List of publications

Online - 2455-3891
Vol 10, Issue 3, 2017 Print - 0974-2441
Research Article

IN VITRO IMMUNOSTIMULATION ACTIVITY OF NIGELLA SATIVA LINN. AND PSORALEA

CORYLIFOLIA LINN. SEEDS USING A MURINE MACROPHAGE CELL LINE

SHEIK NOOR MOHAMED M, JAIKUMAR K, MARIMUTHU S, JOHN WYSON W, ANAND D, SARAVANAN P*

Department of Botany, Ramakrishna Mission Vivekananda College (Autonomous), Mylapore, Chennai - 600 004, Tamil Nadu, India.
Email: sarviveka@[Link]
Received: 18 November 2016, Revised and Accepted: 01 December 2016

ABSTRACT

Objective: The aim of this study is to analyze the phytochemical constituents and the in vitro immunomodulatory potential of ethanol extract of
Nigella sativa and Psoralea corylifolia seeds.

Methods: Phytoconstituents in the ethanol extract of N. sativa and P. corylifolia seeds were analyzed using gas chromatography and mass spectrum
and their immunomodulatory activity was investigated in vitro by cell proliferation assay with J774A.1 cell line.

Results: Thirteen biochemical constituents have been identified with ethanol extract of N. sativa and nine from the P. corylifolia. The maximum relative
activity of 138.77±0.44% cell proliferation was expressed at 25 μg/ml by N. sativa and 80.70±0.42% of proliferation by P. corylifolia at 25 μg/ml.

Conclusion: The results suggest that the ethanol extract of the plant seeds have stimulating activity on macrophage cells and could be useful for
modulating immune functions.

Keywords: Immunomodulatory, Cell proliferation, Macrophage cell line, Nigella sativa, Psoralea corylifolia.

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license ([Link]
org/licenses/by/4. 0/) DOI: [Link]

INTRODUCTION these plants. Therefore the aim of the present study is to identify the
bioactive components of these plants by subjecting the ethanol extracts
The immune system is a remarkably versatile defense system that has
of the plant seeds to gas chromatography and mass spectrum (GC-MS)
evolved to protect animals from invading pathogenic microorganisms
analysis and to investigate their immune stimulation potential using an
and to eliminate diseases. The function and the efficacy of immune
in vitro cell line model.
system may be influenced by many exogenous factors such as food
and pharmaceuticals, physical and psychological stress and hormones. METHODS
Resulting in either immunostimulation or immunosuppression [1].
Suppression in immune response disrupts host’s ability to defend itself Collection of plant materials
against bacteria, viruses, and parasites. Immunomodulation using plant Seeds of N. sativa and P. corylifolia were procured from herbal medicine
material can provide an alternative to conventional chemotherapy for a store in Chennai. The seeds were authenticated at the Department of
variety of diseases, especially when the host defense mechanism has to be Medicinal Botany, National Institute of Siddha, Chennai and a voucher
activated under the condition of impaired immune response [2]. A large specimen (voucher number - NISMB2052015) was submitted.
number of drugs in use are derived from plants, like morphine from
Papaver somniferum, Aswagandha from Withania somnifera, Ephedrine Preparation of plant extracts
from Ephedra vulgaris, Atrophine from Atropa belladonna, etc. [3]. The Fine seeds were cleaned and milled to a powder using a domestic
phytochemical constituents like terpenoids, steroids, proteins, and blender. A total of 25 g of powdered seeds were then subjected to soxhlet
tannins [4] are considered to exhibit this immunomodulatory property. extraction with 250 ml of ethanol as solvent. The seed extract was
concentrated using vacuum rotatory evaporation to void of solvent. The
A large number of plants with therapeutic potentials have been extracts were filter sterilized and stored at 4°C until further use [12,13].
considered. One such is Nigella sativa Linn. (Ranunculaceae),
commonly known as “black cumin,” is an herbaceous plant that grows Cell culture
in Mediterranean countries. The oil and seed constituents have shown J774A.1, a murine macrophage cell line, was procured from National
potential medicinal properties in traditional medicine [5]. The black Centre for Cell Science, Pune, Maharashtra, India. The cells were
seeds contain 36-38% fixed oil, with proteins, alkaloids, saponins, maintained in Dulbecco’s modified Eagle’s medium (DMEM)
and essential oils making up the rest of the composition [6]. Black supplemented with 10% (v/v) inactivated fetal bovine serum (FBS),
seed extract has been reported to possess antimicrobial activity [7], 100 units/ml of penicillin, 100 μg/ml of streptomycin and 3%
antitumor activity [8], and a stimulatory effect on the immune L-glutamine (Himedia Labs, Mumbai). The cells were incubated at 37°C
system [9]. Psoralea corylifolia Linn. commonly known as “Bakuchi” is in a humidified atmosphere containing 5% (v/v) carbon dioxide (CO2).
conventionally used in ayurvedic system of medicine for the treatment The medium was changed every 2 days, and the cells were serially
of various kinds of human disorders but especially for treatment of skin passaged biweekly [14].
disorders [10]. P. corylifolia seed has been reported to contain several
phytoconstituents and possess antibacterial, antifungal, antioxidant, Treatment of cell cultures with ethanol extracts
antiflarial, estrogenic, and immunomodulatory activity [11]. However, A volume of 100 μl of medium containing 1×105 cells were seeded
there is no scientific data on the in vitro immunomodulatory activity of triplicates in 96-well plates. The plates were incubated for 24 hrs at
 Mohamed et al.
Asian J Pharm Clin Res, Vol 10, Issue 3, 2017, 329-332

37°C in 5% CO2. After incubation, the old medium was decanted and all RESULTS
the plates were replenished with fresh DMEM medium supplemented
with 10% FBS. The cells were then treated with varying concentrations The GC-MS analysis of the ethanol extract of N. sativa seeds revealed the
(6.25, 12.5, 25, 50, and 100 μg/ml) prepared by diluting the extract in presence of 13 peaks (Fig. 1) and P. corylifolia showed 9 peaks (Fig. 2)
DMEM and then incubated. The effect of ethanol extracts on macrophage indicating the presence of phytoconstituents whose name, molecular
proliferation was evaluated with wells containing only cells as a weight and structure were identified and characterized on comparison
negative control and compared to cells treated with concanavalin-A with data in the NIST library (Tables 1 and 2).
(Con-A), positive control [15].
Cell proliferation assay
Cell proliferation assay using (3-(4,5-dimethylthiazol-2-Yl)-2,5- Ethanol extract of N. sativa showed a maximum relative activity of
diphenyltetrazolium bromide) (MTT) reagent 138.77±0.44% at 25 μg/ml compared to the standard drug (Con-A)
After incubation, 20 μl of MTT reagent (5 mg/ml in phosphate buffer which expressed a proliferation rate of 51.11±0.96%. 11.30±0.24,
saline) was added to each well and further incubated for 4 hrs at 37°C. 112.03±0.15, 138.77±0.44, 83.46±0.30 and 50.54±0.43% increase in the
Then, dimethyl sulfoxide was added to dissolve the formazon crystals, proliferation of macrophage cells were reported at the dose rate of 6.25,
intensity of the colored reaction product was measured at 540 nm using 12.5, 25, 50, and 100 µg/ml of ethanol extract, respectively (Fig. 3). The
Microplate Reader (BIOTEK, USA) [16]. ethanol extract of P. corylifolia showed a maximum relative activity of
80.70±0.42% at 25 μg/ml compared to the standard (Con-A), resulting
GC-MS analysis in the proliferation range of 10.17±0.76, 28.46±0.46, 80.70±0.42,
GC-MS analysis of the ethanol extract of N. sativa and P. corylifolia 38.02±0.55, and 12.16±0.23% between varying concentrations of 6.25,
was carried out with 2 μl of extract employed on a GC clarus 500 12.5, 25, 50, and 100 μg/ml (Fig. 4).
Perkin Elmer system comprising a AOC-20i auto sampler and
gas chromatograph interfaced to a mass spectrometer (GC-MS) DISCUSSION
instrument employing the following conditions: Column Elite-1
fused silica capillary column (30×0.25 mm ID×1 EM df, composed of The information regarding the chemical constituents of a plant
100% dimethyl polysiloxane), operating in electron impact mode at is generally provided by the qualitative phytochemical screening
70 eV; helium (99.999%) was used as carrier gas at a constant flow of of its extract. Hence, the GC-MS analysis was opted to assess the
1ml/min and an injection volume of 0.5 EI was employed (split ratio phytochemical constituents present in the ethanol extracts of
of 10:1) injector temperature 250°C; ion source temperature 280°C. the seeds. Among the 13 components characterized from the
The oven temperature was programmed from 110°C (isothermal for GC-MS analysis of N. sativa. 9,12-octadecadienoic, ethyl ester was
2 minutes), with an increase of 10°C/minutes, to 200°C/minutes, then identified as a major chemical constituent (50.98%) followed by
5°C/minutes to 280°C/minutes, ending with a 9 minutes isothermal at Hexadecanoic acid, ethyl ester (19.63%) and 8-octadecenoic acid,
280°C. MS were taken at 70 eV; a scan interval of 0.5 s and fragments methyl ester,(E)- (14.56%) in the ethanol extract of N. sativa. Phenol,
from 40 to 550 Da. The MS of the unknown component was compared 4-(3,7-dimethyl-3-ethenylocta-1,6-dienyl)- (45.99%) and Linoleic
with the spectrum of the known components stored in The National acid ethyl ester (45.99%) was identified as the major chemical
Institute of Standards and Technology (NIST) library [17,18]. The constituent in the ethanol extract of P. corylifolia. Other components
name, molecular weight, and structure of the components of the test in trace amounts were also present in the ethanol extract of both
materials were ascertained. plant seeds analyzed.

Statistical analysis The immunomodulatory effect in the cell proliferation model has been
The data were analyzed using one-way analysis of variance (one-way a target of study in the search for new therapeutic agents of natural
ANOVA). Results expressed as mean±standard deviation for triplicate origin [19,20]. Although various components have been screened for
assays using Graph Pad Prism software. Values of p<0.05 were the their immunomodulatory potential using cell line as a model, in this
criteria for statistical significance. study, immune stimulating activity of ethanol extract of N. sativa and

Fig. 1: Chromatogram of ethanol extract of Nigella sativa seed by gas chromatography and mass spectrum

330
 Mohamed et al.
Asian J Pharm Clin Res, Vol 10, Issue 3, 2017, 329-332

Table 1: Chemical composition (%) of ethanol extract of Nigella sativa seed

S. No. Name of the compound Retention time Peak % Molecular weight g/mol Chemical
formula
1 o‑cymol 5.5 1.66 134.21 C10H14
2 Ether, p‑menth‑6‑en‑2‑yl methyl 6.85 1.13 168.27 C11H20O
3 (+)‑Longifolene 10.98 0.99 204.35 C15H24
4 Pentadecanoic acid, 14‑methyl‑, methyl ester 17.08 2.34 270.45 C17H34O2
5 8‑Octadecenoic acid, methyl ester,(E)‑ 18.82 14.56 296.48 C19H36O2
6 9,12‑Octadecadienoic, ethyl ester 19.57 50.98 308.49 C20H36O2
7 10,13‑Eicosadienoic acid, methyl ester 20.63 0.36 322.52 C21H38O2
8 1‑Tetradecene, 2‑decyl‑ 21.23 3.42 336.63 C24H48
9 2,5‑Cyclohexadiene‑1,4‑dione, 2‑methyl‑5‑(1‑methylethyl)‑ 9.27 1.03 164.20 C10H12O2
10 Durohydroquinone 13.7 2.40 166.22 C10H14O2
11 Ethyl 9‑hexadecenoate 17.52 0.90 282.46 C18H34O2
12 Hexadecanoic acid, ethyl ester 17.93 19.63 284.47 C18H36O2
13 Tetradecanoic acid, ethyl ester 15.65 0.83 256.42 C16H32O2

Table 2: Chemical composition (%) of ethanol extract of Psoralea corylifolia seed

S. No. Name of the compound Retention time Peak % Molecular weight g/mol Chemical formula
1 Caryophyllene 11.50 1.81 204.35 C15H24
2 Epoxycaryophyllene 13.32 1.92 220.35 C15H24O
3 Estra‑1,3,5 (10)‑trien‑17a‑ol 17.77 1.13 286.40 C19H26O2
4 9,12‑Octadecadienoic acid (Z, Z)‑, methyl ester 18.75 1.91 294.47 C19H34O2
5 Phenol, 4‑(3,7‑dimethyl‑3‑ethenylocta‑1,6‑dienyl)‑ 19.30 45.99 256.38 C18H24O
6 Linoleic acid ethyl ester 19.45 45.99 308.49 C20H36O2
7 11‑Eicosenoic acid, methyl ester 20.60 0.85 324.54 C21H40O2
8 13‑Docosenoic acid, methyl ester, (Z)‑ 22.53 0.39 352.59 C23H44O2
9 Ethyl 13‑docosenoate (ethyl erucate) 23.28 0.34 366.62 C24H46O2

Fig. 2: Chromatogram of ethanol extract of Psoralea corylifolia seed by gas chromatography and mass spectrum

P. corylifolia have been screened using J774A.1 cell line. Ethanol extract concentration while the minimum concentration would have had a
of N. sativa and P. corylifolia seeds assessed for cell proliferation assay, negligible effect on cell duplication.
induced an increase in cell proliferation depending on the varying
concentrations. In previous reports, 6-carboxymethylthiopurine, Present results indicate that the tested ethanol extract of N. sativa
an anti-inflammatory drug and the hot-water extract of Sargassum and P. corylifolia are capable of stimulating the immune function by
hemiphyllum was reported to stimulate proliferation of J774A.1 increasing the proliferation rate of macrophages. Purification and
cell [21,22]. The proliferation stimuli were dose dependent in earlier isolation of the component responsible for activity were required to
reports, where maximum proliferation was obtained from higher void the toxic component in the crude extract to construct potential
concentration. The reports were in contrast to the current study immunomodulator agent.
where the extract at its maximum and minimum concentration had a
CONCLUSION
varying effect on the macrophage cells. Percentage of proliferation was
comparatively lower at both concentration. The present observation The phytochemical analyze concludes that N. sativa and P. corylifolia
might be due to toxic nature of the analyzed samples at higher seed extract contain various bioactive compounds. The study is evident

331
 Mohamed et al.
Asian J Pharm Clin Res, Vol 10, Issue 3, 2017, 329-332

REFERENCES
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activity of ethanolic extract of leaves of Glycosmis pentaphylla in Swiss
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3. Bhadoriya SS, Mangal A, Mandoriya N. Immunomodulatory effect of
M. tricuspidatum. Int J Curr Pharm Res 2012;4(2):33-6.
4. Biswas K, Chattopadhyay I, Banerjee RK, Bandyopadhyay U.
Biological activities and medicinal properties of neem (Azadirachta
indica). Curr Sci 2002;82(11):1136-345.
5. Salem ML. Immunomodulatory and therapeutic properties of the
Nigella sativa L. seed. Int Immunopharmacol 2005;5(13-14):1749-70.
6. Burits M, Bucar F. Antioxidant activity of Nigella sativa essential oil.
Phytother Res 2000;14(5):323-8.
7. Morsi NM. Antimicrobial effect of crude extracts of Nigella sativa
on multiple antibiotics-resistant bacteria. Acta Microbiol Pol
2000;49(1):63-74.
8. Worthen DR, Ghosheh OA, Crooks PA. The in vitro anti-tumor activity
of some crude and purified components of blackseed, Nigella sativa L.
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Anticancer Res 1998;18(3A):1527-32.
Nigella sativa on J774A.1 cells. The data plotted represent 9. Salem ML, Hossain MS. In vivo acute depletion of CD 8 (+) T cells before
mean±standard deviation of triplicate experiments (n−3). p<0.05 murine cytomegalovirus infection upregulated innate antiviral activity
of natural killer cells. Int J Immunopharmacol 2000;22(9):707-18.
10. Siddhinandan M. Khadirarishta-Kushtharogadhikar. 54th ed. Varanasi:
Bhiashajya Ratnavali Chaukhambha Publication; 2009. p. 353-8.
11. Latha PG, Panikkar KR. Inhibition of chemical carcinogenesis by
Psoralea corylifolia seeds. J Ethnopharmacol 1999;68(1-3):295-8.
12. Chanda S, Kaneria M, Nair R. Antibacterial activity of Psoralea
corylifolia Linn. Seed and aerial parts with various extraction methods.
Res J Microbiol 2011;6(2):124-31.
13. Ozcan M. Inhibitory effects of spice extracts on the growth of
Aspergillus parasiticus NRRL2999 strain. Z Lebensm Unters Forsch A
1998;2079(3):253-5.
14. Musso T, Badolato R, Ravarino D, Stornello S, Panzanelli O, Merlino C,
et al. Interaction of Bartonella henselae with the murine macrophage
cell line J774: Infection and proinflammatory response. Infect Immun
2001;69(10):5974-80.
15. Goel A. Immumological Studies on Fileriaris, Ph.D. Thesis. Lucknow,
Uttar Pradesh, India: Central Drug Research Institute, (CDRI); 1991.
16. Snedecor GW, Cochran WG. Statistical Methods. 7th ed. Ames, Iowa,
USA: The Iowa State University; 1981.
17. Merlin NJ, Parthasarathy V, Manavalan R, Kumaravel S. Chemical
investigation of aerial parts of Gmelina asiatica Linn. by GC-MS.
Pharmacogn Res 2009;1(3):152-6.
Fig. 4: The stimulating effect of the ethanol extract of Psoralea
18. Anand D, Wyson WJ, Saravanan P, Rajarajan S. Phytochemical analysis
corylifolia on J774A.1 cells. The data plotted represent of leaf extract of Eclipta alba (L.) Hassk by GC-MS method. IJPPR
mean±standard deviation of triplicate experiments (n−3). p<0.05 2014;6(3):562-6.
19. Arokiyaraj S, Perinbam K, Agastian P, Balaraju K. Immunosuppressive
that ethanol extract of N. sativa and P. corylifolia played an important effect of medicinal plants of Kolli Hills on mitogen-stimulated
proliferation of the human peripheral blood mononuclear cells in vitro.
role in the modulation of the immune response and thus may have
Indian J Pharmacol 2007;39(4):180-3.
application as an immunomodulatory agent. 20. Sriwanthana B, Treesangsri W, Boriboontrakul B, Niumsakul S,
Chavalittumrong P. In vitro effects of Thai medicinal plants on human
ACKNOWLEDGMENTS lymphocyte activity. Songklanakarin J Sci Technol 2007;29(1):17-28.
21. De Paula ML, Braga FG, Coimbra ES, Carmo AM, Teixeira HC,
The authors are thankful to the Secretary and the Principal, Ramakrishna Da Silva AD, et al. Modulatory effects of 6-carboxymethylthiopurine on
Mission Vivekananda College (Autonomous), Mylapore, Chennai, activated murine macrophages. Chem Biol Drug Des 2008;71(6):563-7.
India for providing all facilities and we especially thank Sophisticated 22. Hwang PA, Chwen-Herng WU, Gau SY, Chien SY, Hwang DF.
Analytical Instrument Facility, Indian Institute of Technology, Chennai, Antioxidant and immune-stimulating activities of hot-water extract
India for carrying out GC-MS studies and validation of the results. from Seaweed Sargassum hemiphyllum. JMST 2010;18(1):41-6.

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 Online - 2455-3891
Vol 10, Issue 3, 2017 Print - 0974-2441
Research Article

IN VITRO IMMUNOSTIMULATION ACTIVITY OF NIGELLA SATIVA LINN. AND PSORALEA

CORYLIFOLIA LINN. SEEDS USING A MURINE MACROPHAGE CELL LINE

SHEIK NOOR MOHAMED M, JAIKUMAR K, MARIMUTHU S, JOHN WYSON W, ANAND D, SARAVANAN P*

Department of Botany, Ramakrishna Mission Vivekananda College (Autonomous), Mylapore, Chennai - 600 004, Tamil Nadu, India.
Email: sarviveka@[Link]
Received: 18 November 2016, Revised and Accepted: 01 December 2016

ABSTRACT

Objective: The aim of this study is to analyze the phytochemical constituents and the in vitro immunomodulatory potential of ethanol extract of
Nigella sativa and Psoralea corylifolia seeds.

Methods: Phytoconstituents in the ethanol extract of N. sativa and P. corylifolia seeds were analyzed using gas chromatography and mass spectrum
and their immunomodulatory activity was investigated in vitro by cell proliferation assay with J774A.1 cell line.

Results: Thirteen biochemical constituents have been identified with ethanol extract of N. sativa and nine from the P. corylifolia. The maximum relative
activity of 138.77±0.44% cell proliferation was expressed at 25 μg/ml by N. sativa and 80.70±0.42% of proliferation by P. corylifolia at 25 μg/ml.

Conclusion: The results suggest that the ethanol extract of the plant seeds have stimulating activity on macrophage cells and could be useful for
modulating immune functions.

Keywords: Immunomodulatory, Cell proliferation, Macrophage cell line, Nigella sativa, Psoralea corylifolia.

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license ([Link]
org/licenses/by/4. 0/) DOI: [Link]

INTRODUCTION these plants. Therefore the aim of the present study is to identify the
bioactive components of these plants by subjecting the ethanol extracts
The immune system is a remarkably versatile defense system that has
of the plant seeds to gas chromatography and mass spectrum (GC-MS)
evolved to protect animals from invading pathogenic microorganisms
analysis and to investigate their immune stimulation potential using an
and to eliminate diseases. The function and the efficacy of immune
in vitro cell line model.
system may be influenced by many exogenous factors such as food
and pharmaceuticals, physical and psychological stress and hormones. METHODS
Resulting in either immunostimulation or immunosuppression [1].
Suppression in immune response disrupts host’s ability to defend itself Collection of plant materials
against bacteria, viruses, and parasites. Immunomodulation using plant Seeds of N. sativa and P. corylifolia were procured from herbal medicine
material can provide an alternative to conventional chemotherapy for a store in Chennai. The seeds were authenticated at the Department of
variety of diseases, especially when the host defense mechanism has to be Medicinal Botany, National Institute of Siddha, Chennai and a voucher
activated under the condition of impaired immune response [2]. A large specimen (voucher number - NISMB2052015) was submitted.
number of drugs in use are derived from plants, like morphine from
Papaver somniferum, Aswagandha from Withania somnifera, Ephedrine Preparation of plant extracts
from Ephedra vulgaris, Atrophine from Atropa belladonna, etc. [3]. The Fine seeds were cleaned and milled to a powder using a domestic
phytochemical constituents like terpenoids, steroids, proteins, and blender. A total of 25 g of powdered seeds were then subjected to soxhlet
tannins [4] are considered to exhibit this immunomodulatory property. extraction with 250 ml of ethanol as solvent. The seed extract was
concentrated using vacuum rotatory evaporation to void of solvent. The
A large number of plants with therapeutic potentials have been extracts were filter sterilized and stored at 4°C until further use [12,13].
considered. One such is Nigella sativa Linn. (Ranunculaceae),
commonly known as “black cumin,” is an herbaceous plant that grows Cell culture
in Mediterranean countries. The oil and seed constituents have shown J774A.1, a murine macrophage cell line, was procured from National
potential medicinal properties in traditional medicine [5]. The black Centre for Cell Science, Pune, Maharashtra, India. The cells were
seeds contain 36-38% fixed oil, with proteins, alkaloids, saponins, maintained in Dulbecco’s modified Eagle’s medium (DMEM)
and essential oils making up the rest of the composition [6]. Black supplemented with 10% (v/v) inactivated fetal bovine serum (FBS),
seed extract has been reported to possess antimicrobial activity [7], 100 units/ml of penicillin, 100 μg/ml of streptomycin and 3%
antitumor activity [8], and a stimulatory effect on the immune L-glutamine (Himedia Labs, Mumbai). The cells were incubated at 37°C
system [9]. Psoralea corylifolia Linn. commonly known as “Bakuchi” is in a humidified atmosphere containing 5% (v/v) carbon dioxide (CO2).
conventionally used in ayurvedic system of medicine for the treatment The medium was changed every 2 days, and the cells were serially
of various kinds of human disorders but especially for treatment of skin passaged biweekly [14].
disorders [10]. P. corylifolia seed has been reported to contain several
phytoconstituents and possess antibacterial, antifungal, antioxidant, Treatment of cell cultures with ethanol extracts
antiflarial, estrogenic, and immunomodulatory activity [11]. However, A volume of 100 μl of medium containing 1×105 cells were seeded
there is no scientific data on the in vitro immunomodulatory activity of triplicates in 96-well plates. The plates were incubated for 24 hrs at
 Mohamed et al.
Asian J Pharm Clin Res, Vol 10, Issue 3, 2017, 329-332

37°C in 5% CO2. After incubation, the old medium was decanted and all RESULTS
the plates were replenished with fresh DMEM medium supplemented
with 10% FBS. The cells were then treated with varying concentrations The GC-MS analysis of the ethanol extract of N. sativa seeds revealed the
(6.25, 12.5, 25, 50, and 100 μg/ml) prepared by diluting the extract in presence of 13 peaks (Fig. 1) and P. corylifolia showed 9 peaks (Fig. 2)
DMEM and then incubated. The effect of ethanol extracts on macrophage indicating the presence of phytoconstituents whose name, molecular
proliferation was evaluated with wells containing only cells as a weight and structure were identified and characterized on comparison
negative control and compared to cells treated with concanavalin-A with data in the NIST library (Tables 1 and 2).
(Con-A), positive control [15].
Cell proliferation assay
Cell proliferation assay using (3-(4,5-dimethylthiazol-2-Yl)-2,5- Ethanol extract of N. sativa showed a maximum relative activity of
diphenyltetrazolium bromide) (MTT) reagent 138.77±0.44% at 25 μg/ml compared to the standard drug (Con-A)
After incubation, 20 μl of MTT reagent (5 mg/ml in phosphate buffer which expressed a proliferation rate of 51.11±0.96%. 11.30±0.24,
saline) was added to each well and further incubated for 4 hrs at 37°C. 112.03±0.15, 138.77±0.44, 83.46±0.30 and 50.54±0.43% increase in the
Then, dimethyl sulfoxide was added to dissolve the formazon crystals, proliferation of macrophage cells were reported at the dose rate of 6.25,
intensity of the colored reaction product was measured at 540 nm using 12.5, 25, 50, and 100 µg/ml of ethanol extract, respectively (Fig. 3). The
Microplate Reader (BIOTEK, USA) [16]. ethanol extract of P. corylifolia showed a maximum relative activity of
80.70±0.42% at 25 μg/ml compared to the standard (Con-A), resulting
GC-MS analysis in the proliferation range of 10.17±0.76, 28.46±0.46, 80.70±0.42,
GC-MS analysis of the ethanol extract of N. sativa and P. corylifolia 38.02±0.55, and 12.16±0.23% between varying concentrations of 6.25,
was carried out with 2 μl of extract employed on a GC clarus 500 12.5, 25, 50, and 100 μg/ml (Fig. 4).
Perkin Elmer system comprising a AOC-20i auto sampler and
gas chromatograph interfaced to a mass spectrometer (GC-MS) DISCUSSION
instrument employing the following conditions: Column Elite-1
fused silica capillary column (30×0.25 mm ID×1 EM df, composed of The information regarding the chemical constituents of a plant
100% dimethyl polysiloxane), operating in electron impact mode at is generally provided by the qualitative phytochemical screening
70 eV; helium (99.999%) was used as carrier gas at a constant flow of of its extract. Hence, the GC-MS analysis was opted to assess the
1ml/min and an injection volume of 0.5 EI was employed (split ratio phytochemical constituents present in the ethanol extracts of
of 10:1) injector temperature 250°C; ion source temperature 280°C. the seeds. Among the 13 components characterized from the
The oven temperature was programmed from 110°C (isothermal for GC-MS analysis of N. sativa. 9,12-octadecadienoic, ethyl ester was
2 minutes), with an increase of 10°C/minutes, to 200°C/minutes, then identified as a major chemical constituent (50.98%) followed by
5°C/minutes to 280°C/minutes, ending with a 9 minutes isothermal at Hexadecanoic acid, ethyl ester (19.63%) and 8-octadecenoic acid,
280°C. MS were taken at 70 eV; a scan interval of 0.5 s and fragments methyl ester,(E)- (14.56%) in the ethanol extract of N. sativa. Phenol,
from 40 to 550 Da. The MS of the unknown component was compared 4-(3,7-dimethyl-3-ethenylocta-1,6-dienyl)- (45.99%) and Linoleic
with the spectrum of the known components stored in The National acid ethyl ester (45.99%) was identified as the major chemical
Institute of Standards and Technology (NIST) library [17,18]. The constituent in the ethanol extract of P. corylifolia. Other components
name, molecular weight, and structure of the components of the test in trace amounts were also present in the ethanol extract of both
materials were ascertained. plant seeds analyzed.

Statistical analysis The immunomodulatory effect in the cell proliferation model has been
The data were analyzed using one-way analysis of variance (one-way a target of study in the search for new therapeutic agents of natural
ANOVA). Results expressed as mean±standard deviation for triplicate origin [19,20]. Although various components have been screened for
assays using Graph Pad Prism software. Values of p<0.05 were the their immunomodulatory potential using cell line as a model, in this
criteria for statistical significance. study, immune stimulating activity of ethanol extract of N. sativa and

Fig. 1: Chromatogram of ethanol extract of Nigella sativa seed by gas chromatography and mass spectrum

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Table 1: Chemical composition (%) of ethanol extract of Nigella sativa seed

S. No. Name of the compound Retention time Peak % Molecular weight g/mol Chemical
formula
1 o‑cymol 5.5 1.66 134.21 C10H14
2 Ether, p‑menth‑6‑en‑2‑yl methyl 6.85 1.13 168.27 C11H20O
3 (+)‑Longifolene 10.98 0.99 204.35 C15H24
4 Pentadecanoic acid, 14‑methyl‑, methyl ester 17.08 2.34 270.45 C17H34O2
5 8‑Octadecenoic acid, methyl ester,(E)‑ 18.82 14.56 296.48 C19H36O2
6 9,12‑Octadecadienoic, ethyl ester 19.57 50.98 308.49 C20H36O2
7 10,13‑Eicosadienoic acid, methyl ester 20.63 0.36 322.52 C21H38O2
8 1‑Tetradecene, 2‑decyl‑ 21.23 3.42 336.63 C24H48
9 2,5‑Cyclohexadiene‑1,4‑dione, 2‑methyl‑5‑(1‑methylethyl)‑ 9.27 1.03 164.20 C10H12O2
10 Durohydroquinone 13.7 2.40 166.22 C10H14O2
11 Ethyl 9‑hexadecenoate 17.52 0.90 282.46 C18H34O2
12 Hexadecanoic acid, ethyl ester 17.93 19.63 284.47 C18H36O2
13 Tetradecanoic acid, ethyl ester 15.65 0.83 256.42 C16H32O2

Table 2: Chemical composition (%) of ethanol extract of Psoralea corylifolia seed

S. No. Name of the compound Retention time Peak % Molecular weight g/mol Chemical formula
1 Caryophyllene 11.50 1.81 204.35 C15H24
2 Epoxycaryophyllene 13.32 1.92 220.35 C15H24O
3 Estra‑1,3,5 (10)‑trien‑17a‑ol 17.77 1.13 286.40 C19H26O2
4 9,12‑Octadecadienoic acid (Z, Z)‑, methyl ester 18.75 1.91 294.47 C19H34O2
5 Phenol, 4‑(3,7‑dimethyl‑3‑ethenylocta‑1,6‑dienyl)‑ 19.30 45.99 256.38 C18H24O
6 Linoleic acid ethyl ester 19.45 45.99 308.49 C20H36O2
7 11‑Eicosenoic acid, methyl ester 20.60 0.85 324.54 C21H40O2
8 13‑Docosenoic acid, methyl ester, (Z)‑ 22.53 0.39 352.59 C23H44O2
9 Ethyl 13‑docosenoate (ethyl erucate) 23.28 0.34 366.62 C24H46O2

Fig. 2: Chromatogram of ethanol extract of Psoralea corylifolia seed by gas chromatography and mass spectrum

P. corylifolia have been screened using J774A.1 cell line. Ethanol extract concentration while the minimum concentration would have had a
of N. sativa and P. corylifolia seeds assessed for cell proliferation assay, negligible effect on cell duplication.
induced an increase in cell proliferation depending on the varying
concentrations. In previous reports, 6-carboxymethylthiopurine, Present results indicate that the tested ethanol extract of N. sativa
an anti-inflammatory drug and the hot-water extract of Sargassum and P. corylifolia are capable of stimulating the immune function by
hemiphyllum was reported to stimulate proliferation of J774A.1 increasing the proliferation rate of macrophages. Purification and
cell [21,22]. The proliferation stimuli were dose dependent in earlier isolation of the component responsible for activity were required to
reports, where maximum proliferation was obtained from higher void the toxic component in the crude extract to construct potential
concentration. The reports were in contrast to the current study immunomodulator agent.
where the extract at its maximum and minimum concentration had a
CONCLUSION
varying effect on the macrophage cells. Percentage of proliferation was
comparatively lower at both concentration. The present observation The phytochemical analyze concludes that N. sativa and P. corylifolia
might be due to toxic nature of the analyzed samples at higher seed extract contain various bioactive compounds. The study is evident

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 NCBH 26

Induction of nitric oxide synthase by methanolic extract of Nigella sativa

seed in RAW 264.7 cells
M Sheik noor Mohamed, K Jaikumar, D Anand, P Saravanan
P.G. & Research Department of Botany, Ramakrishna Mission Vivekananda College
(Autonomous), Mylapore, Chennai - 600 004, India.

ABSTRACT

Nitric oxide (NO) is an important cellular mediator of tissue repair. It is produced in

macrophages by the enzyme inducible nitric oxide synthase (iNOS) during wound healing.
Modulation of nitric oxide (NO) may offer novel approaches in the treatment of a variety of
diseases, such as Alzheimer’s, cardiovascular disease, and diabetes. Herbal medicines may be
used as a strategy in the modulation of NO expression. In the present study, methanolic
extract of N. sativa seed, a traditionally used medicinal plant was investigated for its effect on
the synthesis of nitric oxide by RAW 264.7 cell. Incubation of 3.12µg/ml to 100µg/ml of
seed extract with 1x106 cells in vitro increased the NO production depending on varying
doses. The significant NO production stimulus by seed extract were 54.04%, 38.73%, 27.02%
at 25µg/ml, 12µg/ml, and 6.25µg/ml respectively. The present result is an evident that the
seed extract modulates NO production, thus it could provide a lead for developing a cure to
immune related ailments.

Keywords: Nigella sativa, nitric oxide, macrophages, immunomodulatory, RAW 264.7 cells.