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.
Call Girls Nagpur Just Call 9907093804 Top Class Call Girl Service Available
Ā
Phytochemical and anti proliferative activity of Nigella sativa (Kalonji)
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
3. Introduction
ā¢ Cancer is one of the biggest threat to humanity in modern times and is second highest leading cause of death of death after MI.
ā¢ In 2017 India had 1.04 million new cases of cancer registered with 0.68 million mortalities reported (Sharma et al., 20181)
ā¢ Two of the most prevalent forms of cancer are colorectal cancer and breast cancer(mostly in female).
ā¢ Colorectal cancer begins with formation of polyps on lining of colon, and some polyps change into cancer. (Brenner et al., 20172) .
ā¢ 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 , pagetās 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.
ā¢ 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) .
ā¢ So, scientists have delved into the ethnomedicinal prospects
of different natural products to find answer
ā¢ Nigella sativa of Family Ranunculaceae has been used as an
important medicinal plants for hundreds of years. (Naz, 2011).
ā¢ It originated from South Eastern Asia and also employed
in ancient Egypt, Greece, geographic area and continent.
ā¢ Thymoquinone and Ī± 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
ā¢ Nigella sativa belonging to family Ranunculaceae . Nigella has 14 different species of
which N. sativa is medicinally active.
ā¢ Common names Black cumin, Fennel Flower, Nutmeg Flower, Kalonji, Kalojeera.
ā¢ 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.
ā¢ 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:
ā¢ N.sativa is a Rabi crop.
ā¢ It grows in all types of soil, with just temperature 10-20o
Celsius
ā¢ Two types of plantation possible:
- Indoor Sowing
- Garden Plantation
ā¢ 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.
ā¢ 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)
10. Antiproliferative property of N. sativa
ā¢ The antitumor effects of N. sativa was recognized by Ibn-Sina.
ā¢ 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 200ā300%, was observed (Agbaria et al., 20151).
ā¢ Topical application of N. sativa seed extract inhibited croton oil induced skin
carcinogenesis in mice (Randhawa, 2011).
ā¢ Aqueous and ethanolic extracts of N. sativa were found to inhibit MCF-7 breast
cancer cells
ā¢ Along with single preparation of N.sativa, polyherbal preparation are also
shown to have good cytotoxic activity .
ā¢ 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).
ā¢ Thymoquinone showed promising anti-cancer activity against MCF 7 breast
cancer cell line with IC50 value below 25 Āµg/ml (Shammugun et al., 20162).
ā¢ Ī±-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 Ī±-hederin on breast cancer cells. International Journal of Oncology, 45(2), 757-763..
Fig 5: Thymoquinone
Fig 6: Ī± 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 Ī± hederin as Antiproliferative
agents
ā¢ Poloxin, a thymoquinone derivative, hinders Plk1 causing Plk1 mislocalization, chromosome
congression defects, mitotic arrest (Reindl et al., 20081).
ā¢ 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)
ā¢ 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).
ā¢ One of the substitutes of Ī±-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 Ī± 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
ā¢ Thymoquinone was found to improve the antitumor impact of ifosfamide in mice bearing Ehrlich ascites
carcinoma
ā¢ 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).
ā¢ Thymoquinone improved the anticancer properties of doxorubicin, particularly against HL-60 leukemia cells
and multi-drug resistant MCF-7 cells(IC50 (72hrs)from 30 Āµg/ml to 125 ng/ml)
ā¢ Ī±-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., AlāShabanah, O. A., AlāSawaf, H. A., and AlāBekairi, A. M. (1999). Thymoquinone protects against carbon tetrachloride hetatotoxicity in mice via an antioxidant
mechanism. IUBMB Life, 47(1), 153- 159.
16. Rationale
ā¢ 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.
ā¢ Indigenous system of medicines, has helped in curing different diseases for millenium and
can also serve as an alternative in this problem.
ā¢ N.sativa has been used for different folk and indigenous system of medicine and told to be
āPanaceaā meaning it can cure any disease other than death.
ā¢ 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
ā¢ 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
ā¢ Extraction of N.sativa seeds using solvents of different polarities
ā¢ Preliminary investigation using Phytochemical tests on all the extracts
ā¢ MTT assay of all the extracts on breast cancer cell line.
ā¢ Insilico study of all the previously reported compounds from the same plant.
18. Experiments and Observation
ā¢ 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 .
ā¢ 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 (Āµg/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
ā¢ Methanolic extract was chosen for isolation purposes
ā¢ Column chromatography techniques used for isolation
25. Interaction Diagram of ERĪ±
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
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
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
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
35. Summary
ā¢ Nigella sativa has proven to show antiproliferative property.
ā¢ In invitro assay of T47-D cell line showed IC50 of 8.72Āµg/ml , 8.77Āµg/ml, 4.69 Āµg/ml for
SS1(Pet ether extract), SS2(Chloroform Extract), SS3(methanolic extract) respectively
comparatively.
ā¢ 6 compounds were isolated but due to inadequate spectral data the characterization could
not be completed.
ā¢ 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.
ā¢ 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
ā¢ These phytochemicals provides more possibilities for further research in the field of
antiproliferative drug development.
36. References
ā¢ 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.
ā¢ 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.
ā¢ 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.
ā¢ Izzettin, F. V., Al-taie, A., Sancar, M., & AliustaoÄlu, M. (2017). Influence of Pharmacist Recommendations for Chemotherapy-Related Problems in Diabetic
Cancer Patients. Marmara Pharmaceutical Journal, 21(3).
ā¢ Naz, H. (2011). Nigella sativa: the miraculous herb. Pak. J. Biochem. Mol. Biol, 44(1), 44-48.
ā¢ 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.
ā¢ Randhawa, M. A., & Alghamdi, M. S. (2011). Anticancer activity of Nigella sativa (black seed)āa review. The American journal of Chinese medicine, 39(06),
1075-1091.
ā¢ 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
ā¢ 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.
ā¢ 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 Ī±-hederin on breast
cancer cells. International journal of oncology, 45(2), 757-763.
ā¢ Reindl, W., Yuan, J., KrƤmer, 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.
ā¢ 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.
ā¢ 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.
47. Characterization of Isolated compounds
ā¢ Compound I:
ā¢ Proton Ī“ 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
ā¢ 29 Carbons were also detected from 13C NMR from the range of Ī“ 14.18 to 180.49
ā¢ Compound II was found to be luscious golden yellow luscious oils
ā¢ 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.
ā¢ The DEPT spectrum showed resolved peaks of a single methyl group nineteen methylene
group, eight methine group and one quaternary carbon.
ā¢ Presence of HMBC correlation between proton Ī“ 0.87 with Ī“25.72 , Ī“ 29.13, Proton Ī“1.65
correlated withĪ“ 27.29,Ī“ 29.18, Ī“ 29.25, Ī“ 36.80,Ī“ 39.32., Proton Ī“ 5.40 correlates with Ī“
27.29, Ī“ 127.74, Ī“ 128.15
ā¢ So the compound may be a multisaturated long chain fatty acid containing 29 carbon but due
to inadequate data structures could not be confirmed.
51. Characterization of Isolated compounds
Compound III
ā¢ Proton Ī“ 0.89, 1.20-1.32, 1.4, 1.99, 2.32, 3.58-3.62, 4.16-4.22, 5.32-5.47
ā¢ 21 Carbons were detected in 13C between the range of Ī“ 14.24-130.16
ā¢ Compound IV (SS03) was found to be yellow coloured oily liquid from the methanolic extract.
ā¢ LCMS data molecular ion peak is at m/z 355.29 (m+1) (calculated for C21H38O4 is 354.29)
ā¢ The DEPT spectrum showed resolved peaks of a single methyl group, fourteen methylene
group and six methine group.
ā¢ Presence of HMBC correlation between proton of Ī“ 0.89 with Ī“25.72, Ī“ 29.30, Ī“ 32.29, Ī“
77.30. Proton Ī“ 1.99 is correlated with Ī“ 29.18,Ī“ 127.97, Ī“ 129.41.
ā¢ So the compound may be a multisaturated fatty alcohol containing 21 carbon but due to
inadequate data structures could not be confirmed.