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