Post on 07-Aug-2020
CH2
OCH3
O
O
O
O
CH3
CH3
CH3
(5) Licarin A
C20H22O5, M.W.: 326 (6 mg)
Development of a Standard Procedure for Isolation and Purification of Major
Non-volatile Constituents of Nutmeg Kernel Sharon Chiu, Martin Belski, Thomas Wang, and Ehab A. Abourashed
Department of Pharmaceutical Sciences,
Chicago State University, Chicago, Illinois
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min
-0.12
-0.11
-0.11
-0.10
-0.10
-0.09
-0.09
-0.08
-0.07
-0.07
-0.07
-0.06
-0.06
-0.05
uV(x1,000,000)
Data13:MF23-3-2.lcd Detector A Ch1:270nmData12:MF23-3-3.lcd Detector A Ch1:270nmData11:MF109-1.lcd Detector A Ch1:270nmData10:MF-55-11.lcd Detector A Ch1:270nmData9:MF109-6.lcd Detector A Ch1:270nmData8:MF109-5.lcd Detector A Ch1:270nmData7:MF133-1.lcd Detector A Ch1:270nmData6:MF133-2.lcd Detector A Ch1:270nmData5:MF109-7.lcd Detector A Ch1:270nmData4:MF133-3.lcd Detector A Ch1:270nmData3:MF131-2.lcd Detector A Ch1:270nmData2:MF22-4.lcd Detector A Ch1:270nmData1:DCM-total.lcd Detector A Ch1:270nm
Research reported in this poster was supported by the National Institute On Drug Abuse of the National Institutes of Health
under Award Number R24DA036410. The content is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health.
ABSTRACT
INTRODUCTION
CONCLUSIONS
OBJECTIVES
METHODS
•The developed procedure can reliably be applied for isolation and purification of major
non-volatile constituents from nutmeg.
•Twelve compounds were isolated and identified with yields ranging from 1 mg to 36 mg.
Compound 14 may be new. A few more purified compounds have yet to be identified.
•Purified compounds will be used for biological evaluation and for the development of
specific, accurate, and sensitive analytical procedures for pharmacokinetic studies and
for quality control of nutmeg samples/products.
Acknowledgements
• Develop an efficient and reproducible procedure for isolation and
purification of major non-volatile constituents of nutmeg.
• Establish a supply line of nutmeg constituents for:
• further investigation of their biological activity, and
• use as analytical standards in pharmacokinetic studies and quality
control of nutmeg products.
RESULTS
References
*
Purpose-Nutmeg is a common kitchen spice that has been used traditionally to treat such conditions as
flatulence and diarrhea. Studies have also shown that nutmeg may act as a CNS stimulant, psychotropic,
aphrodisiac and anti-inflammatory agent. Active constituents have been found in both the volatile oil and different
non-volatile extracts of nutmeg. Since nutmeg contains a complex mixture of compounds, the development of a
reproducible procedure for isolation and purification of major non-volatile constituents was conducted to allow for
further investigation of their biological activity and for use as analytical standards in pharmacokinetic studies and
quality control of nutmeg products.
Methods-A sequence of extraction, fractionation and chromatographic purification was adopted and was guided
by analytical HPLC fingerprinting. Finely powdered nutmeg was exhaustively extracted with methanol (MeOH).
The total methanolic extract was frozen and filtered to obtain a defatted total extract (DTE). The DTE was
subjected to successive solvent extraction using hexane, ethyl acetate (EtOAc), and MeOH. Analytical HPLC
revealed 18 potential compounds in the EtOAc extract which was subjected to flash chromatography to yield 12
column fractions. Each of the 12 flash fractions was subjected to preparative HPLC to isolate individual
compounds in a pure form. Spectroscopic methods, mainly mass and NMR spectroscopy, were utilized to identify
each compound.
Results-Of the 18 targeted compounds, 12 compounds were isolated in a pure form in yields ranging from 3 –
36 mg. The isolated compounds were identified as: licarin B (1), methoxylicarin B (2), licarin C (3), malabaricone
B (4), licarin A (5), 5’-methoxylicarin A (7), 2-(3’-allyl-2’,6’-dimethoxy-phenyloxy)-1-acetoxy-(3,4-dimethoxy-
phenyl)-propyl ester (7A), malabaricone C (9), myristicin (13), 2-(3’-allyl-2’,6’-dimethoxy-phenyloxy)-1-methyl-5-
methoxy-1,2-dihydrobenzofuran (14, a putative new compound), isoelemicin (16), and elemicin (17). Other
compounds were isolated in trace amounts and may need further enrichment to be reliably identified.
Conclusion-A significant component of the developed isolation/purification procedure relies on programmed,
automated technics which guarantees a high level of reproducibility. Analytical HPLC was essential for monitoring
the contents of each fraction and purity of the final products. This procedure can reliably be applied for isolation
and purification of major non-volatile constituents from nutmeg. With repeated isolations, these pure compounds
can be prepared in desirable quantities to be subjected to biological testing. The availability of purified
compounds will also allow the development of specific, accurate, and sensitive analytical procedures for
pharmacokinetic studies and for evaluating quality of nutmeg samples.
Nutmeg is the seed kernel of Myristica fragrans
(family Myristicaceae), which is an evergreen tree that is
indigenous to the Moluccas Island and is cultivated in
Indonesia, S. Africa, Malaysia, West Indies, and other
tropical countries. Nutmeg is one of the most commonly
used spices in the world and has been traditionally used as an anti-
diarrheal, anti-flatulence.1 It has also been tested to improve the functions
of the stomach and increase appetite.1 Studies have also shown that
nutmeg may act as a CNS stimulant, psychotropic, aphrodisiac and anti-
inflammatory agent.1 Nutmeg contains about 10% volatile oils, fats,
polysaccharides, and protein.1, 2 The oils consist of terpene hydrocarbons,
terpene derivatives, and phenylpropanoids.1, 2, 3 Of the phenylpropanoids;
myristicin, elemicin, safrol, eugenol and eugenol derivatives comprised of
15 to 20% of the total compound.2 Other constituents found include
copaene, trans- and cis-sabinene hydrate, cis-
piperitol, and others. Non-volatile constituents I
include lignans, catechins and phenolics.3
1. El-Alfy A. et al. “Towards a Better Understanding of Psychopharmacology of Nutmeg: Activities in the Mouse Tetrad Assay” J
Ethnopharmacol. Nov. 12, 2009: 126(2): 280-286
2. Nagaraju B. et al. “Anxiolytic Effect of Myristica fragrans” International Journal Of Phytotherapy Research 2013: 3(1): 1-7
www.earthjournals.org
3. Leung’s Encyclopedia of Common Natural Ingredients. Khan I., Abourashed E. Hoboken, New Jersey. John Wiley & Sons, Inc. 467-469
A. Conceptual Summary of Procedure
B. Extraction, Isolation and Purification Scheme
Crude Material
Total Extract
Fractionation Of Extract
Via ASE
Flash Chromatography
Prep HPLC
Compound Identification
Guided by analytical HPLC
1. HPLC fingerprinting of each flash fraction 2. Preparative HPLC of major constituents of each flash fraction
Isolated compounds were subjected to 1H NMR, 13C NMR, HMQC, and HMBC spectroscopy
12 identified compounds: licarin B (1), methoxylicarin B (2), licarin C (3), malabaricone B (4), licarin A (5), 5’-methoxylicarin A (7), 2-(3’-allyl-2’,6’-dimethoxy-phenyloxy)-1-acetoxy-(3,4-dimethoxy-phenyl)-propyl ester (7A), malabaricone C (9), myristicin (13),
2-(3’-allyl-2’,6’-dimethoxy-phenyloxy)-1-methyl-5-methoxy-1,2-dihydrobenzofuran (14, a putative new compound), isoelemicin (16), and elemicin (17)
Accelerated Solvent Extractor (ASE)
Filtration of
Methanolic Extract
Flash Chromatography
Chromatogram of flash fractions and total extract Preparative HPLC
A. Molecular Formula, Molecular Weight, and Yield of Identified Compounds
Nutmeg Powder (370 g)
Total MeOH Extract (TME) 52.2 g
11.4 g MF-65-2, MF66-2, MF66-5 (17.6 g)
19.3 g
Hexane EtOAc MeOH
1. Freeze filtrate overnight then refilter
2. Concentrate filtrate under vacuum
Defatted Total Extract (DTE, 49.6 g)
1. Mix with 10 g silica 2. ASE run
ca. 120 mL per solvent , concentrate under reduced pressure at 45 oC
MF-79-2 0.07 g
MF-79-3 0.35 g
MF-79-4 0.23 g
MF-79-7 0.13 g
MF-79-8 1.50 g
*MF-79-9 0.48 g
MF-79-5 0.56 g
MF-79-6 1.36 g
* MF-79-10 0.19 g
MF-79-1 0.77 g
MF-79-11 0.13 g
MF-79-12 0.10 g
Flash Chromatogram
Aliquot (8 g) Subjected to flash chromatography
1 2
3 4
5
6 7
8 9 10
11 12 13 14
15 16
17 18 EXTRACT
COMPOUNDS
1
2
3
4
5
6
7A
7
13
14
16
17
O
O
O
CH3
OCH3
CH3
R
O
O
R
CH3
OCH3
CH3
CH3
O
CH3
OOH
OH OH
R
O
O
OH
CH3
OCH3
CH3
CH3
CH2
OCH3
OCH3
O
OCH3
OCH3
O
O
CH3
CH3
O
O
CH2
OCH3
O
CH3O
R
OCH3
CH3
B. Example of Spectroscopic Method for Compound Identification
1H-NMR Spectrum for Compound 14 (400 MHz, acetone-d6) 13C-NMR Spectrum for Compound 14 (100 MHz, acetone-d6)
HMBC Correlations for Compound 14
Selected data and NMR assignments of compound 14
CH3
CH2
OCH3
CH CH
=CH2
ArCH
=CH
CH3
CH2
OCH3
=CH2
=CH CH
CH
ArCH
2 3
4
5 6
7
1’
2’ 4’
6’
(17) Elemicin, R= CH2-CH=CH2
C12H16O3, M.W.: 208 (4 mg)
(14) 2-(4’-allyl-2’,6’-dimethoxy-phenyloxy)-
2-methyl-6-methoxy-2,3-dihydrobenzofuran -
_ C21H24O5, M.W.: 356 (35 mg)
C
C
(1) Licarin B, R= H
C20H20O4, M.W.: 324 (1 mg)
(2) Methoxylicarin B, R= OCH3
C21H22O5, M.W.: 354 (23 mg)
(3) Licarin C, R= OCH3
C22H26O5, M.W.: 370 (26 mg)
(7) 5'-Methoxylicarin A, R=OH
C21H24O5, M.W.: 356 (12 mg)
(13) Myristicin
C11H12O3, M.W.: 192 (6 mg)
(16) Isolemicin, R= CH=CH-CH3
C12H16O3, M.W.: 208 (13 mg)
(7A) 2-(3’-allyl-2’,6’-dimethoxy-phenyloxy)-1-
acetoxy-(3,4-dimethoxy-phenyl)-propyl ester
C24H30O7, M.W.: 430 (18 mg)
(4) Malabaricone B, R=H
C21H26O4, M.W.: 342 (13 mg)
(9) Malabaricone C, R=OH
C21H26O5, M.W.: 358 (36 mg)
OCH3
H
OCH3
H
H
O
O
CH3 H
H
HOCH3
H
H
HH
H
H
5.05
115.2
5.97
137.7
40.2
3.33
136.0
105.7
105.7153.6
133.5
153.6
6.56
6.56
55.6
3.83
55.6
72.9
4.70
1.00 12.6
82.1
4.28
132.4
145.4
114.5
118.6
6.73
6.67
147.2109.6
6.94
55.4 3.78
3.38
125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 500.0 525.0 550.0 575.0 m/z0.0
1.0
2.0
3.0
4.0
5.0
Inten. (x1,000,000)
438
392195357
468413193
252 325 469220 519 581597
549163 299141 506287
MS (M+1 = 357)
1. Suspend in MeOH (2400 mL) 2.Ultrasonicate and filter
Nutmeg fruit with kernel enveloped in
red arillus tissue