Aliphatic and eudesmalolide esters extracted from the roots of Inula racemosa Hook

Abstract

Background:

Phytochemical investigation of hydroalcoholic extract of the root of Inula racemosa Hook.

Materials and Methods:

Open silica gel column chromatographic techniques with different solvent systems was used for isolation of aliphatic and eudesmalolide esters from hydroalcoholic extract of the root of Inula racemosa. The structure elucidation of the compounds was done on the basis of spectral data analysis, chemical reactions and comparision with literature data.

Results:

Phytochemical investigation of the hydroalcoholic extract of the root of Inula racemosa Hook. f. led to the isolation of (5z, 13z)-n-decanyl-n-docos-5, 13-dienoate, a new fatty acid ester, two new sesquiterpenic ester identified as 15-[(13z, 18′z, 20′z)-n-tricos-13, 18, 20-trienyl]-eudesmal-4 (11), 6, 12 (13)-trien-8,14-olide-15-oate and 15- [(16′z), (21′z)-n- tetracos-16′, 21′- dienyl]-eudesmal-4 (11) 6, 12 (13)-trien-8, 14-olide-15-oate, two new eudesmanolide ester i.e. 15-[(16z)-n-monadec-16′- enyl]-eudesmal-4 (11) 6, 12 (13)-trien-8,14-olide-15-oate and 15-[(16′z)-n-tetracos-16′- enyl]-endesmal-4 (11), 6, 12 (13)-trien-8,14-olide-15-oate along with the known compound n-Hexadecanyl n-docosanoate.

Conclusion:

Five new phytoconstituents were identified along with one known compound as aliphatic and eudesmalolide esters from the hydroalcoholic extract of the root of Inula racemosa, as mentioned above.

INTRODUCTION

Inula racemosa Hook. f. commonly known as ‘Pushkarmoola’ of family Asteraceae, with its diverse active components, has been utilized as a therapeutic, since time immemorial in both organized (Ayurveda, Unani) and unorganized (folk, tribal, native) form.[1] The roots of I. racemosa have been used as folk medicine in East Asia and Europe.[2] Native Americans used this plant for the treatment of tuberculosis.[3] The alantolactone obtained from roots of I. racemosa enhances insulin sensitivity, thus proves beneficial in fat reduction.[4] Roots of I. racemosa are also useful in treating pulmonary and cardiovascular disorders.[5] The chemical profiling of the plant roots have shown the presence of alantolactones and isoalontolactones, sitisterol, daucosterol, inunolide, aplotexene, phenylacetonitrite and isoinunal.[6]

The characteristic components of the genus Inula are sesquiterpenes, such as eudesmanes, germacranes, guaianes and bis-sesquiter-penes. Recently, much attention has been paid to Inula species due to their diverse biological activities.[7,8]

During further phytochemical investigation, a new fatty acid ester, two new sesquiterpenic esters and two new eudesmanolide esters were isolated along with known compounds like n-Hexadecanyl n-docosanoate. Herein, we reported the isolation and elucidation of the two new sesquiterpenic esters and two new eudesmanolide esters along with a new fatty acid ester.

MATERIALS AND METHODS

General procedure

Melting points were determined on Perfit melting point apparatus and are uncorrected. Fourier transform infrared spectroscopy (FTIR): Jasco FT/IR-5000; UV: Lambda Bio 20 Spectrophotometer, MeOH; ¹H-NMR (400 MHz): Advance DRY 400, Bruker spectrospin, CDCl₃; ¹³C NMR (75 MHz): Advance DRY 100, Bruker spectrospin, CDCl₃ with TMS as an internal standard; MS: FAB ionization on JEOL-JMS-DX 303; CC: silica gel (Qualigens), 60-120 mesh; TLC: Silica gel G (Qualigens). Spots were visualized by exposure to iodine vapours, UV radiation and by the use of spraying reagents.

Plant material

The root of I. racemosa was collected from the local market of Khari Baoli, Delhi one week before commencing the extraction procedure and identified by Dr. M. P. Sharma, taxonomist, Department of Botany, Faculty of Science, Jamia Hamdard (Hamdard University). A voucher specimen No. PRD/JH/09/18 is deposited in the herbarium of the Faculty of Pharmacy, Jamia Hamdard, New Delhi.

Extraction and isolation

The air-dried root (3 kg) of I. racemosa were coarsely powdered, defatted with petroleum ether and then exhaustively extracted in a Soxhlet apparatus with 90% hydro-methanolic mixture for 72h. The extract was concentrated under reduced pressure to obtain dark brown viscous mass, small portion of the extract was analyzed chemically to determine the presence of different chemical constituents. The viscous dark brown mass was adsorbed on silica gel (60-120 mesh) for column after being dissolved in little quantity of methanol for preparation of slurry. The slurry (255 g) was air dried and chromatographed over silica gel column packed in petroleum ether. The column was eluted successively using solvent mixtures e.g., starting with pure petroleum ether, successive mixtures of petroleum ether and chloroform (9:1, 3:1, 1:1 and 1:3), pure chloroform and finally mixture of chloroform and methanol (99:1, 49:1, 24:1, 95:5, 97:3, 9:1) were used to run the column. Various fractions were collected separately and matched by TLC to check homogeneity. Similar fractions (having same R_f values) were combined and crystallized. The isolated compounds were recrystalized to get pure compounds.

RESULT AND DISCUSSION

Result

IR-1 (n-Decanyl docosdienoate)

Elution of the column with petroleum ether gave colorless amorphous powder of IR-1, recrystallized from acetone-methanol (1:1), 78mg (0.035% yield), R_f : 0.62 (petroleum ether), m.p: 75°C.

UV λ_max (MeOH): 206nm (logε 4.3).

IR γ_max (KBr): 2927, 2856, 1718, 1640, 1460, 1378, 1172, 995, 911cm^-1.

¹H-NMR (CDCl₃): δ 5.79 (1H, m, w_1/2 = 14.3 Hz, H-5), 5.04 (1H, m, w_1/2 = 17.6 Hz, H-6), 4.96 (1H, m, w_1/2 = 13.6 Hz, H-13), 4.91 (1H, m, w_1/2 = 14.1 Hz, H-14), 4.52 (1H, ddd, J = 5.2, 3.6, 10.0Hz, H₂-1′a), 4.30 (1H, ddd, j = 5.2, 5.2, 10.0 Hz, H₂-1’xb), 2.31 (2H, t, J = 7.6 Hz, H₂-2), 2.05 (2H, m, H₂-4), 2.03 (2H, m, H₂-7), 1.76 (2H, m, H₂-12), 1.63 (2H, m, H₂-15), 1.56 (2H, m, CH₂), 1.51 (2H, m, CH₂), 1.36 (8H, brs, 4 × CH₂), 1.28 (6H, brs, 3 × CH₂), 1.24 (20H, brs, 10 × CH₂), 0.94 (3H, t, J = 7.2 Hz, Me-22), 0.87 (3H, t, J = 7.2 Hz, Me-22).

¹³C-NMR (CDCl₃): δ 171.26 (C-1), 138.75 (C-5), 137.81 (C-6), 119.23 (C-13), 114.47 (C-14), 62.41 (C-1’), 33.78 (CH₂), 33.67 (CH₂), 33.53 (CH₂), 31.93 (CH₂), 29.69 (7 × CH₂), 29.36 (CH₂), 29.02 (CH₂), 28.95 (CH₂), 28.89 (CH₂), 28.78 (CH₂), 28.72 (CH₂), 28.70 (CH₂), 28.68 (CH₂), 28.62 (CH₂), 28.55 (CH₂), 28.49 (CH₂), 24.55 (CH₂), 22.69 (CH₂), 14.35 (Me-10’), 10.17 (Me-22).

+ive ESI MS m/z relative intensities: 476 [M]⁺(C₃₂H₆₀O₂)(7.8), 363 (22.3), 337 (29.8), 335 (6.8), 319 (8.9), 249 (7.2) [Figure 1].

Figure 1.

Structure of IR-1 (n-decanyl docosdienoate)

IR-2 (n-Hexadecanyl behenate)

Elution of the column with petroleum ether chloroform (3:1) afforded colorless amorphous powder of IR-2, re-crystallized from chloroform-methanol (1:1), 62 mg (0.027% yield), R_f : 0.5 (petroleum ether-chloroform, 3:1), m.p: 78°C.

UV λ_max (MeOH): 206 nm (logε 3.1).

IR γ_max (KBr): 2997, 2855, 1734, 1457, 1379, 1255, 1173, 723 cm^-1.

¹H-NMR (CDCl₃): δ 4.24 (1H, m, H-1’a), 4.09 (1H, ddd, J = 6.4, 6.0, 10.0 Hz, H₂-1’b), 2.29 (2H, t, J = 7.2 Hz, H-2), 2.02 (2H, m, CH₂), 1.81 (2H, m, CH₂), 1.59 (6H, brs, 3 × CH₂), 1.44 (2H, m, CH₂), 1.27 (8H, brs, 4 × CH₂), 1.23 (46H, brs, 23 × CH₂), 0.86 (3H, t, J = 5.6 Hz, Me-22), 0.82 (3H, t, J = 6.5 Hz, Me-1C’).

¹³C-NMR (CDCl₃): δ 174.39 (C-1), 63.16 (C-1’), 51.42 (C-2), 41.86 (CH₂), 41.11 (CH₂), 36.83 (CH₂), 35.93 (CH₂), 34.12 (CH₂), 31.91 (CH₂), 29.75 (10 × CH₂), 29.67 (7 × CH₂), 29.58 (CH₂), 29.43 (CH₂), 29.34(CH₂), 29.24 (CH₂), 29.14 (CH₂), 29.07 (CH₂), 27.10 (CH₂), 24.71 (CH₂), 22.67 (CH₂), 14.10 (Me-22), 11.97 (Me-16’).

+ive ESI MS m/z relative intensities: 564 [M]⁺(C₃₈H₇₆O₂)(19.3), 323 (46.2), 339 (72.6), 241 (34.2), 225 (87.9)[Figure 2].

Figure 2.

Structure of IR-2 (n-hexadecanyl behenate)

IR-3 (15-tricostrienyl eudesmalolide)

Elution of the column with the petroleum ether-chloroform (1:1) yielded colorless crystals of IR-3 recrystalized from acetone and methanol (1:1), 70 mg (0.031% yield), R_f : 0.6 (Chloroform-methanol), mp: 104-105ΊC.

UV λ_max (MeOH): 207, 278, 375nm (logε 4.5, 1.6, 3.2).

IR γ_max (KBr): 2993, 2842, 1765, 1742, 1640, 1411, 1235, 1050, 922, 778cm^-1.

¹H-NMR (CDCl₃): δ 6.20 (1H, d, J = 3.5 Hz, H-6), 5.21 (1H, brs, H₂-11a), 5.03 (1H, brs, H₂-11b), 4.86 (1H, brs, H₂-13b), 4.80 (1H, brs, H₂-13b), 3.93 (1H, dd, J = 9.5, 9.0 Hz, H-8α), 2.49 (1H, d, J = 3.5 Hz, H-5β), 2.75 (1H, m, H₂-1a), 1.68 (1H, m, H₂-1b), 2.19 (2H, m, H₂-2), 2.02 (1H, dd, J = 6, 7.5 Hz, H₂-3α), 1.93 (1H, dd, J = 4, 10.1 Hz, H₂-3β), 2.23 (2H, d, J = 9.5, H₂-9), 5.36 (2H, m, w_1/2 = 5.5 Hz, H-13’, H-14’), 5.34 (2H, m, w_1/2 = 5.6 Hz, H-18’, H-19’), 5.25 (2H, m, w_1/2 = 5.5Hz, H-20’, H-21’), 4.16 (2H, t, J = 7.5 Hz, H₂-1), 2.78 (2H, m, H₂-15’), 2.47 (2H, m, H₂-17’), 2.16 (2H, m, H₂-12), 1.60 (4H, m, 2CH₂), 1.29 (4H, brs, 2CH₂), 1.24 (12H, brs, 6CH₂), 0.83 (3H, t, 7.5 Hz, Me-23’).

¹³C-NMR (CDCl₃): δ 32.6 (C-1), 31.93 (C-2), 41.01 (C-3), 139.77 (C-4), 52.04 (C-5), 120.20 (C-6), 149.23 (C-7), 85.26 (C-8), 36.28 (C-9), 37.63 (C-10), 109.62 (C-11), 151.24 (C-12), 112.63 (C-13), 170.29 (C-14), 168.46 (C-15), 62.10 (C-1’), 31.53 (C-2’), 30.94 (C-3’), 30.91 (C-4’), 29.70 (C-5’), 29.60 (C-6’), 29.49 (C-7’), 29.27 (C-8’), 29.16 (C-9’), 29.16 (C-10’), 27.20 (C-11’), 47.67 (C-12’), 114.21 (C-13’), 127.12 (C-14’), 45.33 (C-15’), 25.63 (C-16’), 33.77 (C-17’), 127.91 (C-18’), 131.96 (C-19’), 139.11 (C-20’), 130.02 (C-21’), 22.58 (C-22’), 14.09 (C-23’).

+ive ESI MS m/z relative intensities: 576 [M]⁺(C₃₈H₅₆O₄)(2.9), 333 (23.4), 304 (19.4), 252 (98.4), 243 (4.53), 184 (6.8), 149 (4.1) [Figure 3].

Figure 3.

Structure of IR-3 (15-tricosterienyl eudesmolide)

IR-4 (15-nonadecenyl eudesmalolide)

Elution of the column with petroleum ether-chloroform (1:1) gave pale yellow crystals of IR-4, recrystallized from acetone-methanol (1:1), 85mg (0.037% yield), R_f : 0.65 (Chloroform-methanol, 3:2), mp: 120-125°C.

UV λ_max (MeOH): 207, 375nm (logε 4.1, 2.2).

IR γ_max (KBr): 2928, 2841, 1765, 1735, 1638, 1455, 1383, 1258, 1051, 779 cm^-1.

¹H-NMR (CDCl₃): δ 6.21 (1H, d, J = 3.6 Hz, H-6), 5.26 (1H, brs, H₂-13a), 5.06 (1H, brs, H₂-13b), 4.86 (1H, brs, H₂-11a), 4.76 (1H, brs, H₂-11b), 3.95 (1H, t, J = 9.5 Hz, H-8β), 2.79 (1H, m, H₂-1a), 2.75 (1H, m, H₂-1b), 2.49 (1H, d, J = 3.6 Hz, H-5α), 2.26 (2H, d, J = 9.5 Hz, H₂-9), 2.23 (1H, m, H₂-2a), 2.16 (1H, m, H₂-2b), 2.05 (1H, dd, J = 6.5, 14.0 Hz, H₂-3a), 2.01 (1H, dd, J = 6.5, 5.5 Hz, H₂-3b), 5.35 (1H, m, w_1/2 = 5.5 Hz, H-16’), 5.32 (1H, m, w_1/2 = 5.5 Hz, H-17’), 4.14 (2H, t, J = 4.8 Hz, H₂-1’), 2.86 (2H, m, H₂-15’), 2.44 (2H, m, H₂-18’), 1.86 (2H, m, CH₂), 1.72 (2H, m, CH₂), 1.62 (2H, m, CH₂), 1.59 (2H, m, CH₂), 1.52 (2H, m, CH₂), 1.29 (6H, brs, 3 × CH₂), 1.24 (12H, brs, 6 × CH₂), 0.83 (3H, t, J = 6.5 Hz, Me-19’).

¹³C-NMR (CDCl₃): 32.60 (C-1), 31.92 (C-2), 39.82 (C-3), 139.75 (C-4), 52.04 (C-5), 120.19 (C-6), 148.26 (C-7), 85.25 (C-8), 36.26 (C-9), 36.23 (C-10), 109.62 (C-11), 151.93 (C-12), 112.63 (C-13), 171.23 (C-14), 167.85 (C-15), 62.17 (C-1’), 45.13 (C-2’), 30.31 (C-3’), 29.69 (C-4’), 29.34 (C-5’), 31.52 (C-6’), 30. 94 (C-7’), 30.31 (C-8’), 29.69 (C-9’), 29.34 (C-10’), 29.11 (C-11’), 28.82 (C-12’), 27.20 (C-13’), 25.53 (C-14’), 47.63 (C-15’), 130.23 (C-16’), 127.91 (C-17’), 22.57 (C-18’), 14.07 (C-19’).

+ive ESI MS m/z relative intensities: 524 [M]⁺(C₃₄H₅₂O₄)(4.8), 281 (9.1), 252 (39.8), 243 (3.6) [Figure 4].

Figure 4.

Structure of IR-4 (15-nonadecenyl eudesmalolide)

IR-5 (15-Tetracosdienyl eudesmalolide)

Elution of the column with petroleum ether-chloroform (1:3) afforded light brown crystals of IR-5, recrystallized from acetone-methanol (1:1), 90mg (0.04% yield), R_f : 0.5 (Petroleum ether-Chloroform, 1:1), mp: 98-100°C.

UV λ_max (MeOH): 207, 375 nm (logε 4.1, 1.9).

IR γ_max (KBr): 2927, 2855, 1766, 1723, 1640, 1456, 1380, 1258, 1147, 998, 896cm^-1.

¹H-NMR (CDCl₃): δ 6.21 (1H, d, J = 3.6 Hz, H-6), 5.26 (1H, brs, H₂-13a), 5.06 (1H, brs, H₂-13b), 4.88 (1H, brs, H₂-11a), 4.80 (1H, brs, H₂-11b), 3.95 (1H, t, J = 9.2 Hz, H-8β), 2.84 (2H, m, H₂-1), 2.20 (1H, m, H₂-2a), 2.15 (1H, m, H₂-2b), 2.04 (1H, dd, J = 6.0, 8.4 Hz, H₂-3β), 1.97 (1H, dd, J = 5.2, 8.0 Hz, H₂-3α), 2.50 (1H, d, J = 3.6 Hz, H-5α), 2.30 (2H, m, H₂-9), 3.65 (2H, t, J = 5.6 Hz, H₂-1’), 5.48 (1H, m, w_1/2 = 5.6 Hz, H-21), 5.36 (1H, m, w_1/2 = 5.8 Hz, H-16’), 5.31 (1H, m, w_1/2 = 5.5 Hz, H-17’), 5.20 (1H, m, w_1/2 = 5.6 Hz, H-22’), 2.46 (2H, m, H₂-15’), 2.26 (2H, m, H₂-18’), 1.85 (2H, m, H₂-20’), 1.66 (2H, m, H₂-23’), 1.62 (2H, m, H₂-2’), 1.35 (8H, brs, 4 × CH₂), 1.29 (18H, brs, 9 × CH₂), 0.86 (3H, t, J = 6.8 Hz, Me-24’).

¹³C-NMR (CDCl₃): δ 32.48 (C-1), 31.76 (C-2), 41.86 (C-3), 139.64 (C-4), 52.41 (C-5), 119.89 (C-6), 149.95 (C-7), 83.41 (C-8), 37.62 (C-9), 36.83 (C-10), 109.58 (C-11), 151.68 (C-12), 112.25 (C-13), 173.16 (C-14), 170.24 (C-15), 66.03 (C-1’), 31.62 (C-2’), 130.20 (C-16’), 130.04 (C-17’), 125.05 (C-21’), 128.28 (C-22’), 31.34 (CH₂), 47.11 (C-15’), 46.92 (C-18’), 44.78 (C-20’), 29.86 (4 × CH₂), 29.83 (CH₂), 29.76 (CH₂), 29.50 (CH₂), 28.61 (CH₂), 28.53 (CH₂), 27.10 (CH₂), 25.98 (CH₂), 25.52 (CH₂), 24.94 (CH₂), 22.86 (CH₂), 14.05 (Me-24).

+ive ESI MS m/z relative intensities: 592 [M]⁺(C₃₉H₆₀O₄)(2.1), 350 (41.8), 321 (7.1), 294 (5.2), 252 (62.3), 243 (7.6) [Figure 5].

Figure 5.

Structure of IR-5 (15-tetracosdienyl eudesmalolide)

IR-6 (15-tetracosenyl eudesmalolide)

Elution of the column with chloroform gave pale yellow crystals of IR-6, recrystallized from acetone-methanol (1:1), 100 mg (0.04% yield), R_f : 0.7 (Chloroform-methanol, 97:3), mp: 125°C.

UV λ_max (MeOH): 208, 375 nm (logε 4.7, 4.2).

IR γ_max (KBr): 2928, 2855, 1764, 1720, 1645, 1455, 1381, 1259, 1123, 898 cm^-1.

¹H-NMR (CDCl₃): δ 6.20 (1H, d, J = 3.5 Hz, H-6), 5.05 (1H, brs, H-13a), 4.89 (1H, brs, H₂-13b), 4.69 (1H, brs, H₂-11b), 3.95 (1H, t, J = 9.5 Hz, H-9α), 2.75 (1H, m, H-1a), 1.68 (1H, m, H-1b), 2.18 (1H, m, H₂-2a), 2.15 (1H, m, H-1b), 2.02 (1H, dd, J = 5.5, 7.7 Hz, H₂-3α), 1.95 (1H, dd, J = 5.5, 5 Hz, H₂-3β), 2.29 (2H, d, J = 9.5 Hz, H₂-9), 2.48 (1H, d, J = 3.5 Hz, H-5β), 5.33 (1H, m, w_1/2 = 6.5 Hz, H-16’), 5.17 (1H, m, w_1/2 = 7.2 Hz, H-17’), 4.12 (1H, t, J = 7.6 Hz, H-1’), 2.63 (2H, m, H₂-15’), 2.45 (2H, m, H₂-18’), 1.73 (2H, m, CH₂), 1.68 (2H, m, CH₂), 1.55 (2H, m, CH₂), 1.40 (2H, m, CH₂), 1.78 (5H, brs, 10 CH₂), 1.24 (18H, brs, 19 CH₂), 0.85 (3H, t, J = 7 Hz, Me-24’).

¹³C-NMR (CDCl₃): δ 32.60 (C-1), 31.92 (C-2),44.25 (C-3), 139.75 (C-4), 55.32 (C-5), 120.16 (C-6), 149.22 (C-7), 85.29 (C-8), 36.25 (C-9), 37.48 (C-10), 109.60 (C-11), 151.36 (C-12), 112.63 (C-13), 170.22 (C-14), 167.89 (C-15), 62.15 (C-1’), 45.13 (C-2’), 30.93 (C-3’), 30.30 (C-4’), 30.19 (C-5’), 29.69 (C-6’), 29.35 (C-7’), 29.09 (C-8’), 30.19 (C-9’), 29.69 (C-10’), 29.35 (C-11’), 30.93 (C-12’), 30.30 (C-13’), 30.19 (C-14’), 52.02 (C-15’), 130.19 (C-16’), 113.63 (C-17’), 47.62 (C-18’), 29.69 (C-19’),29.35 (C-20’), 29.09 (C-21’),27.20 (C-22’), 22.58 (C-23’), 14.12 (C-24’).

+ive ESI MS m/z relative intensities: 594 [M]⁺(C₃₉H₆₂O₄)(6.8), 351 (5.2), 252 (57.6), 242 (3.2) [Figure 6].

Figure 6.

Structure of IR-6 (15-tetracosenyl eudesmalolide)

DISCUSSION

Compound IR-1, named n-decanyl docosdienoate, was obtained as a colorless crystalline mass from petroleum ether eluent. Its IR spectrum showed distinct absorption bands for ester group (1718 cm^-1), unsaturation (1640cm^-1) and long aliphatic chain (728cm^-1). On the basis of mass and ¹³C-NMR spectra, the molecular ion peak of IR-1 was determined at m/z 476 consistent to the molecular formula of a fatty acid ester, C₃₂H₆₀O₂. The ion peaks arising at m/z 319 [M-C₁₀H₂₁] and 319 [m-OC₁₀H₂₁]⁺ indicated that n-docosdienoic acid was esterified with n-decanol. The ion peaks generated at m/z 363 [C₁₄-C₁₅ fission]⁺, 337 [C₁₂-C₁₃ fission]⁺ and 249 [C₄-C₅ fission, CH₃ (CH₂)₇CH = CH (CH₂)₆CH = CH]⁺ suggested the existence of the vinylic linkages at C-5 (6) and C-13 (14) carbons. The ¹H-NMR spectrum of exhibited four one-proton multiplets at δ 5.79 (w_1/2 = 14.3 Hz), 5.04 (1H, w_1/2 = 17.6 Hz, 4.96 (w_1/2 = 13.6 Hz) and 4.91 (w_1/2 = 14.1 Hz) assigned to trans-oriented vinylic H-5, H-6, H-13 and H-14 protons, respectively. Two one-proton triple doublets at δ 4.52 (J = 5.2, 3.6, 10.0 Hz) and 4.30 (J = 5.2, 5.2, 10.0 Hz) were due to oxygenated methylene H₂-1’ protons. A two-proton triplet at δ 2-31 (J = 7.6 Hz) was ascribed to methylene H₂-2 adjacent to the ester group. The other methylene protons appeared from δ 2.05 to 1.24. Two three-proton triplets at δ 0.94 (J = 7.2 Hz) and 0.87 (J = 6.0 Hz) were accounted to the C-10’ and C-22 primary methyl protons. The ¹³C-NMR spectrum of compound IR-1 displayed signals for ester carbon at δ 171.26 (C-1), vinylic carbons at δ 138.75 (C-5), 137.81 (C-6), 119.23 (C-13) and 114.47 (C-14), methylene carbons between δ 33.78-22.69 and methyl carbons at δ 14.35 (C-10’) and 10.17 (C-22). On the basis of these evidences, the structure of IR-1 has been elucidated as (5z, 13z)-n-decanyl- n- docos-5, 13- dienoate. This is a new fatty acid ester. Earlier (5E, 13E)- docosdienoic acid from Hybanthus enneaspermus whole plant was identified, Meadow foam oil and Eranthis seed oil.[9,10,11]

Compound IR-2, n-hexadecanyl behenate, was obtained as a colorless amorphous powder from petroleum ether chloroform (3:1) eluent. Its IR spectrum showed characteristic absorption bands for ester function (1734 cm^-1) and long aliphatic chain (723 cm^-1). Its mass spectrum displayed a molecular ion peak at m/z 564 corresponding to the molecular formula of a fatty acid ester, C₃₈H₇₆O₂. The ion peaks arising at m/z 323 [CH₃ (CH₂)₂CO]⁺, 241 (M-323)⁺, 339 [CH₃ (CH₂)₂₀COO]⁺, and 225 (M-339)⁺ indicated that behenic acid was esterified with n-hexadecanol. The ¹H-NMR spectrum of IR-2 displayed two one proton signals as a multiplet at δ 4.24 and as a triple doublet at δ 4.09 (J = 6.4, 6.0, 10.0 Hz) assigned to oxygenated methylene H₂-1’ protons. A two proton triplet at δ 2.29 (J = 7.2 Hz) was ascribed to methylene H₂-2 protons adjacent to the ester carbon. The other methylene protons appeared between δ 2.02-1.23. Two three proton triplets at δ 0.86 (J = 5.6 Hz) and 0.82 (J = 6.5 Hz) were accounted to the terminal C-27 and C-16’ primary methyl protons respectively. The ¹³C-NMR spectrum of compound IR-2 exhibited signals for ester carbon at δ 174.39 (C-1), oxygenated methylene carbon at δ 63.16 (C-1’), other methylene carbons from δ 51.42 to 22.67 and methyl carbons at δ 14.10 (C-22) and 11.97 (C-16’). The absence of any signal beyond δ 4.24 in the ¹H-NMR spectrum and between δ 174.39-63.16 supported saturated nature of the molecule. On the basis of these results, the structure of IR-2 has been characterized as n-Hexadecanyl n-docosanoate.

Compound IR-3, named 15-tricosterienyl eudesmolide, was obtained from petroleum ether-chloroform (1:1) eluent. Its IR spectrum showed characteristic absorption bands for lactone ring (1765 cm^-1), ester groups (1742 cm^-1), unsaturation (1640 cm^-1) and long aliphatic chain (778 cm^-1). On the basis of mass and ¹³C-NMR spectra, the molecular ion peak was determined at m/z 576 corresponding to a sesquiterpenic ester C₃₈H₅₆ O₄. The ion peaks arising at m/z 333 [C₁₅-O fission, O (CH₂)₁₂-CH = CH-(CH₂)₃-(CH = CH)₂C₂H₅]⁺ and 243 [M-333]⁺ suggested that the sesquiterpenic acid was esterified with C₂₃ alcohol. The ion fragments forming at m/z 304 [333-C₂H₅]⁺, 252 [333-(CH = CH)₂C₂H₅]⁺, 184 (C_12’-C_13’ fission, O (CH₂)₁₂]⁺ and 149 [333-184] indicated the location of the arylic linkages at C-13’, C-18’ and C-20’ carbons.

The ¹H-NMR spectrum of IR-3 exhibited a one proton doublet at δ 4.86 and 4.80 assigned to vinylic H-6 and to exocyclic methylene H₂-11 and H₂-13, respectively. A one proton double doublet at δ 3.93 (J = 9.5, 9.0 Hz) and a one proton doublet at δ 2.49 (J = 3.5 Hz) were attributed to oxygenated methine H-8β and methine H-5α protons, respectively suggesting olide ring in the molecule. Three two proton multiplets at δ 5.36 (w_1/2 = 5.5 Hz), 5.34 (w_1/2 = 5.6 Hz) and 5.25 (J = 5.5 Hz) were cis oriented vinylic protons of the aliphatic chain. A two proton triplet at δ 4.16 (J = 7.5 Hz) was accounted to oxygenated methylene H₂-1’ protons. A three proton triplet at δ 0.83 (J = 7.5 Hz) was due to terminal C-23’ primary methyl protons. The remaining methylene protons appeared from δ 2.78 to 1.24.

The ¹³C-NMR spectrum displayed signals for lactone carbon at δ 168.46 (C-14) oxygenated methine carbon at δ 62.10 (C-1’), arylic carbons from δ 151.24 to 109.62 methylene and methine carbons between δ 52.04-22.58 and methyl carbon at δ 14.09 (C-23’). The ¹H and ¹³C-NMR spectral values of the eudesmane moiety were compared with the reported spectral data of these compounds.[12,13,14] On the basis of the foregoing discussion the structure of IR-3 has been formulated as 15-[(13z, 18’z, 20’z)-n-tricos-13, 18, 20-trienyl]-eudesmal-4 (11), 6, 12 (13)-trien-8, 14-olide-15-oate. This is a new sesquiterpenic ester.

Compound IR-4, designated as 15-nonadecenyl eudesmalolide, was obtained as a pale yellow crystalline mass from petroleum ether-chloroform (1:1) eluant. Its IR spectrum displayed distinct absorption bands for γ-lactone (1765 cm^-1), ester linkage (1735 cm^-1), unsaturation (1638 cm^-1) and long aliphatic chain (779 cm^-1). On the basis of mass and ¹³C-NMR spectra the molecular ion peak of IR-4 was established at m/z 524 consistent to the molecular formula of a sesquiterpenic ester C₃₄H₅₂O₄. The ion peaks arising at m/z 243 [C₁₅-O fission]⁺ and 281 [M-243, O (CH₂)₁₅CH = CHC₂H₅]⁺ indicated that n-nonadecenyl alcohol was esterified with a sesquiterpenic acid. Expulsion of ethyl group from the mass unit 281 produced an ion fragment at m/z 281 suggesting the existence of the vinylic linkage at C-16’ position. The ¹H-NMR spectrum of IR-4 displayed signals similar to compound IR-3 indicating exocyclic methylene groups at C-4 and C-12, one vinylic linkage at C-6, an olide ring at C-8 (14) position and carboxylate function at C-15. Two one-proton multiplets at δ 5.35 and 5.32 with half-width of 5.5 Hz were accounted to the cis-oriented vinylic H-16’ and H-17’ protons, respectively. A two-proton triplet at δ 4.14 (J = 4.8 Hz) and a three-proton triplet at δ 0.83 (J = 6.5 Hz) were attributed to oxygenated methylene H₂-1’ and primary C-19’ methyl protons, respectively. The remaining methine and methylene protons resonated from δ 2.79 to 1.24. The ¹³C-NMR spectrum of IR-4 showed fifteen carbon signals for the sesquiterpenic unit similar to compound IR-3, oxygenated methylene carbon at δ 62.17 (C-1’), vinylic carbons at δ 130.23 (C-16’) and 127.91 (C-17’) and methyl carbon at δ 14.07 (C-19’). The ¹H and ¹³C NMR spectral data of the eudesmone moiety of IR-4 were compared with the reported values of these sesquiterpenoids.[12,13,14] On the basis of these results the structure of IR-4 has been elucidated as 15-[(16z)-n-monadec-16’- enyl]-eudesmal-4 (11) 6, 12 (13)-trien-8, 14-olide-15-oate. This is a new eudesmanolide ester.

Compound IR-5, named 15-tetracosdienyl eudesmalolide, was obtained as a brown crystalline mass from petroleum ether-chloroform (1:3) eluents. Its IR spectrum showed characteristic absorption bands for γ-lactone (1766cm^-1), ester group (1723cm^-1) and unsaturation (1640cm^-1). The molecular ion peak of IR-5 was established at m/z 592 on the basis of mass and ¹³C-NMR spectra, consistent of the molecular formula of a sesquiterpenic ester C₃₉H₆₀O₄. The ion peaks arising at m/z 243 and 350 (M-243)⁺ due to fission of the C₁₅-OCH₂ linkage indicated that tetracosdienol was esterified with the sesquiterpenic acid. The ion peaks generating at m/z 321 (350-C₂H₅)⁺, 252 [C_17’-C_18’ fission, O (CH₂)₁₅CH = CH]⁺, and 294 [C₂₀-C₂₁, fission, O (CH₂)₁₅CH = CH (CH₂)₃]⁺ suggested vinylic linkages at C-16’ and C-21’ positions.

The ¹H-NMR and ¹³C-NMR spectral data of the sesquiterpenic unit of IR-5 was similar to that of IR-3. A two proton triplet at δ 3.65 (J = 5.6 Hz) was assigned to oxygenated methylene H₂-1’ protons. Four one-proton multiplets at δ 5.48, 5.36, 5.31 and 5.20 with corresponding half widths of 5.6 Hz, 5.8 Hz, 5.5 Hz and 5.6 Hz were ascribed to the cis-oriented vinylic H-21’, H-16’, H-17’ and H-22’, respectively. A three proton triplet at δ 0.86 (J = 6.8 Hz) was due to C-2H’ primary methyl protons. The remaining methine and methylene protons resonated from δ 2.84 to 1.29. The ¹³C-NMR spectrum displayed signals oxygenated carbon at 66.03 (C-1’), vinylic carbons at δ 130.20 (C-16’), 130.04 (C-17’), 125.05 (C-21’) and 128.28 (C-22’), methyl carbon at δ 14.05 (C-24) together with signals for eudesmalolide.

The ¹H-NMR and ¹³C-NMR spectral values of the sesquiterpenic unit were compared with reported data of eudesmane-type sesquiterpenoids.[12,13,14] On the basis of these results the structure of IR-5 has been characterized as 15-[(16’z), (21’z)-n-tetracos-16’, 21’- dienyl]-eudesmal-4 (11) 6, 12 (13)-trien-8, 14-olide-15-oate. This is a new sesquiterpenic ester.

Compound IR-6, named 15-tetracosenyl eudesmalolide, was obtained as a pale yellow crystalline product from chloroform eluents. Its IR spectrum showed characteristic absorption bands for γ-lactone (1764cm^-1), ester function (1720cm^-1) and Unsaturation (1645cm^-1). On the basis of mass and ¹³C-NMR spectra, the molecular ion peak of IR-6 was determined at m/z 594 corresponding to the molecular formula of a sesquiterpenic ester C₃₉H₆₂O₄. The ion peaks arising at m/z 243 and 351 [C₁₅-OCH₂]⁺ fission suggested that a sesquiterpenic acid was esterified with C₂₄ alcohol. The ion frequent arising at m/z 252 [C_17’-C₁₈ fission], O (CH₂)₁₅-CH = CH]⁺ indicated the location of the vinylic linkage at C-16’. The ¹H-NMR signals of the eudesmolide unit of IR-6 were similar to that of IR-3 suggesting identical sesquiterpenic unit in IR-3 and IR-6. Two one proton multiplets at δ 5.33 (w1/2 = 6.5 Hz) and 5.17 (w1/2 = 7.2 Hz) were assigned to cis-oriented vinylic H-16’ and H-17’, respectively. A two proton triplet at δ (w_1/2 = 6.7 Hz) was ascribed to oxygenated methylene H₂-1’. A three proton triplet at δ 0.85 (J = 7 Hz) was accounted to the terminal C-24 primary methyl protons. The ¹³C-NMR spectrum of IR-6 showed signals for vinylic carbons at δ 130.19 (C-16’) and 113.63 (C-17’), oxygenated methylene carbons between δ 14.12 (C-24’) along with the carbon signals of the sesquiterpenic unit similar to that of IR-3. The ¹H and ¹³C-NMR spectral data of IR-6 were compared with the reported data of eudesmalolide compounds.[12,13,14] On the basis of these evidences the structure of IR-6 has been established as 15-[(16’z)-n-tetracos-16’- enyl]-endesmal-4 (11), 6, 12 (13)-trien-8,14-olide-15-oate. This is a new eudesmalolide.

CONCLUSION

On the basis of the above stated evidences, the structure of IR-1 has been elucidated as (5z, 13z)-n-decanyl-n-docos-5, 13-dienoate. This is a new fatty acid ester. Earlier (5E, 13E)-docosdienoic acid from Hybanthus enneaspermus whole plant has been identified.

Also on the basis of these results the structure of IR-2 has been characterized as n-Hexadecanyl n-docosanoate and the structure of IR-3 has been formulated as 15-[(13z, 18’z, 20’z)-n-tricos-13, 18, 20-trienyl]-eudesmal-4 (11), 6, 12 (13)-trien-8, 14-olide-15-oate. This is a new sesquiterpenic ester.

The structure of IR-4 as according to the above disscussed results has been elucidated as 15-[(16z)-n-monadec-16’- enyl]-eudesmal-4 (11), 6, 12 (13)-trien-8, 14-olide-15-oate. This is a new eudesmanolide ester and IR-5 has been characterized as 15-[(16’z), (21’z)-n-tetracos-16’, 21’- dienyl]-eudesmal-4 (11) 6, 12 (13)-trien-8, 14-olide-15-oate. This is a new sesquiterpenic ester.

On the basis of the evidences provided the structure of IR-6 has been established as 15-[(16’z)-n-tetracos-16’- enyl]-eudesmal-4 (11), 6, 12 (13)-trien-8, 14-olide-15-oate. This is a new eudesmalolide.

These compounds have a lot of scope to be studied further and their effects on the living system in relation to the medicinal properties of I. racemosa already known.