Highly potent plant having very useful pharmacological effect especially in anticancer studies and various other diseases. It is even called Panacea because it can cure all diseases except death. Various religious books mention its existence as potent herbal drug.

1. Phytochemical Investigation and Antiproliferative
Activity of Nigella sativa Linn. Seeds
NAME: SAPTARSHI SAMAJDAR
REG. NO.:16mphyto02
COURSE: M.Pharm (Pharmacognosy and Phytochemistry)
Department of Pharmaceutical sciences and Natural Products
CENTRAL UNIVERSITY OF PUNJAB

2. Contents
• Introduction
• Review of Literature
• Rational
• Aim and Objective
• Experiments and Observation
• Summary

3. Introduction
• Cancer is one of the biggest threat to humanity in modern times and is second highest leading cause of death of death after MI.
• In 2017 India had 1.04 million new cases of cancer registered with 0.68 million mortalities reported (Sharma et al., 20181)
• Two of the most prevalent forms of cancer are colorectal cancer and breast cancer(mostly in female).
• Colorectal cancer begins with formation of polyps on lining of colon, and some polyps change into cancer. (Brenner et al., 20172) .
• Breast cancer is most common among women worldwide. As per site of action it can be classified into ductal carcinoma insitu, invasive
ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer , male breast cancer , paget’s disease of nipple, phyllodes tumor
of breast, adenocarcinoma (Hoadley et al., 20143).
Fig 1: Cancer stats
in India
(http://www.moneycontrol.com/gestepahead/curing/article/
india-and-the fight-against-cancer-965373-4.html)
1. Cancer statistics in India on the basis of first report of 29 population-based cancer registries. Journal of Oral and Maxillofacial Pathology, 22(1), 18.
2. Recreational physical activity, sedentary time and the incidence of colorectal polyps in a screening population for colon cancer: AACR.
3. Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin.cell, 158(4), 929-944.

4. Cont.
• Although great advances were made in modern sciences with
development of synthetic chemotherapeutics to stop cancer,
there has been some drawbacks (Izzetin et al., 2017) .
• So, scientists have delved into the ethnomedicinal prospects
of different natural products to find answer
• Nigella sativa of Family Ranunculaceae has been used as an
important medicinal plants for hundreds of years. (Naz, 2011).
• It originated from South Eastern Asia and also employed
in ancient Egypt, Greece, geographic area and continent.
• Thymoquinone and α hederin isolated is known to have
anticancer property
Fig 2: Drawbacks of
chemotherapeutics(adapted from
https://www.pinterest.com/pin/662451426408900480/
accessed on 2/5/18)
1. Influence of Pharmacist Recommendations for Chemotherapy-Related Problems in Diabetic Cancer Patients. Marmara Pharmaceutical Journal, 21(3).
2. Nigella sativa: the miraculous herb. Pak. J. Biochem. Mol. Biol, 44(1), 44-48.

5. Review of Literature
• Nigella sativa belonging to family Ranunculaceae . Nigella has 14 different species of
which N. sativa is medicinally active.
• Common names Black cumin, Fennel Flower, Nutmeg Flower, Kalonji, Kalojeera.
• Native to Southern Europe, North Africa and Southwest Asia and it is cultivated in many
countries in the world like Middle Eastern Mediterranean region, South Europe, India,
Pakistan, Syria.
• Plants grow from 20-90 cm in length having fine segmented leaf. Flowers have 5-10
petal and of various colours. Inside pod of flowers seeds are present. Seeds are dicot
black with spicy smell and a pungent bitter taste.
Fig 3: Morphology of Nigella sativa
(Adapted from Dubey, P. N., Singh, B., Mishra, B. K., Kant, K., & Solanki, R. K. (2016). Nigella ( Nigella sativa L.): A high value seed spice with immense medicinal potential.
Indian Journal of Agricultural Sciences, 86(8), 967-979.))

6. Cultivation methods:
• N.sativa is a Rabi crop.
• It grows in all types of soil, with just temperature 10-20o
Celsius
• Two types of plantation possible:
- Indoor Sowing
- Garden Plantation
• Black cumin does fine with average watering and can
tolerate short periods of drought, but water plants
regularly during long periods without rain to keep the
soil from drying out too much.
• Fertilize plants with any liquid fertilizer, either at the time
of transplanting seedlings grown from seed indoors or
when plants sown directly outside Fig 4: Garden
plantation of N. sativa
(http://www.kisaanhelpline.com/crops/product/19_K
alonji)

7. Phytochemicals reported in N. sativa

8. Cont.

9. Pharmacological Activities
Nigella sativa
Anticancer
activities
Antidiabetic
activity
Cardiovascular activity
Hepatoprotectiv
e activity
Neurological
activity
Gastroprotective
activity
Antibacterial activity
Immunomodulatory
activity
Anticonvulsa
nt activity
Antifungal
activities
Anti allergic property
Anti-
inflammatory
activities
Antiviral activity
Antioxidant activity

10. Antiproliferative property of N. sativa
• The antitumor effects of N. sativa was recognized by Ibn-Sina.
• With regard to modern science, the anticancer activity of N. sativa was
revealed for the first time, when an enhancement of the natural killer (NK) cell
activity, ranging from 200–300%, was observed (Agbaria et al., 20151).
• Topical application of N. sativa seed extract inhibited croton oil induced skin
carcinogenesis in mice (Randhawa, 2011).
• Aqueous and ethanolic extracts of N. sativa were found to inhibit MCF-7 breast
cancer cells
• Along with single preparation of N.sativa, polyherbal preparation are also
shown to have good cytotoxic activity .
• Aqueous and ethanolic extracts of mixtures of N. sativa, H. indicus and S.
glabra were used to treat cancer in Srilanka was confirmed by DEN induced
carcinogenesis in animal (Tavakkoli et al., 2017).
• Thymoquinone showed promising anti-cancer activity against MCF 7 breast
cancer cell line with IC50 value below 25 µg/ml (Shammugun et al., 20162).
• α-hederin obtained from the ethanolic extract of N. sativa has also shown good
result in MCF 7 cell line (Cheung et al., 20143)
1. Anticancer activity of Nigella sativa (black seed) and its relationship with the thermal processing and quinone composition of the
seed. Drug design, Development and Therapy, 9, 3119.
2.Cellular responses with thymoquinone treatment in human breast cancer cell line MCF-7, Pharmacognosy research, 5(3), 200.
3.The anticancer effect and mechanism of α-hederin on breast cancer cells. International Journal of Oncology, 45(2), 757-763..
Fig 5: Thymoquinone
Fig 6: α Hederin

11. Molecular Targets of N. sativa extracts
Khan, A., Chen, H. C., Tania, M., & Zhang, D. Z. (2011). Anticancer activities of Nigella sativa (black cumin). African
Journal of Traditional, Complementary and Alternative Medicines, 8(5S).)

12. Mechanisms of extract on different targets
Fig 7.: Mechanism of Nigella
seed extract on CASPASE 8, 9,
3 and BCL-2 protein
Fig 8:Effect of Nigella on Akt

13. Mechanisms of extract on different targets
Fig 9: Mechanism of Nigella extracts
on NF-kB and IL6 in facilitating
Apoptosis
Fig 10: Effect of N. sativa extract on Superoxide

14. Derivatives of Thymoquinone and α hederin as Antiproliferative
agents
• Poloxin, a thymoquinone derivative, hinders Plk1 causing Plk1 mislocalization, chromosome
congression defects, mitotic arrest (Reindl et al., 20081).
• 4 acylhydrazones and 6-Alkyl derivatives of thymoquinone were likewise tried for inhibition in
human HL-60 leukemia, 518A2 melanoma and MCF-7 breast carcinoma cells( IC50 less than
30ng/ml)
• Analogues of thymoquinone were synthesised by change at carbonyl and benzenoid sites and
tried for their biological activity against pancreatic cancer cell lines (Zaher et al., 20172).
• One of the substitutes of α-hederin, kalopanaxsaponin-I, was found to have anticancer action
1. Inhibition of polo-like kinase 1 by blocking polo-box domain-dependent protein-protein interactions. Chemistry & biology, 15(5), 459-466.
2. Semi-synthetic thymoquinone analogs: new prototypes as potential antihyperlipidemics in irradiated rats. Future medicinal chemistry,
9(13), 1483-1493.

15. Combination Therapy of Thymoquinone and α hederin as
antiproiferative agent
In the attempt to increase their efficacy and limit their adverse effects and, particularly, prevent the development
of resistance, most of the anticancer drugs prescribed in combination
• Thymoquinone was found to improve the antitumor impact of ifosfamide in mice bearing Ehrlich ascites
carcinoma
• It was also shown to augment the antitumor activity of gemcitabine and oxaliplatin against pancreatic cancer.
The study revealed that pre-exposure of cells to thymoquinone followed by gemcitabine or oxaliplatin
resulted in 60% to 80% growth inhibition compared to 15% to 25% when gemcitabine or oxaliplatin were
used alone (Effenberg- Neighnit, 20111).
• Thymoquinone improved the anticancer properties of doxorubicin, particularly against HL-60 leukemia cells
and multi-drug resistant MCF-7 cells(IC50 (72hrs)from 30 µg/ml to 125 ng/ml)
• α-hederin also, in sub-IC50 cytotoxic concentrations, was shown to enhance the cytotoxicity of 5-fluorouracil
in a human colon carcinoma model, HT-29 cells, 3.3-fold
1. Effenberger-Neidnicht, K., & Schobert, R. (2011). Combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin. Cancer chemotherapy and pharmacology, 67(4), 867-874
2. Nagi, M. N., Alam, K., Badary, O. A., Al‐Shabanah, O. A., Al‐Sawaf, H. A., and Al‐Bekairi, A. M. (1999). Thymoquinone protects against carbon tetrachloride hetatotoxicity in mice via an antioxidant
mechanism. IUBMB Life, 47(1), 153- 159.

16. Rationale
• With modern technologies and procedures synthetic drugs had huge development, but it
had some drawbacks like toxicity, MDR, bioavailability and suitability of dosage forms
especially in case of antiproliferative drug development.
• Indigenous system of medicines, has helped in curing different diseases for millenium and
can also serve as an alternative in this problem.
• N.sativa has been used for different folk and indigenous system of medicine and told to be
‘Panacea’ meaning it can cure any disease other than death.
• Extracts of the seeds proved to be very potent against several types of cancer and two
compounds isolated from the seed extracts proved to be equally useful
• More researches on different extracts can pave way many new options for drug
development.

17. Aim and Objectives
The aim and objectives of this dissertation is to perform
• Extraction of N.sativa seeds using solvents of different polarities
• Preliminary investigation using Phytochemical tests on all the extracts
• MTT assay of all the extracts on breast cancer cell line.
• Insilico study of all the previously reported compounds from the same plant.

18. Experiments and Observation
• Collection:
Commercially available seeds were procured from local market of Bathinda
The seeds as they were dry got crushed to a coarse powder using grinder and
stored in a cool dry place away from light and moisture .
• Extraction:
Four different solvents were chosen from lower to higher polarity Pet.
Ether, Chloroform, Methanol and Methanol: Water (80:20 v/v)
method of extratraction were used:
Speed Extractor: Sample taken 2.4Kg
Solvent taken per 3 cycles is 300 ml
Cycles:3 Temp: 1030C Pressure: 5barPet. Ether Chloroform Methanol Methanol+H2O
41g 96g 105g 14g
Fig 11: Speed
extractor

19. Phytochemical Tests:
Tests Name of Test Pet. Ether
Ext.
CHCl3 Ext. Methanolic
Ext.
Water Ext.
Alkaloid Test Hagers test + + + +
Fixed oil Test Oil drop test + + + -
Test for tannin FeCl3 test - - - -
Volatile oil
Test
Sudan III test + + + -
Flavanoid Test Alkaline test - + + +
Glycoside test Fehling test - - - -
Steroid test Salwoski test + + + +
Triterpene test Salwoski test + - + +
Fig 12: Test for
volatile oil

20. Invitro antiproliferative activity
Cell Line Extracts IC50 value (µg/ml)
T47-D (Breast cancer cell line)
SS1(Pet. Ether extract) 8.72
SS2(Chloroform extract) 8.77
SS3( Methanol extract) 4.69
SS4(Methanol+Water extract) NA
Bazedoxifene (standard) 18.29
From the above result we can conclude that three extracts SS1, SS2, SS3 have shown better
IC50 than the standard antiproliferative Bazedoxifene

21. Isolation of Phytochemicals
Compounds Amount (mg) TLC System
I 78 EtOAc : PE 1:5 v/v
II 47.5 EtOAc : PE 1:5 v/v
III 28 EtOAc : PE 3:2 v/v
IV 11 MeOH : EtOAc 3:7 v/v
V 34 EtOAc: HCOOH: H2O
15:3:2 v/v/v
VI 67 MeOH : EtOAc 7:3 v/v
• Methanolic extract was chosen for isolation purposes
• Column chromatography techniques used for isolation

22. Proton and Carbon NMR spectrum of Compound I
Proton (δ) 13C spectrum δ
0.87 14.18
1.19 22.69
1.22-1.29 24.75-2.72
1.51-1.53 27.29-27.75
1.64-1.65 29.18-29.78
1.97 31.63-36.8
1.99-2.06 62.24
2.28 127.74-130.3
2.74-2.76 180.49
5.32-5.35
10.09

23. Proton and Carbon NMR spectrum of Compound III
Proton(δ) 13C Spectrum (δ)
0.89 14.24
1.20 - 1.32 22.80-25.72
1.4 27.30-29.96
1.99 31.63-34.23
2.32 63.39-77.30
3.58-3.63 124.81-130.16
3.92
4.16-4.22
5.32- 5.47

24. In-silico activity of reported compounds from N. sativa
• ER α
Protein used: 3ERT
Inhibitor:4-hydroxytamoxifen(-9.975kcal/mol)
Sl. No. Structures
Dock
score(kcal/mol)
Hbond Lipophilic Electro
1 3ERT standard -9.975 -0.571 -7.957 -0.825
2 longifolene -8.093 0 -4.92 0.027
3 lignoceric acid -8.034 -0.35 -6.401 -0.058
4 cycloartenol -7.947 -1.042 -4.347 -0.496
5 germacrene -7.935 0 -4.916 0.012
6
stigmaglucopyranoside
.
-7.874 -1.92 -4.43 -0.724
7 dihomolinoleic acid. -7.819 -0.886 -5.128 -0.026
8 arachidonic acid. -7.784 0 -5.804 0.153
9 nervonic acid -7.301 -0.637 -6.204 0.007
10 obtusifoliol -7.218 0 -4.826 -0.091

25. Interaction Diagram of ERα
Fig 13: Interaction Diagram of (a)Standard (-9.975 kcal/mol) (b)Longifolene ( -8.093 kcal/mol) (c)Lignoceric
acid(-8.034 kcal/mol)
a b c

26. • Aromatase:
• Protein: 3EQM
• Inhibitor: 4-Androstenedione (-5.214kcal/mol)
Sl. No. Structures
Dock
score(kcal/mol)
Hbond Lipophilic Electro
1 longifolene -6.322 0 -5.189 -0.03
2 germacrene -5.89 0 -4.448 -0.068
3 citronellol. -5.824 -0.742 -3.993 -0.106
4 carvacrol. -5.795 -0.458 -3.932 -0.294
5 thymohydroquinone. -5.784 -0.7 -3.629 -0.23
6 carvone. -5.567 -0.7 -3.238 -0.278
7 P-cymene-8-ol. -5.434 0 -3.602 -0.098
8 α pinene. -5.424 0 -3.801 -0.048
9 terpenen-4-ol. -5.29 -0.575 -4.048 -0.168
10 3EQM standard -5.214 -0.7 -6.918 -0.266

27. Interaction Diagram of Aromatase Receptor
Fig 14: Interaction diagram of a. standard (-5.214kcal/mol) b. longifolene (-6.322 kcal/mol) c.
Germacrene(-5.89 kcal/mol)
a b
c

28. • Progesterone Receptor
• Protein: 1E3K
• Inhibitor: Methyltrienolone (-10.631 kcal/mol)
Sl. No. Structures
Dock score
(kcal/mol)
Hbond Lipophilic Electro
1 Eicosadinoic acid. -11.211 -1.306 -6.261 -0.166
2 Dihomolinoleic acid. -11.039 -1.305 -6.609 -0.184
3 standard 1E3K. -10.631 -0.305 -6.498 -0.229
4 arachidonic acid. -10.065 -1.22 -6.572 -0.239
5 oleic acid. -9.801 -1.303 -5.741 -0.157
6 nervonic acid. -9.135 -1.324 -5.752 -0.254
7 cycloeucalenol. -8.972 -1.635 -7.025 -0.6
8 dithymoquinone. -8.706 0 -6.028 0.051
9 linoleic acid. -8.702 -1.298 -5.912 -0.225
10 longifolene. -8.471 0 -5.267 -0.004

29. Interaction Diagram of Progesterone Receptor
Fig 15: Interaction diagrams of a. Standard (-10.631kcal/mol) b. Eicosadinoic acid(-11.211kcal/mol) ,c.
Dihomolinoleic Acid (-11.039kcal/mol)
a
b c

30. • Glucocorticoid
• Protein: 4P6W
• Inhibitor: Mometasone Furoate (-14.841kcal/mol)
Sl. No. Structures Dock score Hbond Lipophilic Electro
1 standard 4P6W -14.841 -1.191 -8.686 -0.54
2 alpha sitosterol -12.64 -0.7 -8.22 -0.198
3 gramisterol -11.689 -0.754 -7.059 -0.314
4 cycloeucalenol -11.172 -0.7 -7.556 -0.253
5 cycloart -10.938 -0.306 -7.304 -0.101
6 nervonic acid -10.619 -0.993 -7.313 -0.29
7 stigmastanol. -10.514 -1.63 -6.426 -0.567
8 Eicosadinoic acid. -10.139 -1.169 -6.16 -0.336
9 Dihomolinoleic acid. -9.957 -1.226 -5.998 -0.319
10 butyrospermol. -9.729 -0.77 -6.622 -0.149

31. Interaction Diagram of Glucocorticoid Receptors
Fig 16: Interaction diagram of a.standard (-14.481kcal/mol) b.α-sitosterol (-
12.64 kcal/mol) , c. Gramisterol(-11.689kcal/mol)
a
cb

32. • Human Androgen Receptor
• Protein: 2YHD Standard
• Standard: 4-(2, 3 dihydro-1H-pyrimidin-2-yl) benzene-2-diol (-2.466 kcal/mol)
Sl. No. Structures Dock score(kcal/mol) Hbond Lipophilic Electro
1 nervonic acid -4.519 -1.777 -3.353 -0.364
2 stigmaglucopyranoside -3.584 -3.202 -1.115 -0.876
3 lignoceric acid. -3.555 -1.624 -3.01 -0.362
4 nigellidone sulfite. -3.334 -1.007 -2.572 -0.126
5 dihomolinoleic acid. -2.947 -1.467 -2.297 -0.292
6 terpenen-4-ol. -2.893 -0.451 -2.161 -0.177
7 p-cymene-8-ol. -2.831 -0.586 -2.491 -0.219
8 sabinene. -2.694 0 -2.391 0.011
9 α thujene. -2.678 0 -2.585 0.022
10 dithymoquinone. -2.565 -0.425 -1.718 -0.21
14 2YHD Standard -2.466 -1.272 -2.246 -0.245

33. Interaction Diagram of Androgen Receptor
Fig 18: Interaction diagram of a. standard( -2.466 kcal/mol) b.Nervonic acid(-4.519kcal/mol) c.Stigmaglucopyranoside
(-3.584kcal/mol)
a
b
c

34. ADME analysis
Sl. No. Ligand
mol
wt.
H-bond
donor
H- bond
acceptor
QPlog
Po/w
QPlog
HERG
QPP
Caco
QPlogB
B
QPlogK
HSA
%oral
absorption
1 1,8- cineole 154.252 0 0.75 2.46 -2.594 9906.038 0.605 0.224 100
2 alpha pinene 136.236 0 0 3.645 -2.781 9906.038 0.872 0.349 100
3 alpha sitosterol 426.724 1 1.7 7.851 -4.732 3988.738 -0.222 2.199 100
4 alpha thujene 136.236 0 0 3.876 -2.979 9906.038 0.875 0.374 100
5 amyrin 426.724 1 1.7 7.072 -3.769 4371.265 0.171 2.061 100
6 anethole 148.204 0 0.75 3.166 -4.038 9906.038 0.286 0.119 100
7 carvacrol 150.22 1 0.75 3.298 -3.603 3683.827 0.071 0.056 100
8 carvone 150.22 0 2 2.17 -3.331 3577.525 0.127 -0.129 100
9 citronellol 156.267 1 1.7 2.685 -3.729 2894.743 -0.297 -0.01 100
10 cycloart 438.692 2 3.4 6.12 -4.015 1687.933 -0.531 1.512 100
11 cycloeucalenol 370.617 1 1.7 6.787 -4.63 3640.145 -0.257 1.742 100
12 nigellimine 203.24 0 2.5 2.769 -4.04 6660.998 0.289 -0.069 100
13 obtusifoliol. 426.724 1 1.7 7.769 -4.534 3992.679 -0.208 2.163 100
14 nigellone. 193.245 1 2.5 2.681 -3.719 5307.324 0.088 -0.064 100

35. Summary
• Nigella sativa has proven to show antiproliferative property.
• In invitro assay of T47-D cell line showed IC50 of 8.72µg/ml , 8.77µg/ml, 4.69 µg/ml for
SS1(Pet ether extract), SS2(Chloroform Extract), SS3(methanolic extract) respectively
comparatively.
• 6 compounds were isolated but due to inadequate spectral data the characterization could
not be completed.
• Insilico docking study of five receptors with reported compound expressed that multiple
compounds showed better results than the standard inhibitor in three receptors, aromatase,
progesterone and androgen.
• Insilico ADME showed most of the compound had 100% oral bioavailability. Compounds like
longifolene, amyrin, carvone, nigellimine showed not only high oral bioavailability but also
other parameters like stomach lining permeability and serum albumin binding capabilities.
Many other compounds have also shown higher than limit of oral bioavailability
• These phytochemicals provides more possibilities for further research in the field of
antiproliferative drug development.

36. References
• Sharma, S., Satyanarayana, L., Asthana, S., Shivalingesh, K., Goutham, B. S., & Ramachandra, S. (2018). Cancer statistics in India on the basis of first
report of 29 population-based cancer registries. Journal of Oral and Maxillofacial Pathology, 22(1), 18.
• Brenner, D. R., Yannitsos, D. H., Warkentin, M., Shaw, E., Brockton, N. T., McGregor, S. E.,Hilsden, R. J. (2017). Abstract B02: Recreational physical
activity, sedentary time and the incidence of colorectal polyps in a screening population for colon cancer: AACR.
• Hoadley, K. A., Yau, C., Wolf, D. M., Cherniack, A. D., Tamborero, D., Ng, S., Uzunangelov, V. (2014). Multiplatform analysis of 12 cancer types reveals
molecular classification within and across tissues of origin.cell, 158(4), 929-944.
• Izzettin, F. V., Al-taie, A., Sancar, M., & Aliustaoğlu, M. (2017). Influence of Pharmacist Recommendations for Chemotherapy-Related Problems in Diabetic
Cancer Patients. Marmara Pharmaceutical Journal, 21(3).
• Naz, H. (2011). Nigella sativa: the miraculous herb. Pak. J. Biochem. Mol. Biol, 44(1), 44-48.
• Agbaria, R., Gabarin, A., Dahan, A., & Ben-Shabat, S. (2015). Anticancer activity of Nigella sativa (black seed) and its relationship with the thermal
processing and quinone composition of the seed. Drug design, development and therapy, 9, 3119.
• Randhawa, M. A., & Alghamdi, M. S. (2011). Anticancer activity of Nigella sativa (black seed)—a review. The American journal of Chinese medicine, 39(06),
1075-1091.
• Tavakkoli, A., Ahmadi, A., Razavi, B. M., & Hosseinzadeh, H. (2017). Black seed (Nigella sativa) and its constituent thymoquinone as an antidote or a
protective agent against natural or chemical toxicities. Iranian Journal of Pharmaceutical Research, 16, 2-23
• Shanmugam, M. K., Hsu, A., Hui, K. M., Tan, B. K., & Sethi, G. (2016). Thymoquinone inhibits bone metastasis in a breast cancer mouse model by
modulating CXCR4/CXCL12 signaling axis.
• Cheng, L., Xia, T. S., Wang, Y. F., Zhou, W., Liang, X. Q., Xue, J. Q., & Wang, M. (2014). The anticancer effect and mechanism of α-hederin on breast
cancer cells. International journal of oncology, 45(2), 757-763.
• Reindl, W., Yuan, J., Krämer, A., Strebhardt, K., & Berg, T. (2008). Inhibition of polo-like kinase 1 by blocking polo-box domain-dependent protein-protein
interactions. Chemistry & biology, 15(5), 459-466.
• Zaher, N. H., Rashed, E. R., & El-Ghazaly, M. A. (2017). Semi-synthetic thymoquinone analogs: new prototypes as potential antihyperlipidemics in
irradiated rats. Future medicinal chemistry, 9(13), 1483-1493.
• Effenberger-Neidnicht, K., & Schobert, R. (2011). Combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin. Cancer chemotherapy
and pharmacology, 67(4), 867-874.

38. Full Docking Scores of ERα Receptor
Structures Dock score Hbond Lipophilic Electro
1 3ert standard.cdx -9.975 -0.571 -7.957 -0.825
2 longifolene.cdx -8.093 0 -4.92 0.027
3 lignoceric acid.cdx -8.034 -0.35 -6.401 -0.058
4 cycloart.cdx -7.947 -1.042 -4.347 -0.496
5 germacrene.cdx -7.935 0 -4.916 0.012
6 stigmaglucopyranoside.cdx -7.874 -1.92 -4.43 -0.724
7 Dihomolinoleic acid.cdx -7.819 -0.886 -5.128 -0.026
8 arachidonic acid.cdx -7.784 0 -5.804 0.153
9 nervonic acid.cdx -7.301 -0.637 -6.204 0.007
10 obtusifoliol.cdx -7.218 0 -4.826 -0.091
11 Eicosadinoic acid.cdx -7.083 -0.35 -5.931 -0.032
12 cycloeucalenol.cdx -6.754 -0.35 -3.929 -0.228
13 Isofucosterol.cdx -6.575 -0.336 -4.482 -0.155
14 carvacrol.cdx -6.567 -0.683 -3.237 -0.122
15 1,8- cineole.cdx -6.53 0 -3.363 0.032
16 sabinene.cdx -6.413 0 -3.554 0.019
17 nigellimine.cdx -6.329 0 -4.22 -0.01
18 Thymohydroquinone.cdx -6.172 -0.544 -3.22 -0.115
19 alpha thujene.cdx -6.155 0 -3.607 0.006
20 alpha sitosterol.cdx -6.065 0 -5.052 -0.037
21 citronellol.sdf -6.056 -0.828 -2.897 -0.675
22 alpha pinene.cdx -6.017 0 -3.136 0.023
23 limonene.cdx -6.011 0 -3.281 -0.006
24 anethole.cdx -5.996 -0.087 -3.429 -0.015
25 P-cymene-8-ol.cdx -5.962 -0.35 -3.334 -0.234
26 THQ.cdx -5.823 0 -2.721 0.007
27 Gramisterol.cdx -5.769 -0.494 -3.494 -0.176
28 carvone.cdx -5.758 0 -3.062 0.062
29 nigellimine.cdx -5.738 -0.314 -3.711 -0.348
30 linoleic acid.cdx -5.59 -0.644 -4.946 0.041
31 nigellidone sulfite.cdx -5.524 0 -3.057 -0.076
32 oleic acid.cdx -5.427 -0.629 -4.943 0.018
33 nigellone.cdx -5.14 -0.628 -3.103 -0.09
34 myristic acid.cdx -4.957 -0.9 -4.506 0.093
35 Terpenen-4-ol.cdx -4.719 -0.201 -3.018 -0.124
36 nigellicine.cdx -4.215 0 -3.616 -0.112
37 capric acid.cdx -3.797 -0.646 -3.097 0.041
38 nigellone.cdx -3.156 0 -4.14 -0.152
39 Taraxerol.cdx -2.023 -0.35 -1.77 -0.331

39. Full Docking Scores of Aromatase Receptor
Structures Dock score Hbond Lipophilic Electro
1 longifolene.cdx -6.322 0 -5.189 -0.03
2 germacrene.cdx -5.89 0 -4.448 -0.068
3 citronellol.sdf -5.824 -0.742 -3.993 -0.106
4 carvacrol.cdx -5.795 -0.458 -3.932 -0.294
5 Thymohydroquinone.cdx -5.784 -0.7 -3.629 -0.23
6 carvone.cdx -5.567 -0.7 -3.238 -0.278
7 P-cymene-8-ol.cdx -5.434 0 -3.602 -0.098
8 alpha pinene.cdx -5.424 0 -3.801 -0.048
9 Terpenen-4-ol.cdx -5.29 -0.575 -4.048 -0.168
10 3EQM.cdx -5.214 -0.7 -6.918 -0.266
11 nigellimine.cdx -5.208 0 -4.49 -0.298
12 sabinene.cdx -5.176 0 -3.759 0.054
13 alpha thujene.cdx -5.045 0 -3.633 0.034
14 1,8- cineole.cdx -4.801 0 -3.743 -0.02
15 nigellone.cdx -4.668 0 -3.952 -0.054
16 limonene.cdx -4.589 0 -3.47 0.011
17 THQ.cdx -4.515 0 -2.948 -0.041
18 nigellimine.cdx -4.082 0 -4.346 -0.449
19 arachidonic acid.cdx -3.602 -1.469 -6.004 0.085
20 Eicosadinoic acid.cdx -3.57 -0.35 -6.303 0.032
21 capric acid.cdx -3.501 0 -3.368 0.596
22 Dihomolinoleic acid.cdx -3.432 -0.35 -6.252 0.018
23 nigellone.cdx -3.155 0 -4.22 -0.395
24 linoleic acid.cdx -2.506 -0.35 -4.939 -0.028
25 oleic acid.cdx -2.317 -0.257 -5.241 -0.021
26 nervonic acid.cdx -1.969 -0.024 -5.742 0.371
27 myristic acid.cdx -1.766 -0.35 -4.456 0.029
28 anethole.cdx -1.549 0 -3.842 -0.1
29 nigellicine.cdx -1.483 0 -5.007 0.024
30 nigellidone sulfite.cdx -1.346 -0.031 -4.612 -0.239

40. Full Docking Scores of Progesterone Receptor
Structures Dock score Hbond Lipophilic Electro
1 Eicosadinoic acid.cdx -11.211 -1.306 -6.261 -0.166
2 Dihomolinoleic acid.cdx -11.039 -1.305 -6.609 -0.184
3 standard 1E3K.cdx -10.631 -0.305 -6.498 -0.229
4 arachidonic acid.cdx -10.065 -1.22 -6.572 -0.239
5 oleic acid.cdx -9.801 -1.303 -5.741 -0.157
6 nervonic acid.cdx -9.135 -1.324 -5.752 -0.254
7 cycloeucalenol.cdx -8.972 -1.635 -7.025 -0.6
8 dithymoquinone.cdx -8.706 0 -6.028 0.051
9 linoleic acid.cdx -8.702 -1.298 -5.912 -0.225
10 longifolene.cdx -8.471 0 -5.267 -0.004
11 germacrene.cdx -7.649 0 -4.615 -0.003
12 carvacrol.cdx -7.434 -0.675 -3.785 -0.168
13 Thymohydroquinone.cdx -6.925 -0.861 -3.379 -0.406
14 Terpenen-4-ol.cdx -6.745 -0.7 -3.224 -0.364
15 sabinene.cdx -6.664 0 -3.88 -0.005
16 P-cymene-8-ol.cdx -6.553 -0.7 -3.529 -0.181
17 alpha thujene.cdx -6.495 0 -3.887 -0.032
18 nigellimine.cdx -6.489 0 -4.496 -0.139
19 limonene.cdx -6.381 0 -3.686 -0.032
20 1,8- cineole.cdx -6.309 0 -3.712 0.047
21 anethole.cdx -6.306 0 -3.963 -0.038
22 myristic acid.cdx -6.297 -1.235 -4.729 -0.101
23 citronellol.sdf -6.224 -1.501 -3.72 -0.491
24 THQ.cdx -6.185 0 -3.34 0.005
25 alpha pinene.cdx -6.179 0 -3.39 0.021
26 capric acid.cdx -5.978 -0.642 -3.127 -0.185
27 nigellicine.cdx -5.928 0 -4.442 0.115
28 nigellone.cdx -5.836 -0.315 -4.082 -0.145
29 nigellimine.cdx -5.521 0 -4.255 -0.26
30 carvone.cdx -5.22 -0.412 -3.483 -0.169
31 nigellone.cdx -0.713 0 -3.933 -0.372

41. Full Docking Scores of Glucocorticoid Receptor
Structures Dock score Hbond Lipophilic Electro
1 standard 4p6w.cdx -14.841 -1.191 -8.686 -0.54
2 alpha sitosterol.cdx -12.64 -0.7 -8.22 -0.198
3 Gramisterol.cdx -11.689 -0.754 -7.059 -0.314
4 cycloeucalenol.cdx -11.172 -0.7 -7.556 -0.253
5 cycloart.cdx -10.938 -0.306 -7.304 -0.101
6 nervonic acid.cdx -10.619 -0.993 -7.313 -0.29
7 stigmastanol.cdx -10.514 -1.63 -6.426 -0.567
8 Eicosadinoic acid.cdx -10.139 -1.169 -6.16 -0.336
9 Dihomolinoleic acid.cdx -9.957 -1.226 -5.998 -0.319
10 butyrospermol.cdx -9.729 -0.77 -6.622 -0.149
11 arachidonic acid.cdx -9.66 -0.951 -5.818 -0.101
12 Tirucallol.cdx -9.523 -0.731 -6.285 -0.277
13 oleic acid.cdx -9.411 -0.913 -5.168 -0.204
14 Isofucosterol.cdx -8.672 -0.917 -5.597 -0.471
15 myristic acid.cdx -8.64 -1.217 -4.387 -0.167
16 linoleic acid.cdx -8.578 -1.116 -5.937 -0.185
17 obtusifoliol.cdx -7.649 -0.93 -6.031 -0.595
18 nigellidone sulfite.cdx -7.625 0 -5.346 -0.053
19 lupdiol.cdx -7.624 0 -5.849 0.042
20 THQ.cdx -6.93 -0.648 -3.118 -0.222
21 carvone.cdx -6.464 -0.407 -2.972 -0.175
22 capric acid.cdx -6.422 -1.189 -2.626 -0.204
23 longifolene.cdx -6.388 0 -4.568 0.002
24 germacrene.cdx -5.996 0 -4.612 0.009
25 dithymoquinone.cdx -5.92 0 -4.09 0.051
26 carvacrol.cdx -5.861 -0.738 -2.962 -0.323
27 nigellimine.cdx -5.81 -0.358 -3.567 -0.262
28 P-cymene-8-ol.cdx -5.335 -0.892 -3.067 -0.334
29 nigellone.cdx -5.152 -0.592 -3.373 -0.231
30 alpha thujene.cdx -5.115 0 -3.327 0.015
31 limonene.cdx -4.996 0 -3.355 0.032
32 sabinene.cdx -4.939 0 -3.019 -0.072
33 nigellicine.cdx -4.844 0 -3.75 0.162
34 Terpenen-4-ol.cdx -4.829 -0.7 -3.32 -0.159
35 citronellol.sdf -4.777 -1.609 -2.866 -0.561
36 Thymohydroquinone.cdx -4.638 -1.014 -3.186 -0.455
37 alpha pinene.cdx -4.609 0 -2.816 0.018
38 nigellimine.cdx -3.938 0 -3.372 -0.203
39 1,8- cineole.cdx -3.707 0 -3.031 -0.003
40 anethole.cdx -3.324 -0.723 -3.053 -0.324
41 nigellone.cdx 0.199 0 -3.631 -0.135

42. Full Docking Scores of Androgen Receptor
Structures Dock score Hbond Lipophilic Electro
1 nervonic acid.cdx -4.519 -1.777 -3.353 -0.364
2 stigmaglucopyranoside.cdx -3.584 -3.202 -1.115 -0.876
3 lignoceric acid.cdx -3.555 -1.624 -3.01 -0.362
4 nigellidone sulfite.cdx -3.334 -1.007 -2.572 -0.126
5 Dihomolinoleic acid.cdx -2.947 -1.467 -2.297 -0.292
6 Terpenen-4-ol.cdx -2.893 -0.451 -2.161 -0.177
7 P-cymene-8-ol.cdx -2.831 -0.586 -2.491 -0.219
8 sabinene.cdx -2.694 0 -2.391 0.011
9 alpha thujene.cdx -2.678 0 -2.585 0.022
10 dithymoquinone.cdx -2.565 -0.425 -1.718 -0.21
11 carvacrol.cdx -2.542 0 -2.628 -0.009
12 limonene.cdx -2.484 0 -2.387 0.064
13 anethole.cdx -2.476 -0.349 -2.013 -0.199
14 2YHD.cdx -2.466 -1.272 -2.246 -0.245
15 nigellone.cdx -2.423 0 -2.509 -0.013
16 THQ.cdx -2.381 -0.178 -1.812 -0.128
17 Eicosadinoic acid.cdx -2.361 -1.924 -2.303 -0.415
18 alpha hed.cdx -2.3 -1.275 -1.176 -0.925
19 nigellicine.cdx -2.266 0 -2.311 -0.039
20 nigellimine.cdx -2.253 -0.237 -2.207 -0.231
21 carvone.cdx -2.23 -0.415 -1.422 -0.205
22 alpha pinene.cdx -2.104 0 -1.835 -0.003
23 germacrene.cdx -2.095 0 -2.287 -0.031
24 arachidonic acid.cdx -2.065 -0.9 -1.792 -0.192
25 linoleic acid.cdx -2 -1.167 -2.626 -0.303
26 lupdiol.cdx -1.854 -0.414 -1.855 -0.178
27 Taraxerol.cdx -1.846 0 -2.324 0.087
28 cycloart.cdx -1.809 -0.7 -1.232 -0.458
29 cycloeucalenol.cdx -1.77 -0.209 -1.894 -0.191
30 longifolene.cdx -1.715 0 -2.155 0.035
31 nigellimine.cdx -1.715 -0.242 -2.081 -0.116
32 Thymohydroquinone.cdx -1.684 -0.642 -1.054 -0.315
33 citronellol.sdf -1.619 -0.35 -2.246 -0.284
34 butyrospermol.cdx -1.599 -0.35 -1.263 -0.445
35 Tirucallol.cdx -1.561 0 -2.138 0.011
36 capric acid.cdx -1.465 -1.323 -2.359 -0.217
37 obtusifoliol.cdx -1.354 -0.341 -1.777 -0.199
38 amyrin.cdx -1.322 0 -1.591 0.046
39 alpha sitosterol.cdx -1.298 0 -2.095 -0.032
40 Isofucosterol.cdx -1.057 0 -2.075 -0.029
41 Gramisterol.cdx -1.055 0 -1.856 0.012
42 1,8- cineole.cdx -0.924 0 -1.651 -0.021
43 stigmastanol.cdx -0.881 0 -1.577 0.051
44 myristic acid.cdx -0.808 -1.391 -1.732 -0.287
45 oleic acid.cdx -0.311 -1.429 -1.706 -0.328
46 nigellone.cdx -0.059 -0.169 -2.189 -0.16

43. Full ADME DataLigand mol wt. H-bond donor H- bond acceptor QPlog Po/w QPlog HERG QPP Caco QPlogBB QPlogKHSA %oral absorption
alpha hed.cdx 750.965 7 19 2.249 -3.055 4.295 -3.201 -0.195 12.567
1,8- cineole.cdx 154.252 0 0.75 2.46 -2.594 9906.038 0.605 0.224 100
alpha pinene.cdx 136.236 0 0 3.645 -2.781 9906.038 0.872 0.349 100
alpha sitosterol.cdx 426.724 1 1.7 7.851 -4.732 3988.738 -0.222 2.199 100
alpha thujene.cdx 136.236 0 0 3.876 -2.979 9906.038 0.875 0.374 100
amyrin.cdx 426.724 1 1.7 7.072 -3.769 4371.265 0.171 2.061 100
anethole.cdx 148.204 0 0.75 3.166 -4.038 9906.038 0.286 0.119 100
arachidonic acid.cdx 304.472 1 2 5.634 -1.784 558.713 -0.863 0.567 96.144
butyrospermol.cdx 426.724 1 1.7 7.66 -4.371 4360.559 -0.099 2.145 100
capric acid.cdx 172.267 1 2 2.955 -2.161 240.606 -0.911 -0.207 86.867
carvacrol.cdx 150.22 1 0.75 3.298 -3.603 3683.827 0.071 0.056 100
carvone.cdx 150.22 0 2 2.17 -3.331 3577.525 0.127 -0.129 100
citronellol.sdf 156.267 1 1.7 2.685 -3.729 2894.743 -0.297 -0.01 100
cycloart.cdx 438.692 2 3.4 6.12 -4.015 1687.933 -0.531 1.512 100
cycloeucalenol.cdx 370.617 1 1.7 6.787 -4.63 3640.145 -0.257 1.742 100
Dihomolinoleic acid.cdx 306.487 1 2 6.109 -2.941 284.286 -1.336 0.818 93.675
dithymoquinone.cdx 328.407 0 8 1.484 -3.375 639.86 -0.602 -0.528 85.859
Eicosadinoic acid.cdx 308.503 1 2 6.619 -3.831 283.785 -1.563 1.004 96.647
germacrene.cdx 204.355 0 0 5.051 -2.903 9906.038 1.01 0.905 100
Gramisterol.cdx 412.698 1 1.7 7.561 -4.655 3992.971 -0.21 2.061 100
Isofucosterol.cdx 412.698 1 1.7 7.535 -4.703 3377.971 -0.293 2.077 100
lignoceric acid.cdx 368.642 1 2 8.419 -4.446 240.179 -2.191 1.581 100
limonene.cdx 136.236 0 0 3.989 -3.269 9906.038 0.833 0.384 100
linoleic acid.cdx 280.45 1 2 5.515 -2.993 240.935 -1.373 0.631 88.912
longifolene.cdx 204.355 0 0 4.791 -2.659 9906.038 1.025 0.868 100
lupdiol.cdx 442.724 2 3.4 6.006 -3.722 1847.345 -0.352 1.546 100
myristic acid.cdx 228.374 1 2 4.533 -3.037 240.279 -1.292 0.304 96.096
nervonic acid.cdx 366.626 1 2 8.279 -4.413 239.764 -2.103 1.554 100
nigellidinesulfite.cdx
nigellicine.cdx 246.265 1 4 2.274 -1.875 122.553 -0.668 -0.152 77.639
nigellimine.cdx 203.24 0 2.5 2.753 -4.026 6453.321 0.277 -0.071 100
nigellimine.cdx 203.24 0 2.5 2.769 -4.04 6660.998 0.289 -0.069 100
obtusifoliol.cdx 426.724 1 1.7 7.769 -4.534 3992.679 -0.208 2.163 100
nigellone.cdx 193.245 1 2.5 2.681 -3.719 5307.324 0.088 -0.064 100
nigellone.cdx 193.245 1 2.5 2.802 -4.006 5972.38 0.127 -0.037 100
oleic acid.cdx 282.465 1 2 5.282 -1.913 463.171 -1.024 0.413 92.624
P-cymene-8-ol.cdx 150.22 1 0.75 3.167 -3.654 4748.295 0.169 0.042 100
sabinene.cdx 136.236 0 0 3.834 -2.942 9906.038 0.883 0.36 100
standard 6C42.cdx 445.557 2 5 5.19 -7.495 215.375 -0.634 1.24 86.135
stigmaglucopyranoside.cdx 574.84 4 10.2 5.195 -5.585 271.566 -2.134 0.957 75.008
Taraxerol.cdx 396.655 1 1.7 6.761 -4.124 4960.01 0.213 1.893 100
stigmastanol.cdx 416.729 1 1.7 7.624 -4.53 3377.357 -0.353 2.093 100
Terpenen-4-ol.cdx 154.252 1 0.75 2.976 -3.053 5684.019 0.241 0.116 100
TQ.cdx 164.204 0 4 0.754 -3.334 1062.751 -0.332 -0.631 85.526
Thymohydroquinone.cdx 166.219 2 1.5 1.919 -3.471 1421.949 -0.349 -0.173 94.612
Tirucallol.cdx 426.724 1 1.7 7.721 -4.428 4353.669 -0.107 2.179 100

44. 1H and 13C NMR Data of Compound I

45. DEPT 135 and HSQC data of Compound I

46. HMBC and LCMS data of Compound I

47. Characterization of Isolated compounds
• Compound I:
• Proton δ 0.87, 1.18-1.29, 1.51-1.65, 1.97-1.99, 2.06, 2.28, 2.74-76, 5.29-5.40, 10.09 were
detected
• 29 Carbons were also detected from 13C NMR from the range of δ 14.18 to 180.49
• Compound II was found to be luscious golden yellow luscious oils
• In LCMS data molecular ion peak is found to be m/z 446.34 corresponding to molecular
formula C29H50O3, close to reported value 446.39.
• The DEPT spectrum showed resolved peaks of a single methyl group nineteen methylene
group, eight methine group and one quaternary carbon.
• Presence of HMBC correlation between proton δ 0.87 with δ25.72 , δ 29.13, Proton δ1.65
correlated withδ 27.29,δ 29.18, δ 29.25, δ 36.80,δ 39.32., Proton δ 5.40 correlates with δ
27.29, δ 127.74, δ 128.15
• So the compound may be a multisaturated long chain fatty acid containing 29 carbon but due
to inadequate data structures could not be confirmed.

48. 1H and 13C NMR of Compound III

49. DEPT 135 and HSQC of Compound III

50. HMBC and LCMS data of Compound III

51. Characterization of Isolated compounds
Compound III
• Proton δ 0.89, 1.20-1.32, 1.4, 1.99, 2.32, 3.58-3.62, 4.16-4.22, 5.32-5.47
• 21 Carbons were detected in 13C between the range of δ 14.24-130.16
• Compound IV (SS03) was found to be yellow coloured oily liquid from the methanolic extract.
• LCMS data molecular ion peak is at m/z 355.29 (m+1) (calculated for C21H38O4 is 354.29)
• The DEPT spectrum showed resolved peaks of a single methyl group, fourteen methylene
group and six methine group.
• Presence of HMBC correlation between proton of δ 0.89 with δ25.72, δ 29.30, δ 32.29, δ
77.30. Proton δ 1.99 is correlated with δ 29.18,δ 127.97, δ 129.41.
• So the compound may be a multisaturated fatty alcohol containing 21 carbon but due to
inadequate data structures could not be confirmed.