Abstract: Background and Objective: Monodora myristica (M. myristica) seeds are processed locally using various indigenous knowledge systems (IKS) based processing techniques like boiling, roasting and frying for varying lengths of time. It is important to determine the effect of these processing methods on its phytochemical constituents, a step necessary in order to explain its nutritional/medicinal use. This paper determined the effects of different cooking methods (boiling and roasting) and cooking times (10, 20 and 30 min) on the phytochemical constituents of M. myristica seeds using ultra performance liquid chromatograph-mass spectrometer (UPLC-MS). Materials and Methods: M. myristica seeds were thermally processed through boiling and roasting for varying lengths of time. Metabolite profiling using UPLC-MS was utilized to identify phytochemicals in raw and processed seeds. Metabolites were characterized by their UV-vis spectra, retention times relative to external standards, mass spectra and comparison to in-house database, phytochemical dictionary of natural products database and reference literature. Results: A total of 32 metabolites were identified, including terpenoids, sterols, alkaloids, fatty acids, saponins, flavonoids, glycosides and coumarins. Processing induced changes in phytochemical composition, more phytochemicals were identified in the roasted samples and the raw (control) sample had the least (four) number of phytochemicals. Conclusion: These findings are promising as they indicate that suitable processing techniques could be established and then applied in the development of new functional foods from whole M. myristica seeds or its extracts. M. myristica seed can be considered a good source of phytochemicals.
INTRODUCTION
Monodora myristica is a perennial edible plant of the Annonaceae or custard apple family of flowering plants1,2. The seed is a popular spice used in cooking to add flavour and thicken dishes3. It is variously known as Iwor amongst the Itsekiris, Ikposa (Benin), Ehiri or Ehuru (Ibo), Gujiya dan miya (Hausa) and Ariwo, arigbo, Abo lakoshe or eyi naghose (Yoruba), Ehinawosin (Ikale), Uyengben (Edo), Fausse noix de muscade (French)4-6. Studies have shown that almost every part of the tree is important economically and a number of medicinal properties have been ascribed to various parts of this highly esteemed plant7. The most economically important parts are the seeds8. The presence of bioactive compounds in the plant makes it possible for the seeds to be used in traditional medicine as well as a spice in local foods9. The aromatic seeds are antiemetic, astringent, anti-inflammatory, antipyretic, aperient, stimulant, stomachic, tonic and they are added to medicines to impart stimulating properties8,10,11.
The presence of bioactives in edible plants is largely influenced by several factors such as genetic factors, environmental conditions, degree of ripening, variety, processing techniques and storage methods12. Monodora myristica seeds are processed locally (in the sub-Sahara African regions) using various indigenous knowledge systems (IKS) based processing techniques like boiling, roasting and frying for varying lengths of time. They are then dehulled and crushed into flour for use in local dishes, such as the West African "kunu," "tuwo," and "waina". Some natives simply dehull using stone and crush the raw seed for use in local dishes.
Feyisayo and Oluokun13 identified phenolics present in the seeds of M. myristica by gas chromatography (GC) coupled to flame ionization detector (FID). They estimated the total phenolic content as 1478.32 mg/100 g and concluded that M. myristica is a good source of phenolics. Other reports on the phytochemical screening of the seeds of this plant revealed the presence of phenolic compounds, including flavonoids and tannins, cyanogenic glycosides, alkaloids, arocine, lactose, terpene, resins, fiberro-latic oils, anthraquinones, saponins, , steroids, oxalates and phytates14,15. Processing of plants for consumption may cause chemical reactions that result in changes in phytochemical constituents and hence health-benefitting properties of the plant16. A comprehensive review of the literature showed that there is no report on the effect of processing methods on the metabolite composition of M. myristica seed flour. Due to their overwhelming advantages, UPLC-MS based methods are a suitable tool for the investigation of natural products in food17,18. The current study was conducted to characterize the secondary metabolites present in raw and processed M. myristica seeds using UPLC-MS instrument equipped with time of flight (TOF) detectors.
MATERIALS AND METHODS
Reagents and chemicals: Unless otherwise stated, all the chemicals/reagents used were of analytical grade from Sigma-Aldrich Co., Ltd (Steinheim, Germany). Water was purified by using a Milli-Q system (Millipore, Bedford, USA).
Sample: Monodora myristica seeds were purchased from a local market, in Ado-Ekiti, Ekiti State, Nigeria.
Sample preparation: The seeds were cleaned and extraneous materials like dry leaves and stones removed. Samples were divided into seven portions and prepared using the method of Mbah et al.19 with slight modification. The first portion was raw and it served as the control. The second, third and fourth portions were boiled (100°C) in a pot of tap water in a ratio of 1:3 (weights of the seeds to volume of water) for varying times: 10, 20 and 30 min. After boiling, the seeds were oven dried at 100°C for 5 h, dehulled and milled into fine flour. The remaining fifth, sixth and seventh portions were roasted (120°C) for different times (10, 20 and 30 min), dehulled and milled into fine flour. The control seeds were dehulled and milled without any thermal processing. Flour samples were defatted for 4 h using a Buchi 810 Soxhlet Fat Extractor (Flawil, Switzerland) and packaged in labelled polythene bags (CO: raw flour, B10, B20, B30: flour from seeds boiled for 10, 20 and 30 min, respectively, R10, R20, R30: flour samples from seeds roasted for 10, 20 and 30 min, respectively). The packaged flour samples were stored in a cool (±4°C) dry place until required for analysis.
Preparation of methanolic extracts: Flour samples were extracted (1:5 w/v) exhaustively with methanol. The extracts separately were concentrated to dryness under reduced pressure in a rotary evaporator (40°C). Dried extracts were re-dissolved in methanol for further experiments.
Ultra-high performance liquid chromatography mass spectrometry (UPLC-MS) analysis: First, 50 mg of each dried sample was re-dissolved in 5 mL LC-MS grade methanol. The sample solutions were then prepared to a concentration of 200 ppm in methanol and filtered into analysis vial using a filter membrane. The samples were analysed with a UPLC-MS, which consisted of mass spectrometer (Waters Corporation, Micromass MS Technologies, Manchester, UK) coupled to an ultra high performance liquid chromatography System (Alliance 2695 UPLC system, Waters Corporation, Milford, MA, USA). The mass spectrometer was equipped with a UV-VIS and time of flight (TOF) detectors, whose accurate mass measurements provide direct reliable information on molecular formulas. Compounds were separated on an Atlantis T3 C18 column (Waters Corporation, Milford, USA, 100 mm×2.1 mm, 3 μm particle size) using 0.5% aqueous formic acid (solvent A) and 0.5% formic acid in 50/50 v/v acetonitrile:methanol (solvent B). Column temperature was maintained at 40°C. A stepwise gradient from 10-90% solvent B was applied at a flow rate of 0.2 mL min–1 for 26 min. Mass spectra data were recorded for a mass range m/z 100-m/z 1000. Capillary voltage and cone voltage were set at 3 kV and 30 V, respectively. In all searches for elemental composition, 0-100 atoms of carbon, 0-100 atoms of hydrogen, 0-50 atoms of oxygen and 0-50 atoms of nitrogen were included. Experimental and theoretical mass were separated by an error not larger than 5 ppm. Mass spectral data was processed using OpenLynx (Waters Corporation, Milford, MA, USA).
RESULTS AND DISCUSSION
A total of 32 metabolites were identified including triterpenes, alkaloids, saponins, flavonoids and coumarins. Metabolites were identified by their UV-vis spectra, retention times relative to external standards, mass spectra and comparison to in-house database, phytochemical dictionary of natural products database and reference literature. Alkaloids accounted for the highest abundance in the samples (Table 1). Alkaloids are a heterogeneous class of secondary metabolites traditionally defined as basic (alkali like), nitrogen containing organic constituents that occur mainly in plants20.
| Table 1: | Metabolites from the methanolic extracts of raw and processed Monodora myristica seeds |
Alkaloids and extracts of alkaloid containing plants often have pronounced bioactivities and have been used throughout human history as remedies, poisons and psychoactive drugs21,22. The alkaloids identified in this study have been established to have health beneficial properties23,24. Although, alkaloids are considered to be anti-nutrients because of their action on the nervous system and disrupting or inappropriately augmenting electrochemical transmission25, the phytochemical screening of M. myristica seeds revealed safe levels of alkaloids and other anti-nutrients such as phytates, tannins, saponins, cyanogenic glycosides and oxalates14,15. The seed is therefore safe for consumption, hence their use in traditional medicine8,9. Moreover, roasting and boiling are effective in eliminating the anti-nutritional factors in foods26-28.
Only four phytochemicals- Ixoside (C16H19O11), 6α-Hydroxy-7-oxo-ferruginol (C20H23O3), Aromadendrin glucoside derivative (C37H57O12) and Methylsarpagine (C20H25N2O2) were identified in the control (raw) sample. However, these compounds have been established not to be deleterious but beneficial to health29,30. More phytochemicals were identified in the roasted samples compared to the boiled samples. The reason may be attributed to the fact that boiling led to break down of the plant cell wall which permitted the leakage of cell contents including bio-actives31 while roasting is a mere gradual evaporation processes32. These findings suggest that processing significantly released phytochemicals. The same observation was made by De la Parra et al.33 on the phytochemical constituents of corn for production of Masa, Tortillas and Tortilla Chips. Boiling and roasting induced complex changes in phytochemical composition which is accompanied with change in biological activities. Similar result were reported by Wu et al.34 for Sorghum tea.
Data obtained from the analyses of the methanolic extracts of M. myristica seeds are summarised in Table 1. Figure 1-3 shows the chromatogram as affected by processing methods. The next sections present discussions of the chemical characteristics and potential health-benefitting (phytochemical) properties of each of the compounds obtained from the M. myristica seed extracts of the current study (arranged according to their aglycone class) and their occurrence in plant species as reported in the literature.
Two iridoid glycosides were found to occur in the M. myristica seed extracts of this study. A molecular ion at 387.0934 m/z (molecular formula C16H19O11) was identified as ixoside35. This compound was identified in C0, R10, R20 and R30 and has been reported in other species from order Lamiales36,37. Another signal at m/z 377.1814 was observed at retention time 8.60 (peak 2). The same mass data were reported for Scabroside B by Di et al.38 and were identified only in sample B10. Iridoid Glycosides have laxative and anti-microbial properties. They are also widely believed to possess anti-inflammatory properties, but so far the tests have revealed only a very weak anti-inflammatory effect39. Ethanolic extract of M. myristica seed possesses antimicrobial activity against Klebsiella and Bacillus species40 and possess broad spectrum antibacterial activities against selected Gram-positive and Gram-negative bacteria41. It also inhibited Aspergillus niger in "Kunun" beverage42. This generally confirmed that this seed is highly potent to activities of many microorganisms.
Exact mass measurement of the m/z 455.0956, peak 1 confirmed the compound to be C23H19O10, a flavonoid identified in B10. This compound is a rare catechin, (-)-Epicatechin-3-(3’’-O-methyl) gallate described in green tea by Miketova et al.43. The compound with molecular formula C37H57O12 was identified as Aromadendrin glucoside derivative44, another flavonoid which was identified in CO. Flavonoid rich fraction of M. myristica seeds exhibited significant in vitro anti-inflammatory potentials by stabilizing red blood cell membrane exposed to hypotonic and heat induced lysis with maximum percentage stability of 88% in a biphasic mode of response that is comparable with Ibuprofen a standard anti-inflammatory drug45. Antioxidant properties of M. myristica have been reported and attributed mainly to its flavonoids composition46. Feyisayo and Oluokun4 evaluated the antioxidant activity of the flavonoid fraction of the seed extract of M. myristica. The flavonoid fraction exhibited potent and appreciable anti-oxidant potentials with maximum DPPH-radical scavenging activity (41.20%), hydroxyl radical scavenging activity (46.34%). It also exhibited a significant p<0.05 reduction of Fe3+ to Fe2+ (64.64%). It exhibited a dose dependent protective effect against free radical induced haemolysis of red blood cells with maximum protection and inhibition of lipid peroxidation and free radical generation in liver homogenate.
The ion detected at m/z 378.1700 (peak 2) at 8.59 min, according to Zhang et al.47 corresponds to Oxiamycin. It is a dimeric indolo-sesquiterpene identified in R30. Indolo sesquiterpenes are a group of natural products isolated from plants that exhibit various activities, such as antibacterial, anti-parasitic and anti-human immuno deficiency virus (HIV), as well as inhibitory activities against lipid droplet and non-steroidal progestin biosynthesis48-50. The phenolic compounds in M. myristica are responsible for the anti-septic, antifungal or bactericide properties of the plant51. An antibiotic with the molecular formula C10H8NO3 (retention time 8.59 min, peak 2) was identified in B30. It is Mollisianitrile [(3-(3,5-Dihydroxy-4-methoxyphenyl) propiolonitrile, which contains a reactive propiolonitrile moiety believed to be responsible for its antibiotic activities52.
| Fig. 1: | Chromatogram of boiled M. myristica seed extracts |
A signal at m/z 317.1970 was observed at 8.60 min. This compound showed a molecular formula C16H29O6. According to Uchiyama et al.53, it is Linaloyl glucoside, a terpenic glycoside identified in B10 and B30.
Another signal detected at m/z 197.0813 was revealed at retention time 8.60 min and identified in B20. The same mass data was reported for an iridoid aglycone, Cornolactone B, by He et al.54. Cornolactone B is the first natural cis-fused tricyclic dilactone iridoid containing both a five- and a six-membered lactone ring54. Iridoid aglycones have been reported to possess antimicrobial and antitumor properties55. Ukaegbu-Obi et al.56 reported that the seed extracts of M. myristica possess some antimicrobial activities which can be employed in the development of novel therapeutic agents against the test organisms. They assessed the antimicrobial activity of M. myristica seeds on four selected human pathogens, Escherichia coli (E. coli), Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa (P. aeruginosa) using Disc diffusion technique for in vitro antibacterial screening. They observed that the most susceptible bacterium were E. coli, while the most resistant bacterium was P. aeruginosa and the minimum inhibitory concentration result showed that the seed extracts of M. myristica was bacteriostatic. At a retention time of 11.71 min, a signal with m/z 475.2139 was detected. This confirmed the presence of a phenylcoumarin derivative named Lepidotol E57 which was identified in R30.
Nine alkaloids were identified in the processed and raw M. myristica methanolic extracts. Two of them, Melicarpinone (C13H12NO3)25 and Annonamine (C19H22NO2)31 were identified in R20 and R30.
| Fig. 2: | Chromatogram of roasted M. myristica seed extracts |
| Fig. 3: | Chromatogram of raw M. myristica seed extracts |
Compound with molecular formula C20H24NO4 and m/z 342.1713, magnoflorine58 was identified in R10, R20, R30, B20 and B30. The compound with m/z 389.1731 (retention time, 18.67, peak 5), Gelsemium 3, a new gelsedine-type oxindole alkaloid, previously isolated from the stems and leaves of cultivated Carolina jasmine (Gelsemium sempervirens AIT. f.)24 was identified in R10. A compound identified in R30, B10 and B30 presented signals at m/z 265.1540, molecular formula, C14H21N2O3. The mass data was compared with the data obtained by Kim et al.59 and it was identified as Feruloyl putrescine. Amycocyclopiazonic acid (molecular formula C18H21N2O2, m/z 297.1596) was identified in B30. It is a new bacterial indole alkaloid related to the cyclopiazonic acid class, which has previously only been found in fungi29. The metabolite 14β-Hydroxymeloyunine (m/z 311.1748, molecular formula C19H23N2O2), a monoterpenoid indole alkaloid previously isolated from leaves and twigs of Melodinus yunnanensis was identified in R10, R30, B10 and B3060. According to Kumar et al.61 compound with the molecular formula C20H25N2O2 (m/z 325.1927, retention time 24.56min, peak 13) is Methylsarpagine identified in CO, R20 and B20. Compound with a molecular ion 367.2755 and molecular formula C24H35N2O was identified as Granulatamide B, a new tryptamine derivative, marine indole alkaloid62,63. This compound was detected in B20. It had previously been isolated from the 2-propanol extract of the soft coral Eunicella granulate and showed moderate in vitro cytotoxicity against a panel of 16 human tumour cell lines62. Other reports on the phytochemical screening of the seeds of this plant also revealed the presence of phenolic compounds, including flavonoids and tannins, cyanogenic glycosides, alkaloids, arocine, lactose, terpene, resins, fiberro-latic oils, anthraquinones, saponins, steroids, oxalates and phytates14,15. The mechanism of inhibitory action of these alkaloids and phenolic compounds on micro-organisms may be due to impairment of variety of enzyme systems, including those involved in energy production, interference with the integrity of the cell membranes and structural component synthesis64,65.
The ES-MS signals at m/z 201.1 (identified in R10, R30, B20) and m/z 295.2 (identified in B30) were 9,10-Dihydroxy-2-decanoic acid66 and Hydroxy octadecadienoic acid67 respectively. Hydroxy fatty acids are drawing much attention recently due to their anti-inflammatory, antimicrobial and cytotoxic activities68,69. GC-MS analytical report for the chloroform fraction of M. myristica seed showed the presence of n-hexadecanoic acid, 9-Octadecanamide, cis-9-Hexadecenal, acetic acid, cis-vaccenic acid, campesterol and butyl-9-Octadecenoate amongst others70.
The compound 3-Methoxytyramine-BX, at m/z 361.1394, a new betaxanthin (yellow chrome alkaloids) detected at a retention time of 11.73 min (peak 3), was identified in B20 and the same mass spectra was reported for this compound by Schliemann et al.71. Betaxanthins display potent antioxidant, anti-inflammatory and chemo-preventive activity in vitro and in vivo 72. Ishola et al.73 carried out a study to investigate the antinociceptive and anti-inflammatory effects of the hydroethanolic seeds extract of Monodora myristca (HMM) in male albino rats. They reported that HMM possesses antinociceptive effect mediated through interaction with opioidergic, serotonergic and dopaminergic systems and an anti-inflammatory action through inhibition of inflammatory mediator's release. Their study established the scientific basis for its use in the management of pain and inflammatory conditions in traditional medicine. The flavone, Licoflavone B74 was identified in R20, R30 and B30 respectively. A Cholestane glycoside, Dioseptemloside A, (molecular formula C45H75O18, m/z 903.4980)75, was identified in B10, B30 and R30. An antifungal lipopeptide was also detected in this study. According to Ortiz-Lopez et al.76 peak 8 with m/z C47H67N8O10 (retention time, 23.37 min) corresponds to Colisporifungin an antifungal lipopeptide which was identified in B10. M. myristica extracts inhibited the growth of mycelium, the formation of conidial spores and chlamydospores of Sclerotium rolfsii, thereby reducing the number of propagation units of this fungus in the medium65,77. Also, Enabulele et al.5 reported that the aqueous and ethanolic extracts of M. myristica seeds, were active against both Gram-negative and Gram-positive organisms- Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli and Salmonella typhi. Its methanol and dichloromethane extract was active against mites and traditionally used against scabies78. The metabolite, 6α-Hydroxy-7-oxo-ferruginol79 a new phenolic diterpene with the molecular formula C20H23O3 was identified in CO and R10. Gagunin B, (C38H61O11, 693.4197)80 a poly-oxygenated diterpene was identified in R30 and B10. It had been reported to have anti-tumour properties81.
Manoalide (C25H33O4) with m/z 397.2382 (retention time, 24.52 min, peak, 12), a sesterterpene82 and Polyphyllin V, a saponin83 were identified in R20. Manoalide is an analgesic, possesses potent anti-inflammatory activity, irreversibly inhibits human synovial fluid PLA284 as well as bee85 and cobra venom PLA2 and inhibits ornithine decarboxylase86. These activities have led to the use of manoalide in the prevention of post-surgical adhesion of tissues and as a molecular tool in the study of psoriasis and skin cancers82. M. myristica seeds are a natural source of anti-inflammatory agent73. Flavonoid rich fraction of M. myristica seeds inhibited heat induced albumin denaturation with maximum inhibition of 75.38% in a concentration dependent manner that is comparable with aspirin and showed an anti-lipoxygenase activity range from 19-71% which is comparable to that produced by indomethacin45.
Terpecurcumin Q (C36H43O6) with m/z 571.3061 (retention time, 26.05, peak 17) and Gelliusterol E (C25H35O2, m/z 367.2620, retention time, 27.82, peak 19) were identified in R30. Terpecurcumin Q a novel terpene-conjugated curcuminoid exhibited IC50 of 3.9 μM against MCF-7 human breast cancer cells87 while Gelliusterol E inhibited the formation and growth of chlamydial inclusions in a dose-dependent manner with an IC50 value of 2.3 μM88. Essential oils obtained by hydrodistillation of fruits of M. myristica exerted cytotoxic activity against cancer and normal cell lines with more pronounced effect on neoplastic cells in the majority of cases89.
The metabolite 22-Epi-Hippuristan-11-one (C28H45O5, m/z 461.3263), a new highly oxygenated spiroketal steroid hippuristanol, was identified in R20 and B2090. Hippuristanols have been reported to have significant cytotoxicity against several cancer cell lines91. Other reports on the phytochemical screening of the seeds of this plant also revealed the presence of steroids14,15. Amphidinolide T3, a new 19 membered macrolide, which was reported to exhibit cytotoxicity against murine lymphoma L1210 (IC50:7) and human epidermal carcinoma KB cells (IC50: 10)92 was identified in R10 and R20. It presented signals at m/z 423.3103 with molecular formula, C25H43O5. The biological properties, unique structural features and scarcity of supply of Amphidinolides have already attracted immense synthetic interest93.
Unidentified compounds: Molecular ions at m/z 293.1798 (observed exact mass 293.1793), m/z 358.2666 (observed exact mass 358.2670), m/z 623.4253 (observed exact mass 623.4249) and m/z 721.4104 (observed exact mass 721.4078) eluting at 18.88, 22.86, 23.64 and 24.82 min, respectively could not be identified.
CONCLUSION
The combination of the accurate mass spectroscopy (MS) in the determination of elemental composition and the ability of Ultra High Performance Liquid Chromatography (UPLC) to separate isomeric compounds provided a powerful tool for the identification of 32 metabolites present in raw and processed Monodora myristica seed flour. The study revealed that boiling and roasting affected the release of metabolites in the flour. The metabolites identified have been reported to possess phytochemical properties. Furthermore, different unknown substances were reported and other analyses (such as NMR) will be necessary to identify these compounds and to establish if they are phytochemicals. M. myristica seed can be considered a good source of phytochemicals. The phytochemical potential of this plant should be investigated further as the current data indicate that its seeds have abundant and diverse phytochemicals, as well as the fact that the plant is significant in oriental traditional medicine.
SIGNIFICANCE STATEMENT
This study reveals that boiling and roasting can have beneficial effects on the phytochemical constituents of M. myristica. It has also shown that thermal processing induced the release of phytochemicals especially roasting process as the raw extracts had the least number of phytochemicals. The results of this study will help other researchers in the development of new functional foods from whole M. myristica seeds or its extracts.
ACKNOWLEDGMENTS
The authors are thankful to the College of Agriculture, Engineering and Science, University of KwaZulu-Natal, for financial support.