Bio-chemical profiling of black turmeric (Curcuma caesia Roxb.) genotypes

Introduction

Curcuma caesia Roxb.is an erect, perennial, medicinal rhizomatous herb commonly named as 'black turmeric' due to its bluish -black rhizomes. It is an important, lesser known, nonconventional medicinal plant of Zingiberaceae family. Medicinal potentiality of Curcuma caesia Roxb. Is varied, entire plant part has been traditionally used for centuries as a folklore remedy. It is widely used in Ayurvedic, Unani and Siddha herbal system of medicines. Rhizomes have a high economical importance owing to its putative medicinal properties. Blood purifying activity (Arulmozhi et al., 2006) [2] , bronchodilating activity (Paliwal et al., 2011) [23] , anti-oxidant activity (Mangla et al., 2010) [18] , anxiolytic and CNS depressant activity, anticonvulsant (Karmakar et al., 2011) [15] , anthelmintic activity (Gill et al., 2011) [12] , anti-bacterial activity (Rajamma et al., 2012) [25] , anti-ulcer activity (Das et al., 2012) [8] were also noticed. The medicinal and nutritive value of a plant is based on the plethora of chemical substances produced in them. A wide range of variation for essential oil content and its chemical components were reported in Curcuma caesia Roxb, which may be either because of false taxonomic identification of the specimen or due to environmental effects, maturity variations of the various varieties of rhizome or different analytical techniques used for the extraction of oil (Behar et al., 2013) [3] .

The Clarus 680 GC was used in the analysis employed a fused silica column, packed with Elite-5MS (5% biphenyl 95% dimethyl poly siloxane, 30 m × 0.25 mm ID × 250 μm df) and the components were separated using Helium as carrier gas at a constant flow of 1 ml/min. The injector temperature was set at 260 °C during the chromatographic run. The 1μL of extract sample injected into the instrument the oven temperature was as follows: 60 °C (2 min); followed by 300 °C at the rate of 10 °C min -1 and 300 °C, where it was held for 6 min. The mass detector conditions were: transfer line temperature 230 °C; ion source temperature 230 °C; and ionization mode electron impact at 70 eV, a scan time 0.2 sec and scan interval of 0.1 sec. the fragments from 40 to 600 Da. The spectrums of the components were compared with the database of spectrum of known components stored in the GC-MS NIST (2008) library.

Results and discussion

Bioactive compounds present in the plant parts are responsible for varied religious, cosmetic and medicinal values. Curcuma ceasia Roxb. is well known for its social, religious, aesthetic and medicinal purposes. Gas chromatography ("GC") and mass spectrometry ("MS") make a reliable, effective combination for chemical analysis. In the present study an attempt has been made to evaluate the chemical composition of the leaf and rhizome extract of thirty-three black turmeric genotypes through GC-MS analysis. The results are discussed as follows; Leaf and rhizome extracts of genotype GKM-1 had a complex mixture of mono and sesqueterpenes. Leaf extract had recorded a total of eight components, corresponding to 94.552% of the total volume (Table 1). Leaf extract is composed of 2-isopropylidene-5-methyl hex-4-enal (51.562%) being the major component. (+)-2-bornanone (15.946%), camphor (8.952%), iso-bornyl acetate (4.935), 1H,4H-3a,8a-methano azulen-1-one, hexa hydro-(4.729%), (-)-β-caryophyllene oxide (3.290%), (-)-parthenolide (2.940%) and α-santalol (2.198%) were the other volatile compounds present. Tropolone (36.108%) was found to be highest among ten compounds present in the rhizome extract of genotype GKM-1, followed by eucalyptol (12.814%), γ-selinene (7.093%), β-elemene,(-)-(6.800%), (+)-2-bornanone (5.969%), androsterone (6.669%), 1H-indene-1-ethylidene octa hydro-7-a-methyl-cis (6.495%), cis-caryophyllene (3.624%), 3,7-cyclodecadien-1-one,10-(1-methyl ethenyl)-, (E,E)-(3.386%) and bornyl acetate (2.800%). Compounds comprise 91.758% of the total volume of the extract. Results are in congruence with the findings of Mukunthan et al. (2014); Leela and Adheeb (2024) [17,20] . Genotype GKM-2 leaf extract had six compounds, comprises 94.112% of the total volume of the extract, is mainly composed of cyclohexane,1-methyl-2,4-bis(1-methylethenyl)-(63.428%), (+)-2-bornanone (12.133%), 1,3,3-trimethyl-2-hydroxy methyl-3,3-dimethyl-4-(3methylbut-2-enyl)-cyclohexene(9.884%),(-)-βcaryophyllene oxide (4.121%), α-selinene (2.392%) and αsantalol (2.210%). Rhizome extract of GKM-2 had eight phyto-compounds of which major components were tropolone (36.919%), βterpineol (14.171%), β-elemene, (-)-(10.597%), longifolene-(V4) (6.184%), (+)-2-bornanone (5.562%), β-elemenone (4.201%), 4,5,6,7-tetra hydroindazole-3-spirocyclohexane (3.532%), and 17α-methyltestosterone (1.520%). Results are in line with the findings of Pandey and Chowdhari (2003); Behuria & Srivastava (2004) and Mukunthan et al. (2014) [5,20,24] . Total five volatile compounds (amounting 92.522%) were identified in the leaf extract of genotype GKB-3, representing Cyclopentan-1-al, 4-isopropylidene-2-methyl-(55.488%) as a major phyto-compound, followed by isoaromadendrene epoxide (17.902%), (+)-2-bornanone (5.972%), (-)-β-caryophyllene oxide (5.370% and 4.568%) and (-)-parthenolide (3.222%) (Table 3). Seven peaks of GC-MS analysis comprising 82.646% of the rhizome extract of GKB-3 (Table 3). Bicyclo (2.2. [19,22] , Borah et al.

(2019) [6] in the rhizomes. [20,22,24] . GC-MS peaks revealed six compounds in the leaf extract of genotype GKJ-5, which amounting to 90.14% of the total volume of the extract (Table 5). Cyclohexane, 1-methyl-2,4bis(1-methylethenyl)-(53.237%) was higher compounds. (+)-2-Bornanone (14.128%), kauren-18-ol, acetate,(4beta)-(12.136%), (-)-β-caryophyllene oxide (5.029%), α -cadinol (3.209%) and (-)-parthenolide (2.401%) were other phytocompounds present. Methanolic rhizome extract of GKJ-5 had seven phytocompounds, amounting 93.487% of the extract (Table 5). 4-Ethynyl-4-hydroxy-3,5,5-trimethyl-2-cyclohexen-1one (52.704%) was major, followed by 3a,7a-methano-1hinden-1-one, 4,5,6,7-tetrahydro (17.576%), 7-(1,3-dimethyl buta-1,3-dienyl)- [6,24,26] for γelemene. GMV-6 leaf extract is composed of six bioactive compounds, cis-dihydrocarvone (42.375%) and (+)-2bornanone (11.077%) were the major compounds. (1S, 4R)p-mentha-2,8-diene, 1-hydroperoxide (5.188%), (-)-βcaryophyllene oxide (4.112%), ledol (3.603%) and iso bornyl acetate (3.078%) were the other components (Table 6). Rhizome extract had eight peaks, out of which 4,5,6,6atetrahydro-1H-pentalen-2-one (26.345%) was the major compound, followed by [ [20,21,22] observed similar compounds in the rhizome and leaf extract of Curcuma caesia Roxb. Abundance of monoterpenoid alcohol cis-dihydrocarvone in the leaf extract, reflects the strong perfumery activity of the leaves. Traces of isobornyl acetate will have a synergetic correlation with major compound and enhances the perfumery efficiency (Borah et al., 2019) [6] . [20] and Borah et al.

(2019) [6] confirmed β-elemene and camphor in the leaves. [6,22,28] for camphor in C. caesia leaf oil. 8). 1,E-8,Z-10-tridecatriene (48.917%) was the highest observed compound in the leaf extract of genotype GGR-10, followed by bicyclo [2.2.1] heptan-2-one, 1,7,7-trimethyl-, (1S)-(11.896%), 5,8-decadien-2-one, 5,9-dimethyl-, (E)-(5.744%), bornyl acetate (4.329%), (-)-β-caryophyllene oxide (3.268%) and 2-methyl-3-(3-methyl-but-2-enyl)-2-(4methyl-pent-3-enyl)-oxetane (3.065%). Six peaks comprise 77.219 % of the total volume of the leaf extract (Table 9). [10,13,19] .

1,E-8,Z-10-dodecatriene and ent-16-kauren-19-ol acetate were the major components of the leaf extract of GAB-11, comprises 40.853% and 34.034% of the total volume of the extract respectively (Table 10). 2019) [9] . Total eight phyto-compounds found in the leaf extract of genotype GAB-12, amounting to 93.673% of the total volume (Table 11). About 50.676% of leaf extract was constituted by 1-silacyclohexa diene -2,5-. Followed by 1 [13,17] . GC-MS analysis of genotype GAK-14 leaf extract had ten compounds comprise 92.558% of the total volume of the extract (Table 13).1,E-8,Z-10-dodecatriene (41.630%) was the major compound, followed by 1H,4H-3a,8amethanoazulen-1-one, hexahydro-(10.287%), (1S,4R)-pmentha-2,8-diene, 1-hydroperoxide (9.384%),bicyclo [2.2.1] heptan-2-one,1,7,7-trimethyl-,(1S)-(7.994%), santalol, ecis,epi-β-(6.185%), (-)-β-caryophyllene oxide (4.800%), 3methyl-4-(phenylthio)-2-prop-2-enyl-2,5-dihydrothiophene 1,1-dioxide (4.782%), bornyl acetate (2.451%) and 5,9,13pentadecatrien-2-one, 6,10,14-trimethyl-, (E,E)-(1.431%) were other volatile compounds present. (5.851%), iso bornyl acetate (5.580%) and 5-pentadecen-7yne, (Z)-(0.793%) were other components. Seven compounds comprise 95.571% of the total volume of the leaf extract (Table 14). Norethynodrel (24.004%) and 5,6-decadien-3-yne,5,7diethyl(23.298%) were found to be the highest observed compounds in the rhizome extract of genotype GMW-15 (Table 14). Ten compounds constitute 87.528% of the extract. Other volatile compounds were cyperene (9.896%), eucalyptol (9.743%), elemene (9.549%), bornyl acetate (3.076%), azulene, 1,2,3,4,5,6,7,8-octahydro-1,4-dimethyl-7-(1-methylethenyl)-, (1S,4S,7R)-(2.890%), germacrol (2.323%), camphene (1.427%) and γ-elemene (1.322%). 2019) [9,20,24,28] . Genotype GOK-18 leaf had heighest 1,E-8,Z-10dodecatriene (46.040%), followed by [20,22,24] The GC-MS analysis of leaf extract had seven peaks (Table 18), GOK-19 had cyclohexane, 1-methyl-2,4-bis (1methylethenyl)-(45.158%), 1-cycloheptene, 1,4-dimethyl-3-(2-methyl-1-propene-1-yl)-4-vinyl (20.116%), 2undecanone,6,10-dimethyl (6.708%), 5,9,13-Pentadecatrien-2-one, 6,10,14-trimethyl-, (E,E)-(6.336%), aromandendrene (3.912%), (-)-β-caryophyllene oxide (3.656%) and bornyl acetate (3.424%). The results denote abundance of sesquiterpenes and cyclo alkene (20.116%) in the leaves of genotype GOK-19. A Sesqueterpene tropolone (49.703%) was found to be the major bioactive compound of genotype GOK-19 rhizome extract (Table 18), followed by patchulane (10.661%), bicyclo [2.2.1] heptan-2-one,1,7,7-trimethyl-, (1S)-(6.095%), bornyl acetate (4.886%), retinal (4.757%), anobin (3.338%), selina-4(15),7(11)-diene (3.138%) and γelemene (2.454%). 21). Other components were 3,7-cyclodecadien-1one, 10-(1-methylethenyl)-, (E,E)-(15.490%), elemene (7.205%), α-caryophyllene (4.541%), curzerene(3.141%) and γ-elemene (3.111%) (Jyotirmayee Lenka et al., (2025) [13] . 4-Ethynyl-4-hydroxy-3,5,5-trimethyl-2-cyclohexen-1-one (22.894%), 3-keto-isosteviol (12.179%), methandrostenolone (11.813%), 7-(1,3-dimethylbuta-1,3dienyl)-1,6,6-trimethyl-3,8-dioxatricyclo[5.1.0.0(2,4)]octane (10.686%), verrucarol (6.523%), 5,6-decadien-3-yne,5,7diethyl-(5.828%),γ-elemene (0.956%) and cyclohexane, 1ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, (1α,2β,4β)-(0.878%) were the phyto compounds present in the rhizome extract of GMS-24. (Table 22). GC-MS analysis revealed five and four bioactive symmetrical peaks for leaf and rhizome extract of GNK-25 respectively (Table 23). 1,E-8Z-10-tridecatriene (47.802%) and 4,5,6,6a-tetrahydro-1H-pentalen-2-one (42.648%) were found to be highest observed compounds in leaf and rhizome respectively. The highest percentage of 2,6,10,14-hexadecatetraen-1-ol, 3,7,11,15-tetramethyl-, acetate, (E,E,E)-(44.521%) was identified in the leaf extract GNU-26 (Table 24). Bicyclo [

410%), cis-Z-alpha-bisabolene epoxide (6.157%), iso-bornyl acetate (5.624%), santalol cis alpha (4.715%) and +-aromadendrene (1.041%) were other compounds present. Norbornane, 2-methyl-3-methylene -2-(4-methyl-3pentenyl)-(40.031%) was found to be the highest phytocompound in the GNU-26 rhizome extract, followed by cyclohexene,1,5,5-trimethyl-6-(2-propenylidene)-(15.929%), benzofuran, 6-ethenyl-4,5,6,7-tetrahydro-3,6dimethyl-5-isopropenyl-, trans-(10.265%), eucalyptol (9.256%), γ-elemene (5.929%), isobornyl acetate (4.409%) and verrucarol (4.304%). Genotype GNF-27 leaf characterized by seven peaks (Table 25). 4-Isopropenyl-4,7-dimethyl-1-oxaspiro [2.5] octane (41.034%), 4,5,6,6a-tetrahydro-1H-pentalen-2-one (15.422%), eucalyptol (11.653%), 18-oxokauran-17-yl acetate (3.193%), 4-methoxy-6-methyl-6,7-dihydro(4H) furo[3,2-c] pyran (2.780%) and iso-bornyl acetate (2.743%) were the other phyto-compounds present. Rhizome extract had five symmetric peaks,4-ethynyl-4-hydroxy-3,5,5trimethyl-2-cyclohexen-1-one (26.639%) was found to be the major compound, followed by 1H indene, 1ethylideneoctahydro-7a-methyl-cis (15.306%), androstan -17-one,3ethyl-3-hydroxy-,(5alpha) (12.222%),cyclohexane,1ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, (1α,2β,4β)-(1.865%) and iso bornyl acetate (1.762%) (Table 25). 27). Followed by bicyclo [2.2.1] heptan-2-one, 1,7,7-trimethyl-, (1S)-(23.653%), 4,5,6,6a-tetrahydro-1Hpentalen-2-one (12.427%), (-)-β-caryophyllene oxide (3.520%), iso bornyl acetate (2.867%) and 6-10,14-Trimethyl pentadecan-2-one (2.762%). Similarly, rhizome had benzene, ethoxy-(42.703%) followed by [2,2-dimethyl-4-(3-methylbut-2-enyl)-6-methylidenecyclohexyl] methanol (15.342%), cyclohexene, 1,5,5-trimethyl-6-(2propenylidene)-(12.725%), γ-elemene (5.368%) and bornyl acetate (2.886%). These phytochemicals comprise of 94.292% and 82.968% of the total volume of the leaf and rhizome extracts respectively (Table 27). Similarly, rhizome had 4,5,6,6a-tetrahydro-1H-pentalen-2one (40.448%) being the major compound identified, followed by curzerene (12.526%),patchulane (10.532%), γelemene (6.121%), alpha santalol (6.057%) and verrucarol (4.304%) are other volatile compounds identified in the genotype GNP-36.

Overall GC-MS analysis of leaf and rhizome extract of thrity three black turmeric genotypes revealed wide variation in the chemotypes and chemical composition of individual genotypes. The results are in accordance with the findings of Mukunthan et al. (2014) [20] , Singh et al. (2020) and Borah et al. (2020) [7,27] . Besides, work carried out by Behura and Srivastava (2004) and Borah et al. (2019) [6] provides a strong base to further explore and repurpose leaf and rhizome extracts based on geographical collection (Leela and Adheeb (2024)) [17] . The results on identification of new compounds from leaf and rhizome extract of Curcuma caesia Roxb. Genotypes are in congruence with the reports Kumar et al. (2020) [16] and Borah et al.(2020) [7] .

Conclusion

The study showed less similarity in the composition of both leaf and rhizome extract of different genotypes explained intraspecific diversity with respect to chemical components. Bioactive constituents of black turmeric are dependent on its geographical distribution also gave an insight of diverse pharmacological potentiality of each genotype. GC-MS analysis of leaf and rhizome extract revealed that a wide variation was observed in the chemotypes and chemical composition of the genotypes. GAB-13, GAK-14 and GMI-22 genotypes leaf extract had heighest number of phyto-compounds (10 No's), followed

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