CHEMICAL COMPOSITION AND ANTIOXIDANT ACTIVITIES OF BLACK SEED OIL (NIGELLA SATIVA L.)HTML Full Text
CHEMICAL COMPOSITION AND ANTIOXIDANT ACTIVITIES OF BLACK SEED OIL (NIGELLA SATIVA L.)
S. Dinagaran1, S. Sridhar *1 and P. Eganathan2
Department of Botany 1, Government Arts College, Tiruvannamalai- 606 603, Tamil Nadu, India.
Meta Procambial Biotech Private Limited 2, Thanneer Pandal Palayam, Peria Semour PO, Erode- 638 004, Tamil Nadu, India.
ABSTRACT: Nigella sativa L. is an annual herb and cultivated largely in the East Mediterranean region. Seeds used in traditional folk medicine for the treatment of various purposes in the systems of Unani, Ayurveda, Chinese and Arabic. Nigella sativa seed oil was isolated using soxhlet hexane extraction process. GC-MS analysis identified a total of 32 compounds among which 9-eicosyne (63.04%), linoleic acid (13.48%), palmitic acid (9.68%) were the major constituents. Saturated aliphatic fatty acid accounted 63.04% of the seed oil extract. Fatty acid and monoterpene hydrocarbon constituted 23.26% and 4.91% respectively. Also the seed oil included compounds of alkanes and sesquiterpene hydrocarbons that constituted 2.84%, and 0.30% respectively. The seed oil was estimated for its chemical compounds and antioxidant activity using in vitro assays such as DPPH, ABTS, nitric oxide, hydrogen peroxide and total antioxidant scavenging capacity. Higher antioxidant scavenging activity of TAC and ABTS was found in seed oil. The seed oil contains higher percentage of fatty acids and exhibit antioxidant activity which are useful for preparation of pharmaceutical products
fatty acid, antioxidant activity
INTRODUCTION: Nigella sativa L. is an annual herb belonging to the Ranunculaceae family and cultivated in various parts of the globe, in particular in the East Mediterranean region 1. Seeds of Nigella sativa have been used in traditional folk medicine for the treatment of various purposes in ancient medical systems of Unani, Ayurveda, Chinese and Arabic for quite long time 2. The extracts of seeds have been reported to possess anti-inflammatory and antioxidant activities, and also suppress coughs, disintegrate renal calculi, retard carcinogenic process, treat abdominal pain, diarrhea, flatulence and polio 3, 4.
Activity of ingredients was reported in Nigella sativa seeds against various types of cancers including cervical 5, blood 6, hepatic 7, colon 8, skin 9, fibrosarcoma10, renal 11, prostate 12 and breast 3.
Nigella sativa seed contains fixed oil that ranges between 28 to 36% and chiefly composed of unsaturated fatty acids that are arachidonic, eicosadienoic, linoleic and linolenic and saturated fatty acids that includes palmitic, stearic and myristic 13. The seed oil contains compounds such as cholesterol, campesterol, stigmasterol, β-sitosterol, α-spinasterol, (+)-citronellol, (+)-limonene, p-cymene, citronellyl acetate, carvone, nigellone, arachidic, linolenic, linoleic, myristic, oleic, palmitic, palmitoleic and stearic acids 14. Seed oil contains fixed oils like linoleic acid (55.6%), oleic acid (23.4%) and palmitic acid (12.5%) and volatile oils like trans-anethole (38.3%), p-cymene (14.8%), limonene (4.3%), and carvone (4.0%) 15.
A literature search revealed that a complete chemical profiling of Nigella sativa seed oil has not yet been reported. The present investigation was therefore undertaken to obtain analyse the chemical composition of Nigella sativa seed oil and its antioxidant activities.
MATERIALS AND METHODS:
Collection of seed:
Seeds of Nigella sativa were purchased from the herbal plant and powder shop in Triplicane, Chennai, Tamil Nadu, India. The seed material was sieved and false and small seeds and inert material removed.
Extraction of seed oil:
The seeds were coarsely ground using a table top mixture and seed oil extracted using hexane in a soxhlet apparatus for 2 hours and stored in an amber glass screw cap bottle at room temperature until use.
Gas Chromatography-Mass Spectrometry analysis:
GC-MS analysis were conducted using Agilent MSD (5975B-inert XL MSD) apparatus equipped with reference libraries (NIST); column DB-5MS (J&W Scientific) cross-linked fused-silica capillary column (30 m × 0.25 mm × 0.25 µm thickness), coated with 5% phenyl-polymethylsiloxane; column temperature, 80°C for 0 min, rising to 150°C at 10°C/min, then 250°C at 5°C/min, then rising to 270°C at 20°C held for 6 min. injector temperature 270°C, injection mode, split; split ratio 1:20; volume injected, 2 µl of the seed oil. Helium was used as a carrier; interface temperature 270°C; acquisition mass range, m/z 55-550. The compounds of the oil were identified by comparing their retention indices (RI), with NIST (National Institute of Standards and Technology) library.
ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) scavenging assay:
The ABTS radical cationdecolorization method is based on the reduction of ABTS•+ radicals by antioxidants of the essential oil tested. The tubes containing ABTS and APS were incubated at room temperature for a period of 16 hours. 20 µl of various concentrations of 10 mM PBS pH 7.4 test solutions was added to 230 µl of ABTS radical solution (0.238 mM). The absorbance values were recorded immediately at 734 nm using shimadzu UV 1800 spectrophotometer 16.
ABTS scavenging activity (%)= [Acontrol - Asample/ Acontrol] × 100
DPPH (1, 1-diphenyl-2- picrylhydrazyl) radical scavenging assay:
Free radical scavenging activity of the seed oil was measured in terms of radical scavenging ability using the stable free radical DPPH 16. Different concentrations (10µl, 20µl, 30 µl, 40µl & 50µl) of sample were taken and 50µl of 0.659 mM DPPH dissolved in methanol solution was added to make up to one using double distilled water. The tubes were incubated at 25ºC for 20 minutes.The absorbance value was recorded at 510 nm using shimadzu UV 1800 spectrophotometer. The same procedure was followed for control without the sample.
DPPH Scavenging ability (%) = [Acontrol - Asample/ Acontrol] × 100
Hydrogen peroxide radical scavenging assay:
The ability of the oil to scavenge hydrogen peroxide was determined according to the method described by Rajamanikandan et al 17. 0.6ml of 40mM of hydrogen peroxide was prepared using 50mM phosphate buffer (pH 7.4).Different concentrations (10µl, 20µl, 30 µl, 40µl & 50µl) of sample were added to hydrogen peroxide solution.The tubes were incubated for 10 minutes. The absorbance values were recorded at 230 nm using shimadzu UV 1800 spectrophotometer.
Hydrogen peroxide activity (%) = [Acontrol - Asample/ Acontrol] × 100
Nitric oxide scavenging assay:
Nitric oxide scavenging assay was determined using method by Jain & Agrawal 16. Different concentrations (10µl, 20µl, 30 µl, 40µl & 50µl) of sample were taken and added 50µl of 10mM sodium nitroprusside dissolved in 0.5M phosphate buffer (pH 7.4). The tubes were incubated under fluorescent light at room temperature for 15 minutes. After the incubation period, 125 µl of Griess reagent was added [Griess reagent: 0.1% of N-1-Naphthyl-ethylene diamine dissolved in water, 1% Sulphanilic acid dissolved in 5% Orthophosphoric acid]. The tubes were incubated again at room temperature for 10 minutes. The absorbance values were recorded at 546 nm using shimadzu UV 1800 spectrophotometer.
Total antioxidant capacity assay:
Total antioxidant capacity assay was determined as described by Rajamanikandan et al 17. Different concentrations (10µl, 20µl, 30 µl, 40µl & 50µl) of extracts were taken and 1ml of reagent solution was added.[Reagent solution: 0.6M sulphuric acid, 28mM sodium phosphate and 4 mM ammonium molybdate].
The tubes were capped and incubated in thermal block at 95ºC for 90minutes. After the time interval the tubes were cooled down at room temperature. The absorbance was recorded at 695 nm using shimadzu UV 1800 spectrophotometer.
Results were documented as mean ± standard deviation (n = 3) and focused on one-way analysis of variance (ANOVA). The significance of the difference between means was determined by Duncan’s multiple range test (P<0.05) using SPSS 17.0 statistical software (SPSS South-Asia Pvt Ltd, Bangalore).
RESULTS AND DISCUSSION:
Chemical constituents of Nigella sativa:
GC-MS chemical profile of the hexane extract of seed oil contains a total of 32 compounds and 9-eicosyne (63.04%) was a major chemical constituent present in Nigella sativa seed oil. The other chemical constituents present were linoleic acid (13.48%) followed by palmitic acid (9.68%), p-cymene (2.54%) and thymoquinone (1.86%) and cs-7-dodecen-1-yl acetate (1.11%) (Table1). The chromatogram of GC-MS chemical profile of seed oil is presented in Fig. 1.
TABLE 1: GC-MS CHEMICAL CONSTITUENTS OF NIGELLA SATIVA SEED OIL
|S.no.||Retention Time||Chemical Name||Area %|
|32||36.146||Linoleic acid ethyl ester||0.29|
Saturated aliphatic fatty acid 63.04%
Fatty acid 23.26%
Monoterpene hydrocarbon 4.91%
Alkanes hydrocarbon 2.84%
Sesquiterpene hydrocarbon 0.30%
FIG. 1: GC-MS CHROMATOGRAM OF SEED OIL OF NIGELLA SATIVA
The present report of seed oil contains higher percentage of 9-eicocyne (63.04%). Similarly, 9-eicosyne compound has been reported from other plant species 23.64% in Blepharismaderaspatensis 18, 19.61% in Borassus flabellifer 19, 11.91-23.32% in Portulacaoleracea 20, 5.30% in Stevia rebaudiana 21, 2.74% in Syzygium calophyllifolium 22, 2.24% in Melia azedarach 23, 1.74% in Cassia auriculata 24, 1.47% in Andrographis paniculata 25, 0.756% in Buxus microphylla 26, 0.36% in Atractylodes macrocephala 27. Thymoquinone is an important bioactive constituent of the volatile oil of Nigella sativa and reported to exert several pharmacological activities such as antioxidant, antihistaminic, chemotherapeutic and anti inflammatory activities 28, 29, 30, 31, 32.
Major group of components were present in the seed oil and included aliphatic fatty acid (63.04%), fatty acid (23.26%), monoterpene hydrocarbon (4.91%), alkanes hydrocarbon (2.84%) and sesquiterpene hydrocarbon (0.30%) (Table1). Nigella sativa seed oil contains anethole, p-cymene, limonene, carvone and thymoquinone15. Seed oil consists of four saturated fatty acids (17.0%) and four unsaturated fatty acids (82.5%). Linoleic acid (55.6%), oleic acid (23.4%) and palmitic acid (12.5%) are its major components 15. The present investigation of Nigella sativa seed oil posses higher percentage of fatty acids (86.30) and few chemical compounds are also similarly reported fatty acids 33 and other chemical constituents 34 but the percentages of chemical composition differs.
The present analysis indicates that higher composition of fatty acids (86.30%) was present in the hexane seed oil extract. Fatty acids are known as important nutrients in both human and animal diets, and also possess various health benefits 35, 36, and are used in the pharmaceutical industry 37. Saturated and unsaturated fatty acids from various seed oil sources showed good antioxidant activities 38. Seed oil of Nigella sativa was analyzed for antioxidant assays. Higher ABTS scavenging activity was found in 10 and 20 µg/ml of lower concentration of seed oil (Fig. 2A).
The activity of seed oil decreases proportionally with the concentration of the sample and is comparable with that of the standard. Seed oil of Nigella sativa showed scavenging activities of DPPH, H2O2 and NO that increased scavenging activity in lower concentration and decreases in higher concentration (Fig. 2B, 2C and 2D). Seed oil of Nigella sativais capable of scavenging higher percentage of TAC molecule compared to ascorbic acid (Fig. 2E) and also higher scavenging activity compared to ABTS.
FIG. 2A: ABTS SCAVENGING OF HEXANE EXTRACT OF NIGELLA SATIVA SEED OILCOMPARED TO THAT OF ASCORBIC ACID. EACH VALUE IS EXPRESSED AS MEAN ± STANDARD DEVIATION (n=3)
FIG. 2B: DPPH SCAVENGING OF HEXANE EXTRACT OF NIGELLA SATIVA SEED OILCOMPARED TO THAT OF ASCORBIC ACID. EACH VALUE IS EXPRESSED AS MEAN ± STANDARD DEVIATION (n=3)
FIG. 2C: HYDROGEN PEROXIDE RADICAL SCAVENGING OF HEXANE EXTRACT OF NIGELLA SATIVA SEED OIL COMPARED TO THAT OF ASCORBIC ACID. EACH VALUE IS EXPRESSED AS MEAN ± STANDARD DEVIATION (n=3)
FIG. 2D: NITRIC OXIDE SCAVENGING OF HEXANE EXTRACT OF NIGELLA SATIVA SEED OIL COMPARED TO THAT OF ASCORBIC ACID. EACH VALUE IS EXPRESSED AS MEAN ± STANDARD DEVIATION (n=3)
FIG. 2E: TOTAL ANTIOXIDANT SCAVENGING OF HEXANE EXTRACT OF NIGELLA SATIVA SEED OIL COMPARED TO THAT OF ASCORBIC ACID. EACH VALUE IS EXPRESSED AS MEAN ± STANDARD DEVIATION (n=3)
All assays uniformly showed that higher scavenging activity was found in lower concentrations of the seed oil (10 and 20 µg/ml) and increasing concentrations (30, 40 and 50 µg/ml) showed decreased scavenging activities. The hexane extract of Nigella sativa seed oil showed higher radical scavenging activity in lower concentration against ABTS and TAC free radical. The present analysis of seed oil contains 1.86% of thymoquinone. The thymoquinone (20.32%) exhibited strong antioxidant activity (14.0±0.7 µg/ml) in essential oil and has been found to be the most active compound to decrease oxidation and NO excretion 39, anti-eicosanoid and antioxidant activity 33. Also higher composition of fatty acids plays higher scavenging biological activities. Consumption of fatty acids as a dietary supplement or as a food ingredient has the potential to provide health benefits 40.
CONCLUSION: From the present study Nigella sativa seed oil is found to contain higher percentage of fatty acid and higher scavenging activities in lower concentrations. There is a need for further exploration for their medicinal properties and utilization.
ACKNOWLEDGEMENT: The authors thank to Meta Procambial Biotech Private Limited, Erode for their laboratory facility.
CONFLICT OF INTEREST: The authors declare no conflict of interest.
- Hedrick UP: Sturtevant's Edible Plants of the World. Dover, New York, pp. 388-389. 1972.
- Randhawa MA and Alghamdi MS: Anticancer activity of Nigella sativa (black seed)-a review.Am J Chin Med 2011; 39(06): 1075-1091.
- Ahmad A, Husain A, Mujeeb M, Khan SA, Najmi AK, Siddique NA, Damanhouri ZA and Anwar F: A review on therapeutic potential of Nigella sativa: A miracle herb.Asian Pac J Trop Biomed 2013; 3(5): 337-352.
- Al-Khalaf MI and Ramadan KS: Antimicrobial and anticancer activity of Nigella sativa oil-a review.Aus J Basic ApplSci 2013; 7(7): 505-514.
- Effenberger K, Breyer S and Schobert R: Terpene conjugates of the Nigella sativa seed‐oil constituent thymoquinone with enhanced efficacy in cancer cells.ChemBiod 2010; 7(1): 129-139.
- El‐Mahdy MA, Zhu Q, Wang QE, Wani G and Wani AA: Thymoquinone induces apoptosis through activation of caspase‐8 and mitochondrial events in p53‐null myeloblastic leukemia HL‐60 cells. Int J Cancer 2005;117(3): 409-417.
- Thabrew MI, Mitry RR, Morsy MA and Hughes RD: Cytotoxic effects of a decoction of Nigella sativa, Hemidesmusindicus and Smilax glabra on human hepatoma HepG2 cells.Life Sci 2005; 77(12): 1319-1330.
- Chehl N, Chipitsyna G, Gong Q, Yeo CJ and Arafat HA: Anti‐inflammatory effects of the Nigella sativa seed extract, thymoquinone, in pancreatic cancer cells.HPB(Oxford) 2009; 11(5): 373-381.
- Salomi MJ, Satish CN and Panikkar KR: Inhibitory effects of Nigella sativa and saffron (Crocus sativus) on chemical carcinogenesis in mice. Nutr Cancer 1991; 16(1): 67-72.
- Awad EM: In vitro decreases of the fibrinolytic potential of cultured human fibrosarcoma cell line, HT1080, by Nigella sativaPhytomedicine 2005; 12(1): 100-107.
- Khan N and Sultana S: Inhibition of two stage renal carcinogenesis, oxidative damage and hyperproliferative response by Nigella sativa. Eur J Cancer Prev 2005;14(2): 159-168.
- Yi T, Cho SG, Yi Z, Pang X, Rodriguez M, Wang Y, Sethi G, Aggarwal BB and Liu M: Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing AKT and extracellular signal-regulated kinase signaling pathways.Mol Cancer Therapeu 2008; 7(7): 1789-1796.
- Hajhashemi V, Ghannadi A and Jafarabadi H: Black cumin seed essential oil, as a potent analgesic and antiinﬂammatory drug. Phytother Res 2004; 18(3): 195-199.
- Rastogi RP and Mehrotra BN: Compendium of Indian Medicinal Plants, reprinted edn, Vol. 3,CSIR, New Delhi,1993; pp. 452-453.
- Nickavar B, Mojab F, Javidnia K and Amoli MA: Chemical composition of the fixed and volatile oils of Nigella sativa from Iran. Z Natutforsch C 2003; 58(9-10): 629-631.
- Jain PK and Agrawal RK: Antioxidant and free radical scavenging properties of developed mono and polyherbal formulations. Asian J ExpSci 2008; 22(3): 213-220.
- Rajamanikandan S, Sindhu T, Durgapriya D, Sophia D, Ragavendran P and Gopalakrishnan VK: Radical scavenging and antioxidant activity of ethanolic extract of Mollugonudicaulisby in vitro Indian J Pharm Edu Res 2001; 45(4): 310-316.
- Suriyavathana M and Indupriya S: GC-MS analysis of phytoconstituents and concurrent determination of flavonoids by HPLC in ethanolic leaf extract of Blepharismaderaspatensis (L) B. Heyne ex Roth. World J Pharm Res 2014;3(9): 405-414.
- Sahni C, Shakil NA, Jha V and Gupta RK: Screening of nutritional, phytochemical, antioxidant and antibacterial activity of the roots of Borassusflabellifer (Asian Palmyra Palm).J Pharmacog Phytochem 2014; 3(4): 58-68.
- Zhu H, Wang Y, Liang H, Chen Q, Zhao P and Tao J: Identification of Portulacaoleracea from different sources using GC–MS and FT-IR spectroscopy.Talanta 2010; 81(1): 129-135.
- Verma RN and Batra A: Isolation and analytic characterization of rebaudioside A and GC-MS analysis of methanolic leaves extract of Stevia rebaudiana Annal Phytomed 2013; 2(1): 108-114.
- Vignesh R, Puhazhselvan P, Sangeethkumar M, Saranya J, Eganathan P and Sujanapal P: GC-MS analysis, antimicrobial, scavenging ability and cytotoxic activity of leaves of Syzygiumcalophyllifolium J Bioly Act Prod from Nat 2013; 3(2): 121-129.
- Sen A and Batra A: Chemical composition of methanol extract of the leaves of Melia azedarach.Asian J Pharm Clin Res 2012; 5(3): 42-45.
- Senthilrani S and Renuka Devi P: Biological activities and chemical composition of Cassia auriculata. Asian J Biochem 2014; DOI: 10.3923/ajb.2014
- Thangavel M, Umavathi S, Thangam Y, Thamaraiselvi A and Ramamurthy M: GC-MS analysis and larvicidal activity of Andrographispaniculata (Burm. F) Wall. Ex Nees. against the dengue vector Aedesaegypti (L)(Diptera: Culicidae).Int J CurrMicrobiolApplSci 2015; 4(7): 392-403.
- Zhang ZF, Ye Y and Chen Z: Molecular characteristics of woody extracts of Buxusmicrophylla. Pakistan J Pharm Sci 2014;27(6): 2073-2078.
- Peng W, Han T, Xin WB, Zhang XG, Zhang QY, Jia M and Qin LP: Comparative research of chemical constituents and bioactivities between petroleum ether extracts of the aerial part and the rhizome of Atractylodesmacrocephala.Med Chem Res 2011; 20(2): 146-151.
- El Gazzar M, El Mezayen R, Marecki JC, Nicolls MR, Canastar A and Dreskin SC: Anti-inflammatory effect of thymoquinone in a mouse model of allergic lung inflammation.Int Immunopharmacol 2006; 6(7): 1135-1142.
- Khalife KH and Lupidi G: Nonenzymatic reduction of thymoquinone in physiological conditions.Free Rad Res 2007; 41(2): 153-161.
- Kanter M, Coskun O and Uysal H: (2006). The antioxidative and antihistaminic effect of Nigella sativa and its major constituent, thymoquinone on ethanol-induced gastric mucosal damage.Arch Toxicol 2006; 80(4): 217-224.
- Norwood AA, Tucci M and Benghuzzi H: A comparison of 5-fluorouracil and natural chemotherapeutic agents, EGCG and thymoquinone, delivered by sustained drug delivery on colon cancer cells.Biomed SciInstru 2006; 43: 272-277.
- Tekeoglu I, Dogan A and Demiralp L: Effects of thymoquinone (volatile oil of black cumin) on rheumatoid arthritis in rat models.Phytother Res 2006; 20(10): 869-871.
- Houghton P J, Zarka R, de las Heras B and Hoult JR: Fixed oil of Nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation.Planta Med 1995; 61(1): 33-36.
- Mozaffari FS, Ghorbanli M, Babai A and Sepehr MF: The effect of water stress on the seed oil of Nigella sativaJ Essent Oil Res 2000; 12(1): 36-38.
- Liu JH, Zschocke S, Reininger E and Bauer R: Inhibitory effects of Angelica pubescens biserrata on 5-lipoxygenase and cyclooxygenase. Planta Med 1998;64(6): 525-529.
- Singh S and Majumdar DK: Evaluation of antiinflammatory activity of fatty acids of Ocimum sanctum fixed oil.Indian J ExperiBiol 1997; 35(4): 380-383.
- Weidner MS: Novel pharmaceuticals, dietary supplements and cosmetics containing fatty acids Zingiber extract for the treatment or prevention of inflammation, hypersensitivity or pain. PCT Int. Appl. 2000; AN 2000, 627966.
- Laneuville O, Breuer DK, Dewitt DL, Hla T, Funk CD and Smith WL: Differential inhibition of human prostaglandin endoperoxide H synthases-1 and-2 by nonsteroidal anti-inflammatory drugs.J Pharmacol Exp Therapeu 1994; 271(2): 927-934.
- Kazemi M: Phytochemical composition, antioxidant, anti-inflammatory and antimicrobial activity of Nigella sativa essential oil. J Essen Oil Bear Plants2014; 17(5): 1002-1011.
- Henry GE, Momin RA, Nair MG and Dewitt DL: Antioxidant and cyclooxygenase activities of fatty acids found in food.J Agricul Food Chem 2002; 50(8): 2231-2234.
How to cite this article:
Dinagaran S, Sridhar S and Eganathan P: Chemical composition and antioxidant activities of black seed oil (Nigella sativa L.). Int J Pharm Sci Res 2016; 7(11): 4473-79.doi: 10.13040/IJPSR.0975-8232.7(11).4473-79.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
S. Dinagaran, S. Sridhar * and P. Eganathan
Department of Botany, Government Arts College, Tiruvannamalai, Tamil Nadu, India
24 May, 2016
05 July, 2016
27 July, 2016
01 November, 2016