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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

65  

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

66  

(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.

69  

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

71  

Figure 3b: Infra Red Spectrum of TQ

Figure 3c: 1HNMR Spectrum of TQ

72  

Figure 3d: 13CNMR Spectrum of TQ

73  

Figure 4a: Total Ion Current (TIC) Chromatogram of ATQ

Figure 4b: Mass Spectrum of ATQ

74  

Figure 4c: Infra Red Spectrum of ATQ

Figure 4d: 1HNMR Spectrum of ATQ

75  

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.

76  

Figure 5a: ORTEP Structure of ATQ

Figure 5b: Packing Arrangement of ATQ in Crystal Lattice

77  

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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