FTIR and GCMS Analysis of Antidiabetic Compounds in Ethyl acetate Seed Extracts of Momordica charantia

 

Renuka R1, Jeyanthi G.P2

1Department of Biochemistry, Sri Ramakrishna College of Arts and Science for Women,

Coimbatore, Tamil Nadu, India.

2Research and Consultancy, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore, Tamil Nadu, India.

*Corresponding Author E-mail: renukabio@srcw.ac.in

 

ABSTRACT:

Momordica charantia L., which belongs to the Cucurbitaceae family is also known as bitter gourd, bitter melon, karela and paharkai in Tamil has been widely used as a vegetable and also as herbal medicine for diabetic patients. Identification of bioactive compounds in the plant material may be useful for proper standardization of herbs and their formulations. The present study was conducted to analyze the antidiabetic compounds present in Momordica charantia seed extracts using FTIR (Fourier Transform Infra-Red spectrophotometry) and GCMS (Gas Chromatography Mass Spectroscopy) analysis. FT-IR analysis of ethyl acetate seed extracts of Momordica charantia confirmed the presence of alcohols, phenols, alkanes, alkynes, aldehydes, aromatic compounds, aromatic amines and aromatic hydrocarbons. GC-MS analysis of ethyl acetate seed extracts of Momordica charantia revealed the presence of about forty compounds in  that four were found to be biologically important as per literature studies because of their anti diabetic, antioxidant, antimicrobial and anticancer properties.

 

KEYWORDS: Momordica charantia, FTIR, GCMS, Anti diabetic compounds, Diabetes.

 

 


INTRODUCTION:

Herbal plants are very common in use in our daily life. These herbs are used as a nutrient or as a source of food. Vegetables, spices, fruits, seeds, flowers, pulses and even tea leaves come under the category of herbs. All these are being consumed daily directly or indirectly and these materials can prove to be useful in the treatment of diabetes1.

 

Traditionally, Diabetes mellitus is treated with diet, physical exercise and herbal remedies. Herbal drugs are acceptable and are believed to cause less adverse effects, more effective and cost less. Herbal extracts have shown to possess both antioxidant and anti diabetic activities2.Traditional Indian medicines have great potential for scientists to find active compounds and develop new drugs for treatment of diabetes.

 

 

Thus, there is an increasing requirement and the feasibility to screen and obtain active compounds including plant extracts from Indian traditional medicinal plants for the treatment of diabetes and its complications3. For centuries, indigenous drugs either alone or in combination have been advocated in the traditional system of medicine for the treatment of various ailments. India is rich in plant wealth and has excellent base of utility of plants with ayurvedic science4.

 

Momordica charantia grows in tropical and sub tropical areas in east Africa, Asia, Caribbean and South America, and it is used as a food and as well as a medicine. Although the seeds, leaves and vines of bitter melon have all been used, the fruit is the safest and most prevalent part of the plant which is used medicinally5. Momordica charantia posses wide range of pharmacological activities and it has been used as drug in India as folk remedy in the form of decoctions and infusions to treat the deficiency disease such as anti-hyperglycaemic activity6.

 

The anti diabetic effect of plants is attributed to the combination of phytochemicals or single component of the plant extracts. The phytochemicals responsible for anti diabetic properties mainly are alkaloids, phenolic acids, flavonoids, glycosides, saponins, polysaccharides, stilbenes and tannins7. Herbal plant sources consist of important phyto-constituents such as polyphenols, flavonoids, alkaloids and other components which can against defective cellular metabolism and regulate its functional property. The variance in the cellular products helps to boost the body or cells against immune-complications caused by diabetes mellitus. The active compounds from the plants could be recorded and systematically validated to increase the plant-based medicines with fewer side effects or without side effects8.Antidiabetic medicines derived from plant sources have lesser side effects and offered cost effective management of diabetes through nutrient supplementation9.

 

Currently available treatment options for diabetes in modern medicine have several adverse effects. Hence, there is a need to develop safe and effective treatment modalities for diabetes10.Treating diabetes mellitus with plant derived compounds which are accessible and do not require laborious pharmaceutical synthesis seems highly attractive. Isolation and identification of active compounds from these plants, preparation of standardized dose and dosage regimen can play a significant role in improving the hypoglycaemic action11.

 

The extraction of bioactive constituents from the plants and their quantitative and qualitative estimation is important for exploration of new molecules to be used by pharmaceutical and agrochemical industry directly or can be used as a lead molecule to synthesize more potent molecules12. Hence the present study was conducted to analyze the bioactive compounds present in Momordica charantia seed extracts using FTIR and GCMS Analysis.

 

MATERIALS AND METHODS:

Preparation of the Plant Extract:

Five grams of Momordica charantia seed powder was macerated with 50 mL of ethyl acetate for 48 hours filtered and collected. The solution was evaporated in water bath shaker to get dry extract. TLC is a simple, quick and inexpensive process that can be used for the analysis of mixtures13. Ethyl acetate seed extracts of Momordica charantia was separated by Preparative Thin Layer Chromatography (TLC) and potent fraction having highest alpha amylase inhibition was subjected to FTIR (Fourier Transform Infra-Red spectrophotometry) and GCMS (Gas Chromatography Mass Spectroscopy) analysis for identification of the active compounds.

Fourier Transform Infra-Red (FT- IR) spectrophotometry analysis 14:

Fourier transform infrared spectroscopic imaging technique with infrared array detectors has recently emerged as a powerful characterization tool. IR spectroscopy is an important technique which utilizes electromagnetic radiations in the infrared region for the determination and identification of molecular structure15. FT-IR is the most powerful tool for identifying the types of chemical bonds and functional groups present in the phytochemicals. The wavelength of light absorbed is primary feature of the chemical bond as can be seen in the annotated spectrum. By interpreting the IR absorption spectrum, the chemical bonds in a compound can be determined. Dried powder of Momordica charantia seed extract was used for FTIR analysis. Ten mg of the Momordica charantia dried extract powder was encapsulated in 100 mg of KBr pellet, in order to prepare translucent sample discs. The powdered sample of each extract was loaded in FT-IR spectroscope (Shimadzu, Japan), with a scan range from 400 to 4000 cm-1 with a resolution of 4 cm-1.

 

Gas Chromatography Mass Spectroscopy (GCMS) analysis 16:

GC-MS analysis was carried out on a GC clarus 500 Perkin Elmer system and gas chromatograph interfaced to a mass spectrometer (GC-MS). Column Elite-1 fused silica capillary column (30mm x 0.25mm ID x 1 μ Mdf, composed of 100% dimethyl poly siloxane), operating in electron impact mode at 70eV; Helium (99.999%) was used as carrier gas at a constant flow of 1ml /min and an injection volume of 1 ml was employed (split ratio of 10:1); Injector temperature 2500C; Ion-source temperature 2800C. The oven temperature was programmed from 1100C (isothermal for 2 min) with an increase of 100C / min, to 2000C then 50C / min, to 2800C, ending with a 9 min Isothermal at 2800C. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds and fragments from 45 to 450 Da. Total GC running time was 47 minutes for Momordica charantia seed extract. The relative percentage of each compound was calculated by comparing its average peak area to the total area. Software adopted to handle mass spectra and chromatograms was a Turbo Mass Ver 5.2.0.

 

RESULTS:

FTIR spectrum of potent TLC fraction of Momordica charantia seed extract was analyzed and the results of FTIR spectrum profile are depicted in Figure1 and in Table 1.

 

Figure1: FT-IR spectrum of Momordica charantia seed extract

 

FTIR analysis was used to identify the functional group of active compounds based on the peak values in the region of infrared radiation. Ethyl acetate extracts of seeds of Momordica charantia showed characteristic absorption bands at 3456.44 cm-1 for N-H stretching and O-H stretch, 2931.80 cm-1 and 2862.36 cm-1 for C-H stretch, 2306.86 cm-1 for N Ξ N stretch,1735.93 cm-1 for-CHO,1566.20 cm-1 for aromatic nitro compounds or carboxylate,1373.32 cm-1 for aliphatic nitro compounds C-O-C –Cyclic ethers, 1242.16 cm-1 and 1172.72 cm-1 for sulphate ions, C-O-C stretch in esters, aromatic amines-CN stretch, 20 amine NH stretch, 933.55 cm-1 for CH= CH2  and CH2 , 840.96 cm-1  for R-NH2  primary amine aromatic CH in plane bend, 786.96 cm-1  for meta substituted aromatic compounds and 732.95 cm-1  for mono substituted aromatic hydro carbons.

 

Table 1: Interpretation of FT- IR absorptions of Momordica charantia seed extract

Group frequency wave number (cm-1)

Bond and functional groups

3456.44

> N-H Aromatic 20 amine NH stretch

- OH Normal polymeric OH stretch-Alcohols/Phenols

2931.80

2862.36

> CHMethylene asymmetric/symmetric stretch

> CH- Methyne C-H stretch

2306.86

N Ξ N Diazonium salts N Ξ N Stretch

1735.93

-CHO Aldehyde or ester, six membered lactone

1566.20

Aromatic nitro compounds/carboxylate

1373.32

Nitrate ions, aliphatic nitro compounds

C-O-C –Cyclic ethers

1242.16,

1172.72

Sulphate ions, C-O-C stretch in esters

Aromatic amines-CN stretch, 20 amine NH stretch

933.55

CH= CH2 in vinyl groups

CH2  out of plane wag

840.96

R-NH2  primary amine

Aromatic CH in plane bend

786.96

Meta substituted aromatic compounds

732.95

-(CH2) Mono substituted aromatic hydro carbons

GCMS profile of potent TLC fractions of Momordica charantia seeds:

Potent TLC fractions of ethyl acetate extracts of Momordica charantia seeds were subjected to gas chromatography and mass spectrum analysis, GC-MS running time was 47 minutes. The GC-MS chromatogram of Momordica charantia seed extract is presented in Figure 2.

 

Figure 2: GCMS profile of potent TLC fractions of Momordica charantia seeds

 

The present study on GC-MS revealed the presence of about forty compounds. The identity of the components in the extract was assigned by the comparison of their retention indices and mass spectra fragmentation patterns with the National Institute of Standards and Technology (NIST) 08 LIB and WILEY8 LIB library sources for matching the identified compounds from the plant material.

 

Among the forty compounds identified four of them were found to be biologically important as per literature studies because of their anti diabetic, antioxidant, antimicrobial and anticancer properties. The active compounds with their retention time, peak area and nature of the compound, molecular formula and molecular weight are depicted in Table 2.

 


 

Table 2: Compounds identified at various retention times from Momordica charantia seed extracts

Retention time

Peak Area

Name of the Compound

Nature of the compound and common name

Molecular formula

Molecular weight

8.769

337899

1-Methyl-4-(1-methylethenyl)cyclo hexene

Cyclic terpene Essential oil Limonene

C10H16

136

25.122

59104

2,6,10-Trimethyl dodecane

Sesquiterpene Farnesane

C15H32

212

26.012

6593923

1-Nonadecene

Alkene

C19H38

266

37.022

282371

1,2-Benzenedicarboxylic acid

Aromatic dicarboxylic acid

C8H5NO6

211

 


DISCUSSION:

Functional group analysis plays a vital role in understanding the overall physicochemical properties of the extract. Identification of the functional group helps to study their structure–activity relationships. FT-IR spectral analysis of the root bark extracts of Mammea suriga showed the presence of phytochemicals carrying hydrogen bonded –OH functional group. This hydroxyl functionality is an integral part of most of the phenolic phytochemicals such as flavonoids and tannins17.The functional groups of carboxylic acids, amines, amides, sulphur derivatives, polysaccharides, organic hydrocarbons and halogens that is responsible for various medicinal properties was studied in Aerva lanata18. FTIR spectrum analysis of Ichnocarpus frutescens plant extract showed the presence of the functional groups of nitriles, alkanes, ketones, phenols, alkyl halides and aliphatic amines and FTIR spectroscopy is proved to be a reliable and sensitive method for detection of biomolecular composition19.

 

D-limonene (1-methyl-4-(1-methylethenyl) cyclo hexane) reduces the oxidative stress in STZ-induced diabetic rats by decreasing lipid peroxidation and sparing the activities of antioxidant enzymes. Therefore, D-limonene can be considered as a safe food supplement with a potential as an anti diabetic agent in diabetic complications20.Antioxidants are our first line defence against free radical damage and critical for maintaining optimum health and well being21.

 

GC-MS analysis of Petrea volubilis root revealed the presence of 1, 2- benzenedicarboxylic acid, mono (2- ethyl hexyl) ester which might be responsible for anti diabetic activity through α-amylase inhibition22.GCMS analysis of Tamilnadia uliginosa fruit extracts showed the presence of 1-nonadecene and the compound possess antioxidant, antimicrobial and anticancer properties23. Enhydrin, a major sesquiterpene lactone from Smallanthus sonchifolius leaves was found to be effective to reduce post-prandial glucose and found to be useful in the treatment of diabetic animals24. The ethyl acetate extracts of seeds of Momordica charantia showed potent free radical scavenging activity, alpha- amylase inhibition and the mechanism of inhibition was found to be non competitive25. Qualitative phytochemical analysis of Momordica charantia confirmed the presence of phytochemicals like flavonoids, saponins, terpenoids, coumarins, emodins, alkaloids, proteins, etc 26.

 

Previously reported studies has confirmed that alpha amylase inhibitory potentials of Momordica charantia seed extracts showed highest inhibition in one of the Thin layer chromatographic fractions with 96 % inhibition and High Performance Thin Layer analysis of the ethyl acetate extracts of seeds of Momordica charantia and Trigonella foenum graecum showed the presence of phenols and flavonoids and these compounds might be responsible for antidiabetic properties27. Momordica charantia has been used successfully in ayurvedic medicine from ancient times, more clinical trials should be conducted to support its therapeutic use. It is also important to recognize that Momordica charantia extracts may be effective not only when used singly, but may actually have a modulating effect when given in combination with other herbs or drugs28.

 

CONCLUSION:

In the present study FT-IR analysis of Momordica charantia seed extracts confirmed the presence of alcohols, phenols, alkanes, alkynes, aldehydes, aromatic compounds, aromatic amines and aromatic hydrocarbons. GCMS analysis of potent TLC fractions identified 1-Methyl-4-(1-methylethenyl) cyclo Hexane, 2,6,10-Trimethyl dodecane, 1-Nonadecene and 1,2-Benzenedicarboxylic acid. These compounds identified in Momordica charantia seeds might be responsible for the anti diabetic and alpha amylase inhibitor activities. Hence they can be further purified and have a promising future scope in formulation of new drugs in the management and treatment of diabetes mellitus without any side effects.

 

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Received on 02.10.2020            Modified on 31.12.2020

Accepted on 04.02.2021           © RJPT All Right Reserved

Research J. Pharm.and Tech 2021; 14(12):6705-6709.

DOI: 10.52711/0974-360X.2021.01158