Comparison Study for the Phytochemical Constituents of two Curcuma Species by GC-MS Technique

Curcuma , a major Zingiberaceae genus, contains approximately 110 species throughout the Asia-Pacific region. The present work aimed to study the two Indian Curcuma species , Curcuma caesia Roxb. and Curcuma angustifolia Roxb., whose rhizomes are extracted using ultrasound-assisted extraction (UAE) with chloroform solvent. The extracts are subjected to phytochemical screening and analysed employing gas chromatography-mass spectrometry (GC-MS) procedures. UAE studies of these two Curcuma species' rhizomes using chloroform as a solvent have been conducted for the first time. The chloroform extracts of Curcuma caesia and Curcuma angustifolia contain twenty-six and thirty-two components, respectively. The component with the highest area percentage in both species was 2-cyclohexen-1-one, 4-ethynyl-4-hydroxy-3,5,5-trimethyl (2CEHT), a cyclic unsaturated ketone having anticancer effects. The GC-MS measurement data and phytochemical screening results provide an update on the physiologically active phytoconstituents detected in rhizome extracts, which may be used to standardise crude plant extracts and understand the species' chemical composition and medicinal potential.


INTROdUCTION
Curcuma caesia Roxb.and Curcuma angustifolia Roxb.are two species belonging to the Curcuma genus, which is a part of the Zingiberaceae family.Curcuma angustifolia Roxb. is distributed in the Northeast, Madhya Pradesh, Uttar Pradesh, and Himachal Pradesh, while Curcuma caesia Roxb. is found in the Indian states of West Bengal, Orissa, Uttar Pradesh, Madhya Pradesh, Sikkim, and Chhattisgarh.In terms of importance and use, Curcuma angustifolia Roxb.is used in the production of arrowroot powder, and its rhizomes are used to make food.It is also used to treat diarrhoea, fever, and pain, and is a demulcent and blood-clotting agent. 1 Curcuma caesia Roxb. is utilised as a folk medicine and has antiasthmatic, anticancer, antiallergy, and anti-inflammatory qualities.The rhizome has been used as a condiment and food preservative. 2,3dicinal plants are appreciated for their therapeutic phytoconstituents, which may lead to the development of new medications.Because of the phytochemicals found in medicinal plants, as well as the pharmaceutical and cosmetic industries' shift towards organic products, medicinal plant research is just as essential as traditional drug research. 4hytoconstituents from plants must be extracted and measured in order to discover novel compounds or use them as a lead molecule in the production of more effective medicinal molecules. 5Extraction is critical in phytochemical processing for identifying and evaluating bioactive phytoconstituents from plant sources.Common extraction methods include decoction, hot continuous extraction, percolation, maceration, infusion, and others. 6Modern extraction methods, such as supercritical fluid extraction (SFE), microwave-assisted extraction (MAE), and ultrasound-assisted extraction (UAE), are constantly being developed to improve production while lowering costs. 4Heat extraction has the potential to destroy thermolabile natural substances.Ultrasound-Assisted Extraction (UAE) accelerates and cools the extraction process.Ultrasound cavitates and ruptures cell walls, accelerating the extraction of active plant components from the matrix and enhancing mass transfer. 7lvent plays a vital part right through the extraction process and the type of solvent to be used is determined based on the nature of phytoconstituents to be extracted.For the extraction of the nonpolar secondary metabolites from plants, non-polar solvents are usually used.[10][11] The use of UAE in chloroform extracts of Curcuma caesia Roxb.rhizomes are reported in this study for the first time.There have been no reports of Curcuma angustifolia Roxb.rhizome extracts being investigated by the UAE.This study of the phytoconstituents of a UAE rhizome chloroform extract sheds insight on its therapeutic potential The information presented in this study will help reaffirm the usage of Curcuma caesia Roxb.and Curcuma angustifolia Roxb.rhizomes as reserves for medicinal phytoconstituents and can provide a pathway for developing herbal products based on the identification of the components and understanding its nature from the present study.

Plant collection
The Curcuma caesia Roxb.rhizome was gathered in March 2020 and 2021 from the ICAR-IISR (Indian Institute of Spices Research Kozhikode), located in Kerala.ICAR-IISR authenticated and maintained the rhizome under accession number Acc. 292 (IC 349014).
Curcuma angustifolia Roxb.rhizome was obtained from Jorhat, Assam, in the month of February every year between 2020-2021.Plant authentication with Accession No. was given by CSIR-National Botanical Research Institute (NBRI) Herbarium (LWG) 109910.

Chemicals
Analytical grade chloroform was used for the study.Whatman Filter Paper 41 and MDI 0.45 micron nylon syringe filters were used for the filtration of extracts.

Sample preparation GC-MS Analysis
The rhizomes underwent a thorough scrubbing with water, followed by division into small pieces and subsequent drying for five days in shade.They were then powdered in a mixer grinder and sifted to obtain a fine powder.The powdered rhizomes were stored in covered jars and used for upcoming experiments.The rhizome powder and the solvent (chloroform) were taken in a ratio of 1:25 and sonicated for 30 min using an ultrasound sonicator water bath. 13The extraction was performed in a pulsed manner over 11 and 19 min with 2-3 min breaks in between.The temperature was maintained between 25°C and 30°C.Following sonication, ashless filter paper 41 from Whatman was used to sift the extracts, which were then filtered again through a nylon syringe filter of 0.45 micron before being introduced into the GC-MS instrument.

Phytochemical screening
The extracts filtered through Whatman Filter Paper 41 were utilized in the qualitative phytochemical screening studies.

Instrumentation Sonicator
The Dakshin, 200H sonicator had a stainless steel tank with a capacity of 6.5 litres, an ultrasonic frequency of 36±3 kilohertz (KHz), and an ultrasonic power of 200 watts.It required an electric supply of 230 volts A.C, 50 Hz, and could operate at a maximum temperature of 60°C.

GC-MS
The GC-MS is a Shimadzu GCMS-QP2010 Plus model coupled with the GCMS-QP2010 Ultra Mass Spectrometer.

GC condition
The analysis by GC-MS was carried out on a capillary column, Restek Rtx-5MS, having dimensions measuring 30 metres x 0.25 millimetres ID, 0.25 micron with helium being the carrier gas and pressure as the flow control mode set at 83.5 kilopascals (kPa).The oven was programmed to heat at 80°C during the first 5 min then upstretched to 150°C at a scale of 10°C each minute and kept for 2 min afterwards elevated to 220°C at an amount of 10°C for every minute and placed constant for 2 min and ultimately enhanced to 290°C at the same proportion of 10°C every single minute and stationed for 5 minutes.The entire duration of the run was 35 minutes. 14The temperature of the injector port was 250°C.The volume injected was 2 microlitres (µL) with a split ratio of 90.0.

MS condition
The ion source and interface were heated to 220°C and 260°C, respectively.The detector gain was set at 1.03 kilovolts (kV).The solvent cut time was maintained at 2.0 min and the scan start time and end time were 2.0 min and 34.0 min respectively, with a start mass to charge (m/z) of 35.0 and an end m/z of 700.0.The scan speed was set at 2500.The data processing of mass spectra and chromatograms was performed using LabSolutions' GCMS Solution Version 2.70 software.The NIST 11 library database was used for identifying the chemical components.

GC-MS method conditions
By carefully establishing the analysis method conditions, the extracts underwent GC-MS screening in order to achieve uniform peak responses, optimal peak separation, and peak resolution.The detectable peaks were identified by name, mass, and structure.A nonpolar column, Restek Rtx-5MS, with an optimum length of 30 m, a moderate ID of 0.25 mm, and a thin film thickness of 0.25 micron, was used for the study.Helium was used as the carrier gas due to its inertness, safety, and its ability to provide good separations.
A gradient temperature programme was finalised for faster elution of components from the column.A constant pressure mode was used for reduced consumption of carrier gas. 18The initial column oven temperature was 100 degrees lower than the injection surface temperature, as this difference encouraged the analyte concentration to reach the column head at the earliest. 19Direct injection was used for injecting the sample, as the heat at the injection interface vaporised the sample mixture before it entered the column.
To minimise the quantity of plant extract entering the column, a split ratio of 90 was used, resulting in narrow and sharp peaks.Ion source temperatures of 220°C were vital for EI ionisation.

GC-MS assessment findings
Six components were found to be common in the GC-MS data of both curcuma species.The area percentages of these phytoconstituents were equated graphically for better understanding in Figure 3. From the graphical representation, it is evident that the area concentration of 2CEHT is present in higher amounts as compared to other phytoconstituents in both species.This phytoconstituent is reported to be present in the essential oil of a plant species that demonstrates anticancer activity. 20e data from GC-MS examination of the rhizome chloroform extract of Curcuma caesia Roxb.prepared by UAE from this investigation were judged with the GC-MS statistics of the chloroform extract generated by Soxhlet extraction by Atom et al., 12 In contrast to the twenty-six components discovered in the GC-MS study of the of Curcuma caesia Roxb.rhizome chloroform extract prepared by the UAE, twenty components were detected in the chloroform extract of Curcuma caesia Roxb.rhizomes prepared by Soxhlet extraction.Phenol, 2,4-bis(1,1-dimethylethyl)-, a phenolic component, was found common in both studies.
The ability of plants or herbs to treat disease depends on the phytochemical makeup of those substances, which displays a variety of intriguing and unique biological functions.It has been found that the various phytochemicals identified in this study have a wide range of biologic functions. 214][35][36][37][38][39][40] The detection of bioactive phytoconstituents in the GC-MS examination of rhizome chloroform extracts of Curcuma caesia Roxb.and Curcuma angustifolia Roxb.extracted by UAE defines a straightforward approach for the extraction of phytoconstituents from different plant parts with minimal processing time.

Fig. 3 .
Fig. 3. Graphical comparison of area percentages of common phytoconstituents found in Curcuma caesia Roxb.and Curcuma angustifolia Roxb.chloroform extracts Th e s t u d y a s s e s s e d c h l o r o fo r m ultrasonic-assisted extracts of Curcuma caesia Roxb.and Curcuma angustifolia Roxb.rhizomes by GC-MS analysis and phytochemical screening.Results showed significant phytoconstituent extraction, highlighting the need for recurring usage of UAE in plant standardisation studies.The extracts exhibited potential therapeutic use that can be converted to prospective novel medications by performing further studies for the isolation and separation of the bioactive c o m p o n e n t s fo u n d in t h e s t u dy.O ve ra ll , Curcuma caesia Roxb.and Curcuma angustifolia Roxb.could be crucial sources of medicine in contemporary treatment.

Table 4
lists the bioactivities reported for the phytoconstituents detected in the GC-MS evaluation of Curcuma caesia Roxb.andCurcuma angustifolia Roxb.rhizomes extracted in chloroform by UAE.As certain phytoconstituents detected inCurcuma caesia Roxb.andCurcuma angustifoliaRoxb.rhizomes demonstrate biological activity, the chloroform extracts of both these species can be standardised further and used as herbal medications for repor ted ailments.These phytoconstituents can be used as marker compounds and quality control tools in the standardisation of plant extracts.The data presented in this study gives scientists the chance to investigate phytoconstituents that have not yet been linked to any biological activity.