin

20

Streptomycin

21

Trypsin

22

EDTA

23

Glucose in PBS

Table 4: Interpretation of FTIR spectrum

S. No.

Frequency Region (cm-1)

Functional Group

Class of Phytochemicals

1.

3287

O-H

Alcohols/Phenols

2.

2922

O-H

Carboxylic acids

3.

2361

C=N

Nitriles

4.

1651

C=C

Alkenes

5.

1558

N-O

Nitro compounds

6.

1316

C-C

Aromatics

7.

948

C-H

Alkenes/Aromatics

8.

878 and 773

C-X

Alkyl halides

 

Chemical Characterization:

Infrared spectroscopic analysis:

The method followed for IR studies is potassium bromide pressed pellet technique. The spectral regions were scanned between 4000 cm-1 and 667 cm-1 in mid IR region. The functional groups and finger prints of an unknown isolated compound was detected.

 

Nuclear Magnetic Resonance Spectroscopic Analysis:

The study was carried with proton magnetic resonance. The solvent utilized was deuterium oxide since the compound is derived by water. The running time of instrument was found as 30 minutes. Interpretation was done with obtained spectrum.

 

LC/MS Analysis:

The isolated molecule was finally characterized for determining its molecular weight and molecular formula. It was achieved by liquid chromatography and mass spectroscopic analysis using electron spray ionization method. The different fragmented ion peaks were found in the spectrum.

 

Biological Characterization: 

MTT Assay:

The serial two fold dilutions from 100 µM to 3.125 µM were prepared to utilize for the study. The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 x 105 cells/ml using respective media containing 10% FBS. To each well of the 96 well micro titer plates, 100 µl of the diluted cell suspension (50,000cells/well) was added. After 24h, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100 µl of different test concentrations of test drugs were added on to the partial monolayer in micro titer plates.

 

The plates were then incubated at 37oC for 24 h in 5% CO2 atmosphere. After incubation, the test solutions in the wells were discarded and 100µl of MTT (5mg/10 ml of MTT in PBS) was added to each well. The plates were incubated for 4 h at 37oC in 5% CO2 atmosphere. The supernatant was removed and 100 µl of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a micro plate reader at a wavelength of 590 nm.

 

The percentage growth inhibition was calculated and concentration of test drug needed to inhibit cell growth by 50% (IC50) values is generated from the dose-response curves for each cell line.


 

Table 5: Interpretation of 1H NMR spectrum

S. No.

Number of Signals

Position of Signals 

(δ – ppm)

Ratio of protons

Spin – Spin coupling

Description of Proton

1

13

1.101

1.702

Singlet

R CH3 - alkyl (methyl)

2

1.214

4.572

Singlet

R CH2 R - alkyl (methylene)

3

1.289

1.306

3.766

Doublet

4

1.844

1.883

1.950

2.000

2.008

2.023

10.487

Sextet

R3C H - alkyl (methine)

5

2.197

1.044

Singlet

R CO CH3 - α to carbonyl (C is next to C=O)

6

2.376

2.648

Singlet

Ar CH3 - benzylic    (C is next to Ph)

7

2.571

4.381

Singlet

RC ≡ C H - alkynyl

8

2.651

2.860

Singlet

R2N CH3 - α to nitrogen (C is attached to N)

9

2.691

3.975

Singlet

R CH2 X - α to halogen (C is attached to Cl, Br, I)

10

3.230

4.564

Singlet

ROH – (alcohol)

11

3.375 – 4.214 (3.991)

91.450

9 multiplets

RO CH3 - α to oxygen (C is attached to O)

12

5.216

5.070

Triplet

R2C = CHR - vinylic (H is attached to alkene C)

13

8.424

0.960

Singlet

R CO NHR – (amide)

 

Figure 1: Graphical Abstract

 

Figure 2: FTIR spectrum of Isolated compound

 


RESULTS AND DISCUSSION:

The yield of hydroalcoholic extract was as much as high to carry out each step of analysis in a systematic manner. The percentage yield of extract was 28.91 %w/w.

 

Phytochemical tests and TLC analysis reported that the extract composed of proteins, carbohydrates and amino acids as primary metabolites and alkaloids, flavonoids, tannins, saponins, triterpenoids and glycosides as secondary metabolites.

 

The existence of maximum secondary metabolites of the plant leads isolation and characterization of them. The amount of isolated fractions collected close to 1.0 – 20.0 %w/w from the extract.

 

The fractions were checked for its purity by carrying out infrared analysis. The highly pure substances were evaluated to know their anticancer property. FTIR analysis reported that the selected isolated compound would contain the functional group like hydroxyl of phenolic and carboxylic moieties and the finger print of the spectrum showed to have aromatic nucleus in it. The vibrational frequency range for each group is illustrated in the table 4 and the respective spectrum is being focused in the figure 2.

 

NMR spectral studies on the analyte resulted to exist with methyl, methylene, vinyl, amide, carbonyl and aromatic protons in the chemical structure. The chemical shift values for each kind of proton are enumerated in the table no. 5 and the spectrum is shown in the figure 3.

 

 

Figure 3: 1H NMR spectrum of Isolated compound

LC/MS interpretation has given information on molecular weight of the moiety as 1677.60 g/mol for its parent ion peak. M+1 peak for 13C isotope at m/z of 114.39, base peak with 100% intensity at m/z of 338.42, M+5 peak for chloride or bromide or hetero atoms at 699.01 and m+ (metastable ion) peak at m/z of 1037.15. The values are depicted in the figure 4.


 

 

Figure 4: LCMS spectrum of Isolated compound

 


 

 

Figure 5: Microtiter plate of Sample at 10μg/ml

Figure 6: Microtiter plate of Sample at 320μg/ml

 

 

 

Figure 7: Calibration chart of inhibitory activity of Sample and its extract

 

 

Figure 8: IC50 values of Sample and its extract

 

MTT colorimetric assay revealed the significant inhibitory activity on MCF – 7 breast cancer cell lines. IC50 value of the compound was determined as 173.1 μg/ml against control. The activity was found more comparing with that of extract with numerous molecules. Microtiter plates, calibration chart and inhibitory concentration of the lead molecule along with its extract are given in the figures 5, 6, 7 and 8 respectively.

 

CONCLUSION:

From the chemical and biological characterization of a lead molecule of isolated compound from Indian water spinach, the functional group expected for pharmacological activity is existed with the compound. The moiety has hydroxyl group might be a flavonoid since it has shown positive results for preliminary characterization studies itself. Hence, a novel flavonoid group of molecule with anticancer activity will meet Global requirement to treat Breast cancer in women, which is the common immunological disease found in them, for sure as a natural remedy, which a human prefers for his long term medication to avoid maximal side effects.

 

ACKNOWLEDGEMENT:

This research was defended by the Management and Principal of Karpagam Educational Institutions, Coimbatore. We express gratitude to our colleagues from Karpagam College of Pharmacy and Faculty of Pharmacy, Karpagam Academy of Higher Education, Coimbatore who provided wisdom and expertise that incredibly assisted the study.

 

CONFLICT OF INTEREST:

It is declared none.

 

REFERENCES:

1.      Sasikala M, Mohan S, Ajitha Vasudevan Nambiar, Dona S, Karpakavalli M and Sundaraganapathy R. High Performance Thin Layer Chromatographic Determination of Phytoconstituents in Hydroalcoholic and Methanolic Whole Plant Extract of Peristylus goodyeroides (D. Don) Lindl. World J Pharma Res. 2017, 6 suppl 10: 1405-16.

2.      Sasikala M, Mohan S, Haritha T H, Manoj K, Karpakavalli M and Sundaraganapathy R. Extraction and Phytochemical Screening of Whole Plant of Peristylus goodyeroides (D. Don) Lindl. from Southern Whestern Ghats of India. World J Pharma Res. 2017, 6 suppl 10: 1394-404.

3.      Sasikala M and Sundaraganapathy R. Optimization of Extraction Method for Ipomoea aquatica Forssk. [Indian River Spinach] From Its Whole Plant, Int J Rec Sci Res, 2017, 8 suppl 5: 16967-971.

4.      Sasikala M, Sundara Ganapathy R, Karpakavalli M and Mohan S. Chemotaxonomical and Pharmacological Review on Medicinal Plants in Temperate Region, Int J Pharm and Tech, 2017, 9 suppl 1: 29165-172.

5.      Sasikala M and Sundaraganapathy R. Qualitative Analysis of Alkaloids Exist in the Hydroalcoholic Extract of Ipomoea aquatica Forssk. in Tamil Nadu. Int J Chem Tech Res, 2017, 10 suppl 7: 446-54.

6.      Sasikala M and Sundaraganapathy R. Fluorescence Detection of Phytoconstituents in Hydroalcoholic Extract of Ipomoea aquatica Forssk. by Thin Layer Chromatographic Analysis, Int J Pharmacog and Phytochem Res, 2017, 9 suppl 7: 990-6.

7.      Sasikala M and Sundaraganapathy R. Preliminary Phytochemical Evaluation of Hydroalcoholic Extract of Ipomoea Aquatica Forssk. from Aliyar Riverinein South India, Int J Pharma Bio Sci, 2017, 8 suppl 3: 356-65.

8.      Sasikala M, Karpakavalli M, Ranjithkumar D, Prakash G, Vanathi P and Mohan S. In-vitro Free Radical Scavenging Activity Studies of Extracts and Isolated Compounds of Eugenia jambolana Lam. Seeds, World J Pharma Res. 2016, 5 suppl 6: 2021-30.

9.      A.H. Beckett and J.B. Stenlake. Practical pharmaceutical chemistry. Thin layer chromatography. CBS Publishers. 4th Edition. 2005: 115-28.

10.   S.S. Khadabadi, S.L. Deore and B. A. Baviskar. Experimental Phytopharmacognosy – A Comprehensive guide. Nirali Prakashan, Pune, Maharashtra. Second edition, 2013. p.3.1-3.13, A 1.1-2.8.

11.   Harborne JB. Phytochemical Methods. Chapman and hall Ltd., London: U.K., 1973. 49-188.

12.   Y.R. Sharma, Elementary Organic Spectroscopy: Principles and Chemical Applications, S. Chand Publishers, Mumbai, 2010.

13.   Gunti Gowtham Raj, Hyma Sara Varghese, Sarita Kotagiri, B. M. Vrushabendra Swamy, Archana Swamy and Rafi Khan Pathan. Anticancer Studies of Aqueous Extract of Roots and Leaves of Pandanus odoratissimus f. ferreus (Y. Kimura) Hatus: An In Vitro Approach. J Tradit Complement Med. 2014 Oct-Dec; 4 suppl 4: 279–84.

14.   Sumitra Chanda and Krunal Nagani. In vitro and in vivo Methods for Anticancer Activity Evaluation and Some Indian Medicinal Plants Possessing Anticancer Properties: An Overview. J Pharmacog and Phytochem. 2013, 2 suppl 2:140-52.

15.   P.A. Shah, J.M. Parekh, P.S. Shrivastav. Assessment of critical extraction and chromatographic parameters for the determination of Bupropion and its three primary metabolites in human plasma by LC-MS/MS. Microchem. J., 2017, 135: 81-90.

16.   Kumaran S, Nazeema Banu B, Julie J, Abirami J, Kumareasan R, Muthukumaran T, Rajasree S and Jeya Jothi K. Anti-cancer activity of Datura metel in MCF-7 cell line. Asian J Pharma and Clini Res. 2014, 7 suppl 1: 181-183.

17.   Mukundam Borah, Swarnamoni Das and Shagufa Ahmed. Antibacterial activity of the ethanolic extract of leaves of Citrus maxima (Burm.) Merr. on Escherichia coli and Pseudomonas aeruginosa. Asian J Pharma and Clini Res. 2013, 6 suppl 4: 136-139.

 

 

 

 

 

Received on 29.01.2019         Modified on 19.02.2019

Accepted on 29.03.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(7):3371-3376.

DOI: 10.5958/0974-360X.2019.00569.9