Post on 08-Jun-2018
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CHAPTER 3
EXTRACTION, ISOLATION AND CHARACTERIZATION OF
THYMOQUINONE FROM COMMERCIAL Nigella sativa OIL AND
SYNTHESIS OF ITS AMINO-DERIVATIVE WITH SINGLE
CRYSTAL X-RAY STRUCTURE
3.1 Plants and Phytochemicals as Medicines
Nature has been a source of our basic needs from times immemorial and our
understanding of nature has lead to use of natural resources in almost all the aspects of
our lives. Their properties have been documented in various civilizations like Egyptian -
Ebers Papyrus, Chinese - Shennong Herbal, Tang Herbal, Indian – Charaka Samhita;
Sushruta Samhitas and Arabic - The Royal Book of All Medicine by Ali Ibn Abbas al-
Majusi and Canon of Medicine by Ibn Sina 1-5.
Phytochemicals became the major source of treating various ailments and
diseases. Drug discoverers have been always fascinated by the compounds found in
nature and these researchers have drawn their inspiration from natural products. This
strategy has led to development of blockbuster molecules and their use in treatment of
human sufferings. World War II laid the foundation of large scale production of penicillin
and the industries which were producing penicillin for the wartime started looking for the
newer antibiotics 6. Further breakthrough discoveries of streptomycin, gentamicin,
tetracycline and other antibiotics triggered off massive funding in large scale research and
development schemes in industries and institutes 7. Pharmaceutical industries and
researchers did not focus only on antibacterial agents but they also explored the
possibilities of finding active phytochemicals against other diseases. Two compounds
compactin 8 and mevinolin 9 were reported with potential to inhibit cholesterol
biosynthesis and these reports led to development of statin therapeutics and their
successful implementation.
Recently Newmann and Cragg summarized all approved drugs from 1981 to 2010
for all diseases all over the world and gave a detailed classification (Table 1). This
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analysis shows the astounding impact of natural products on the process of drug design
and discovery 10.
Table 1: Approved Drugs Inspired from Natural Products 10 (Newman et al. J Nat Prod 2012;75:311-335)
Symbol Type of Drug
(on the basis of origin)
Percent of 1073 approved drugs in last 30 years
against cancer
N An unmodified Natural Product 6% (59 out of 1073)
ND A modified Natural Product 28% (299 out of 1073)
S* A synthetic compound with a Natural Product Pharmacophore
5% (55 out of 1073)
S*/NM
A synthetic compound with a Natural Product Pharmacophore showing competitive inhibition of the natural product substrate
11% (122 out of 1073)
S A synthetic compound without Natural Product conception
36% (387 out of 1073)
S/NM A synthetic compound showing competitive inhibition of the natural product substrate
14% (146 out of 1073)
NB Botanical ‘‘defined mixtures’’ recognized as drug entities by the FDA and similar organizations
0.004% (5 out of 1073)
3.2 Isolation of Thymoquinone
Thymoquinone (TQ), also known as 2-isopropyl-5-methyl-1,4-benzoquinone is an
important constituent of oil obtained from seeds of Nigella sativa 11-13. Ghosheh and co-
workers developed method for analyzing oil of Nigella sativa seeds through high
performance liquid chromatography (HPLC). The constituents from the oil were isolated
by using C18 PrepSep mini columns and quantification of these recovered constituents by
HPLC was completed on a reversed-phase μBondapak C18 analytical column. Isocratic
mobile phase of water:methanol:2-propanol (50:45:5% v:v) at flow rate of 2 ml/min and
254 nm radiation was used for detection of TQ 14.
Ashraf and co-workers reported isolation of TQ from seeds of Nigella sativa by
subjecting 20 g of finely powdered seeds to Soxhlet extractor with hexane and solvent
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was removed under vacuum followed by stream of nitrogen. The extract was loaded on
silica gel column and eluted with hexane, 15% diethyl ether in hexane, diethyl ether and
methanol 500 mL each and analyzed on HPLC after evaporation and reconstitution in
methanol. HPLC analysis showed 368 mg/g of thymoquinone in hexane fraction and 658
mg/g in 15% diethyl ether in hexane fraction 15. Supercritical fluid carbon dioxide
extraction (SCFE-CO2) of Nigella sativa oil at 150 bar and 40ᵒC for 120 min produced
4.09 mg of thymoquinone per ml of CO2 extract as reported by Solati 16.
3.3 Experimental Aspects
Solvents and reagents were procured from SD Fine Chemicals Limited and
Standard Sample of TQ was supplied by Aldrich, India. Oil of Nigella sativa was
obtained from Mahida and Sons, Mangrol, Gujrat, India. All the solvents used were
purified by procedures described in Vogel’s Text book of Practical Organic Chemistry 17.
TLC was checked on Pre-Coated Silica Gel 60 G 254 plates from Merck India Limited.
HPLC grade methanol was used for HPLC experiment without further purification.
Column chromatography was used for purification of compounds with petroleum ether
and ethyl acetate as solvents.
3.3.1 Extraction of TQ from Nigella sativa
Extraction of TQ from commercially available Nigella sativa oil was performed
by sonication as this is reported by Velho-Pereira and colleagues 18. Nigella sativa oil
was obtained from Mahida and Sons, Mangrol, Gujrat, India for isolation of TQ. They
market oil under the name of Herbal Kalonji Oil. 5 gm of oil sample was taken in a 25 ml
volumetric flask with 10 ml of methanol and sonicated for 20 minutes. Methanol layer
was separated from oil and evaporated. Concentrated viscous liquid was loaded on silica
gel column (Mesh size 60-120) and eluted with petroleum ether (60ᵒC-80ᵒC). The fastest
moving yellow colour spot was concentrated after elution and found to be matching with
standard TQ sample (Aldrich) on TLC plate in 10% chloroform in petroleum ether. This
sample was subjected to HPLC analysis and compared with standard sample of TQ
obtained from Sigma Aldrich with methanol as the solvent. Retention time of 3.39
minutes shown by purified column fraction matched with that of standard TQ sample
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(Figure 1 and 2). The weight of TQ obtained from column was 1.03 g after drying. Thus,
the w/w percentage of TQ obtained from Nigella sativa oil sample was found to be 20.6%
which is less than the reported value of 36.7% during GC-MS analysis in other report 19.
Figure 1: HPLC of TQ Fraction after Isolation and Purification
Figure 2: HPLC of Standard TQ Sample (Aldrich)
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3.3.2 Synthesis of 3-amino-5-isopropyl-2-methylcyclohexa-2,5-diene-1,4-dione
3-amino Thymoquinone (2) was synthesized with the procedure described by
Moore and co-worker with modifications in molar ratio of reactants and acid catalyst 20.
A mixture of TQ (1 mmol, 0.164 g) and sodium azide (1.3 mmol 0.084 g) in ethanol was
refluxed for 3 Hrs in the presence of 3 ml of glacial acetic acid. Reaction was followed by
TLC in CHCl3. Reaction was worked up by neutralization of acetic acid with NaHCO3
and extracted with chloroform (20 ml × 2). CHCl3 was evaporated under vacuum and
residue was taken up for purification by column chromatography starting with petroleum
ether and gradual increase of polarity up to 10% ethyl acetate in petroleum ether. Eluted
compound 2 was obtained by evaporation of solvent as viscous oily red liquid. It was
dissolved in HPLC grade methanol and solution is kept for slow evaporation at room
temperature which lead to red crystals of compound 2 in 45% yield.
The single crystal X-ray structure of the ATQ was determined through
measurements on a deep red colored crystal of 0.4508×0.2591×0.1939 mm3 dimension.
The crystallographic parameters and selected bond lengths and bond angles are listed in
Table 2, 3 and 4. The ORTEP drawing together with the numbering scheme and the unit
cell packing arrangement are shown in Figures 5a and 5b respectively.
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3.3.3 Structural Characterization
Thymoquinone (TQ): 2-isopropyl-5-methylcyclohexa-2,5-diene-1,4-dione
Yield 20.6%. FTMS Peak for mass number 187.22, which is sodiated TQ adduct with
100% intensity in accordance with M.F. C10H12O2 + sodium peak. IR (cm-1): 2967 (C-
H), 1637 (C=O), 1610 (C=C), 1H NMR (500 MHz, DMSO-D6): ppm = 1.06 (d, J=7Hz,
6H), 1.9 (s, 3H), 2.86 (m, J=7Hz, 1H), 6.5 (s, 1H), 6.7 (s, 1H); 13C NMR (125 MHz
DMSO): ppm = 14.77, 21.03, 26.01, 130.1, 133.36, 144.92, 153.95, 187.08 [i1], 188.09
[i1]. (i stand for interchangable). (Figure 3a-3d)
3-amino thymoquinone (ATQ): 3-amino-5-isopropyl-2-methylcyclohexa-2,5-diene-1,4-dione
Yield 45%. LCMS : RT = 1.94 min. M+ peak 179.09 (M=179.1 in accordance with MF
C10H13NO2). IR (cm-1): 3462-3332 (-NH2), 1645 (C=O), 1H NMR (500 MHz, DMSO-
D6): ppm = 1.06 (d, J=7Hz, 6H), 1.71 (s, 3H), 2.85 (m, J=7Hz, 1H), 6.26 (s, 1H), 6.42 (s,
2H); 13C NMR (125 MHz DMSO): ppm = 8.5, 21.0, 25.8, 106.6, 131.9, 145.1, 148.9,
183.6 [i1], 184.7[i1]; (Figure 4a-4e)
3.3.4 Result and Discussion
FTMS of TQ shows two peaks of m/z 187.07 and 187.22. m/z peak of 187.22
with 100% relative abundance appears as peak of sodiated adduct of TQ [TQ-Na]+ where
expected m/z peak of 164.20 appears with added 23Na at 187.22 (Figure 3a).
1HNMR spectrum of TQ shows first peak as a doublet at 1.06ppm which belongs
to two methyl groups of iso-propyl moeity present at second position of quinone ring,
with splitting constant of 7Hz. Methine hydrogen of the same moeity appears as multiplet
due to neighbouring methyl groups at 2.86 ppm and 7 Hz splitting constant. Methyl group
at fifth position of quione ring appears as a singlet at 1.9 ppm. Hydrogen at third position
appear at 6.5 ppm and hydrogen at sixth position appears slightly downfield with shift of
6.7 ppm as singlet which appear to be in agreement with the reported value of 1HNMR by
earlier group, which used advance NMR techniques like Two-Dimensional Heteronuclear
Single Quantum Coherence Transfer Spectra (2D HSQCT) on Bruker Avance AQS 500
MHz 21.
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13C NMR of TQ showed aliphatic carbon atoms at shielded positions. Methyl
group at fifth position of quionone ring appears at 26.01 ppm, methyl groups of iso-
propyl moeity appear at 14.77 ppm with –CH appearing at 21.03 ppm. Doubly bonded
carbon atoms of quinone ring appear in deshielded region with carbon bearing methyl
group appearing at 144.92 ppm (145.1 ppm reported 21) and its neighbouring –CH
appearing at 133.36 ppm. Carbon bearing iso-propyl group appearing at 153.95 ppm
(156.4 ppm reported 21) and its neighbouring -CH at 130.1 ppm. Carbonyl carbon at
fourth position appears at 188.09 ppm [i1] (188.3 ppm reported 21) and carbonyl carbon at
first position appears at 187.08 ppm [ i1] (187.3 ppm reported 21). These values are in
agreement with reported literature values 21. The chemical shifts of two carbon pair can
not be atributed with certanity and they are mentioned as interchangable values with [i]
and reported 13C chemical shifts of TQ in parenthesis. IR spectrum shows C=O peak at
1637 cm-1 with a shoulder and C-H streching of methyl groups at 2967 cm-1.
LCMS shows Total Ion Current (TIC) chromatogram of ATQ as intense peak at
1.94 minutes and mass shows m/z peak of 179.09. 1HNMR spectrum of ATQ shows first
peak for six hydrogen atoms as a doublet at 1.06 ppm, which belongs to two methyl
groups of iso-propyl group present at fifth position of quinone ring, with splitting
constant of 7Hz. One hydrogen of the same moeity appears as multiplet due to
neighbouring methyl groups at 2.85 ppm and 7 Hz splitting constant. Methyl group at
second position of quione ring appears as a singlet at 1.71 ppm which appears slightly
upfield as compared to TQ because of electron donating –NH2 group on the adjacent
carbon at third position. Hydrogen at sixth position appears as a singlet at 6.26 ppm and
this position is slightly shielded as in comparision with parent TQ. Hydrogen atoms of –
NH2 group appear as singlet at 6.42 ppm and integration shows that the peak is for two
hydrogen atoms.
In 13C NMR of ATQ, methyl group at second position of quionone ring appears at
25.8 ppm. Methyl groups of iso-propyl moeity appear at 8.5 ppm with –CH at 21.07 ppm.
Doubly bonded carbon atoms of quinone ring appear in deshielded region with carbon
bearing methyl group appearing at 106.6 ppm and its neighbouring carbon bearing –NH2
at 148.9 ppm which matches with reported value of similar alkyamino derivatives of
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benzoquinone 22. Carbon bearing iso-propyl group appears at 145.1 ppm and its
neighbouring -CH at 131.9 ppm. Carbonyl carbon at first position appears 183.6 ppm and
carbonyl carbon at fourth position appears at 184.7 ppm and these values may be
interchangable [i1].
Infra red spectrum of ATQ showed some prominent peaks like 3405 cm-1 and
3465 cm-1 for –NH2 group 23. Observed carbonyl stretching peaks are in typical range 24
at 1643 cm-1 and 1667 cm-1 with some shoulder, which appear to be in agreement with
reported values by Raschi et al. The band at 1446 cm-1 appears for -CH3 antisymmetric
bending modes and 1397 cm-1 band for symmetric mode appears to be in agreement with
reported values 25.
Figure 3a: High Resolution Mass Spectrum of Isolated TQ
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Figure 3b: Infra Red Spectrum of TQ
Figure 3c: 1HNMR Spectrum of TQ
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Figure 3d: 13CNMR Spectrum of TQ
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Figure 4a: Total Ion Current (TIC) Chromatogram of ATQ
Figure 4b: Mass Spectrum of ATQ
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Figure 4c: Infra Red Spectrum of ATQ
Figure 4d: 1HNMR Spectrum of ATQ
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Figure 4e: 13CNMR Spectrum of ATQ
3.4 Single Crystal X-Ray Crystallography
The crystal was kept at 110.00(10) K during data collection. Using Olex2 26 the
structure was solved with the XS structure solution program using Direct Methods and
refined with the XL refinement package using Least Squares minimization 27. ATQ
molecule has an electron donating –NH2 group at C3 (Figure 5a), which exhibits the
electron donating resonance effect. The C1-C2 bond length (1.449(2) Å) is smaller than
C1-C6 bond length (1.487(2) Å) and C1-O bond length (1.2378(18) Å) is more than C4-
O bond length (1.2255(18) Å), these differences can be attributed to asymmetric charge
distribution in the molecule 28. The same factor generates a considerable difference
between C2-C3 bond length (1.362(2) Å) and C5-C6 bond length (1.333(2) Å). The C=O
bond length for C1-O and C4-O are in the range of typical carbonyl bond lengths and the
bond angles for the carbon atoms of quinone rings are in range of sp2 hybrid carbon 29.
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Figure 5a: ORTEP Structure of ATQ
Figure 5b: Packing Arrangement of ATQ in Crystal Lattice
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Table 2: Crystal Data and Structure Refinement for ATQ
Identification code akdk1110
Empirical formula C10H13NO2
Formula weight 179.21
Temperature/K 110.00(10)
Crystal system Monoclinic
Space group P21/c
a/Å 9.2525(4)
b/Å 12.6323(4)
c/Å 9.1496(4)
α/° 90.00
β/° 118.064(5)
γ/° 90.00
Volume/Å3 943.67(6)
Z 4
ρcalcmg/mm3 1.261
m/mm-1 0.088
F(000) 384
Crystal size/mm3 0.4508 × 0.2591 × 0.1939
2Θ range for data collection 5.94 to 57.96°
Index ranges -12 ≤ h ≤ 7, -16 ≤ k ≤ 17, -11 ≤ l ≤ 11
Reflections collected 4099
Independent reflections 2144[R(int) = 0.0238]
Data/restraints/parameters 2144/0/170
Goodness-of-fit on F2 1.070
Final R indexes [I>=2σ (I)] R1 = 0.0455, wR2 = 0.1090
Final R indexes [all data] R1 = 0.0587, wR2 = 0.1194
Largest diff. peak/hole / e Å-3 0.243/-0.217
Flack Parameter N/A
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Table 3: Selected Bond Lengths [Å] for ATQ
Atom Atom Length/Å
C1 C2 1.449(2)
C1 C6 1.487(2)
C1 O1 1.2378(18)
C2 C3 1.362(2)
C2 C7 1.503(2)
C3 C4 1.506(2)
C3 N1 1.349(2)
C4 C5 1.486(2)
C4 O2 1.2255(18)
C5 C6 1.333(2)
C5 C8 1.512(2)
C8 C9 1.537(2)
C8 C10 1.527(2)
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Table 4: Selected Bond Angles [°] for ATQ
Atom Atom Atom Angle/˚
C2 C1 C6 119.64(13)
O1 C1 C2 121.86(14)
O1 C1 C6 118.48(14)
C1 C2 C7 118.37(14)
C3 C2 C1 118.53(14)
C3 C2 C7 123.09(15)
C2 C3 C4 121.56(14)
N1 C3 C2 125.69(14)
N1 C3 C4 112.74(13)
C5 C4 C3 118.98(13)
O2 C4 C3 119.34(14)
O2 C4 C5 121.68(14)
C4 C5 C8 117.12(13)
C6 C5 C4 117.56(14)
C6 C5 C8 125.30(14)
C5 C6 C1 123.48(14)
C5 C8 C9 109.38(13)
C5 C8 C10 112.59(14)
C10 C8 C9 110.62(14)
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3.5 Summary and Conclusion
In this chapter isolation of thymoquinone from commercial sample of Nigella
sativa oil, its purification, characterization by FTMS, IR, 1HNMR, 13CNMR and
synthesis of its amino derivative (ATQ) is reported along with characterization by
LCMS, IR, 1HNMR, 13CNMR and Single Crystal X-Ray Crystallography.
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