HPLC-Guided Isolation, Purification and Characterization of Phenylpropanoid and Phenolic Constituents of Nutmeg Kernel (Myristica fragrans)

Abstract

Many studies on the biological activities of nutmeg continue to appear in the literature. The most common targets include GIT, CNS, oxidative stress and inflammation. However, results obtained from most studies are often inconsistent due to the variability of utilized samples, lack of standardized nutmeg products or insufficient amounts of pure compounds for comprehensive follow-up investigation. To address the consistency and supply issue we utilized available technology to develop a reproducible procedure for preparation of specific extracts and isolation of the major phenolic constituents present in nutmeg kernel. A well-defined and reproducible sequence of extraction, fractionation and chromatographic purification was adopted and was guided by HPLC fingerprinting. Spectroscopic methods, mainly NMR, and mass spectrometry were utilized to identify each compound. Thirteen compounds were isolated in a pure form and identified as: elemicin (1), isoelemicin (2), myristicin (4), surinamensin (5), malabaricone C (6), 2-(3′-allyl-2′,6′-dimethoxy-phenyloxy)-1-acetoxy-(3,4-dimethoxyphenyl)-propyl ester (7), methoxylicarin A (8), licarin A (9), malabaricone B (10), licarin C (11), 5′-methoxylicarin B (12), licarin B (13), and 2-(3′-allyl-2′,6′-dimethoxy-phenyloxy)-1-methyl-5-methoxy-1,2-dihydrobenzofuran (3, a new compound). With repeated isolation runs, these pure compounds can be prepared in quantities sufficient for biological evaluation as needed. The availability of purified compounds will also allow the development of specific, accurate, and sensitive analytical procedures for pharmacokinetic studies and for quality control of nutmeg products. Both aspects are essential for nutmeg-focused drug discovery. The same approach can also be adapted to other medicinal plants of potential interest.

Chemical diversity and pharmacological significance of the secondary metabolites of nutmeg (Myristica fragrans Houtt.)

“…Among the commonly encountered benzofuranoid neolignans are licarins A, B, C & E and their related analogs ( 35 - 39 , Figure 5A) which are present in nutmeg as well as other genera such as Aristolochia , Machilus , Nectandra , and others (Aiba et al 1977, Giang et al 2006, Leon-Diaz et al 2010). Additionally, the two benzofuranoid neolignans (7 R ,8 R )-7,8-dihydro-7-(3,4-dihydroxyphenyl)-3’-methoxy-8-methyl-1’-( E -propenyl)benzofuran ( 40 ) and 3-(4'-allyl-2',6'-dimethoxy-phenoxy)-2-methyl-6-methoxy-2,3-dihydrobenzofuran ( 41 ) were isolated and reported as new by Duan et al and by Chiu et al, respectively (Chiu et al 2016, Duan et al 2009). …”

“…Two recent reports utilized high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC) for qualitative analysis of nutmeg samples. Tripathi and Dwivedi utilized normalphase HPTLC for fingerprinting and standardization of nutmeg samples from India (Tripathi and Dwivedi 2015); while Chiu et al employed reversed-phase HPLC to guide the isolation and identification of thirteen volatile (phenylpropanoids) and non-volatile constituents (neolignans and malabaricones) from nutmeg samples purchased in the United States (Chiu et al 2016). As for quantitative analysis, the most recent method employed ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) to determine 16 constituents in nutmeg fruit extracts and Indian polyherbal formulations (Pandey et al 2015).…”

Phenolic compounds from nutmeg (Myristica fragrans Houtt.) inhibit the endocannabinoid-modulating enzyme fatty acid amide hydrolase

“…Each extract was dried under vacuum to yield four separate fractions. The total methanolic extract and its solvent fractions were fingerprinted by an analytical HPLC method developed in our laboratory . The most active of the four fractions, ethyl acetate fraction, was further subjected to flash chromatography using a n ‐hexane‐ethyl acetate gradient to generate 10 column fractions.…”

“…The most active of the four fractions, ethyl acetate fraction, was further subjected to flash chromatography using a n ‐hexane‐ethyl acetate gradient to generate 10 column fractions. The major constituents in each fraction were purified by semipreparative reversed‐phase HPLC (Shimadzu Prominence System, Kyoto, Japan) to yield 13 compounds as described in an earlier report . Evaluation of the isolated compounds for FAAH and MAGL inhibition was performed using enzyme assay inhibitor screening kits purchased from Cayman Chemical Company (Ann Arbor, USA).…”

Raw Material from Nutmeg (Myristica fragrans) as Effective Fungicide against Fusarium oxysporum and the Oleoresin Profile of Nutmeg

“…Further, antioxidant activities of licarin B that isolated from nutmeg have been evaluated [21]. Many compounds including licarin A, B, C and methoxylicarin A have been isolated from nutmeg utilizing spectroscopic methods, mainly NMR, and mass spectrometry [22]. Phenol, 4-[(2S, 3S)-2,3-dihydro-7-methoxy-3-methyl-5-(1E)-1-propenyl-2-benzofuranyl]-2,6-dimethoxy has been detected from methanol crude extract of dried mace [23].…”