Identification and Analysis of Chemical Constituents of Rasam by Gas Chromatography–Mass Spectrometry (GC-MS)

 

Agilandeswari Devarajan^1,2, MK Mohan Maruga Raja^2*

¹Department of Pharmaceutics, Hillside College of Pharmacy & Research Centre, Bengaluru, Karnataka, India.

²Pharmacy Department, Centre for Research & Development, PRIST University, Thanjavur, Tamil Nadu, India.

 

ABSTRACT:

Unavae marunthu” (meaning “food is medicine”) is a classical and extraordinary discovery of Siddha system of medicine. Rasam, is a very popular spice soup and it is consumed on daily basis in every South Indian home especially in Tamil Nadu. Literatures are available for the ingredients used in the preparation of rasam, but the details of chemical constituents present in rasam were not available. Hence, the present study was planned to study rasam in a stage wise manner and to analyse its chemical constituents by gas chromatography–mass spectrometry. Rasam was prepared in a five stage process to evaluate the significance of the traditional processing. Four samples (RS1, RS2, RS3 and RS4) at different stages of preparation were studied by gas chromatography–mass spectrometry. Stage wise preparation of rasam from RS1 to RS4 showed completely altered chemical composition. The only chemical constituent retained from RS1 to RS4 was 1,2-benzenedicarboxylic acid diethyl ester. 5-(propenyl-2)-1,3,7-nonatriene and di-n-octyl phthalate were retained from RS1 to RS3 but both the constituents were not present in RS4. 4h-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl and mome inositol were retained from RS3 to RS4. The major constituent in RS4 was found to be guanosine. Altered chemical composition may be due to breakdown of certain chemical constituents and/or formation of new chemical entities (NCEs).

 

 

 

INTRODUCTION:

Siddha, a system of traditional medicine originated in ancient Thamilakam in South India (10,000 to 4,000 BCE).¹ “Unavae marunthu” (meaning “food is medicine”) is a classical and extraordinary discovery of Siddha system of medicine. Rasam, is a very popular spice soup. It is consumed on daily basis in every South Indian home especially in Tamil Nadu. It is also called as rasam or chaaru or saaru in South Indian languages viz. Tamil, Malayalam, Telugu and Kannada. Rasam is mostly consumed with rice. In a traditional South Indian meal, it is preceded by a sambar rice course and is followed by curd rice.

 

Rasam, is a traditional functional food as its ingredients are medicinally claimed for various ailments. It is traditionally prepared using tamarind juice as a base, with a variety of spices which are considered to be good for health and improving the digestion.² The main spices used in rasam preparation are coriander, garlic, curry leaves, tamarind, cumin, black pepper, mustard, turmeric, red chilli and asafoetida.³ In South India, it is considered as an effective home remedy for common cold, cough and an antidote for flu or fever.^{4, 5} Devi and Priyadharshini, 2014 have reported that rasam is traditionally used for the treatment of cold, cough and diabetes.⁶ Mani et al., 1997 have reported that the glycemic index (GI) and the triacyglycerol response in ninety normal volunteers after consuming South Indian meals with rasam significantly controlled diabetes.⁷ Bolla et al., 2015 have reported that South Indian diet with rasam everyday showed a significant reduce in the blood sugar levels of 40 volunteers between 30 and 60 years.⁸ Rajan et al., 2001 have reported that rasam is given daily in the evening to nursing mothers for inducing more secretion of milk.⁹ Rasam has been reported for anti-microbial¹⁰ and anti-platelet activity.¹¹ Exhaustive literatures are available for the ingredients used in the preparation of rasam, but the details of chemical constituents present in rasam were not available. Hence, the present study was planned to study rasam in a stage wise manner and to analyse its chemical constituents by gas chromatography–mass spectrometry.

 

MATERIALS AND METHODS:

Materials:

All ingredients of rasam were purchased from Arokya organic shop, Vellore, Tamil Nadu. All utensils used for the preparation of rasam were of Stainless Steel 316 grade (SS 316). All other chemicals and solvents were obtained from SD Fine Chemicals (Mumbai, India) and of analytical grade.

 

Preparation of rasam:

Rasam was prepared in five stages as mentioned below;

1. Tamarind fruit pulp mixture (T1): 6.88 g of tamarind fruit pulp was immersed in 450 mL of water for 10 min, then it was hand crushed for 45 times and strained. The strained liquid was rinsed with 5 mL water, to which 0.4 g of turmeric powder and 4 g of sea salt was added.

2. Tomato fruit mixture (T2): 82.44 g of fresh tomato fruits were cut and hand crushed for 60 times. The crushed fruit was rinsed with 5 mL of water.

3. Spice mixture (T3): 1.33 g of pepper drupes was crushed in a SS 316 mortar and pestle for 85 times. 2.67 g of cumin fruits was added over to the crushed pepper drupes and crushed for 100 times. To the above crushed mixture 0.82 g of chili pepper was added and crushed for 50 times. To the above mixture 9.63 g of garlic cloves was added and crushed for 90 times.

4. All mixture (T4): Tomato fruit mixture (T2) was rinsed with 10 mL of water and spice mixture (T3) was rinsed with 10 mL of water. Both rinsing were added to tamarind fruit pulp mixture (T1), this was designated as sample RS1.

5. Final product (T5): 4 ml of Indian sesame oil was heated at 60°C for 2 min. After 5 seconds 0.82 g of mustard seeds were added. After 3 seconds 1.53 g of whole chili pepper was added. After 2 seconds 0.61 g of curry leaves was added, this was designated as sample RS2. Immediately all mixture (T4) was rinsed with 20 mL of water and added. The whole liquid was allowed to boil for a 5 min. After 5 min 1.50 g of coriander leaves was added, this was designated as sample RS3. When the liquid frothed, 0.05 g of as a foetida was added and the heating was switched off to yield the final product, this was designated as sample RS4 ^{[10, 11]}.

 

Gas chromatography–mass spectrometry study:

GC Programme:

Column: Elite-1 (100% Dimethyl poly siloxane), 30m X 0.25mm ID X 1μm df

Equipment: GC Clarus 500 Perkin Elmer

Carrier gas: Helium 1 ml/min

Detector: Mass detector-Turbo mass gold-Perkin Elmer

Software: Turbo mass 5.1

Sample injected: 2 μl

Split: 10:1

 

Oven Temperature Programme:

110 deg C-2 min hold

Up to 280°C at the rate of 5 deg/min-9min hold

Injector temp: 250°C

Total GC time: 30 min

 

Table 1: Biological source of the ingredients and its quantity used in the preparation of rasam

Common names	Morphological part used	Nature of the material	Botanical name	Family	Quantity used
Tamarind	Ripped fruit pulp	Dried	Tamarindus indica L.	Fabaceae	6.00 g
Turmeric	Rhizome powder	Dried	Curcuma longa L.	Zingiberaceae	0.40 g
Sea salt	NA	Solid	NA	NA	4.00 g
Tomato	Ripped fruit	Fresh	Solanum lycopersicum L.	Solanaceae	82.44 g
Chili pepper	Crushed fruit of long chilli pepper	Dried	Capsicum annuum L.	Solanaceae	0.82 g
Cumin	Ripped fruit	Dried	Cuminum cyminum L.	Apiaceae	2.67 g
Garlic	Cloves	Dried	Allium sativum L.	Amaryllidaceae	9.63 g
Black pepper	Unripe drupe	Dried	Piper nigrum L.	Piperaceae	1.33 g
Indian sesame oil	Seed	Oil	Sesamum indicum L.	Pedaliaceae	4 mL
Black mustard	Seed	Dried	Brassica nigra L.	Brassicaceae	0.82 g
Chili pepper	Whole fruit of long chili pepper	Dried	Capsicum annuum L.	Solanaceae	1.53 g
Curry leaves	Leaves	Fresh	Murraya koenigii (L.) Sprengel	Rutaceae	0.61 g
Portable water	NA	Liquid	NA	NA	500 mL
Coriander	Leaves	Fresh	Coriandrum sativum L.	Apiaceae	1.50 g
Asafoetida	Dried latex (oleogum resin) exuded from the rhizome or tap root	Powder	Ferula assa-foetida L.	Apiaceae	0.05 g

 

 

 

MS Programme:

Library used: NIST 11 and WILEY8

Inlet line temperature: 200°C

Source temperature: 200°C

Electron energy: 70 eV

Mass scan: (m/z) 45-450

Total MS Time: 30 min

 

Preliminary qualitative phytochemical screening:

The prepared rasam was studied for the presence and absence of secondary metabolites such as alkaloids, tannins, saponins, flavonoids, terpenoids, steroids, glycosides, and volatile oil.^12,13

 

RESULTS AND DISCUSSION:

The biological source and quantity of the ingredients used in the preparation of rasam are shown in Table 1.

 

The stage wise samples RS1, RS2, RS3 and RS4 in the preparation of rasam were studied to evaluate the significance of the traditional processing. The GC-MS spectra of RS1, RS2, RS3 and RS4 are shown in Figure 1, 2, 3 and 4.

 

Figure 1: GC-MS spectra of RS1

 

Figure 2: GC-MS spectra of RS2

 

Figure 3: GC-MS spectra of RS3

 

Figure 4: GC-MS spectra of RS4

 

There is clear evidence that the stage wise preparation of rasam from RS1 to RS4 has shown completely altered chemical composition (Table 2). The only chemical constituent retained from RS1 to RS4 was 1,2-benzenedicarboxylic acid diethyl ester. 5-(propenyl-2)-1,3,7-nonatriene and di-n-octyl phthalate were retained from RS1 to RS3 but both the constituents were not present in RS4. 4h-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl and mome inositol were retained from RS3 to RS4 (Table 2). Mome inositol is reported as anti-alopecic, anti-cirrhotic, anti-neuropathic, cholesterolytic, lipotropic and a sweetener.¹⁴ The major constituent in RS4 was found to be guanosine. The currently used antiviral drug aciclovir, often used in herpes treatment¹⁵, and the anti-HIV drug abacavir¹⁶, are analogues of guanosine. The preliminary qualitative phytochemical analysis of rasam results confirmed the presence of alka­loids, tannins, saponins, flavanoids, terpenoids, steroids, glycosides, and volatile oil. The processing in the preparation of rasam involved heating the spices in water and oil. The traditional processing of rasam has provided a completely altered chemical composition in the final product, rasam which is evident from the GC-MS study. Altered chemical composition may be due to breakdown of certain chemical constituents and/or formation of new chemical entities (NCEs). As RS3 and RS4 involved heating process, the volatile constituents would have disappeared, and that also might be the reason for altered chemical composition of rasam.

 

Table 2: Name of the chemical constituent and its retention time in stage wise samples of rasam (RS1, RS2, RS3 and RS4)

RS1		RS2		RS3		RS4
RT (min)	Chemical constituent	RT (min)	Chemical constituent	RT (min)	Chemical constituent	RT (min)	Chemical constituent
10.6	Benzaldehyde, 4-(1-methylethyl)	17.6	Tricyclo[3.3.1.13,7] decane, [2,2-dimethyl-3-(2-methyl-1-propenyl)cyclopropylidene]	9.6	Terpinen-4-ol	6.0	2-furancarboxaldehyde, 5-methyl-
11.4	5-(propenyl-2)-1,3,7-nonatriene	20.9	Butanoic acid, nonyl ester	9.8	5,7-octadien-2-ol, 2,6-dimethyl	6.3	2,4-dihydroxy-2,5-dimethyl-3(2h)-furan-3-one
13.7	Sucrose	21.0	1,3-dioxolane, 4-methyl-2-phenyl	11.12	1,3-dioxolane, 2,4,5-trimethyl	7.4	Benzeneacetaldehyde
13.8	Cycloserine	21.05	1,3-dioxolane, 4-methyl-2-phenyl	11.15	4h-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl	8.0	5-methyloctene-1
15.5	1,2-benzenedicarboxylic acid, diethyl ester	21.1	Trisiloxane, 1,1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]	11.4	5-(propenyl-2)-1,3,7-nonatriene	9.1	4h-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-
15.6	Pyrrol-2(5h)-one, 4-acetyl-5-(2-fluorophenyl)-1-(2-furfuryl)-3-hydroxy	21.2	3-[3-(benzoyloxy)propoxy] propyl benzoate	13.6	Xanthosine	10.4	5-hydroxymethylfurfural
17.6	Octadecanoic acid, 2-oxo-, methyl ester	21.4	Galactitol	13.7	4,5,5-d3-trans-3,4-dihydroxy-cyclopentene	10.6	1,2,3-propanetriol, 1-acetate
18.2	Undecanal	22.8	n-methyladrenaline, tri-tms	13.90	Oxirane, 2-ethyl-3-propyl-, cis-	10.8	7-oxabicyclo[4.1.0]heptan-2-one, 6-methyl-3-(1-methylethylidene)
18.72	1,2-benzenedicarboxylic acid, diundecyl ester	23.2	1,2-bis(trimethylsilyl)benzene	13.97	2,2,4-trimethyl-3-pentanol	10.9	4-dimethylsilyloxypentadecane
18.79	2,3-dimethylfumaric acid	23.3	1,1,1,3,5,7,9,9,9-nonamethylpentasiloxane	14.0	1,6,6-trideuterocyclohexa-2-en-1-ol	11.1	1,4-dioxane, 2,3-bis(isopropyloxy)-
19.1	Octadecanoic acid, methyl ester	23.4	Ethane, 1,1,1-triethoxy	14.1	2,3-dehydro-4-oxo-.beta.-ionol	11.2	Heptanoic acid, 6-oxo-
19.3	S(-)-cathinone, n-acetyl	25.1	3,6-dioxa-2,7-disilaoctane, 2,2,4,5,7,7-hexamethyl	15.5	1,2-benzenedicarboxylic acid, diethyl ester	13.6	Guanosine
19.5	[dodecanoyl(methyl) amino] acetic acid	26.62	1-di(tert-butyl)silyloxy-2-phenylethane	16.7	Mome inositol	13.7	2,4-dimethylhexanedioic acid
21.2	Methyl 4-hydroxybutanoate	26.69	Cyclotetrasiloxane, octamethyl	16.9	Docosanoic acid	13.8	1,2-cyclopentanediol, 3-methyl-
26.6	Hexahydropyridine, 1-methyl-4-[4,5-dihydroxyphenyl]	26.9	Diisopropyl(ethoxy)silane	17.6	1-t-butyl-4-(adamantyl-1)benzene	13.9	Silane, (2-ethoxyethoxy)trimethyl-
26.7	2,4,6-cycloheptatrien-1-one, 3,5-bis-trimethylsilyl	27.6	Silane, triethyl(2-phenylethoxy)	18.7	1-hexadecanol	15.5	1,2-benzenedicarboxylic acid, diethyl ester
26.9	2,4,6-cycloheptatrien-1-on, 3,5-bis-trimethylsilyl	28.2	4-[(2,2,2-trifluoroacetyl)amino]-2-cyclohexen-1-yl trifluoroacetate	19.1	Hexanoic acid, 2-methyl-	16.7	Mome inositol
27.4	9,12,15-octadecatrienoic acid, 2-[(trimethylsilyl)oxy]-1 (trimethylsilyl)	28.29	3,4-dimethylbenzoic acid, trimethylsilyl ester	19.5	Undecanoic acid	16.9	diethylene glycol monododecyl ether
28.7	Di-n-octyl phthalate	28.7	1,2-benzenedicarboxylic acid, diisooctyl ester	20.8	1-hexadecanol	17.0	3-o-methyl-d-glucose
29.3	4-deuterio-trans-3,4-dihydroxy-cyclopentene	29.3	3-(methylsulfanyl)-1-phenyl-3-(2-pyridinylamino)-2-propen-1-one	28.7	Di-n-octyl phthalate	20.8	1-heneicosanol

RT-retention time

 

CONCLUSION:

The traditional processing in the preparation of rasam had altered the chemical composition of the final product. A better understanding of chemistry of the constituents during the preparation processes may provide;

1.    New leads based on structure activity relationship (SAR) studies of chemical constituents.

2.    Interesting leads can be obtained on the novel chemical structure of NCEs from the formulation. 

3.    Interactions between chemical constituents of different ingredients will provide unique combination for studying interaction between organic and inorganic constituents.

The concept of classical phytotherapy using herbal drug combinations with superior efficacy and lesser side effects in comparison with single isolated constituents of plant extracts has been repeatedly assessed clinically as well as pharmacologically. Such plant extracts that hit more than one biological target may offer a better pharmacological approach.¹⁷ A study on rasam, which is being consumed from time immemorial, is only an approach of “drug rediscovery”. In view of all the above facts, rasam should be extensively explored with the modern scientific approaches to identify its pharmaceutical potential beyond its culinary and nutritional effect.

 

CONFLICT OF INTEREST:

Author(s) declare that there is no conflict of interest.

 

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Accepted on 11.10.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(12): 4183-4187.