ABSTRACT
Phytochemicals are chemical compounds often referred to as secondary metabolites. Twenty eight bioactive phytochemical compounds were identified in the methanolic extract of Nigella sativa. The identification of phytochemical compounds is based on the peak area, retention time molecular weight, and molecular formula. Gas chromatography-mass spectrometry (GC-MS) analysis of Nigella sativa revealed the existence of the ß-Pinene, D-Glucose, 6-O-α-Dgalactopyranosyl, O-Cymene, DL-Arabinose, Trans-4-methoxy thujane, 2-Propyl-tetrahydropyran-3-ol, Terpinen-4-ol, α- D-Glucopyranoside, O-α-D-glucopyranosyl-(1.fwdarw.3)-ß-D-fruc, Thymoquinone, 2-Isopropylidene-5-methylhex-4-enal, Limonen-6-ol, pivalate, Longifolene, 2-(4-Nitrobutyryl)cyclooctanone, ß-Bisabolene, 1,1-Diphenyl-4-phenylthiobut-3-en-1-ol, Phenol, 4-methoxy-2,3,6-trimethyl, Pyrrolidin-2-one-3ß-(propanoic acid, methyl ester),5-methylene-4α, Cholestan-3-ol, 2-methylene-,(3ß,5α), l-(+)-Ascorbic acid 2,6-dihexadecanoate, 9,12-Octadecadienoic acid (Z,Z)-, methyl ester, 1-Heptatriacotanol, 10,13-Eicosadienoic acid, methyl ester, E,E,Z-1,3,12-Nonadecatriene-5,14-diol, 9-Octadecenamide,(Z), 2H-Benzo[f]oxireno[2,3-E]benzofuran-8(9H)-one,9-[2-(dimethylar, Phthalic acid, decyl oct-3-yl ester, 1,2-Benzenedicarboxylic acid, bis(8-methylnonyl) ester and Stiqmasterol.
Key words: Gas chromatography-mass spectrometry, Fourier-transform infrared spectroscopy, Nigella sativa, phytochemicals.
Nigella sativa L. (Ranunculaceae) is an annual herbaceous plant native to (and cultivated in) Southwest Asia, and cultivated and naturalized in Europe and North Africa (Al-Johar et al., 2008). The seeds and seed oil have been used as a diuretic, appetitizer, hemorrhagic and anti-dandruff therapy in folk medicine (Al-Othman et al., 2006). N. sativa L. commonly known as Kalonji in Hindi, a member of Ranunculaceae family, also known as the black cumin seeds is one of the most revered medicinal seeds in history. It is an annual aromatic plant and its cultivation is traced back more than 3000 to the kingdom of the Assyrians and ancient Egyptians (Ashraf et al., 2006; Hameed et al., 2015a). N. sativa taxonomic classification (Ashraf, 2011), depicts it is a flowering dicotyledon plant belonging to family Ranunculaceae under kingdom plantae. Morphology N. sativa is an annual medicinal herb, about 30 to 60 cm high (Boskabady et al., 2007; Jasim et al., 2015), with finely divided, linear leaves. The flowers are usually pale blue and white, with 5 to 10 petals. The fruit is a large inflated capsule that composed of 3 to 7 united follicles, each containing numerous black trigonal seeds (Chaudhry and Tariq, 2008; Hameed et al., 2015b).
The black kalonji seeds possess the anthelmintic, insecticidal, antimalarial, antibacterial, antifungal, and antitumor effects. There are also reports that black seeds possess diuretic, carminative, digestive and antiseptic properties (Burits and Burcar, 2000; Ali and Blunden, 2003; Saleh, 2006; Abdulelah and Abidin, 2007; Ali et al., 2008). The seeds have also been used traditionally for centuries in the Middle East, Far East, and some Mediterranean and European countries for the treatment of different ailments, such as diabetes, hypertension, cardiac diseases, hemorrhoids, and sexual diseases and as an abortifacient (Kanter, 2008; Iqbal et al., 2010).
Seeds of N. sativa are reported to contain amino acids, carbohydrates, fixed and volatile oils. The yield of black seed fixed oil ranges from 22.0 to 40.35% (Ali and Blunden, 2003; Cheikh-Rouhou et al., 2007; Altameme et al., 2015a). The extracts of N. sativa (Black seeds) have been used by patients to suppress coughs, disintegrate renal calculi (Hashem and El-Kiey, 1982), retard the carcinogenic process, treat abdominal pain, diarrhea, flatulence, and polio (Enomoto et al., 2001), exert choleretic and uricosuric activities, anti-inflammatory and antioxidant effects (Mansour et al., 2002; Altameme et al., 2015b). Besides, the essential oil was shown to have antihelminthic, antischistosomal (Mahmoud et al., 2002), antimicrobial (Aboul-Ela et al., 1996), and antiviral.
Preparation of extract
N. sativa were purchased from local market in Hilla city, middle of Iraq. After thorough cleaning and removal of foreign materials, the seeds were stored in airtight container to avoid the effect of humidity and then stored at room temperature until further use (Hameed et al., 2015c; Hamza et al., 2015). N. sativa seeds are washed with water, dried and ground into powder. N. sativa seeds powder was macerated using methanol for 1 × 24 h. Whatman No.1 filter paper was used to separate the extract of plant. The filtrates were used for further phytochemical analysis. It was again filtered through sodium sulphate in order to remove the traces of moisture (Hussein et al., 2015).
Gas chromatography-mass spectrum (GC-MS) analysis
The GC-MS analysis of the plant extract was made in a Agilent 7890 A instrument under computer control at 70 eV. About 1 μl of the methanol extract was injected into the GC-MS using a micro syringe and the scanning was done for 45 min. As the compounds were separated, they eluted from the column and entered a detector which was capable of creating an electronic signal whenever a compound was detected (Imad et al., 2014a; Hameed et al., 2015d). The greater the concentration in the sample, the bigger the signal obtained which was then processed by a computer. The time from when the injection was made (Initial time) to when elution occurred is referred to as the retention time (RT). While the instrument was run, the computer generated a graph from the signal called chromatogram. Each of the peaks in the chromatogram represented the signal created when a compound eluted from the gas chromatography column into the detector (Mohammed and Imad, 2013). The x-axis showed the RT and the y-axis measured the intensity of the signal to quantify the component in the sample injected. As individual compounds eluted from the gas chromatographic column, they entered the electron ionization (mass spectroscopy) detector, where they were bombarded with a stream of electrons causing them to break apart into fragments. The fragments obtained were actually charged ions with a certain mass .The mass/charge (M/Z) ratio obtained was calibrated from the graph obtained, which was called as the mass spectrum graph which is the fingerprint of a molecule (Imad et al., 2014b). Before analyzing the extract using GC-MS, the temperature of the oven, the flow rate of the gas used and the electron gun were programmed initially. The temperature of the oven was maintained at 100°C. Helium gas was used as a carrier as well as an eluent. The flow rate of helium was set at 1 ml/min. The electron gun of mass detector liberated electrons having energy of about 70eV.The column employed here for the separation of components was Elite 1(100% dimethyl poly siloxane). The identity of the components in the extracts was assigned by the comparison of their retention indices and mass spectra fragmentation patterns with those stored on the computer library and also with published literatures. Compounds were identified by comparing their spectra to those of the Wiley and NIST/EPA/NIH mass spectral libraries (Kareem et al., 2015; Imad et al., 2014c).
Preparation of the extract was done by maceration method. Maceration was done using the appropriate solvent with several times shaking or stirring at room temperature (Rader et al., 2007). Maceration is a method that is suitable for compounds that do not withstand heating at high temperatures (Ramaa et al., 2006). The aim is to attract the chemical components based on the principle of mass transfer of substance into the solvent component, where the movement began to occur at the interface layer and then diffuses into the solvent (Sethi et al., 2008). Identification of the structure using a mass spectrometer conducted to determine the compounds contained in the samples analyzed can be seen from the relative abundance of mass fragments of molecules (m/e) of the molecular ion (M +). The more stable a molecular fragment that is formed is, then the fragment will be at a relative abundance of large and have a longer lifespan (Vuorela et al., 2004; Shama et al., 2009).
Gas chromatography and mass spectroscopy analysis of compounds was carried out in methanolic extract as shown in Table 1. The GC-MS chromatogram of the 28 peaks of the compounds detected are shown in Figure 1. Chromatogram GC-MS analysis of the methanol extract of N. sativa showed the presence of 28 major peaks and the components corresponding to the peaks were determined as follows. The first setup peak were determined to be ß-Pinene (Figure 2).
The second peak showed D-Glucose, 6-O-α-Dgalactopyranosyl (Figure 3). The next peaks was considered to be O-Cymene, DL-Arabinose, Trans-4-methoxy thujane, 2-Propyl-tetrahydropyran-3-ol, Terpinen-4-ol, α- D-Glucopyranoside, O-α-D-glucopyranosyl-(1.fwdarw.3)-ß- D-fruc, Thymoquinone, 2-Isopropylidene-5-methylhex-4- enal, Limonen -6-ol, pivalate, Longifolene, 2-(4- Nitrobutyryl) cyclooctanone, ß-Bisabolene, 1,1-Diphenyl-4-phenylthiobut-3-en-1-ol, Phenol, 4-methoxy-2,3,6-trimethyl, Pyrrolidin -2-one-3ß-(propanoic acid, methyl ester),5-methylene-4α, Cholestan-3-ol, 2-methylene-,(3ß,5α), l-(+)-Ascorbic acid 2,6-dihexadecanoate, 9,12- Octadecadienoic acid (Z,Z)-, methyl ester, 1-Heptatriacotanol, 10,13-Eicosadienoic acid, methyl ester, E,E,Z-1,3,12-Nonadecatriene-5,14-diol, 9-Octadecenamide,(Z), 2H-Benzo[f]oxireno[2,3-E]benzofuran-8(9H)-one,9-[[[2-(dimethylar, Phthalic acid, decyl oct-3-yl ester, 1,2-Benzenedicarboxylic acid, bis(8-methylnonyl) ester and Stiqmasterol (Figures 4 to 30). Plants are considered to be one of the natural bases for the production of bioactive compounds, many of which are used to support health and fight against pathological conditions and many of them are marketed as food or herbal medicines (Shama et al., 2009). The usage of herbal medicine has amplified dramatically for various diseases amongst general people over last few years not only because of their easy accessibility without prescription, low cost and appointment to the health care specialists and more with the belief that natural remedies have less lethal effects as compared to synthetic medicines (Ashraf et al., 2011). A qualitative investigation of N. sativa has revealed the presence of sterols, triterpenes, tannins, flavanoids, cardiac glycosides, alkaloids, saponins, volatile oils, volatile bases, glucosinolates and anthraquinones (A1-Yahya, 1986). Qualitative evaluation of the black seed oil via capillary GC-MS technique has enabled the identification of 67 compounds, when classified into various functional groups corresponding with the following data: monoterpenes (~46%); carbonyl compounds (~25%); phenols (~1.7%); alcohols (~0.9%) and esters (~16%) (Abu-Jadayil et al., 1999).
N. sativa seed is a promising source for active ingredients that would be with potential therapeutic modalities in different clinical settings. The efficacy of the active ingredients, however, should be measured by the nature of the disease. It contains chemical constitutions which may be useful for various herbal formulation as anti-inflammatory, analgesic, antipyretic, cardiac tonic and antiasthamatic.
The authors have not declared any conflict of interests.
The authors thank Dr. Abdul-Kareem Al-Bermani, Lecturer, Department of Biology, for valuable suggestions and encouragement.
REFERENCES
|
Al-Yahya MA (1986). Phytochemical studies of the plants used in traditional medicine of Saudi Arabia. Fitoterapia 57(3):179-182.
|
|
|
|
Abdulelah HA, Abidin BA (2007). In vivo anti-malarial tests of Nigella sativa (black seed) Different extracts. Am. J. Pharm. Toxic. 2:46-50.
crossref
|
|
|
|
Aboul-Ela MA, El-Shaer NS, Ghanem NB (1996). Antimicrobial evaluation and chromatographic analysis of some essential and fixed oils. Pharmazie 51:993-4.
|
|
|
|
Abu-Jadayil S, Tukan SKH, Takruri HR (1999). Bioavailability of iron from four different local food plants in Jordan. Pl. Foods. Hum. Nutr. 54:285–294.
crossref
|
|
|
|
Ali A, Alkhawajah, Randhawa MA, Shaikh NA (2008). Oral and interaperitoneal LD50 of thymoquinone an active principal of Nigella sativa in mice and rats. J. Ayub Med. Coll. 20(2):25-27.
|
|
|
|
Ali BH, Blunden G (2003). Pharmacological and toxicological properties of Nigella sativa. Phytother Res. 17(4):299-305.
crossref
|
|
|
|
Al-Johar D, Shinwari N, Arif J, Al-Sanea N, Jabbar AA, El-Sayed R, Mashhour A, Billedo G, El-Doush I, Al-Saleh I (2008). Role of Nigella sativa and a number of its antioxidant constituents towards azoxymethane-inducedgenotoxic effects and colon cancer in rats. Phytother. Res. 22:1311-1323.
crossref
|
|
|
|
Al-Othman AM, Ahmad F, Al-Orf S, Al-Murshed SK, Arif Z (2006). Effect of dietary supplementation of Ellataria cardamomum and Nigella sativa on the toxicity of rancid corn oil in Rats. Int. J. Pharmacol. 2:60-65.
crossref
|
|
|
|
Altameme HJ, Hameed IH, Idan SA, Hadi MY (2015a). Biochemical analysis of Origanum vulgare seeds by Fourier-transform infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). 7(9):221-237.
|
|
|
|
Altameme HJ, Hameed IH, Kareem MA (2015b). Analysis of alkaloid phytochemical compounds in the ethanolic extract of Datura stramonium and evaluation of antimicrobial activity Afr. J. Biotechnol. 14(19):1668-1674.
|
|
|
|
Ashraf M, Ali Q, Iqbal Z (2006). Effect of nitrogen application rate on the content and composition of oil, essential oil and minerals in black cumin (Nigella sativa L.) seeds. J. Sci. Food Agric. 86:871-876.
crossref
|
|
|
|
Ashraf R (2011). Plant (Garlic) Supplement with standard Antidiabetic agent provides better diabetic control in Type-II diabetes patients. Pak. J. Pharmaceut. Sci. 24(4):565-570.
|
|
|
|
Boskabady MH, Javan H, Sajady M, Rakhshandeh H (2007). The possible prophylactic effect of Nigella sativa seed extract in asthmatic patients. Fundam. Clin. Pharmacol. 21(5):559-566.
crossref
|
|
|
|
Burits M, Burcar F (2000). Antioxidant activity of Nigella sativa essential oil. Phytother. Res. 14(5): 323-8.
crossref
|
|
|
|
Chaudhry N, Tariq P (2008). In vitro antibacterial activities of Kalongi, Cumin and Poppy seed. Pak. J. Bot. 40(1):461-467.
|
|
|
|
Cheikh-Rouhou S, Besbes S, Hentati B, Blecker C, Deroanne C, Attia H (2007). Nigella sativa L.: Chemical composition and physicochemical characteristics of lipid fraction. Food Chem. 101:673–681.
crossref
|
|
|
|
Enomoto S, Asano R, Iwahori Y, Narui T, Okada Y, Singab AN, Okuyama T (2001). Hematological studies on black cumin oil from the seeds of Nigella sativa L. Biol. Pharm. Bull. 24:307–10.
crossref
|
|
|
|
Hameed IH, Hussein HJ, Kareem MA, Hamad NS (2015a). Identification of five newly described bioactive chemical compounds in methanolic extract of Mentha viridis by using gas chromatography-mass spectrometry (GC-MS). J. Pharmacogn. Phytother. 7 (7):107-125.
crossref
|
|
|
|
Hameed IH, Ibraheam IA, Kadhim HJ (2015b). Gas chromatography mass spectrum and Fourier-transform infrared spectroscopy analysis of methanolic extract of Rosmarinus oficinalis leaves. J. Pharmacog. Phytother. 7(6):90-106.
crossref
|
|
|
|
Hameed IH, Jasim H, Kareem MA, Hussein AO (2015c). Alkaloid constitution of Nerium oleander using gas chromatography-mass spectroscopy (GC-MS). J. Med. Plants Res. 9(9):326-334.
crossref
|
|
|
|
Hameed IH, Hamza LF, Kamal SA (2015d). Analysis of bioactive chemical compounds of Aspergillus niger by using gas chromatography-mass spectrometry and Fourier-transform infrared spectroscopy. J. Pharmacog. Phytother. 7(8):132-163.
crossref
|
|
|
|
Hamza LF, Kamal SA, Hameed IH (2015). Determination of metabolites products by Penicillium expansum and evaluating antimicobial activity. J. Pharmacogn. Phytother. 7(9):194-220.
crossref
|
|
|
|
Hussein AO, Hameed IH, Jasim H, Kareem MA (2015). Determination of alkaloid compounds of Ricinus communis by using gas chromatography-mass spectroscopy (GC-MS). J. Med. Plants Res. 9(10):349-359.
crossref
|
|
|
|
Imad H, Mohammed A, Aamera J (2014a). Genetic variation and DNA markers in forensic analysis. Afr. J. Biotechnol. 13(31):3122-3136.
crossref
|
|
|
|
Imad H, Mohammed A, Cheah Y, Aamera J (2014b). Genetic variation of twenty autosomal STR loci and evaluate the importance of these loci for forensic genetic purposes. Afr. J. Biotechnol. 13:1-9.
crossref
|
|
|
|
Imad H, Muhanned A, Aamera J, Cheah Y (2014c). Analysis of eleven Y-chromosomal STR markers in middle and south of Iraq. Afr. J. Biotechnol. 13(38):3860-3871.
crossref
|
|
|
|
Iqbal MS, Qureshi AS, Ghafoor A (2010). Evaluation of Nigella sativa L., for genetic variation and Ex-situ conservation. Pak. J. Bot. 42(4):2489-2495.
|
|
|
|
Jasim H, Hussein AO, Hameed IH, Kareem MA (2015). Characterization of alkaloid constitution and evaluation of antimicrobial activity of Solanum nigrum using gas chromatography mass spectrometry (GC-MS). J. Pharmacogn. Phytother. 7(4):56-72.
|
|
|
|
Kanter M (2008). Effects of Nigella sativa and its major constituent, thymoquinone onsciatic nerves in experimental diabetic neuropathy. Neurochem. Res. 33:87-96.
crossref
|
|
|
|
Kareem MA, Hussein AO, Hameed IH (2015). Y-chromosome short tandem repeat, typing technology, locus information and allele frequency in different population: A review. Afr. J. Biotechnol. 14(27):2175-2178.
crossref
|
|
|
|
Mansour MA, Nagi MN, El-Khatib AS, Al-Bekairi AM (2002). Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: a possible mechanism of action. Cell. Biochem. Funct. 20:143–51.
crossref
|
|
|
|
Mohammed A, Imad H (2013). Autosomal STR: From locus information to next generation sequencing technology. Res. J. Biotechnol. 8(10):92-105.
|
|
|
|
Rader JI, Delmonte P, Trucksess MW (2007). Recent studies on selected botanical dietary supplement ingredients. Anal. Bioanal. Chem. 389:27-35.
crossref
|
|
|
|
Ramaa CS, Shirode AR, Mundada AS, Kadam VJ (2006). Nutraceuticals an emerging era in the treatment and prevention of cardiovascular diseases. Curr. Pharm. Biotechnol. 7:15-23.
crossref
|
|
|
|
Saleh S (2006). Protection by Nigella sativa (Black seed) against hyperhomo-cysteinemia in rats vascular disease. Preventia. 3:73-78.
|
|
|
|
Sethi G, Ahn K, Aggarwal B (2008). Targeting nuclear factor-kappa B activation Pathway by thymoquinone: Role in suppression of antiapoptotic gene products and enhancement of apoptosis. Mol. Cancer Res. 6:1059-1070.
crossref
|
|
|
|
Sharma NK, Ahirwar D, Jhade D, Gupta S (2009). Medicinal and pharmacological potential of Nigella sativa: A review. Ethnobot. Rev. 13:946-55
|
|
|
|
Vuorelaa P, Leinonenb M, Saikkuc P, Tammelaa P, Rauhad JP, Wennberge T, Vuorela H (2004). Natural products in the process of finding new drug candidates. Curr. Med. Chem. 11:1375–1389.
crossref
|
