Phytochemical Screening, GC-MS, FT-IR Analysis of Methanolic Extract of Curcuma caesia Roxb (Black Turmeric)

Pakkirisamy, Kalakandan, and Ravichandran: Phytochemical Screening, GC-MS, FT-IR Analysis of Methanolic Extract of Curcuma caesia Roxb (Black Turmeric)

Authors

INTRODUCTION

Curcuma Linn. is a large genus belonging to the family Zingiberaceae. It comprises about 70 species of rhizomatous herbs distributed mostly in Southeast Asia as wild and cultivated plants. Curcuma caesia is a perennial herb with bluish-black rhizomenative to Northeast and Central India. Curcuma caesia (Black Turmeric) is also sparsely originated in Papi Hills of East Godavari, West Godavari, and Khammam Districts of Andhra Pradesh. The rhizomes of Curcuma caesia have a high economic importance because of its putative medicinal properties. The rhizomes are used in the treatment of smooth muscle relaxant activity1 haemorrhoids, leprosy, asthma, cancer, epilepsy, fever, wound, vomiting, menstrual disorder, anthelmentic, aphrodisiac, inflammation, gonorrhoeal discharges, etc.2

The rhizomes of the plant are aromatic in nature. The inner part of the rhizome is bluish-black in colour and emits a characteristic sweet smell, due to presence of essential oil.3 Traditionally, the rhizomes of Curcuma caesia Roxb. Are used in treating leucoderma, tumors , asthma, piles, bronchitis etc. The paste is applied on bruises, contusions and rheumatic pains.4 Fresh rhizome decoction is used as antdiarrhoeiaric and to get relief from stomach ache. The fresh rhizome paste of curcuma caesiais Roxb applied during the snake bite and scorpion bite.5,6

The advances in analytical techniques, including GC-MS and FT-IR that were powerful tools for identification and determination of phytochemicals compounds. The present study was carried out the bioactive compounds present in the Curcuma caesia Roxb in methanol extract with the aid of GC-MS and FT-IR techniques, which may provide an insight in its use of traditional medicine.

MATERIAL AND METHODS

Plant material collection and extraction

Rhizomes of Curcuma caesia Roxb. were collected from Thanjavur, Tamil Nadu, and India. The collected Rhizome was shade dried, powered and extracted with methanol using Soxhlet apparatus for 8 hours. The extracts were filtered and filtrates were concentrated under reduced pressure at 40° C using a rotary flash evaporator and stored at 4°C until use for phytochemical screening.

Phytochemical Screening

Phytochemical analysis was carried out for identification of tannins, terpenoids, flavonoid, alkaloid, phenol, phytosterol, Quinones and saponins according to standard methods.7,8

GC –MS Analysis Preparation of extract

Rhizome powder of Curcuma caesia Roxb were shade dried. 20 g of the powdered tubers were soaked in 95% ethanol for 12 h. The extract was then filtered through Whatmann filter paper No.41 along with 2 gm sodium sulfate to remove the sediments and traces of water in the filtrate. Before filtering, the filter paper along with sodium sulphate was wetted with 95% ethanol. The filtrate was then concentrated by bubbling nitrogen gas into the solution. The extract contained both polar and non-polar phytocomponents of the plant material used.

GC Condition and Identification of Compounds

The sample was investigated through Gas Chromatography Mass Spectrometry/Mass Spectrometry Electron Ionization (GC-MS/EI) mode. The GC-MS/MS is a Scion 436- GC Bruker model coupled with a Triple quadruple mass spectrophotometer with fused silica capillary column BR-5MS (5% Diphenyl/95% Dimethyl polysiloxane) and Length: 30m; Internal diameter: 0.25 mm; Thickness: 0.25 μm. Helium gas (99.999%) was used as the carrier gas at a constant flow rate of 1 ml/min and an injection volume of 2 μl was employed (split ratio of 10:1). The injector temperature 250°C; ion-source temperature 280°C. The oven temperature was programmed from 110°C (isothermal for 2 min), with an increase of 10°C/min, to 200°C, then 5°C/min to 280°C, ending with a 9 min isothermal at 280°C and total GC running time was 41 min.9 This last increase was to clean the column from any residues. The mass spectrometer was operated in the positive electron ionization (EI) mode with ionization energy of 70eV. The solvent delay was 0-3.0 min. A scan interval of 0.5 seconds and fragments from m/z 50 to 500 Da was programmed. The inlet temperature was set at 280 °C, source temperature 250 °C. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was MS Work station 8. The NIST Version 2.0 library database of National Institute Standard and Technology (NIST) having more than 62,000 patterns was used for identifying the chemical components. The GC-MS/MS was performed by Food Safety and Quality Testing Laboratory, Institute of crop processing technology, Thanjavur.

FTIR Spectroscopic Analysis

Fourier transform infrared spectrophotometer (FTIR) is perhaps the most powerful tools for identifying the types of chemical bonds (functional groups) present in compounds. Dried powders of different solvent extracts of each plant material were used for FTIR analysis. 10mg of the dried extract powder was encapsulated in 100 mg of KBr pellet, in order to prepare translucent sample disc. The powdered sample of each plant specimen was loaded in FTIR Spectroscope (Shimadzu, IR Affinity1, Japan), with a scan range from 400 to 4000 cm-1 with a resolution of 4cm-1.

RESULTS AND DISCUSSION

Plants are very important source of potentially useful bioactive principles for the development of new chemotherapeutic agents.10 The biological and pharmacological properties of many plants are still unknown. World over, the scientists are exploring the potential of utilizing pharmacologically active compounds from medicinal plants.11 Herbal medicines are used by 80% of the people worldwide due to its high efficiency, cheap cost, nonnarcotic nature and fewer side effects.12

In the present study, the exploration of phytochemical screening with Methanol extract of Curcuma caesia revealed the presence of carbohydrate, flavonoid, steroid, phenol, alkaloid, tannin, amino acid, terpenoid and glycoside compounds which are known to have remedial activity against diseases producing pathogen. Therefore it can be used pharmacologically to develop new compounds for health benefit (Table 1). Phytochemical constitutes of plants serves as defense mechanism against by many microorganisms. The therapeutic properties of medicinal plants are possibly due to the presence of various secondary metabolites.13 Thus the preliminary screening test may be useful in the detection of the bioactive principles and subsequently may lead to the drug discovery and improvement.

Table 1

Phytochemical constituents present in Methanolic extracts of Curcuma caesia Roxb

S.NoPhytochemicalsCurcuma caesia Methanol Extract
1Tannins+
2Terpenoids+
3Flavonoid+
4Phenol+
5Phytosterol+
6Saponins+

The compounds present in the methanolic extract of Curcuma caesia, were identified by GC-MS analysis (Figure 1). The active principles with their retention time (RT), molecular formula, molecular weight (MW) and concentration (%) are presented in Table 2. Seventeen compounds were identified in methanolic extract by GC-MS. The major components present in the Curcuma caesia (Black turmeric) were α-Santalol (46.90%), Retinal (10.72%), Ar-tumerone(10.38%), Alloaromadendrene (5.93%), Megastigma-3,7(E),9-triene (4.80%), Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl(4.38%), 5,8,11,14,17-Eicosapentaenoic acid, methyl ester, (all-Z)-(4.26%) Tricyclo [8.6.0.0(2,9)]hexadeca-3,15-diene, trans-2,9-anti-9,10-trans-1,10(3.26%) and various other compounds were identified as low level . These phytochemicals are responsible for various pharmacological actions like antimicrobial and anti-oxidant anti-inflammation, Anti-cancer, Hepato protective, Diuretic, Antiasthma activities etc (Table 3). Curcuma caesia has medicinal value the presence of these major constituents.14

Figure 1

GC-MS analysis of Curcuma caesia Roxb of methanol extract.

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Table 2

Phytocomponents identified in the Methanolic extract of the Curcuma caesia Roxb By GC-MS

S.NoRTName of the compoundMolecular formulaMWPeak area (%)
14.14(+)-2-BornanoneC10H16O1520.91
24.39IsoborneolC10H18O1540.40
37.85AlloaromadendreneC15H242040.63
49.13Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl-C15H222024.38
59.33Tricyclo[8.6.0.0(2,9)]hexadeca-3,15-diene, trans-2,9-anti-9,10-trans-1,10-C16H242163.26
69.71trans-Sesquisabinene hydrateC15H26O2221.39
710.74α-SantalolC15H24O22046.90
811.46Ar-tumeroneC15H20O21610.38
911.93Megastigma-3,7(E),9-trieneC13H201764.80
1013.185,8,11,14,17-Eicosapentaenoic acid, methyl ester, (all-Z)-C21H32O23164.26
1115.57Retinal, 9-cis-C20H28O28410.72
1216.29AndrostenediolC19H30O22902.85
1317.15(+)-2-BornanoneC24H40O43921.03
1417.32IsoborneolC24H32O43842.17
1520.66AlloaromadendreneC23H32O64045.93
Table 3

Activity of phyto-components identified in Curcuma caesia Roxb by GC-MS.

S.NOName of the CompoundCompound natureActivity
1(+)-2-BornanoneMonoterpene oxideAnti-tumor, Analgesic Antibacterial, Anti-inflammatory Sedative, Fungicide
2IsoborneolMonoterpene alcoholAnti-tumor, Analgesic Antibacterial, Anti-inflammatory Fungicide, Sedative, Antipyretic, Antifeedent, Candidicide, Hepatoprotective, Pesticide, Perfumery
Tranquilizer, Myorelaxant, Antibronchitic
3AlloaromadendreneSesquiterpeneAnti-tumor, Analgesic Antibacterial, Anti-inflammatory Sedative, Fungicide
4Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl-Color pigmentsSkin care products Anti-inflammatory
Anticancer, Antioxidant
5Tricyclo[8.6.0.0(2,9)]hexadeca-3,15-diene, trans-2,9-anti-9,10-trans-1,10-SteroidAntimicrobial, Anti-inflammatory
Anticancer, Diuretic, Antiasthma
Haepatoprotective
6trans-Sesquisabinene hydrateSesquiterpene alcoholAnti-tumor, Analgesic Antibacterial, Anti-inflammatory Fungicide, Sedative, Antipyretic, Antifeedent, Candidicide, Hepatoprotective, Pesticide, Perfumery, Tranquilizer, Myorelaxant, Antibronchitic
7α-SantalolSesquiterpene oxideAnti-tumor, Analgesic Antibacterial, Anti-inflammatory Sedative, Fungicide
8Ar-tumeroneKetone compoundAnti-tumor, Analgesic Antibacterial, Anti-inflammatory Sedative, Fungicide, Antiarthritic Antidote for arsenic, Anti HIV Anticancer,
9Megastigma-3,7(E),9-trieneAlkene compoundAnticancer, Antitumor, Expectorant, Memory enhancer
105,8,11,14,17-Eicosapentaenoic acid, methyl ester, (all-Z)-Unsaturated fatty acid esterCardio protective
11Retinal, 9-cis-Vitamin A precursorAnticancer, Eye protective
12AndrostenediolSteroidAntimicrobial, Anti-inflammatory, Anticancer, Diuretic, Antiasthma
Haepatoprotective
13(+)-2-BornanoneAcidic compoundReduce gallstone formation, Chemo preventive
14IsoborneolSteroidCytotoxic steroid, Anti-inflammatory
15Alloaromadendrenecardiac glycosideInhibit membrane protein, Strengthen the failing heart

The FT-IR spectrum was used to identify the functional groups of the active components present in extract based on the peaks values in the region of IR radiation. When the extract was passed into the FT-IR, the functional groups of the components were separated based on its peaks ratio. The results of FT-IR analysis confirmed the presence of N-H, O-H, C=C, C-H, C-O and CH3 functional groups (Figure 2 and Table 4). FTIR spectroscopy is proved to be a reliable and sensitive method for detection of bio molecular composition.

Figure 2

FTIR analysis of Curcuma caesia Roxb.

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Table 4

FTIR Peak Values of Methanolic Extract of Curcuma caesia Roxb.

S. NOPEAK VALUESFUCTIONAL GROUPS
13287.28N-H
22923.88O-H
31635.03C=C
41324.97C-H
51149.68C-O
61077.21C-O
71016.05CH3

CONCLUSION

The present work has been performed to establish the various Phytochemical, GCMS and FTIR parameters, which could serve as important and has commercial interest in both research institutes and pharmaceuticals companies for the manufacturing of the innovative drugs. This primary information will facilitate in conducting further studies on discovery of bioactive constituents, resolve of their efficacy by in vivo studies and demonstration of their safety and efficacy in clinical trials.

Acknowledgements

The authors are thankful to Dr. C. Anandharamakrishnan, Director, Indian Institute of Food Processing Technology, Thanjavur, Tamil Nadu, India for providing all the facilities to conduct this research work and also thankful to the members of Department of Food Safety and Quality Testing division of for their support.

Notes

[1] Conflicts of interest There are no conflicts of interest.

ABBREVIATION USED

GC MS

Gas chromatography–mass Spectrometry

FT-IR

Fourier transform- infrared

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ABOUT AUTHORS

Dr. K.Suresh Kumar, Associate Professor and Head i/c of Department of Food Safety and Quality Testing, Indian Institute of Food Processing Technology, Thanjavur,Tamil Nadu, India.

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Dr.P.Muthukumaran, Senior Research Fellow, Department of Food Safety and Quality Testing, Indian Institute of Food Processing Technology, Thanjavur, Tamil Nadu, India.

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Dr.R.Karthikeyan, Technical Assistant, Department of Food Safety and Quality Testing, Indian Institute of Food Processing Technology, Thanjavur, Tamil Nadu, India.

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