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

 

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.

 

 

 

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 property^1-3.

 

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 them^1-6.

 

 

 

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 R_f, qualitative parameter in TLC to detect the compound comparing it with reference standard^7-10.

 

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 samples^11-13.

 

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 elucidation^11-13.

 

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 lines^13-17.

 

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 37^oC 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 37^oC 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

 

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.

 

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Received on 29.01.2019         Modified on 19.02.2019

Accepted on 29.03.2019         © RJPT All right reserved