63 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
22
Embed
CHAPTER 3 EXTRACTION, ISOLATION AND …shodhganga.inflibnet.ac.in/bitstream/10603/93493/11/11_chapter3.pdf · silica gel column and eluted with hexane, 15% diethyl ether in hexane,
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
63
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
64
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)
67
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.