Page 1 of 24 r Fo er Pe w vie Re ly On 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research URL: http://mc.manuscriptcentral.com/gnpl Page 3 of 24 LC-MS analysis of oils of Monodora myristica and Monodora tenuifolia and isolation of a novel cyclopropane fatty acid Al-Tannak, N.F1,2*, Ibrahim Khadra2, Igoli N.P3 and Igoli J.O4 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University. 1 2Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, United Kingdom. Fo Centre for Food Technology and Research, Benue State University Makurdi, Benue State Nigeria. 3 rP Department of Chemistry, University of Agriculture Makurdi, PMB 2373, Benue State, Nigeria. 4 Author and co-authors emails: Naser Al-Tannak, Email; Dr_altannak@hsc.edu.kw Ibrahim Khadra, Email: Ibrahim.khadra@strath.ac.uk Ngozi Igoli, Email: Ngozi_Igoli@yahoo.com John Igoli, Email: igolij@gmail.com iew ev - rR ee On *Corresponding author: Dr. Naser Al-Tannak, Email; Dr_altannak@hsc.edu.kw, Tel: +(965) 2463-6070, 0096599139913 Fax: +(965) 2463-6898 ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research 1 URL: http://mc.manuscriptcentral.com/gnpl Natural Product Research Abstract: Seeds of Monodora myristica and M. tenuifolia were extracted with hexane and the extracts were subjected to column chromatography, LC-MS and NMR analysis. In addition to masses of previously isolated compounds, other masses corresponding to unidentified compounds from the plants were detected. Using 2D NMR techniques, one of the fractions from M. tenuifolia was characterised as a novel 13-(2-butylcyclopropyl)-6,9-dodecadienoic acid. However, none of the compounds detected in LC-MS corresponded to the ones previously identified by GC-MS. Keywords: LC-MS analysis, NMR, Phytochemical analysis, Chromatography, Nigeria iew ev rR ee rP Fo On ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 4 of 24 2 URL: http://mc.manuscriptcentral.com/gnpl Page 5 of 24 1. Introduction Monodora myristica and Monodora tenuifolia are the only two Monodora species commonly used as spices in Nigeria. Several phytochemical and GC-MS studies have been carried out on their seed oils (Ukaegbu-Obi et al., 2015, Agiriga A.N, Siwela M, 2018). Previous studies have identified some volatile as well as non-volatile compounds (Ngouana T.K, 2015). The Monodora species are important food spices in most parts of West Africa and Asia. Monodora myristica Geartn. and Monodora tenuifolia Benth (Simo et al., 2018). (family Anonnacea) are widely distributed from Africa to Asia, Central and South America and Australia (Omobuwajo et al., 2003). They are native to West central and East Africa, extending from Sierra Leone to Uganda, Kenya Congo and Angola. Thus Monodora myristica is called African nutmeg. It is one of the most important spice trees of the evergreen forest of West Africa and mostly prevalent in the Southern part of Nigeria (Ravindra and Kallupurackac, 2001). Almost every part of the tree has economic importance. Nutritional value of M. myristica and M. tenuifolia centers on their usefulness as seasonings because of their aromatic flavour. However, the seeds which are embedded in the fruits are of major interest (Uhegbu et al., 2011, Njoku et al, 2012, Ezenwali et al, 2010). The seeds are also used in traditional medicine to relieve tooth ache, dysentery, diarrhea, dermatitis, headache and vermifuge (Ezenwali et al, 2010, Ishola et al., 2016). The plant extracts are reported to have good anti-oxidant activity and could be important in the management and treatment of stress induced diseases (Njoku et al. 2012, Akinwunmi and Oyedapo, 2015, Moukette et al., 2015). Several phytochemical studies to isolate the chemical constituents have been carried out on these plants and other Monodora species (Onyiriuka and Nwaji, 1972; Spiff et al., 1984; Mayunga et al., 2004, Igoli et al., 2011, Ntie-Kang et al., 2014), however, the diversity of the compounds isolated imply there could be much more interesting ones, also taking into account the wide range of bioactivities observed for the plant extracts. Though GC-MS studies of the seed oils (Adewole et al., 2013) have identified myristicin, caffeic acid, safrole, methyl eugenol, catechin, elemicin, quercetin, kaempferol, methyl isoeugenol, eugenol. These studies have not adequately identified all the useful compounds especially the non-volatile ones in the seed oils or non-polar extracts. LC-MS and NMR based metabolomics presents a new way of investigating natural products or plant extracts. It is holistic and unbiased and provides the most functional information about extracts or natural product mixtures. Dereplication studies (Roessner and Dias, 2013) of plant extracts is promising as previously known compounds are easily identified based on the intensities of their NMR or mass spectral peaks. It can also indicate the presence of unknown compounds based on chemical shifts in NMR and unknown elemental composition in MS. This makes for further studies of the extracts or the isolation of such novel compounds. However, no LC-MS studies or column purification which could identify or yield non-volatile and polar compounds have been reported for the seed oils of these plants. The aim therefore is to analyse the hexane extracts and purify by column chromatography the seeds oils of M. myristica and M. tenuifolia, and identify the constituents by LC-MS and NMR spectroscopy. iew ev rR ee rP Fo On ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research 2. Results and Discussion The yield of light brown oils was 42.0 g from M. myristica and 30.0 g from M. tenuifolia. Column fractions were oily but the more polar ones yielded white solids. The Characterization of MXHE-29 as 13-(2-butylcyclopropyl)-6,9-Dodecadienoic acid was achieved by NMR analysis and the fraction MXHE-29 on evaporation yielded compound 1 as a white solid and it gave a particularly interesting proton nmr spectrum (Figure S1). The proton spectrum indicated it was a single unsaturated fatty acid containing a cyclopropane 3 URL: http://mc.manuscriptcentral.com/gnpl Natural Product Research ring (Marcel et al., 1997). It also showed signals for olefinic (δH 5.37), allylic (δH 2.05) and bis-allylic (δH 2.79) protons hence the fatty acid chain must be doubly unsaturated (Knothe and Kenar, 2004, Marcel et al., 1997). A terminal methyl group was observed at 0.90 ppm. The cyclopropane methylene protons were the most shielded and were observed at -0.31 and 0.58 while the cyclopropane methine protons were identical and observed at 0.67 ppm. The rest of the proton signals was an envelope of CH2 protons from various parts of the fatty acid side chain. The 13C-DEPT spectrum (Figure S2) showed one carbonyl carbon at δC 180.4, two signals for olefinic doubled bonded carbons at 129.9 and 128.0, cyclopropane carbons at 10.9 (methylene) and 15.8 (methine) and a methyl group carbon at 14.1 ppm. Using 2D NMR experiments (Figures S4- S8) such as COSY, TOCSY, HSQC, HMBC and NOESY, the structure was deduced as follows: correlations in the COSY spectrum identified the vicinal protons in the fatty acid chain as well as the germinal ones on the cyclopropane ring. TOCSY was used to identify the spin systems in the various sections of the chain while HMBC was used to connect the sections and the positions of the carboxylic acid group and the olefinic double bonds. The HSQC was used to identify the carbons bearing the protons (Table S2). The CH3 group must be terminal as it showed correlations to only two carbons (C-17 and C18) in the HMBC and the carboxylic acid group must also be terminal as the two protons H-2 and H-3 that showed correlations to it were vicinal. The olefinic bonds must be on the carboxylic acid part of the chain as H-2 which showed HMBC correlations to it also showed correlations to C-4 and its proton H-4 showed correlations to C-6. Similarly, the cyclopropane ring must be on the terminal CH3 part of the chain as H-17 (attached to C-17) and H-16 which are vicinal showed correlations to C-20. Correlations from the bis-allylic protons H-8 confirmed the presence of adjacent double bonds. The linkage of the olefinic section to the cyclopropane ring was confirmed by 3J correlations from H-12 to C-14 and H20 to C-13. The compound was thus identified to be 13-(2-butylcyclopropyl)-6,9dodecadienoic acid (1) and confirmed by the mass at 306.2 in its LC-MS (Figure S9). The full chemical shift assignments are given in Table S2 and the structure in Figure S1. The retention times, masses in EI (positive and negative modes) and inferred compounds from the masses are given in Tables S3 and 4. This is based on previously isolated compounds from these plant materials or other Monodora species. iew ev rR ee rP Fo Previous phytochemical reports on the constituents of the seed oils of these plant materials were by GC-MS (Esuoso et al., 2000, Adewole et al., 2013). However, GC-MS reports on the constituents of the seed oils did not identify any of the alkaloids inferred from the LC-MS results (Tables S3 and S4). The major compounds identified under GC-MS were terpenes, fatty acids and triglycerols (Esuoso et al., 2000). The compounds identified under LC-MS in this study were mostly non-volatile and could not have been identified by GC-MS. The novel fatty acid is quite interesting and cyclopropane fatty acids (CFA) have been isolated from several sources such as millipedes (Oudejans, 1971), molluscs (Fenical et al., 1979), molds (Saito and Ochiai, 1998) and a lot of them have been synthesized (Shah et al., 2014, Arai et al., 1983). CFAs are typically found in microorganisms, seed oils of subtropical plants, protozoa and less commonly, within fats and phospholipids produced by animals. They are biosynthesized by methylenation of cis-unsaturated fatty acids via esterification to phospholipids with S-adenosylmethionine and catalyzed by CFA synthases (Shah et al., 2014). On ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 6 of 24 4 URL: http://mc.manuscriptcentral.com/gnpl Page 7 of 24 3. Experimental 3.1 General Experimental Procedures The plant seeds were purchased from Markets in Makurdi, Benue State Nigeria and were identified at the Forestry and Wild Life Department, University of Agriculture Makurdi. Voucher specimen were deposited at their herbarium and assigned voucher numbers UAM/FH/0324 for M. myristica and UAM/FH/0325 for M. tenuifolia. LC-MS was carried out using a Dual source LCMS, an Agilent 6130 with 1200 series LC and UV at 254 nm. NMR spectra were obtained using a Bruker Avance III (400 MHz) spectrophotometer using CDCl3 and TMS as internal standard. Spectra were processed using Mnova software. Column chromatography were carried out using silica gel MN-60 (Macherey-Nagel GmbH & Co. KG) in glass columns eluting gradient wise with hexane, hexane-ethyl acetate, ethyl acetate and ethyl acetate-methanol mixtures. TLC analysis was performed on pre-coated silica gel aluminium plates and spots were visualized using anisaldehyde-sulfuric acid reagent. Fo 3.2 Extraction of oils rP The ground seeds of M. myristica (200 g) and M. tenuifolia (150 g) were extracted with 500 mL of hexane each. The solvent was removed using a rotary evaporator to yield the oils. ee 3.3 Column chromatography About 3.0 g of the seed oils were separately adsorbed onto silica gel and the resultant slurry dried as much as possible. Glass columns (45 cm by 2.0 cm internal diameter) were packed with 150 g silica gel in hexane and allowed to settle. Thereafter, the dry slurries of the oils were separately loaded onto the packed columns and eluted gradient-wise using 5% incremental amounts of ethyl acetate in hexane, ethyl acetate and then methanol in ethyl acetate until 10% methanol in ethyl acetate. About 25 mL fractions were collected to obtain 70 fractions altogether. The fractions were allowed to dry, examined by TLC and similar fractions were combined and analysed by NMR. Compound 1 was obtained as fraction MXHE-29 and was not purified further. Its proton NMR showed interesting properties, it was therefore subjected to further 2D NMR analysis to confirm its structure. iew ev rR 3.4 NMR analysis On Extracts and fractions were dissolved in CDCl3 and transferred into NMR tubes. Their proton spectra were acquired and examined. Compound 1 (fraction MXHE-29) with interesting proton spectrum was subjected to carbon-13 and 2D (COSY, HMBC, HSQC, DEPT, NOESY and TOCSY) experiments to deduce their structures. ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research 3.5 LC-MS analysis About 1.0 mg of each extract was dissolved in methanol and used for the LC-MS analysis. LC-MS was acquired using the following parameters: Flow rate was 1.00 mL/min, injection volume was 10.00 µL. Run time and solvent composition are given in Table S1. For the MS, ionization mode was MM-ES+APCI in both positive and negative polarity and mass range was from 100-1000 mass units. 4. Conclusion 5 URL: http://mc.manuscriptcentral.com/gnpl Natural Product Research Oils from the seeds of Monodora myristica and Monodora tenuifolia were analysed by LCMS and NMR to identify the constituents. The compounds identified by LC-MS were mainly alkaloids of the indole and benzoisoquinoline type while the compounds identified by NMR were long chain unsaturated fatty acids and their triglycerides. A new cyclopropane fatty acid of the type 20:2 (6, 9) was isolated and identified as 13-(2-butylcyclopropyl)-6,9dodecadienoic acid. This study has also confirmed the presence of a CFA in the seed oil of a tropical plant. Conflict of interest The authors declare no conflict of interest. Fo Acknowledgements Authors are grateful to SIPBS, University of Strathclyde Glasgow, Scotland for the NMR and LC-MS analysis. iew ev rR ee rP On ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 8 of 24 6 URL: http://mc.manuscriptcentral.com/gnpl Page 9 of 24 References Adesomoju A, Ekundayo OO, Eramo TL, Into HR. 1991. Alkaloids of Monodora tenuifolia. Planta Medica. 57(4): 393-4. Adewole E, Ajiboye BO, Idris OO, Ojo OA, Onikan A, Ogunmodede OT, Adewumi DF. 2013. Phytochemical, Antimicrobial and GC-MS of African Nutmeg (Monodora myristica). International Journal of Pharmaceutical Science Invention. 2(5): 25-32. Agiriga A.N, Siwela M. 2018. Characterisation of Phytochemicals in Raw and Processed Monodora myristica (Gaertn.) Dunal Seeds by UPLC-MS. Pakistan Journal of Nutrition, 17: 344-354. Akinwunmi, K, Oyedapo O. 2015. In vitro anti-inflammatory evaluation of african nutmeg (Monodora myristica) seeds. Eur. J. Medi. Plants 8: 167-174 rP Fo Arai Y, Shimoji K, Konno M, Konishi Y, Okuyama S, Iguchi S, Hayashi M, Miyamoto T, Toda M. 1983. Synthesis and 5-Lipoxygenase Inhibitory Activities of Eicosanoid Compounds. Journal of Medicinal Chemistry. 26: 72-78. ee Esuoso KO, Lutz H, Bayer E and Kutubuddin M. 2000. Unsaponifiable Lipid Constituents of Some Underutilized Tropical Seed Oils J. Agric. Food Chem. 48: 231-234 rR Fenical W, S1eeper, HL, Pau1 VJ, Sta11ard MO, Hao H, Sun HH. 1979. Defensive chemistry of navanax and related opisthobranch molluscs. Pure and Applied Chemistry. 51: 1865-1874. ev Igoli JO, Gray AI, Clements CJ, Mouad H. 2011. Anti-Trypanosomal activity and cytotoxicity of some compounds and extracts from Nigerian medicinal plants. In Phytochemicals-Bioactivities and impact on Health. Intec Publishers Croatia. Chapter 16: p. 375-388 iew Ishola, I.O., V.O. Ikumawoyi, G.O. Afolayan and O.J. Olorife. 2016. Antinociceptive and anti-inflammatory properties of hydroethanolic seed extract of Monodora myristica (Annonaceae) in rodents. West Afr. J. Pharm. 27: 22-32. On Knothe, G, Kenar JA. 2004. Determination of the fatty acid profile by Spectroscopy. European Journal of Lipid Science and Technology. 106: 88–96 1H-NMR ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research Magoria N, Nyandoro SS, Munissi JJ, Heydenreich EM. 2013. Antimycobacterial and cytotoxicity evaluation of the constituents of Monodora carolinae. Tanz. J. Sci. 39: 12-18 Marcel SF, Jie LK, Mustafa J. (1997) High-Resolution Nuclear Magnetic Resonance Spectroscopy-Applications to Fatty Acids and Triacylglycerols. Lipids 32 (10), 1019-1034. Moukette B.M, Pieme C.A, Njimou J.R, Nya Biapa C.P, Marco B, Ngogang Y. 2015. In vitro antioxidant properties, free radicals scavenging activities of extracts and polyphenol composition of a non-timber forest product used as spice: Monodora myristica. Biol Res. 48(1): 15. 7 URL: http://mc.manuscriptcentral.com/gnpl Natural Product Research Ngouana, T.K.; Mbouna, C.D.J.; Kuipou, R.M.T.; Tchuenmogne, M.A.T.; Zeuko’o, E.M.; Ngouana, V.; Mallié, M.; Bertout, S.; Boyom, F.F. 2015. Potent and Synergistic Extract Combinations from Terminalia Catappa, Terminalia Mantaly and Monodora tenuifolia Against Pathogenic Yeasts. Medicines. 2, 220-235 Nkunya M H, Makangara JJ, Jonker, SA. 2004. Prenylindoles from Tanzanian Monodora and Isolona Species. Natural Product Research. 18(3): 253-258 Ntie-Kang F, Lifongo LL, Simoben CV, Babiaka SB, Sippl W, Mbaze LM. 2014. The uniqueness and therapeutic value of natural products from West African medicinal plants. Part I: Uniqueness and Chemotaxonomy RSC Advances. 4: 28728-28755 Nwaji MN, Onyiriuka SO, Taylor DAH. 1972. 6-(3-Methylbuta-1 ,3-dienyl)indole from Monodora tenuifolia. Journal Chemical Society Chemical Communications. p 327 Fo Oudejans RCHM, van der Horst DJ, van Dongen JCPM. 1971. Isolation and Identification of Cyclopropane Fatty Acids from the Millipede Graphidostreptus tumuliporus (Karsch) (Myriapoda: Diplopoda) Biochemistry. 10(26): 4938-4941 ee rP Simo M.K, Nguepi M.D, Sameza M.L, Toghueo R.K, Fekam F.B, Froldi G. 2018. Cameroonian medicinal plants belonging to Annonaceae family: radical scavenging and antifungal activities, Natural Product Research. 32:17, 2092-2095 rR Ukaegbu-Obi, K.M., M.O. Meribe and C.E. Odo. 2015. Assessment of antimicrobial activity of aqueous and ethanolic extracts of Monodora myristica (Ehuru) seeds. Mint. J. Pharm. Med. Sci. 4: 1-2. ev Roessner U, Dias DA. (Eds) (2013). Metabolomics Tools for Natural Product Discovery: Methods and Protocols. Methods in Molecular Biology 1055. Springer New York. 311 p iew Saito T, Ochiai H (1998). Fatty Acid Composition of the Cellular Slime Mold Polysphondylium pallidum. Lipids. 33: 327–332 On Shah S, White JM, Williams SJ. 2014. Total syntheses of cis-cyclopropane fatty acids: dihydromalvalic acid, dihydrosterculic acid, lactobacillic acid, and 9,10methylenehexadecanoic Acid. Organic and Biomolecular Chemistry. 12: 9427 ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 10 of 24 Spiff AI, Duah FK, Slatkin, DJ, Schiff L Jr. 1984. Alkaloids of Monodora tenuifolia. Planta Medica. 50(5): 455 8 URL: http://mc.manuscriptcentral.com/gnpl Page 11 of 24 LC-MS analysis of oils of Monodora myristica and Monodora tenuifolia and isolation of a novel cyclopropane fatty acid Al-Tannak, N.F1,2*, Ibrahim Khadra2, Igoli N.P3 and Igoli J.O4 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, Jamal Abdul Nasser St, Kuwait. 1 2Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, United Kingdom Centre for Food Technology and Research, Benue State University Makurdi, Benue State Nigeria 3 Fo Department of Chemistry, University of Agriculture Makurdi, PMB 2373, Benue State, Nigeria. 4 rR ee rP Abstract Seeds of Monodora myristica and M. tenuifolia were extracted with hexane and the extracts were subjected to column chromatography, LC-MS and NMR analysis. In addition to masses of previously isolated compounds, other masses corresponding to unidentified compounds from the plants were detected. Using 2D NMR techniques, one of the fractions from M. tenuifolia was characterised as a novel 13-(2-butylcyclopropyl)-6,9-dodecadienoic acid. However, none of the compounds detected in LC-MS corresponded to the ones previously identified by GC-MS. iew ev On Keywords: LC-MS analysis, NMR, Phytochemical analysis, Chromatography, Nigeria ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research *Corresponding author: Dr. Naser Al-Tannak, Email; Dr_altannak@hsc.edu.kw, Tel: +(965) 2463-6070, 0096599139913 Fax: +(965) 2463-6898 URL: http://mc.manuscriptcentral.com/gnpl Natural Product Research Table S1. Solvent composition for LC-MS analysis. Run time (min) Solvent A (water with 5mM Solvent B (Acetonitrile with 5mM ammonium acetate) % 0.00 95.0 5.0 1.48 95.0 5.0 8.50 0.0 100.0 13.50 0.0 100.0 16.50 95.0 5.0 18.00 95.0 5.0 iew ev rR ee rP ammonium acetate) % Fo On ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 12 of 24 URL: http://mc.manuscriptcentral.com/gnpl Page 13 of 24 Table S2. Chemical shift assignments for compound 1 in CDCl3 Position 1H 1 - 2 δ ppm (mult, J (Hz) δ ppm 13C HMBC (2J, 3J) COSY 180.4 (C) - - 2.36 (t, 7.5) 34.1 (CH2) C-1, C-3, C-4 H-3 3 1.66 (p, 7.1) 24.6 (CH2) C-1. C-4 H-2, H-4 4 1.36 (m) 29.4 (CH2) C-6, H-5, H-6 5 2.05 (dq, 13.1, 6.6) 27.2 (CH2) C-6/7, H-6, H-7 6 5.37 (m) 129.9 (CH) C-5, C-8 H-5, H-8 7 5.37 (m) 129.9 (CH2) C-8, C-9 H-5, H-8 8 2.79 (t, 6.3) 25.7 (CH2) C-6/7, C-9/10 H-7, H-9 9 5.37 (m) 128.0 (CH2) C-11 H-8, H-11 10 5.37 (m) 128.0 (CH2) C-11, C-12 H-8, H-11 11 2.05 (dq, 13.1, 6.6) 27.2 (CH2) C-9/10, C-12 H-10, H-12 12 1.36 (m) 29.4 (CH2) C-14, C-13 H-11, H-13 13 1.17 (m) 28.8 (CH2) C-15 H-15, H-17 14 0.67 (p, 5.6, 4.7) 15.8 (CH) C-15 H-12, H-20 15 0.67 (p, 5.6, 4.7) 15.8 (CH) C-14, C-20 16 1.17 (m) 28.8 (CH2) C-15, C-16, C-17, H-15, H-17 iew ev rR ee rP Fo On C-20 H-16, H-20 ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research 17 1.38 (m) 31.9 (CH2) C-15, C-19, C-20 H-16, H-18 18 1.31 (m) 22.8 (CH2) C-17, C-19 H-17, H-19 19 0.90 (t, 6.7) 14.1 (CH3) C-17, C-18 H-18 20 -0.31 (td, 5.3, 3.9) 10.9 (CH2) C-13, C-14/15 H-14, H-15 0.58 (m) URL: http://mc.manuscriptcentral.com/gnpl Natural Product Research Table S3. LC-MS retention times and masses for Monodora myristica oil Retention time m/z (mass) Inference or Compound* 1.376-1.908 145.2 Indole-5-carbaldehyde 5.020 152.2 Eucalyptol 6-(4-oxobut-2-enyl)-indole or 6-(3-methylbuta-1,3-dien- 10.286-10.801 184.2 1-yl)-indole or 3-Dimethylallylindole 5.050 Fo 236.9 9-Hexadecinal 270.2 Heptadecanoic acid 5.321-5.440 272.2 Liriodenine 8.408 297.0 10.806 340.2 9.323-9.853 367.2 5.384 ee rP Stepharine Laurelliptine rR Annonidine ev *Based on SciFinder data base hits for compounds previously isolated from Monodora spp iew On ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 14 of 24 URL: http://mc.manuscriptcentral.com/gnpl Page 15 of 24 Table S4. LC-MS retention times and masses for Monodora tenuifolia Retention time m/z Inference or Compound* 0.607 145.2 Indole-5-carbaldehyde 10.275 184.2 6-(4-oxobut-2-enyl)-indole or 6-(3-methylbuta-1,3-dien1-yl)-indole 5.297 Liriodenine Kaempferol 297.0 Stepharine 5.934 302.2 Quercetin 10.804 340.2 10.805 341.9 9.326-10.787 367.2 Annonidine 10.804 340.2 Sparsiflorine 10.805 341.9 Magnoflorine or Laurelliptine 12.473 560.4 Cannabisin B 5.325-5.390 306.2 Compound 1 8.405 rP 288.2 ee 6.591 273.8 Fo Sparsiflorine Magnoflorine or Laurelliptine iew ev rR On *Based on SciFinder data base hits for compounds previously isolated from Monodora spp ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research URL: http://mc.manuscriptcentral.com/gnpl Natural Product Research 1 20 13 9 11 5 7 3 OH 1 15 19 17 Figure S1. Structure of isolated cyclopropane fatty acid. iew ev rR ee rP Fo On ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 16 of 24 URL: http://mc.manuscriptcentral.com/gnpl O 3400 3200 3000 2800 2600 2400 2200 2000 I (t) 2.79 J(6.33) F (p) 1.65 J(7.10) H (t) 2.36 J(7.52) J (m) 5.37 C (p) 0.67 J(5.57, 4.74) E (m) 1.31 G (dq) 2.05 J(13.07, 6.61) B (m) 0.58 1800 A (td) -0.31 J(5.28, 3.90) K (m) 1.17 1400 1000 800 600 400 200 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) D138824.1.fid — Person 100-16 — MXHE-29 — @proton CDCl3 {C:\NMRdata} AIG 27 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 iew ev rR Figure S2. Proton spectrum for compound 1 in CDCl3 On ly URL: http://mc.manuscriptcentral.com/gnpl -200 1.00 62.44 1.59 8.69 2.15 0.99 6.46 3.73 6.01 0.76 2.26 0 ee rP 11.5 1600 1200 D (m) 0.90 Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research 5.38 5.37 5.36 5.36 5.35 2.79 2.38 2.36 2.34 2.33 2.08 2.06 2.04 2.02 1.69 1.69 1.67 1.65 1.63 1.61 1.43 1.42 1.41 1.40 1.39 1.38 1.36 1.36 1.35 1.34 1.34 1.33 1.32 1.32 1.31 1.30 1.28 1.20 1.19 1.18 1.17 1.16 1.15 0.93 0.92 0.92 0.91 0.91 0.90 0.89 0.89 0.88 0.87 0.68 0.66 0.65 0.61 0.60 0.58 -0.29 -0.30 -0.31 -0.32 -0.32 Page 17 of 24 -0.5 34.11 31.92 30.18 29.69 29.67 29.63 29.59 29.44 29.35 29.24 29.13 29.06 29.03 28.72 28.70 27.21 25.62 24.67 22.68 15.76 14.06 10.90 130.15 129.96 129.68 128.06 127.89 Page 18 of 24 400000 350000 300000 250000 200000 150000 100000 50000 0 -50000 Fo -100000 -150000 rP -200000 -250000 ee 180 170 160 150 140 130 120 110 100 90 f1 (ppm) D138824.2.fid — Person 100-16 — MXHE-29 — @deptq135 CDCl3 {C:\NMRdata} AIG 27 80 70 60 50 40 30 20 Figure S3. 13-Carbon spectrum for compound 1 in CDCl3 iew ev rR On ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 180.42 Natural Product Research URL: http://mc.manuscriptcentral.com/gnpl 10 Page 19 of 24 10 20 30 40 50 70 80 90 rP 4.5 4.0 3.5 120 130 3.0 2.5 f2 (ppm) rR 5.0 110 ee 5.5 100 2.0 1.5 1.0 0.5 D138824.3.ser — Person 100-16 — MXHE-29 — @HSQC CDCl3 {C:\NMRdata} AIG 27 Figure S4. HSQC spectrum for compound 1 in CDCl3 iew ev On ly URL: http://mc.manuscriptcentral.com/gnpl 0.0 -0.5 f1 (ppm) 60 Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research Natural Product Research -0.5 0.0 0.5 1.0 1.5 2.5 3.0 3.5 rP 4.0 4.5 ee 5.0 4.5 4.0 5.5 6.0 rR 5.5 5.0 3.5 3.0 2.5 f2 (ppm) 2.0 1.5 1.0 0.5 0.0 D139493.3.ser — Person 100-16 — MXHE-29 — @NOESY CDCl3 {C:\NMRdata} AIG 9 iew ev Figure S5. NOESY spectrum for compound 1 in CDCl3 On ly URL: http://mc.manuscriptcentral.com/gnpl -0.5 f1 (ppm) 2.0 Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 20 of 24 Page 21 of 24 -0.5 0.0 0.5 1.0 1.5 2.5 3.0 3.5 4.0 rP 5.0 4.5 5.0 5.5 ee 5.5 4.5 4.0 3.5 6.0 3.0 2.5 f2 (ppm) 2.0 1.5 1.0 0.5 D139493.4.ser — Person 100-16 — MXHE-29 — @TOCSY CDCl3 {C:\NMRdata} AIG 9 rR Figure S6. TOCSY spectrum for compound 1 in CDCl3 iew ev On ly URL: http://mc.manuscriptcentral.com/gnpl 0.0 f1 (ppm) 2.0 Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research Natural Product Research 10 20 30 40 50 60 70 90 100 110 120 130 140 rP 5.0 4.5 160 170 ee 5.5 150 4.0 3.5 180 3.0 2.5 f2 (ppm) 2.0 1.5 1.0 0.5 0.0 D138824.10.ser — Person 100-16 — MXHE-29 — @HMBC CDCl3 {C:\NMRdata} AIG 27 rR Figure S7. HMBC spectrum for compound 1 in CDCl3 iew ev On ly URL: http://mc.manuscriptcentral.com/gnpl -0.5 f1 (ppm) 80 Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 22 of 24 Page 23 of 24 -0.5 0.0 0.5 1.0 1.5 2.5 3.0 3.5 4.0 rP 5.0 4.5 5.0 ee 5.5 ag/John_MXHE-29 — 4.5 4.0 3.5 3.0 5.5 2.5 f2 (ppm) 2.0 1.5 1.0 0.5 rR Figure S8. COSY spectrum for compound 1 in CDCl3 iew ev On ly URL: http://mc.manuscriptcentral.com/gnpl 0.0 -0.5 f1 (ppm) 2.0 Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Natural Product Research Natural Product Research iew ev rR ee rP Fo Figure S9. LC-MS total ion chromatogram and spectrum for compound 1 On ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 24 of 24 URL: http://mc.manuscriptcentral.com/gnpl