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Chemical constituents from Myristica fragrans fruit a

b

ac

K. Sajin Francis , Eringathodi Suresh & Mangalam S. Nair a

Chemical Sciences & Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram, 695019, KeralaIndia b

CSIR-Central Salt & Marine Chemicals Research Institute, Bhavnagar, GujaratIndia c

Academy of Scientific and Innovative Research (AcSIR), New Delhi, India Published online: 10 Jul 2014.

To cite this article: K. Sajin Francis, Eringathodi Suresh & Mangalam S. Nair (2014) Chemical constituents from Myristica fragrans fruit, Natural Product Research: Formerly Natural Product Letters, 28:20, 1664-1668, DOI: 10.1080/14786419.2014.934236 To link to this article: http://dx.doi.org/10.1080/14786419.2014.934236

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Natural Product Research, 2014 Vol. 28, No. 20, 1664–1668, http://dx.doi.org/10.1080/14786419.2014.934236

Chemical constituents from Myristica fragrans fruit K. Sajin Francisa, Eringathodi Sureshb and Mangalam S. Naira,c*

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a Chemical Sciences & Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram 695019, Kerala, India; bCSIR-Central Salt & Marine Chemicals Research Institute, Bhavnagar, Gujarat, India; cAcademy of Scientific and Innovative Research (AcSIR), New Delhi, India

(Received 9 April 2014; final version received 10 June 2014) A neolignan, erythrosurinamensin and a diaryl phenyl propanoid, virolane were isolated from Myristica fragrans for the first time. Apart from these two, previously known steroids, other lignans and neolignans were isolated from the fruit pericarp of M. fragrans. The structures of the compounds were identified by employing various spectroscopic methods. Keywords: Myristica fragrans pericarp; Myristicaceae; surinamensin; virolane; neolignans

1. Introduction Myristica fragrans Houtt. (Myristicaceae) commonly known as ‘nutmeg’ is a well-known aromatic evergreen tree found in India, Indonesia, Sri Lanka and South Africa (Jaiswal et al. 2009). Nutmeg mace is well known all over the world as an important spice. Nutmeg has been shown to possess analgesic (Sonavane et al. 2001), antifungal (Nadkarni 1998), antimicrobial (Takikawa et al. 2002), antiinflammatory (Olajide et al. 1999), as well as hepatoprotective (Morita et al. 2003) activities in various in vitro and in vivo studies. With regard to its medicinal and commercial value, the dried kernel (seed) and mace/aril are the most exploited parts. About 30 non-volatile compounds have been reported from seed and mace of M. fragrans so far. Among these, licarin A (Aiba et al. 1973) has been found to have anti tuberculosis activity (Leon-Diaz et al. 2010), while guaiacin (Hattori et al. 1986) has been found to have anticancer activity (Lee et al. 2007). Several neolignans isolated from the seed and mace have shown antibacterial activity (Hattori et al. 1988). Another important part of M. fragrans is the fruit pericarp, which is used in food preparation. Whereas the essential oil of nutmeg fruit pericarp has been studied in detail (Choo et al. 1999), the non-volatile constituents from the fruit pericarp have not been studied so far. Herein, we report the isolation of eight known compounds viz., accuminatin, licarin A, 7-methoxy-3methyl-5-((E)-prop-1-enyl)-2-(3,4,5-tri methoxy phenyl)-2,3-dihydrobenzofuran, licarin B, 0 guaiacin, elimicin, erythro-(7S,8R)-D8 -7-acetoxy-3,4,30 ,50 -tetramethoxy-8-O-40 -neolignan, ery0 thro-(7S,8R)-D8 -7-hydroxy-3,4,30 ,50 -tetramethoxy-8-O-40 -neolignan as well as that of virolane and surinamensin which have previously not been reported from any part of M. fragrans. Surinamesin has been found to have anti-leishmanial activity against Leishmania donovani (Barata et al. 2000).

*Corresponding author. Email: [email protected] q 2014 Taylor & Francis

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2. Results and discussions Acetone extract of nutmeg pericarp was subjected to column chromatography on silica gel and neutral alumina which afforded accuminatin (1) (Yu et al. 2000), licarin A (2), 7-methoxy-3-methyl-5-((E)prop-1-enyl)-2-(3,4,5-tri methoxy phenyl)-2,3-dihydrobenzofuran (3) (Dean & Cooks 1982), licarin B (4) (Aiba et al. 1973), stigmasterol, b-sitosterol, guaiacin (7), elimicin (8) (Tommy et al. 1998), 0 virolane (9) (Filho et al. 1973), erythro-(7S,8R)-D8 -7-acetoxy-3,4,30 ,50 -tetramethoxy-8-O-40 0 neolignan (10) (Isogai et al. 1973), erythro-(7S,8R)-D8 -7-hydroxy-3,4,30 ,50 -tetramethoxy-8-O-40 neolignan (11) (Isogai et al. 1973) and surnamensin (12) (Barata et al. 1978) along with palmitic acid (Figure 1). To the best of our knowledge, this is the first report on the isolation and identification of compounds 9 and 12 from M. fragrans. All the compounds were identified by the comparison of their spectral data with that in literature. Structure of 3 was also confirmed by single crystal X-ray structure determination, which also further helped to confirm the stereochemistry of compounds 1, 2 and 4 (Figure 2). Molecular formula of compound 9 was found to be C17H18O4 from HR-MS (287.1274 [M þ H]þ). The compound showed a broad absorption at 3416 cm21 in IR spectrum which indicated the presence of hydroxyl group. The 1H NMR spectrum of compound 9 indicated the presence of three methylene groups [d 1.87 (m), 2.54 (t), 2.58 (t)]. The protons at d 2.54 and d 2.58 showed a correlation with protons at d 1.87 ppm in 1H– 1H correlation spectroscopy which suggested that there is a propane system attached to two aromatic rings. The 13C NMR and DEPT spectra suggested the presence of 17 carbons, which include a methyl, 4 methylene, 6 methine, and 6 γ R1

6

β

α′

1′

α

4′

O

β′

H

OCH3

2

R2

γ′ H

H

R3

H

H H

H

HO

1. R1=H, R2=R3=OCH3

H

HO 5. β-sitosterol

2. R1=H, R2=OH, R3=OCH3

6. stigmasterol

3. R1=R2=R3=OCH3 4. R1=H, R2=R3=O-CH2-O H3CO HO

1

8

OCH3

9 H3CO

5 6′ 5′

8'

9′

2

H3CO

2′

6

8 7

H3CO 9

1

3′

2 OH 6″

8. elimicin

OCH3

3

6′ 5′

9. virolane

OH 7. guaiacin OH H3CO H3CO

2

R 7

8 9

H3CO OCH3

6 O 4′

H3CO 10. R=OAc

H3CO

2′

OCH3

8′

5 6′

7′

11. R=OH

Figure 1. Structures of isolated compounds 1 –12.

OCH3 O

9′

12. surinamensin

2″

O O

5″

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Figure 2. ORTEP diagram of the compound 3 (40% probability factor for the thermal ellipsoids).

quaternary carbons. Among them, the signals at d 55.3 and 100.3 could be attributed to the methoxy and methylenedioxy carbons respectively. Based on the above data, the structure was arrived as virolane (9) and confirmed from comparison of spectral data of reported in the literature. Molecular formula of the compound 12 (C22H28O6) was established from HR-MS (411.1773 [M þ Na]þ). IR spectrum showed broad absorption at 3501 cm21 suggesting the presence of hydroxyl group. 1H NMR spectrum of compound 12 showed the presence of two methyl groups [d 1.19 (d), 1.89 (dd)], four methoxy groups [d 3.82 (s), 3.85 (6H, s), 3.90 (s)], a hydroxyl group [d 3.59 (s) exchangeable with D2O], five aromatic protons and two olefinic protons [d 6.20 (1H, dq), J ¼ 16, 1.5 Hz and d 6.38 (1H, dd), J ¼ 16 and 6.5 Hz]. The protons at d 1.89 showed a correlation with protons appearing at d 6.20 in 1H – 1H correlation spectrum. A doublet at d 4.82 integrating for one proton and a doublet of quartet at d 4.36 integrating for one proton having correlation in 1H – 1H in correlation spectrum with J ¼ 3 Hz indicated that they are erythro to each other. The 13C NMR and DEPT spectra showed the presence of 22 carbons which include six methyl, nine methine and seven quaternary carbons. Among them, signals at d 55. 8, 55.8, 55.9 and 60.8 could be attributed to methoxy groups. Thus, the structure of the compound was confirmed as surinamensin (12) by comparing with the reported data. 3. Experimental 3.1. General TLC was carried out on Si gel 60 F254 (Merck, Bangalore, India) and CC on Si gel 100– 200 mesh (Merck), CC on neutral alumina, 60 –325 m (Spectrochem). 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded on a Bruker AMX 500 NMR Spectrometer (Bruker AMX, Fallanden, Switzerland) using CDCl3 with tetramethylsilane as internal standard. IR spectra were recorded on Bruker FT-IR spectrometer (Bruker AXS, The Helios, Singapore). Mass spectra were recorded under ESI/HR-MS at 61,800 resolution using Thermo Scientific exactive mass spectrometer (Thermo Fischer Scientific, Bremen, Germany). 3.2. Plant material M. fragrans fruits were collected in January 2013, from Ernakulam in Kerala State, India. Voucher specimen (TBGRI-60679) has been deposited in a herbarioum of Jawaharlal Nehru

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Tropical Botanical Garden and Research Institute, Palode, Trivandrum in Kerala State. The pericarp, seed and mace were separated and used for the study. 3.3. Extraction and isolation Of the dry and powdered fruit pericarp, 750 g was extracted with acetone at room temperature, which yielded 32 g of crude extract. This was then suspended in ethyl acetate and stirred for an hour. The ethyl acetate soluble part (19 g) was subjected to CC on silica gel (100 –200 mesh) and eluted with hexane – ethyl acetate mixtures of increasing polarities to give 15 fraction pools. Fraction pools 1– 3 (2.05 g) on crystallisation in hexane yielded palmitic acid (486 mg). Fraction pool 4 was subjected to CC on silica gel (100 –200 mesh) using hexane – ethyl acetate (9:1) as eluant and followed by crystallisation in ethyl acetate/hexane afforded licarin B (280 mg). Fraction 5 was submitted to CC on silica gel by eluting with hexane –ethyl acetate (7:1) which afforded elimicin (156 mg). Fraction 7 when subjected to CC on silica gel by eluting with hexane – ethyl acetate (9:1) gave six sub-fraction pools, named 7a –7f. The fraction 7b on crystallisation in hexane afforded mixture of stigmasterol and b-sitosterol (150 mg). Fraction 7c when subjected to CC on neutral alumina, afforded accuminatin (44 mg), 7-methoxy -3-methyl5-((E)-prop-1-enyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihydrobenzofuran (20 mg), virolane (6 mg) and licarin A (15 mg). Fraction 7d on crystallisation in hexane –ethyl acetate afforded guaiacin (10 mg). Fraction 8 when submitted to CC on neutral alumina by eluting with hexane – 0 ethyl acetate (9:1) yielded erythro-(7S,8R)-D8 -7-acetoxy-3,4,30 , 50 -tetramethoxy-8-O-40 0 neolignan (30 mg) and erythro-(7S,8R)-D8 -7-hydroxy-3,4,30 ,50 -tetramethoxy-8-O-40 -neolignan (10 mg). Fraction 9 when submitted to CC on neutral alumina by eluting with hexane – ethyl acetate (8:2) yielded erythro surinamensin (50 mg). 3.4. X-ray crystallography of compound 3 Single crystal with suitable dimension was chosen under an optical microscope and mounted on a glass fibre for data collection. Intensity data for all the four crystals were collected using MoKa ˚ ) radiation on a Bruker SMART APEX diffractometer (Bruker AXS, Madison, (l ¼ 0.71073 A WI, USA) equipped with CCD area detector at 293 K for compound 3. The data integration and reduction were processed with SAINT software. An empirical absorption correction was applied to the collected reflections with SADABS. The structures were solved by direct methods using SHELXTL and were refined on F 2 by the full-matrix least-squares technique using the program SHELXL-97. All non-hydrogen atoms were refined anisotropically till convergence is reached. Hydrogen atoms attached to the organic moiety are either located from the difference Fourier map or stereo chemically fixed. Crystal data for compound 3: molecular formula: C22H26O5, formula weight: 370.43, crystal ˚ , b ¼ 8.721(2) A ˚ , c ¼ 27.664(7) A ˚, system: orthorhombic, space group: P212121, a ¼ 8.220(2) A ˚ 3 , Z ¼ 4, density Dx V ¼ 1983.1(8) A ¼ 1.241 Mg m 3, absorption coefficient ( m ) ¼ 0.087 mm 21, reflections collected ¼ 9053, independent reflections ¼ 3862, Rint ¼ 0.0299, number of parameters ¼ 250, goodness-of-fit on F 2 ¼ 1.048, R indices (all data) R1 ¼ 0.0945, wR2 ¼ 0.1586, final R indices [(I $ 2s(I)] R1 ¼ 0.0662, wR2 ¼ 0.1450. The crystal structure of compound 3 has been deposited with the Cambridge Crystallographic Data Centre with deposition number CCDC 984377. 4. Conclusion Non-volatile chemical constituents (1 –12) were isolated from M. fragrans fruit pericarp for the first time. Compounds 9 and 12 are reported from this plant for the first time. The structures of

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these compounds were established on the basis of spectroscopic techniques and X-ray crystallographic data. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S14. Acknowledgements

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Mrs Soumini Mathew and Mrs Viji S. for NMR and mass spectral studies. Financial support from CSIR, New Delhi.

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Chemical constituents from Myristica fragrans fruit.

A neolignan, erythrosurinamensin and a diaryl phenyl propanoid, virolane were isolated from Myristica fragrans for the first time. Apart from these tw...
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