SUPPLEMENTARY MATERIAL
Curcuma ecalcarata- New natural source of pinocembrin and piperitenone
K. B. Rameshkumara*, D. B. Alan Sheejab, Mangalam S. Nairb and V. Georgec
a Phytochemistry and Phytopharmacology Division
Jawaharlal Nehru Tropical Botanic Garden and Research Institute
Palode, Thiruvananthapuram- 695 562, Kerala, India
bOrganic Chemistry Section
CSIR-National Institute for Interdisciplinary Science and Technology
Thiruvananthapuram- 695 019, Kerala, India
*Corresponding author. Email:
Phytochemical analysis of the rhizome extract of C. ecalcarata, a hitherto uninvestigated south Western Ghats endemic species, resulted in the isolation and identification of the diaryl heptanoid trans, trans-1,7-diphenyl-5-hydroxy-4,6-heptadiene-3-one (1), the steroid -sitosterol (2), the flavanone pinocembrin (4) and the monoterpenoids piperitenone (3) and 8-hydroxy piperitone (5). HPTLC estimation of pinocembrin in the rhizome revealed the plant as a rich source of pinocembrin (0.37% dry wt.). The rhizome essential oil was isolated by hydrodistillation and analysed by GC-FID, GC-MS and 13C NMR. Among the 30 constituents identified in the oil, monoterpenoids predominated (94.2%) followed by sesquiterpenoids (5.8%). The major compound consisting 65.2% of the oil was isolated and identified as piperitenone (3). The study highlights the plant as a rich source of the flavanone pinocembrin and the volatile aroma compound piperitenone.
Keywords: Curcuma ecalcarata;pinocembrin; HPTLC; essential oil; piperitenone; GC- MS
Experimental
General experimental procedures
Specific rotations were recorded using Rudolf Autopol IV polarimeter. UV spectra were recorded using Shimadzu UV-1800 spectrophotometer. The FT-IR spectra were taken on ABB spectrometer using KBr pellet method. NMR spectra were taken on JEOL GSX (300 MHz) FT-NMR using TMS as internal standard. Mass spectra were recorded using JEOL JMS-600 spectrometer. Silica gel 60-120 mesh and Silica gel G (Acme Synthetic Chemicals, Mumbai) were used for column chromatography and PTLC respectively.
Plant material
Fresh rhizomes were collected from Kulathuppuzha, Thiruvananthapuram district, Kerala state, India during August, 2008 and voucher specimen (No. 50922) was deposited in the Herbarium of JNTBGRI (TBGT).
Extraction and Isolation of chemical constituents
Shade dried rhizome powder (2.2 kg) were extracted with methanol (3 x 5L) at room temperature and the pooled methanol extract was concentrated using rotavapor and 5g of the concentrated extract was subjected to column chromatography (silica gel 60-120 mesh, 250g; hexane:ethylacetate gradient elution). The fractions eluted with hexane: EtOAc (9.8: 0.2) gave compound 1 and 2 on sub-column chromatography. Compound 3and 4 werepurified from fractions eluted with hexane: ethyl acetate 9.5: 0.5 and 9.0:1.0 respectively, while fractions eluted with hexane: ethyl acetate (8.0:2.0) gavecompound 5.
Estimation of pinocembrin by HPTLC
5g of the dried, powdered rhizomes was extracted with methanol using Soxhlet apparatus for 6h. and made up to 200 mL using methanol. Camag (Switzerland) HPTLC system was used for the HPTLC analysis. The extracts were filtered through nylon 0.45 μm membrane filters (PALL Gelman Laboratory, India) and 1.5 μL of the extract was applied to HPTLC plate (20 x 20 cm dimension and 0.2 mm layer thickness, 60F254, E. Merck, Germany). Standard stock solution of pinocembrin (Sigma-Aldrich, USA) was made by dissolving pinocembrin in methanol (1mg/ml) and different concentrations were tested to find the linear response of the standard. The spots were applied with automatic Linomat V sample applicator, fitted with a Camag microsyringe under N2 flow. The plates were developed using the mobile phase, chloroform: methanol (9:1, v/v), after saturating the twin trough chamber with the same solvent system for 15 min. After chromatographic development, the peak areas of the bands were measured at 289 nm (max for authentic pinocembrin) densitometrically using Camag TLC scanner 3 and the data were analysed using WinCATS Software version 4.03. The experiments were done in triplicate and the average area of pinocembrin was taken. The calibration curves were prepared by plotting the amount of pinocembrin spotted (ng/band) as independent variable (X) and the respective peak area as dependent variable (Y). Specificity of the method was studied by comparing Rf values of standard pinocembrin on repeated applications (n=4), and also comparing the Rf value with that of pinocembrin in the extract. Instrumental precision was studied by scanning four bands of pinocembrin (200 ng/spot) by the proposed method. The coefficient of variation of peak areas of spots was used to evaluate the instrumental precision.
Essential oil isolation and analysis
400g of fresh rhizomes were hydrodistilled using a Clevenger type apparatus for 3 h and the oil obtained was stored in refrigerator at 4oC till further analysis. The GC-FID analysis was carried out on a Varian CP-3800 gas chromatograph equipped with a flame ionization detector (FID) and a CP Sil 8CB fused silica capillary column (30m × 0.32mm, film thickness- 0.25m). Nitrogen was used as carrier gas at flow rate 1mL/min. The oven temperature 60 to 246oC at 3oC/min., injector temperature 220oC and detector temperature 250oC. The GC/MS analysis was done on a Hewlett Packard 6890 gas chromatograph fitted with a cross-linked 5% phenyl methyl siloxane HP-5 MS capillary column (30m × 0.32mm, film thickness- 0.25m) coupled with a 5973 series selective mass detector. 1.0L each of the essential oils were injected. Helium was used as the carrier gas at 1.4mL/min. constant flow mode, with injector temperature 220oC and oven temperature 60oC to 246oC (3oC/min). Mass spectra at electron impact (EI+) mode were taken at 70eV. 13C NMR spectra of the oils were recorded at 125 MHz in CDCl3 (1:4, v/v), using tetramethylsilane as an internal standard on a JEOL GSX (300 MHz) FT-NMR spectrometer. The major compound of the oil was isolated by preparative thin layer chromatography (PTLC) using chloroform: methanol (9:1) solvent system. The major compound appeared at Rf value 0.50 was eluted using chloroform and further purified by column chromatography using hexane: ethyl acetate (9.5:0.5) isocratic elution.
Identification of the oil constituents
The oil constituents were identified by MS library search (WILEY 275), comparison of the relative retention indices calculated with respect to homologous of n-alkanes (C6-C30, Aldrich Chem. Co. Inc.) (Van & Kratz 1963) and comparison of mass spectrum reported in the literature (Adams2007). The percentage composition was determined by using single area percentage method, without considering corrections for response factors. The Identification by 13C NMR was based on comparison of the 13C NMR signals of the total oil to the 13C NMR signals for pure compound available in the literature (Formacek Kubeczka 1982).
Trans, trans-1,7-diphenyl-5-hydroxy-4, 6-heptadiene-3-one(1):Pale yellow crystals from acetone/hexane, mp (uncorrected) 70-72oC. []D: +10.9 (c 0.1, MeOH). UV max MeOH (nm): 342, 229 and 204 nm. IR (KBr, cm-1): 3406, 1646, 1593, 1420, 1361, 1321, 1122, 970, 698. 1H NMR (300 MHz, CDCl3): 7.58 (d, J 15.9 Hz, H-1), 6.45 (d, J 15.9Hz, H-2), 5.63 (s, H-4), 3.05 (2H, t, H-6), 2.73 (2H, t, H-7), 7.17–7.61 (10H, m, H-1’-H-6’’). 13C NMR (75 MHz, CDCl3): 31.6 (CH2), 42.4 (CH2), 101.2 (CH), 123.1 (CH), 126.6 (CH), 128.3 (CH), 128.7 (CH), 128.9 (CH), 129.3 (CH), 130.3 (CH), 135.4 (C), 140.2 (CH), 141.2 (C), 177.1 (C). EIMS (70 eV) m/z (%): 278 (M+, 98), 260 (11), 256 (7), 187 (54), 173 (100), 155 (19), 145 (54), 131 (98), 115 (19), 103 (40). HRMS, EI [M+]: 278.1348, C19H18O2 required 278.1307.
-Sitosterol(2):Colourless crystalls from hexane / ethyl acetate, mp (uncorrected) 138-140oC. TLC: Chloroform (100%) Rf: 0.46. IR (KBr, cm-1): 3346, 2953, 2935, 2904, 2866, 1465, 1379, 1058, 958, 839 and 800 [fig.3.10]. 1H NMR (300 MHz, CDCl3): 3.51 (m, H-3), 5.35 (m, H-6),0.68 (s, H-18), 1.01 (d, H-19), 0.92, d (J = 6.6 Hz, H-21). EIMS (70 eV) m/z (rel. int. %): 414 (M+, 100), 396 (58), 381 (38), 329 (38), 303 (39), 271 (32), 273 (31), 255 (57), 231 (29), 213 (60), 199 (31), 173 (35), 159 (68), 145 (74), 133 (63), 119(64), 105 (82).
Piperitenone (3): IR (CH2Cl2, max, cm-1): 2924, 1659, 1630, 1613, 1437, 1378, 1299, 1215, 1179, 1067, 1017. 1H NMR (300 MHz, CDCl3): 5.81 (1H, s), 2.58 (2H, t, J= 6 Hz), 2.22 (2H, t, J = 6 Hz), 2.02 (3H, s), 1.86 (3H, s), 1.78 (3H, s). 13C NMR (125 MHz, CDCl3): 191.7, 159.7, 142.4, 128.9, 128.7, 31.8, 27.9, 23.7, 22.8, 22.4.
Pinocembrin(4): Yellow crystals from acetone/ hexane, mp (uncorrected) 204oC. []D: – 41.9 (c 0.1, MeOH). UV max MeOH (nm): band-I: 325 nm, band-II: 289 nm, IR (KBr cm-1): 3094, 1632, 1585, 1489, 1466, 1435, 1358, 1304, 1169, 1087, 825, 713 cm-1.1H NMR (300 MHz, CDCl3): 5.47 (1H, dd, J 12.6, 3.0 Hz, H-2), 3.09 (1H, dd, J 17.1, 12.6, H-3), 2.76 (1H, dd, J 17.1, 3.0, H-3), 5.92 (1H, d, J, 2.1 Hz, H-6), 5.94 (1H, d, J 2.1 Hz, H-8), 7.3 to 7.5 (5H, m, H-2’-H-6’). 13C NMR (75 MHz, CDCl3): 40.9 (CH2), 76.6 (CH), 93.6 (CH), 94.6 (CH), 100.2 (C), 124.7 (CH x 2), 126.9 (CH x 3), 136.9 (C), 161.1(C), 162.0 (C), 165.2(C), 193.7(C=O). EI-MS (70 eV) m/z (rel. int. %): 256 (M+, 100), 179 (63), 152 (63), 124 (54).
8-Hydroxypiperitone (5): IR (CH2Cl2, max, cm-1): 3395, 2925, 1720, 1642, 1454, 1380, 1209, 1155, 1017. 1H NMR (300 MHz, CDCl3): 5.80 (1H, s), 5.18 (1H, s), 2.27-2.22 (3H, m), 2.00-1.98 (1H, m), 1.88 (3H, s), 1.16 (3H, s), 1.13 (3H, s). 13C NMR (125 MHz, CDCl3): 203.6, 163.7, 127.3, 72.6, 54.7, 31.4, 28.3, 25.4, 24.5, 24.1.
Table S1. Essential oil composition of Curcuma ecalcarata rhizome
RRI / Compound / Percentage966 / Camphene / 1.2
984 / -Pinene / tr
995 / Myrcene / 0.1
1005 / -Phellandrene / 0.1
1009 / Pseudolimonene / 0.2
1015 / -Terpinene / 0.2
1022 / p-Cymene / 0.5
1026 / Limonene / 0.4
1030 / 1, 8-Cineole / 13.1
1087 / Isoterpinolene / 3.0
1103 / Linalool / 0.2
1131 / Camphor / 5.7
1134 / Camphene hydrate / 1.1
1140 / Isoborneol / 0.2
1147 / Borneol / 0.1
1157 / Terpinen-4-ol / 0.3
1165 / p-Cymene-8-ol / 0.8
1190 / -Terpineol / 1.3
1223 / Piperitone / 0.3
1323 / Piperitenone / 65.2
1348 / Piperitenone oxide / 0.2
1404 / Methyl eugenol / tr
1399 / trans-Caryophyllene / 1.0
1434 / -Humulene / 0.3
1496 / -Cadinene / 0.2
1510 / -Cadinene / 0.4
1578 / d-Nerolidol / 2.1
1590 / Caryophyllene oxide / 0.6
1657 / epi -Cadinol / 0.8
1671 / -Cadinol / 0.4
RRI: Relative retention index; tr: traces (<0.1%)
Figure S1. Chemical structures of compounds 1-5 from Curcuma ecalcarata rhizome
Figure S2. Gas chromatogramof Curcuma ecalcarata rhizome oil
Figure S3.13C NMR spectrum of Curcuma ecalcarata rhizome oil
Figure S4.13C NMR spectrum of piperitenone
Figure S5. HPTLC densitogram of Curcuma ecalcarata rhizome methanol extract (1.5L, track 1) and standard pinocembrin (50-300ng, tracks 2,3,4,5 and 6) at 289nm.
References
Adams RP. 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry. 4th edition. Allured Pub. Co.: Carol Stream (IL).
Formacek V,Kubeczka KH. 1982. Essential oils analysis by capillary gas chromatography and carbon-13 NMRspectroscopy. Wiley: Chichester.
Van DH, Kratz PD. 1963. A generalization of the retention index system including linear temperature programmed gas Liquid partition chromatography. J Chromatogr A. 11: 463-471.