Chemical Constituents From the Roots of Oenothera biennis L.

Shahnaz Sultana1,2, Mohammed Ali1*, Showkat Rasool Mir1

1Phytochemistry Research Laboratory, Faculty of Pharmacy, Jamia Hamdard, New Delhi, 110 062, India

2Present address: College of Pharmacy, Jazan University, Jazan, Saudi Arabia

Received: 10-Sep-2017 , Accepted: 13-Apr-2018

Keywords: Oenothera biennis, Roots, Chemical constituents, Isolation, Characterization



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Oenothera biennis L. (Onagraceae), is a biennial herb, native to eastern and central North America. It is cultivated in temperate regions of the world and in Indian gardens. The plant parts are used to treat gastro-intestinal disorders, eczema, whooping cough, asthma, blood disorders, laziness, obesity, piles and boils. The dried root powder was exhaustively extracted with methanol and the extract concentrated to yield a dark brown viscous mass. It was dissolved in small quantity of methanol and adsorbed onto silica gel (60 - 120 mesh) for preparation of a slurry. The air dried slurry was subjected to chromatography over silica gel column packed in petroleum ether. The column was eluted successively with petroleum ether,   chloroform  and methanol in order of increasing polarity to  isolate the new phytoconstituents characterized as 3,11,15-trimethyl-14β-hydroxy-n-hexadeca-7-en-4,18-olide (phyt-7-enyl-14β-ol 4β,18-olide, 2), 3-methyl-6α,8β-dihydroxy-7-carboxylic acid tetralin-1,9β-olide (3), 3,7-dimethyl-11-hydroxymethylene dodec-3α,6α-diol- 11-enyl  2′,3′,6′-benzene triol (4), 1,9,10-trimethoxy-3,11-dihydroxy-13-(18,19-dihydroxyprenyl)-anthracene (5) and  α-D-glucopyranosyl–(4→1′)-α-D-glucopyranosyl -6′-cetoleate (6) along with benzoic acid (1). The structures of all the isolated phytoconstituents have been established on the basis of spectral data analysis and chemical reactions.

1 Introduction

Oenothera biennis L., syn., O. muricata L., O.  suaveolens Desf., Brunyera biennis Bubani, Onagra biennis (L.) Scop. (Onagraceae), known as  evening-primrose, evening star and  sun drop, is a native to eastern and central North America and grown in Indian gardens1. It is a biennial, 30 – 150 cm high herb with lanceolate leaves, erect, stout and softly-hairy stem, oblong and hairy pods with precious seeds. Its roots and leaves are edible.

A tea prepared  from the leaves is taken as a dietary aid,  stimulant and  to treat cough, blood disorders,  laziness and obesity. A root paste is applied  to subside  piles and boils and  to improve muscle strength. The leaves, stem bark, flowers and seed oil are used to treat gastro-intestinal disorders,  eczema, whooping cough and asthma. Evening primrose oil from the seeds  relieves abdominal bloating,  acne, allergies, Alzheimer`s  disease, asthma, attention deficit hyperactivity disorder,  autoimmune conditions, breast pain, cardiovascular disease, cirrhosis of the liver, cramps, diabetic neuropathy,  depression, dysmenorrhea, eczema, endometriosis, fatigue syndrome, fibrocystic breasts, hot flushes, gastrointestinal symptoms, menstrual  hyperchlolesterolemia, hypertension, impotence, female infertility, inflammation,migraine, multiple sclerosis, myalgia, obesity, nerve damage,   neurodermatitis, osteoporosis, premenstrual problems, psoriasis, rheumatoid arthritis ,  rosacea,  schizophrenia, tension and whooping cough2-4.

The seeds are a potential source of unsaturated fatty acids including γ-linoleic (8–14%), ω-6 polyunstaturated fatty, oleic, palmitic and stearic acids5-8, phytosterols9, 10   and phenolic compounds11. The roots contained aryl, lipid and triterpenic constituents12, lanosterols, tetralin-1, 9-olide and phytyl lactone13,14. The leaves possessed flavonoids15,16 and andoenothin17. The manuscript describes isolation and characterization of  diterpenic and  tetraline lactones, dodecenyl benzene triol, prenyl anthracene and an acyl diglucoside as new chemical constituents from the roots of O. biennis.

2 Materials and Methods

2.1 General procedures

Melting points were determined on a Perfit melting apparatus (Ambala, Haryana, India) and are uncorrected. UV spectra were measured with a Lambda Bio 20 spectrophotometer (Perkin Elmer-Rotkreuz, Switzerland) in methanol. Infrared spectra were recorded on Bio-Rad FTIR 5000 spectrophotometer (FTS 135, Kawloon, Hong Hong) using KBr pellets; γmax values are given in cm-1. The 1H and 13C NMR spectra were screened on Advance DRX Bruker spectrospin 400 and 100 MHz, respectively, instruments (Karlesruthe, Germany) using CDCl3 or DMSO-d6 as a solvent and TMS as an internal standard. Mass spectra were scanned by effecting ionization at 70 eV on a JEOL-JMS-DX 303 spectrometer (Japan) equipped with direct inlet probe system. Column chromatography was performed on silica gel (60-120 mesh; Qualigen, Mumbai, India). TLC was run on silica gel G (Qualigen). Spots were visualized by exposing to iodine vapours, UV radiation and spraying with ceric sulphate solution. 

2.2 Plant material

The roots of O. biennis were collected from Delhi and identified by Dr. M. P. Sharma, Department of Botany, Jamia Hamdard. A voucher specimen has been retained in the Phytochemistry Research Laboratory, Jamia Hamdard.

2.3 Preparation of extract

The dried roots (1.0 kg) were coarsely powdered, defatted with n-hexane and exhaustively extracted in a Soxhlet apparatus with methanol. The methanolic extract was concentrated under reduced pressure to yield a dark brown viscous mass (11.8 g). A small portion of the extract was analyzed chemically to determine the presence of different chemical constituents.

 2.4 Isolation of phytoconstituents

The viscous dark brown extract (150 g) was dissolved in small quantity of methanol and adsorbed onto silica gel (60 - 120 mesh) for preparation of a slurry. The slurry was air dried and subjected to chromatography over silica gel column packed in petroleum ether. The column was eluted successively with petroleum ether, mixture of petroleum ether -  chloroform (9:1, 3:1, 1:1, 1:3), chloroform and the mixture of chloroform - methanol (99:1, 97:3, 95:5, 92:8, 9:1, 3:1, 1:1, 1:3). Various fractions were collected separately and matched by TLC to check the homogeneity. Similar fractions having the same Rf values were combined and crystallized. The isolated compounds were recrystallized to get the following compounds:  

2.5 Benzoic acid(1)

Elution of the column with chloroform furnished colourless crystals of 1, recrystallized from methanol; 227 mg, m. p.: 221 – 222 oC, UV λmax (MeOH): 210, 270 nm; IR γmax(KBr): 3368, 2950, 2841, 1702, 1656,   1543, 1433, 1329, 1268, 1025 cm-11H NMR (CDCl3): δ 6.99 (2H, m, H-2, H-6), 6.97 (2H, m, H-3, H-5), 6.94 (1H, m,  H-4);  13C NMR (CDCl3): δ 145.38 (C-1), 138.61 (C-2, C-6), 120.97 (C-3, C-5), 109.41 (C-4), 169.47 (C-7);  +ve ion FAB MS m/z(rel. int.): 122 [M]+ (C7H6O2) (10.6).

2.6 Phyt-7-enyl-14β-ol-4β,18-olide (2)

Further elution of the column with chloroform afforded a pale yellow semisolid mass of 2,  167 mg,  UV λmax (MeOH): 213, 229 nm; IR γmax(KBr):  3411,  2922, 2847, 1737,  1646,   1447, 1389, 1258, 1041 cm-11H NMR (CDCl3): δ 5.35 (1H, m, H-8), 4.06 (1H, brm, w1/2 = 17.2 Hz,  H-4α), 3.67 (1H, brm, w1/2 = 14.2 Hz,  H-14α), 2.72 (2H, m, H2-6), 2.36 (2H, m, H2-9), 2.23 (1H, m, H-11), 2.05 (1H, m, H-3α), 2.01 (2H, m, H2 -5), 1.80 – 1.19 (9H, m, H-5, 4 x CH2), 1.15 (3H, d, J = 6.5 Hz, Me-16), 1.10 (3H, d, J = 6.7 Hz, Me-20), 1.03 (3H, d, J = 6.6 Hz, Me-17), 0.99 (3H, d, J = 6.7 Hz, Me-19), 0.85 (3H, d, J = 6.3 Hz, Me-1); 13C NMR (CDCl3): δ 14.48  (C-1), 22.19 (C-2), 39.51 (C-3), 78.85 (C-4), 58.25 (C-5), 50.04  (C-6), 140.22 (C-7), 120.15 (C-8), 30.51 (C-9), 30.53 (C-10), 42.73 (C-11), 21.23  (C-12), 21.43 (C-13), 68.19 (C-14), 31.49 (C-15), 18.33 (C-16), 16.51 (C-17), 166.42 (C-18), 20.17 (C-19), 18.27 (C-20);  FAB MS m/z ( rel. int.): 324 [M]+  (C20H36O3) (12.7), 281 (14.5), 267 (23.8), 251(4.7),  239 (19.2), 157  (8.3), 129 (6.7), 57 (13.2). 

2.7 3-Methyl-6α,8β-dihydroxy-7-carboxylic acid tetralin-1,9β-olide (3) 

Elution of the column with chloroform – methanol (49 : 1) gave pale yellow crystals of 3, recrystallized from chloroform – methanol (1: 1);  131 mg, m. p.: 118 - 119 oC, UV λmax (MeOH): 211, 241 nm; IR γmax(KBr):  3448, 3288, 3236,  2951, 2846, 1765, 1702, 1626, 1547,  1443, 1359, 1247, 1031 cm-11H NMR (CDCl3):  δ 7.06 (1H, d, J = 1.5 Hz, H-2), 7.02 (1H, d, J = 1.5 Hz, H-4), 4.23 (1H, d, J  = 10.2 Hz,  H-9α), 3.36 (1H, d, J = 5.1 Hz, H-6β), 3.32 (1H, dd, J = 5.1, 10.2 Hz, H-8α), 2.79 (1H, dd, J = 10.2, 5.1 Hz, H-7β), 2.33 (3H, s, Me-12);  13C NMR (CDCl3): δ  146.61 (C-1), 110.19 (C-2), 110.12 (C-3), 121.55 (C-4), 139.85 (C-5), 68.52 (C-6), 52.47 (C- 7), 66.72 (C-8), 77.86 (C-9), 121.53 (C-10), 169.17 (C-11), 28.55 (C-12), 179.35 (C-13);  FAB MS m/z ( rel. int.):  264 [M]+  (C13H12O6) (10.4), 246 (15.3) , 219 (8.9), 201 (19.2). 

2.8 3,7-Dimethyl-11-hydroxymethylene dodec-3α,6α-diol- 11-enyl  2′,3′,6′-benzene triol (4)

Elution of the column with chloroform - methanol (48 : 1) produced yellow crystals of 4, recrystallized from chloroform – methanol (1: 1); 208 mg, m. p. : 141 - 143 oC, UV λmax (MeOH): 213, 273 nm; IR γmax(KBr): 3411, 3283, 3248, 2925, 2856, 1643, 1510,  1456, 1325, 1267, 1176, 1033 cm-11H NMR (CDCl3):  δ 7.29 (1H, d, J = 10.8 Hz, H-4′), 7.25 (1H, d, J = 10.8 Hz, H-5′), 7.23 (1H, s, H-12), 3.88 (2H, s, H2-15), 3.79 (1H, ddd, J = 4.8, 5.1, 5.9 Hz, H-6β), 2.27 (1H, dd, J = 9.3, 9.9 Hz, H2-10a), 2.16 (1H, dd, J = 9.2, 11.8 Hz, H2-10b), 2.08 – 1.28 (13H, , 6 x CH2, CH), 1.25 (3H, s, Me-13), 0.99 (3H, d, J = 6.9 Hz,  Me-14), 0.87 (3H, t, J = 6.3 Hz, Me-1);  13C NMR (CDCl3):  δ 14.36 (C-1), 22.97 (C-2), 72.10 (C-3), 38.27 (C-4), 32.25 (C-5), 62.42 (C-6), 52.40 (C-7), 41.55 (C-8), 29.97 (C-9), 56.71 (C-10), 125.55 (C-11), 107.75 (C-12), 28.12 (C-13), 19.48 (C-14), 61.17 (C-15),   132.91 (C-1′), 153.41 (C-2′), 167.87 (C-3′), 131.16 (C-4′), 129.21 (C-5′), 166.97 (C-6′);   FAB MS m/z ( rel. int.):  382 [M]+  (C21H34O6) (37.1), 309 (12.7), 281 (5.7), 201 (11.5),  131 (15.4) , 125 (7.8), 101 (12.3), 73 (8.1). 

2.9 1,9,10-Trimethoxy-3,11-dihydroxy-13-(18,19-dihydroxyprenyl)-anthracene (5)

Elution of the column with chloroform–methanol (19 : 1) yielded yellow crystals of 5, recrystallized from chloroform – methanol (1: 1);  121 mg, m. p.: 221-223 oC, UV λmax (MeOH): 241, 259, 324  nm; IR γmax(KBr): 3315, 2927, 2848, 1638, 1529, 1476, 1418, 1353, 1203, 1095 cm-11H NMR (CDCl3):  δ 7.79 (1H, s,  H-6), 7.66 (1H, d, J = 2.4 Hz, H-2), 7.61 (1H, s, H-8), 7.19 (1H, d, J = 2.4 Hz, H-4), 4.23 (3H, s, OMe), 4.20 (3H, s, OMe), 4.15 (3H, s, OMe), 3.16 (2H, d, J = 5.6 Hz, H2-18), 3.11 (2H, d, J = 5.6 Hz, H2-19), 2.35 (2H, m,  H2-15), 1.75 (2H, m, H2-16), 1.17 (1H, m, H-17);  13C NMR (CDCl3): δ 158.62 (C-1), 141.29 (C-2), 158.31 (C-3), 120.92 (C-4), 112.45 (C-5), 120.56 (C-6), 112.41 (C-7), 116.74 (C-8), 155.57 (C-9), 155.09 (C-10), 165.02 (C-11), 117.05 (C-12), 145.07 (C-13), 113.25 (C-14), 30.09 (C-15),   23.09 (C-16), 39.86 (C-17), 62.39 (C-18), 62.36 (C-19), 57.45 (OMe), 57.26 (OMe), 57.22 (OMe);  FAB MS m/z ( rel. int.):  402 [M]+  (C22H26O7) (3.1), 387 (35.7), 372 (5.9), 357 (22.5).               

2.10  α-D-glucosyl-(4→1′)-α-D-glucosyl-6′-cetoleate (6)

Elution of the column with chloroform-methanol (9:1) gave a yellow semisolid mass of 6,purified bypreparative TLC(chloroform: methanol, 3:1); 153 mg,  UV lmax (MeOH): 217 nm (log ε 4.2); IR λmax (KBr):  3427, 3396, 3312, 2925, 2849, 1725, 1650, 1458, 1379, 1273, 1163, 1075,  723 cm-11H NMR (MeOD):  d 5.22 (1H, d, J = 3.7 Hz, H-1α), 4.09 (1H, m, H-5), 3.96 (1H, m, H-2), 3.81 (1H, m, H-3), 3.65 (1H, m, H-4), 3.13 (2H, d, J = 11.5 Hz, H2-6), 5.15 (1H, d, J = 3.9 Hz, H-1′α), 4.06 (1H, m, H-5ʹ), 3.89 (1H, m, H-2ʹ), 3.79 (1H, m, H-3ʹ), 3.51 (1H, m, H-4ʹ), 3.46 (2H, d, J = 9.0 Hz, H2-6ʹ), 5.30 (2H, m, H-9′′, H-10′′), 2.32 (2H, m, H2-2′′), 1.85 (2H, m, H2-8), 1.81 (2H, m, H2-11′′),  1.50 (2H, m, H2-3′′), 1.38 (2H, m, H2-7′′),   1.23 (22H, brs, 11 x CH2), 0.78 (3H, t, J= 6.8 Hz, Me-20′′); 13C NMR (DMSO-d6): d 103.73 (C-1), 74.59 (C-2), 72.05 (C-3), 71.85 (C-4), 76.99 (C-5), 61.66 (C-6),  103.54 (C-1ʹ), 73.95 (C-2ʹ), 71.95 (C-3ʹ), 70.53 (C-4ʹ), 76.87 (C-5ʹ), 65.34 (C-6ʹ),  173.16 (C-1′′), 53.86 (C-2′′), 133.40 (C-9′′), 129.71 (C-10′′),  34.89 – 31.87 (6 x CH2), 29.97 – 29.31 (5 x CH2), 27.25 (CH2), 26.24 (CH2), 25.14 (CH2), 22.78 (CH2), 13.46 (C-20′′);  +ve FAB MS m/z (rel. int.): 634 [M]+ (C32H58O12) (14.6), 325 (14.8), 309 (12.1), 179 (8.7), 163 (19.5).

3 Results and Discussions

Compound 1 was the known compound identified as  benzoic acid. 

Compound 2 showed IR absorption bands for hydroxyl group (3411 cm-1), δ-lactone (1737  cm-1) and unsaturation (1646 cm-1). On the basis of mass and 13C NMR spectra, its molecular weight was determined at m/z 324 corresponding to a molecular formula of an acyclic diterpenic lactone, C20H36O3. The ion peaks generating at m/z 281 [C14 – C15 fission, M - C3H7]+, 251  [C13 – C14 fission]+, 239 [C11 - C12 fission]+   and 129 [C10 - C11 fission]+ suggested location of the hydroxyl group at C-14. The ion fragments produced at m/z 57, 267 [C3 - C4 fission]+ and 157 [C8 – C9 fission]+  indicated the existence of the δ-lactone ring at C-4(18) and the vinylic linkage at C7 position. 

The 1H NMR spectrum of 2 showed a one - proton multiplet at δ 5.35 assigned to vinylic H-8 proton. A one-proton broad multiplet at δ 4.06 with half width of 17.2 Hz was ascribed to α-oriented oxymethine H-4 proton. A one-proton multiplet at δ 3.67 with half-width of 14.2 Hz was attributed to carbinol H-14α proton. Four three-proton doublets at δ 1.15 (J = 6.5 Hz), 1.10 ( J = 6.7 Hz), 1.03 (J = 6.6 Hz) and  0.99 (J = 6.7 H) and a three-proton triplet at δ 0.85 (J = 6.3 Hz) were associated with the secondary C-16, C-20, C-17 and C-19 and primary C-1 methyl protons, respectively, all attached to saturated carbons. The remaining methine and methylene protons appeared from δ 2.72 to 1.19.  The 13C NMR spectrum of  2 showed the presence of 20 carbon atoms and the important signals appeared for lactone carbon at δ 166.42 (C-18), oxymethine carbon at δ 78.85 (C-4), carbinol   carbon at δ 68.19 (C-14), vinylic carbons at δ 140.22 (C-7) and 120.15 (C-8) and methyl carbons at δ 14.48 (C-1), 18.33 (C-16), 16.51 (C-17), 20.17 (C-19) and 18.27 (C-20). The DEPT spectrum of 2 displayed the presence of five methyls, seven methylene, six methine and two quaternary carbons. The 1H-1H COSY spectrum of  2 exhibited correlations of H-4 with H-3, H2-5 and H2-6; H-8 with H2-6 and H2-9; and H-14 with H2-13, H-15, Me-16 and Me-20. The HMBC spectrum of 2 showed that H-3 and H2-5 interacted with C-4; H2-6 and H-8 interacted with C-7; and H2-13, H-14, Me-16 and H-20 interacted with C-14. The HSQC experiment showed important interactions between the vinylic proton H-8 at δ 5.35 with the C-8 carbon signal at δ 120.15, carbinol H-14 proton at δ 3.67 with C-14 carbon signal at δ 68.19 and oxymethine H-4 at δ 4.06 with C-4 carbon signal at δ 78.85. On the basis of spectral data analysis, the structure of 2 has been established as 3,11,15-trimethyl-14β-hydroxy-n-hexadeca-7-en-4,18-olide (phyt-7-enyl-14β-ol 4β,18-olide), a new diterpenic lactone.

Compound 3 produced effervescences with sodium bicarbonate indicating the presence of a carboxylic function. Its IR spectrum showed absorption bands for hydroxyl groups (3448, 3288 cm-1), carboxylic group (3236, 1702 cm-1), five membered lactone ring (1765 cm-1) and aromatic ring (1626, 1547, 1031 cm-1). On the basis of mass and 13C NMR spectra its molecular formula was determined at m/z 264 consistent with a molecular formula of a tetrahydronaphthalene - type lactone C13H12O6 . Elimination of a carboxylic group from the molecular ion peak yielded an ion fragment at m/z 219. Removal of a water molecule and  subsequently the carboxylic group generated the ion fragments at m/z 246 and 201,  respectively.  The 1H NMR spectrum of 3 showed two one - proton doublets at δ 7.06 (J = 1.5 Hz) and 7.02 ( J = 1.5 Hz) assigned to meta-coupled  aromatic H-2 and H-4 protons, respectively.  Two one-proton doublets at δ 4.23 (J = 10.2 Hz) and 3.36 (J = 5.1 Hz) were attributed correspondingly to oxymethine H-9α and carbinol H-6β protons. Two one-proton double doublets at δ 3.32 (J = 5.1, 10.2 Hz, H-8α) and 2.79 (J = 10.2, 5.1 Hz, H-7β), and a three-proton singlet at δ  2.33 (Me-12) were ascribed to carbinol H-8α, methine H-7β and C-12 methyl protons, respectively.   The 13C NMR spectrum of 3 exhibited the presence of 13 carbon signals and the important carbon signals appeared for carboxylic carbon at δ 179.35 (C-13), lactone carbon at δ 169.17 (C-11), carbinol carbons at δ 68.52 (C-6) and 66.72 (C-8), oxymethine  carbon at δ  77.86 (C-9),  methyl carbon at δ 28.55   and aromatic carbons between δ 146.61 - 110.12. The DEPT spectrum of 3 showed the presence of one methyl and six each of methine and quaternary carbons. The 1H-1H COSY spectrum of 3 showed correlations of H-4 with H-2, H-6 and Me-12; and H-8 with H-7 and H-9. The HMBC spectrum of 3 exhibited that H-2 interacted with C-11; Me-12, H-4 and H-2 interacted with C-3; H-7 interacted with C-13; and H-8 and H-9 interacted with C-10.  The HSQC experiment showed important correlations between the aromatic proton signals at δ 7.06 (H-2) and 7.02 (H-4) with the respective  carbon signals at δ 110.19 (C-2) and 121.55 (C-4); carbinol protons at δ 3.36 (H-6) and 3.32 (H-8) with carbon signals at δ 68.51 (C-6) and 66.72 (C-8), respectively; oxymethine proton at δ 4.23 (H-9)  with carbon signal at δ 77.86 (C-9) and methyl H3-12  proton at δ 2.33 with the carbon signal at δ 28.55 (C-12). On the basis of these evidences the structure of 3 has been elucidated as 3-methyl-6α,8β-dihydroxy-7-carboxylic acid tetralin-1,9β-olide, a new tetralin lactone.

Compound 4 gave positive tests of phenols and showed IR absorption bands for hydroxyl groups (3411, 3283, 3248 cm-1) and aromatic ring (1643, 1510, 1033 cm-1). On the basis of mass and 13C NMR spectra its molecular ion peak was determined at m/z 382 consistent with a molecular formula of a  sesquiterpenic benzene triol C21H34O6 . The ion peaks arising at m/z 73 [C3 – C4 fission, C4H9O]+,  309 [M – 73]+,  101 [C5 – C6 fission, C6H13O]+,  281 [M – 101]+ and 131 [C6 – C7 fission, C7H15O2]+ suggested existence of the  hydroxyl groups  at C3 and C6 carbons.  The ion fragments produced at m/z  201 [C10 – C11 fission, C12H25O2]+ and 125 [C12 – C1′ fission, C6H5O3]+ indicated the presence of the C-15 hydroxymethylene  group  and a vinylic linkage located  at C11 and a benzene triol ring at C12 carbons. The 1H NMR spectrum of 4 showed two one-proton doublets at δ 7.29 (J = 10.8 Hz) and 7.25 (1H, d, J = 10.8 Hz) assigned to ortho-coupled aromatic H-4′ and H-5′ protons, respectively.  A one-proton singlet at δ 7.23 ,  a two-proton singlet at δ 3.88  and a  one-proton  triplet doublet at δ 3.79 (J = 4.8, 5.1, 5.9 Hz)  were attributed correspondingly to vinylic H-12 linked to the aromatic ring, hydroxymethylene H12 protons located on the unsaturated C11 carbon and carbinol C-6β proton. Three three-proton signals appearing as a singlet at δ 1.25, as a doublet at δ 0.99 (J = 6.9 Hz) and as a triplet at δ 0.87 (J = 6.3 Hz) were accounted to tertiary C-13, secondary C-14 and primary C-1 methyl protons, respectively, all attached to the saturated carbons. The remaining methine and methylene protons resonated from δ 2.27 to 1.28.   The 13C NMR spectrum of 4 displayed  the presence of 21 carbon signals and the important  signals appeared for benzene carbons between  δ 167.87 – 129.21, vinylic carbons at δ 125.55 (C-11) and 107.75 (C-12), carbinol carbons at δ 72.10 (C-3) and 62.42 (C-6), hydroxymethylene carbon at δ  61.17 (C-15) and  methyl carbons  at δ 14.36 (C-1), 28.12 (C-13)    and 19.48 (C-14).  The DEPT spectrum of 4 showed the presence of three methyl, seven methylene, five methine  and six quaternary carbons. The 1H-1H COSY spectrum of 4 showed correlations of Me-13 with H2-2  and  H2-4; H-6 with H2-5, H-7,  Me-14 and H2-8; H-12 with H2-10 and H2-15; and H-4′ with H-5′. The HMBC spectrum of 4 exhibited that Me-1, H2-2, H2-4 and Me-13 interacted with C-3;  H2-5 and H-7 interacted with C-6; H2-10, H-12 and H2-15 interacted with C-11; and H-4′ and H-5′  interacted with C-3′. 

The HSQC experiment displayed important correlations between the aromatic proton signals at δ 7.29 (H-4′) and  7.25 (H-5′) with the carbon signals at δ 131.16 (C-4′) and 129.21 (C-5′); vinylic  proton at δ 7.23 (H-12) with the carbon signal at δ 107.75 (C-12), carbinol proton at δ 3.79 with carbon at δ 62.42 (C-6), hydroxymethylene proton at δ 3.90 (H2-15) with carbon signal at δ 61.17 (C-15) and methyl proton signals at δ 0.87 (Me-1), 1.25 (Me-13) and  0.99 (Me-14)  with their respective carbon signals at δ  14.36 (C-1), 28.12 (C-13) and 19.48 (C-14). On the basis of these spectral data analysis the structure of 4 has been established as 3,7-dimethyl-11-hydroxymethylene dodec-3α,6α-diol- 11-enyl  2′,3′,6′-benzene triol, a phenolic substituted sesquiterpene.  

Compound 5 responded positively to phenolic tests and showed UV absorption maxima at 241, 259, 324  nm for aromaticity. Its IR spectrum had absorption bands for hydroxyl groups (3315 cm-1) and aromatic ring (1638, 1529, 1095 cm-1). On the basis of mass and 13C NMR spectra its molecular formula was established at m/z 402 consistent with a molecular formula of a  prenylated  anthracene C22H26O7 . The ion peaks arising at m/z 387 [M - Me]+,  372 [387 – Me]+and 357 [372 - Me]+  suggested the presence of three  methoxy groups in the molecule.  The 1H NMR spectrum of 5 showed two one-proton doublets at δ 7.66 (J = 2.4 Hz) and 7.19 ( J = 2.4 Hz), and two one –proton singlets at δ 7.79 and 7.61 assigned to aromatic H-2, H-4, H-6 and H-8 protons, respectively, three methoxy protons as three-proton singlets  at δ 4.23, 4.20  and  4.15, methylene protons as two-proton multiplets at δ 2.35 and 1.75, a methine proton as a one-proton multiplet at δ 1.17 and hydroxymethylene protons as two-proton doublets at δ  3.16 (J = 5.6 Hz) and 3.11 (J = 5.6 Hz) accounted to H2-18 and  H2-18 protons, respectively.  The 13C NMR spectrum of 5 displayed  the presence of 22 carbon signals and the important  signals appeared for aromatic carbons between  δ 165.02  – 112.41 , methoxy carbons at δ  57.45, 57.26 and 57.22, hydroxymethylene carbons at δ  62.39 (C-18) and  62.36 (C-19),   methylene  carbons  at δ 30.09 (C-15) and  23.09 (C-16) and methine carbon at δ 39.86 (C-17).

The DEPT spectrum of 5 showed the presence of three methoxy, four methylene, five methine and ten quaternary carbons. The 1H-1H COSY spectrum of 5 showed correlations of H-17 with H2-15, H2-16,   H2-18 and   H2-19; and H-2 with H-4. The HMBC spectrum of 5 exhibited interactions of  H2-18, H2-19 and H2-16  with C-17;  H2-15 with C-13; H-2 and H-4  with C-3; and H-8  with C-9.  The HSQC experiment showed important correlations between the aromatic proton signals at δ  7.66 (H-2), 7.19 (H-4), 7.79 (H-6) and 7.61 (H-8) with their respective carbons at δ 141.29 (C-2),  120.92 (C-4), 120.56 (C-6) and 116.74 (C-8); methylene proton signals at δ 2.35 (H2-15) and  1.75 (H2-16) with the carbon signals at δ 30.09 (C-15) and   23.09 (C-16), respectively, and hydroxymethylene proton signals at δ 3.16 (H2-18)  and 3.11 (H2-19) with the corresponding carbon signals at δ 62.39 (C-18) and  62.36 (C-19). On the basis of these spectral data analysis the structure of 5 has been established as 1,9,10-trimethoxy-3,11-dihydroxy-13-(18,19-dihydroxyprenyl)-anthracene, a new anthracene derivative.

Compound 6 gave positive tests for glycosides and displayed characteristic IR absorption bands for hydroxyl groups (3427, 3396, 3312 cm-1), ester function (1725 cm-1) and long chain aliphatic hydrocarbon (723 cm-1). The molecular ion peak of 6 was determined at m/z 634 on the basis of mass and 13C NMR spectra corresponding to a molecular formula of a diglycosyl ester, C32H58O12. The ion fragments generating at m/z 163 [C6H11O5]+,  179 [C6H11O6]+, 325 [C6H12O6-C6H10O4]+ and 309 [M - 325, CH3(CH2)9-CH=CH-(CH2)7COO+] suggested that a dihexose unit was  esterified with cetoleic acid. 

The 1H NMR spectrum of 6 exhibited a two-proton multiplet at δ 5.30 assigned to vinylic  H-9′′ and  H-10′′ protons. Two one-proton doublets at δ  5.22 ( J = 3.7 Hz) and 5.15 (J = 3.9 Hz) were ascribed to α-oriented anomeric  H-1 and H-1′ protons, respectively.

The other sugar protons appeared from δ 4.09 to 3.13. A three-proton triplet at δ 0.78 (J= 6.8 Hz) was accounted to terminal C-20′′ primary methyl protons.  The remaining methylene protons resonated between δ 2.32 – 1.23. The 13C NMR spectrum of 6 exhibited signals for the ester carbon at δ 173.16 (C-1′′), anomeric carbons at δ 103.73 (C-1) and δ 103.54 (C-1ʹ), other sugar carbons in the range from δ 76.99  to 61.66, methylene carbons between δ 53.86 - 22.78 and methy1 carbon at δ 13.46 (C-14′′). The shifting of  1H NMR  H-4 signal in the deshielded region at δ 3.65 and 13C NMR C-4 signal at δ 71.85  indicated (4→1′) linkage of  the sugar units.

The presence of 1H NMR signal for oxymethylene  H2-6ʹ in the deshielded region at δ 3.46  and 13C NMR signals for C-6′ at δ 65.34 suggested the attachment of ester linkage C-6′.  The 1H-1H COSY spectrum of 6 displayed correlations of H-1ʹ with  H-2ʹ, H-3ʹ and H-4;  H-5ʹ with H-4ʹ, H-3ʹ and H2-6′;  and H-9ʹʹ with H2-8ʹʹ, H-10′′ and H2-11ʹʹ. The HMBC spectrum of 6 showed that H-4 and H-2ʹ interacted with C-1′;  H2-6ʹ and  H2-2′ʹ  interacted with C-1′ʹ; and H2-8′ʹ, H-9ʹʹ and H2-11ʹʹ correlated with C-10ʹʹ.

The HSQC spectrum of 6 exhibited correlations of anomeric protons at δ 5.22 (H-1) and 5.15 (H-1′) and vinylic protons at δ 5.30 (H-9′′, H-10′′) with their respective 13C NMR signals at δ 103.73 (C-1), 103.54 (C-1ʹ), 133.40 (C-9′′) and 129.71 (C-10′′).  Acid hydrolysis of 6 yielded cetoleic acid and D-glucose (Rf 0.12, n-butanol-acetic acid-water, 4:1:5). On the basis of this discussion the structure of 6 has been elucidated as α-D-glucopyranosyl–(4→1′)-α-D-glucopyranosyl -6′-cetoleate, a  new ester glycoside.

4 Conclusion

Phytochemical investigation of a methanolic extract of the roots of O. biennis yielded benzoic acid, diterpenic and  tetraline lactones, dodecenyl benzene triol, prenyl anthracene diol and an acyl diglucoside for the first time. This work has enhanced understanding about the phytoconstituents of the plant. These secondary metabolites can be utilized as effective analytical markers for identity, purity and quality control of this plant in future.

5 Conflicts of Interests

The authors hereby declare that there are no conflicts of interests.

6 Author’s contributions

SS and MA carried out isolation, data analysis/interpretation and manuscript preparation. SRM worked on the structural formulae, research conception/design and data acquisition.

7 Acknowledgements

The authors are thankful to the instrumentation centers, Central Drug Research Institute, Lucknow and Jawaharlal Nehru University, New Delhi for recording spectral data of the compounds.

8 Conflict of interest

The authors declared that there are no conflicts of interest.

9 Author’s contributions

SS, MA and SRM performed the experimental work and drafted the manuscript.

8 References

  1. Anonymous. The Wealth of India-Raw Materials. CSIR, New Delhi. 1966; 7; 90. 
  2. Montserrat-de la Paz S, García-Giménez MD, Ángel-Martín M, Pérez-Camino MC. Long-chain fatty alcohols from evening primrose oil inhibit the inflammatory response in murine peritoneal macrophages. Journal of Ethnopharmacology. 2014; 151; 131–136.
  3. Farzaneh F, Fatehi S, Sohrabi M,Alizadeh K. The effect of oral evening primrose oil on menopausal hot flashes: a randomized clinical trial. Archives of Gynecology and Obstetrics. 2013; 288; 1075– 1079.   
  4. Rodrigues RF, Costa IC, Almeida FB, Cruz RAS, Ferreira AM, Vilhena JCE, Florentino AC, Carvalho JCT, Fernandes CP. Development and characterization of evening primrose (Oenothera biennis) oil  nanoemulsions. Revista Brasileira de Farmacognosia. 2015; 25; 422- 425.  
  5. Granica S, Czerwinska ME, Piwowarski JP, Ziaja  M, Kiss AK. Chemical composition, antioxidative and anti-inflammatory activity of extracts prepared from aerial parts of Oenothera biennis L. and Oenothera paradoxa Hudziok obtained after seeds cultivation. Journal of Agriculture and Food Chemistry. 2013; 61; 801–810.
  6. Yunusova SG, Yakupova LR, Ivanova AV, Safiullin RL, Galkin EG, Yunusov  MS. Fatty acid composition of Oenothera biennis seed oil during storage, antioxidant activity. Chemistry of Natural Compounds. 2010; 46; 278–282.  
  7. Hudson BJF. Evening primrose (Oenothera spp.) oil and seed. Journal of American Oil Chemical Society. 1984; 61; 540-543.
  8. Wettasinghe  M, Shahidi  F, Amarowickz R. Identification and quantification of low molecular weight phenolic antioxidants in seeds of evening primrose (Oenothera  biennis L.). Journal of Agriculture and Food Chemistry. 2002; 50; 1267–1271.  
  9. Fedeli E, Paganuzzi V, Tiscornia E. Chemical composition of Chinese Oenothera (O. biennis) oil. Rivista Italiandelle Sostanze Grasse. 1976; 53; 25 – 32.  
  10. Montserrat-de la Paz S, Fernández-Arche  A, Ángel-Martín  M, García-Giménez, MD. The sterols isolated from evening primrose oil modulate the release of proinflammatory mediators. Phytomedicine. 2012; 19; 1072–1076.  
  11. Shahidi F, Amarowicz R, He Y, Wettasinghe M. Antioxidant activity of phenolic extracts of evening primrose (Oenothera biennis): a preliminary study. Journal of Food  Lipids.1997; 4 (2):75–86.   
  12. Shukla YN, Srivastava A, Kumar A. Aryl, lipid and triterpenoid constituents for Oenothera biennis.Indian Journal of Chemistry. 1999; 38B; 705-708. 
  13. Ahmad A, Ali M, Tandon S. New oenotheralanosterol A and B from the Oenothera biennis roots.Chinese Journal of Chemistry. 2010; 28;  2474 - 2478.   
  14. Ahmad A, Tandon S, Ahmad  J. New chemical constituents from the Oenothera biennis Roots. Asian Journal of Chemistry.  2012; 24(12); 5424-5426.  
  15. Kowalewski Z, Kowalska M, Skrzypezakowa L. Flavonoids of Oenothera biennis L. Dissertationes Pharmaceuticae et Pharmacologicae.1968; 20; 573-575.   
  16. Howard GZ, Mabry TJ, Raven HP. Distribution of flavonoids in twenty-one species of Oenothera. Phytochemistry. 1972; 11; 289−291.
  17. Yoshida T, Chou T, Matsuda M, Yasuhara T, Yazaki K, Hatano T, Nitta A, Okuda T, Woodfordin D, Oenothin A. Trimeric hydrolysable tannins of macro-ring structure with antitumor activity.Chemical and Pharmaceutical Bulletin.1991; 39;1157 – 1162.