GC-MS, FTIR and Antiulcer Screening of Aqueous Seed Extract and Oil of Nigella sativa in Wistar Rats

1 Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria. 2 Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences. Federal University of Oye-Ekiti, Ekiti state. 3 Department Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria. 4 Department of Laboratory Science, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.


INTRODUCTION
Nigella sativa L, known as black cumin or black seed (family, Ranunculaceae) is used as a spice in Indian and Middle Eastern cuisine. In Asia, Middle East, and Africa, it is used as an herb and oil. Gastrointestinal tract (GIT) is responsible for the absorption of nutrients, digestion of food, and excretion of unabsorbed waste products 1 . It is the organ that provides a continuous supply of water, electrolyte, and nutrients to the whole part of the body. Peptic ulcer disease or PUD is a pathological condition of this organ and refers to the painful spot characterized by the presence of an ulcer in any part of GIT which is exposed to acid and pepsin. Nigella sativa L ethnomedicinal properties include antimicrobial, antidiabetic, antifertility, antioxytocic, analgesic, anthelmintic, antihypertensive, antiulcer, circulatory and immune system support 2 . A Gastro duodenal lesion due to hyperacidity leads to peptic ulcerations which are erosions of the mucosal epithelial lining of the gastrointestinal tract 3 . Despite the pathophysiology of peptic ulceration not been completely elucidated, it is known that an imbalance between aggressive factors (acid and pepsin secretion) and cytoprotective factors of the gastric mucus membrane (mucus and bicarbonate secretion) result in gastric ulceration 4 . The most common symptoms of peptic ulcer include epigastric and nocturnal pain which are alleviated by either food intake or antacids; other symptoms include indigestion, nausea, loss of appetite, fatty food intolerance, and heartburn. 5,6 The aetiology of gastric ulcer involves environmental factors such as alcoholic beverages and non-steroidal anti-inflammatory drugs (NSAIDs) usage, Helicobacter pylori, genetic factors amongst others 7.8 . The chemical composition of N. sativa seeds contains unsaturated fatty acids with terpene alcohols, traces of alkaloids (isochinoline alkaloids and pyrazol alkaloids). In the ISSN: 2250-1177 [49] CODEN (USA): JDDTAO essential oil contains thymoquinone, p-cymene, pinene, dithymoquinone and thymohydroquinone 2 . Peptic ulcer of the stomach and duodenum was known in the 1700s, there is a revolution of understanding and management of PUD after the discovery of Helicobacter pylori. Several treatments are available for peptic ulcers such as antacids, H2 blockers, proton-pump inhibitors, antibiotics, and combination therapy. Adverse effects and decreased efficacy over time have limited the use of some anti-ulcer drugs. Reports on clinical evaluation of conventional anti-ulcerogenic drugs showed that there are incidences of relapses, adverse effects and danger of drug interactions during ulcer therapy 9 . The high cost of newly available drugs for peptic ulcer disease is responsible for the persistence, morbidity and mortality of the disease in third world countries due to low per capita income 10 . As a result, the search for an ideal antiulcer drug continues and has also been extended to herbs for new and novel molecules that afford better protection and reduction in the incidence of relapse 10 . Herbal drugs are used widely even when their biologically active compounds are unknown because of their assumed effectiveness, availability, lesser side-effects and relatively low cost 11 . The study was aimed to evaluate the GC-MS, FTIR and antiulcer screening of aqueous seed extract and oil of Nigella sativa in wistar rats

Sample preparation
The Nigella sativa seeds were collected from Roban stores Awka in September, 2020. Samples were authenticated by a taxonomist in the department of botany Mr Okeke Ebele in Nnamdi Azikiwe University Awka, air-dried in a dark room without sunlight, grinded and then they were stored in a cool room (4 °C) for subsequent experiments.

Seed processing
Digestion method was used in extracting the seed. Digestion is an extraction method that involves the use of moderate heat during extraction process. The solvent of extraction is poured into a clean container followed by powdered drug material. The mixture is placed over water bath or in an oven at a temperature about 50 o C 12 .

Oil extraction
Hydro-distillation method was used in extracting the oil. The air-dried seeds of Nigella sativa were grinded right before the extraction process. In order to protect the plant materials from overheating or charring by direct steam, grinded seeds were immersed in distilled water in a round bottom flask on a heater 13 . Essential oil of the seeds was extracted by hydrodistillation Clevenger apparatus method (Fig. 1) at the boiling range of water and atmospheric pressure. The extraction process was optimized with respect to time, heating power (250, 300, 350 W) and solid (g) to solvent (ml) ratio (1:3, 1:10, 1:12).

Figure 1: Hydro-distillation Clevenger apparatus system
Extracted essential oil was dried over anhydrous sodium sulfate to remove all the water and then stored it in darksealed-vial at 4 °C for further tests. The yield of essential oil was calculated by the following equation: Where y is the yield of essential oil (%), V is the volume of collected essential oil (ml) and W is the weight of the plant material (g).

Microscopic examination
Microscopic studies were carried out by preparing thin sections of leaf. The thin sections were further washed with water, staining was done by clearing in chloral hydrate solution then heat fixed and allowed to cool, then mounted using glycerine. The specimen was gently covered with a cover slip and placed on the stage of the microscope for observation (40x) 14 .

Chemomicroscopic examination
Examination of the powder for lignin, starch, mucilage, calcium oxalate crystals, cellulose, fatty oil and protein were carried out using standard techniques 15 .

Physicochemical analysis
The parameters which were studied are moisture content, ash values and extractive values 16,17 .

Qualitative phytochemical analysis
The

Acute Toxicity
The acute toxicity study of Nigella sativa was carried out according to the method employed by Lorke's method 18 but modified, using a total of 21 rats 19,20 .

Animal husbandry
Thirty Wistar Rats (120-150 g) were obtained from the animal house, Department of Pharmacology and Toxicology, Nnamdi Azikiwe University Awka. They were fed with grower mash (vital feed, grand cereal) and water and kept for 2 weeks to acclimatize with the animal house conditions (a crossventilated room with temperature between 25 °C and 32 °C, 12 h light/12 h dark cycle) before the commencement of the study. The research was conducted in accordance with the Nnamdi Azikiwe University Research and Ethical Committee guidelines, the ARRIVE guidelines (reporting of in vivo experiment), and the National Institutes of Health (NIH) guide for the CARE and use of laboratory animals (NIH Publications No. 8023, revised 1978).

Experimental Design:
The thirty (30) Wistar rats were randomly divided into six groups of five rats each and fasted for 18 h before administration of extract. Rats in Group 1 were pretreated with 1 ml/kg Distilled water, the rats in Group 2 were pretreated with 30 mg/kg Famotidine, while those in Groups 3 and 4 were pretreated with aqueous extract of N. sativa at 250 mg/kg and 500 mg/kg. Group 5 and 6 were pretreated with Black seed oil at 1 ml and 1.5 ml, 30 mins before the administration of 1 ml 80% ethanol. All the rats were sacrificed 1 h after 80% ethanol administration using ketamine injection, and the stomachs were cut open, the stomach content were deposited in a beaker, the ulcer index was determined. The gastric content was collected in test tube and centrifuged at 3000 rpm for 10 min, Total acidity (m Eq/L), Gastric acid volume (ml) and pH of the supernatant was measured using digital pH meter 21,22,23 .

Histopathology procedure
The stomachs were fixed in 10% neutral buffered formalin, dehydrated in graded series of alcohol, cleared in xylene, and embedded in paraffin wax. The tissues were sectioned at 5 µm with a rotary microtome and stained with hematoxylin and eosin (H and E) and cresyl violet stain 24 .

Fourier Transform Infrared Spectroscopic (FTIR) Analysis
Buck scientific M530 USA FTIR was used for the analysis. This instrument was equipped with a detector of deuterated triglycine sulphate and beam splitter of potassium bromide. The software of the Gram A1 was used to obtain the spectra and to manipulate them. An approximately of 1.0g of samples, 0.5mi of nujol was added, they were mixed properly and placed on the salt pallet. During measurement, FTIR spectra was obtained at frequency regions of 4,000-600cm^1 and coadded at 32 scans and at 4cm^1 resolution. FTIR spectra were displayed as transmitter values 25 .

GC/MS Analysis
The Gas chromatography-mass spectrometry (GC-MS) analyses of N. sativa aqueous seed extract was carried out in Agilent Technologies (Wilmington, Delaware, USA) equipment with a column HP-5MS (30 m × 0.25 mm 1D X 0.25). A sample of 1 µL of each extract was injected. For detection, an ionization system with energy of 70 eV was used. The flow rate of carrier gas was constant and it was maintained at 1.1 mL/min. The injection temperature was 250 °C. The warming program of the oven was isothermal for 5 min at 60 °C followed by a warming of 5 °C/min up to 100 °C/min (2 min), and 10 °C/min up to 250 °C/min (5 min). The interpretation of the mass spectra was made using the National Institute of Standard and Technology (NIST) library 26,27,28 .

Statistical analysis
Data obtained from the study were analyzed using Statistical Package for Social Sciences (SPSS-21). Results were presented as mean ± Standard error of mean (SEM) of sample replicates. Raw data were subjected to one way analyses of variance (ANOVA) followed by post hoc turkey's test. p<0.05 were considered to be statistically significant.

DISCUSSION
Gastric ulcer is due to an imbalance between protective and aggressive factors in the stomach, which is caused by the infection of Helicobacter pylori 29 , non-steroid antiinflammatory drugs, stress condition, and alcohol consumption 30,31 . Microscopic and pharmacognostic standardization will help in the identification and authentication of the genuine plant materials, the presence of starch grains, Lignified tissues, cellulose, proteins, and oil globules, as shown in tables 1 and 2. A previous result of the macroscopic characteristics of N. sativa seed includes epidermal cells are irregular in shape, trichomes are nonglandular, and stomata are actinocytic with a broad base. Some previous studies also show similar results 32 . The leaf epidermal features which include types of stomata, epidermal cells, and hairs are significant tools in delimiting the taxonomy in many plants 33,34,35,36 . Ahmad et al 37 reported variations in the pattern of the epidermal cells that can be used as an important microscopic tool to identify many closely related species. The study of trichomes has been found useful by many researchers 38,39 .
The proximate composition of Nigella sativa seed extract contains moisture content 5.3 %, total ash 4.8 %, acid insoluble ash 1.0 %, water-soluble ash 1.3 %, acid insoluble ash 1%, water-soluble extractive value 8.5 %, and alcohol soluble extractive value 7.0 % as shown in table 3. In this study, the increased value of water-soluble extract indicates that Nigella sativa seed extract dissolves more in water than alcohol and also should extract more secondary metabolites. Fluorescence analysis is also required to evaluate the purity of plant crude 40 . The crude drug moisture content should not be greater than 14% w/w 41 . The growth of microorganism (yeast and fungi) is enhanced by high moisture content of crude drugs, causing the breakdown of crucial bioactive compounds. The high value of ash is due to contamination and the presence of impurities 42 . Pharmacognostic and physicochemical studies of the plant parts act as a reliable tool for detecting adulteration and plant identification 43,44,45 .
The phytochemical screening of Nigella sativa aqueous seed extract showed the presence of saponins, alkaloids, flavanoids, tannins, glycosides, carbohydrates, and reducing sugar. Saponins, flavonoids, alkaloids, fats and oils are present in huge amounts, as shown in table 4. Previous studies of the phytochemical screening of the P. nitida seed extract indicate the presence of alkaloids, saponins, flavonoids, cardiac glycoside, terpenoids, protein, and carbohydrates. Some of the phytochemical compounds detected such as glycoside, saponins, tannins, flavonoids, terpenoids, and alkaloids have been reported to have antimicrobial activity 42,46 . It is significant to note that alkaloids contribute to plant species fitness for survival. Some alkaloids and saponins have been found to possess antimicrobial activity 47 . Flavonoids have been shown to have antibacterial, anti-inflammatory, antiallergic, antiviral, antineoplastic, antiulcer activity 48,49 .
The result of the acute toxicity after 24 hours of administration of the various doses (10, 100 and 1000 mg/kg body weight) of the aqueous extract of Nigella sativa in the first phase and the second phase, three-dose ranges were also used 2000, 3000, 4000 and 5000 mg/kg body weight and there was no death after 24 hours. Therefore, the LD50 was taken to be above 5000 mg/kg practically did not cause any death nor any sign of toxicity observed. Okoye et al 50 carried out the LD50 of combined aqueous extracts of Ocimum gratissimum and Anacardium occidentale (1:1) at a dose of 5000 mg/kg orally which practically did not cause any death nor any sign of toxicity observed.
High concentrations of ethanol induce vascular endothelium injury of the gastric mucosa, which become edematous, and congestive, present point and scattered bleeding lesions, focal hemorrhage, necrosis, and giant deep ulcers were visible. The aqueous extract (250 mg/kg and 500 mg/kg) and oil extract (1 ml and 1.5 ml) showed a significant reduction (p < 0.05) in ulcer index, total acidity, Gastric acid volume, and pH of gastric secretion, when compared with the negative control Distilled water (1 ml/kg) as shown in Table 6. The effect of aqueous and oil extracts are dose-dependent. The rats pretreated with N. sativa aqueous extract (500 mg/kg and 250 mg/kg) and oil extract (1.5 ml/kg and 1 ml/kg) showed a significantly reduction in Ulcer index (4.05±0.9, 8.68 ± 0.54 and 4.62 ± 0.19, 8.87 ± 0.4), than those of rats pretreated with the negative control Distilled water (1 ml/kg) (13.00±0.54) at p < 0.05. The maximum effect was observed at 500mg/kg of the aqueous extract with ulcer index of (4.05±0.9) and 1.5ml of the oil gave an ulcer index of (4.62±0. 19), as compared with the negative control Distilled water (1 ml/kg) with ulcer index of 13.00±0.54 is an indication that the extract can protect the stomach lining from ethanol-induced mucosal damage.
Ethanol induces gastric mucosal damage by promoting disturbances of mucosal microcirculation, ischemia, endothelin release, degranulation of mast cells, inhibition of prostaglandins and decrease of gastric mucus production 51,52 . Ethanol rapidly penetrates the gastric mucosa causing injury characterized by membrane damage, erosive hemorrhagic lesions with diffuse coagulative cell necrosis, cell exfoliation, multiple superficial erosions, marked vascular congestion and ulcer formation 53 Histological examination of the stomach of rats pretreated with negative control showed irregularity in gastric mucosa, such as severe desquamation (Red arrow) and loss of surface epithelial cell (blue arrow), necrosis, vacuolization, edema and dilated gastric glands along with infiltration of inflammatory cells (yellow arrow). The stomach of rats pretreated with the positive control (Famotide) revealed a few superficial surfaces of mucosa (black arrow), gastric glands appeared normal without inflammatory cells infiltration (yellow arrow).
Histoarchitecture not affected. The stomach of rats pretreated with N. sativa aqueous extract (250 mg/kg) showed the mucosa was infiltrated by inflammatory cells (Black arrows) that almost displayed extensive edema. The stomach of rats pretreated with N. sativa aqueous extract (500 mg/kg) showed the mild appearance of hemorrhage (Green arrow). The stomach of rats pretreated with N. sativa oil extract (1 ml) displayed few superficial surfaces of mucosa (Red arrow), gastric glands necrosis cannot be seen histoarchitecture moderately affected. The stomach of rats pretreated with N. sativa oil extract (1.5 ml) showed that gastric mucosa exhibited focal loss of superficial gastric epithelium (blue arrow). The gastric glands were almost normal in appearance (black arrow). The healing process of gastric ulcer including several processes in gaster mucous e.g. congestive, hemorrhagic, edema, necrosis, inflammation, erosion, ulceration and dysplastic change 56 . Evaluation for the healing process in the clinical setting was based on visual endoscopy, but this study was based on microscopic evaluation and gastric ulcer determination. Microscopic evaluation showed gastric glands dilatation, increase of connective tissue, increase of micro vascularization, and recovery of a sensory nerve. It could be the basis for evaluating the quality of the healing process of gastric ulcers 57,58 .
Fourier Transformed Infrared (FTIR) technique is an important tool used to identify the characteristic functional groups, which are instrumental in the determination of functional groups and organic compounds inherent in any given sample. The peak value 819.0908cm -1 was assigned to the C-CI stretching vibration of the halogeneous compound. GC-MS analysis is followed by library search of non-polar extract of the seed identified five compounds such as glycerin, n-Hexadecanoic acid, 9, 12-octadecadienoic acid-methyl ester, 9, 12-octadecadienoic acid and 9, 12-octadecadienoyl chloride. The compounds from the GC-MS investigation indicate the biological properties and in turn the pharmaceutical values of the study plant extract.
The GC-MS techniques have been proven to be suitable for the chemical profiling of medicinal plants 60 , these techniques provide a sufficient profile and identification of the compounds analyzed 61 .
Glycerin is known as glycerol, it is widely used in FDAapproved wound and burn treatments. It has been identified with retention times 6.545 and 7.075, it has laxative and antibacterial properties 27 . Glycerol is a simple polyol compound. It is a colorless, odorless, viscous liquid that is sweet-tasting and non-toxic. The glycerol backbone is found in lipids known as glycerides. Glycerol has been detected in a good number of propolis samples from different regions around the world for example in Turkish propolis, Canadian propolis and Brazilian geopropolis 62, 63, 64 .
n-Hexadecanoic acid (palmitic acid) is a long-chain saturated fatty acid with a 16-carbon backbone commonly found in milk products, meat, and oil. It is the most common saturated fatty acid found in animals, plants and microorganisms. n-Hexadecanoic acid has been identified with a retention time of 12.357, it also has the following properties; nematicide, pesticide, lubricant, anti-androgenic, flavor, hemolytic 5-alpha reductase inhibitor, antioxidant and hypo-cholesterolemic features 65 .

CONCLUSION
The pharmacognostic properties can act as a reliable tool for the standardization and quality evaluation of the plant part. The anti-ulcerogenic properties exhibited by the plant extracts are due to the presence of secondary metabolites, this study suggests that aqueous and oil extract possesses antiulcer properties and thus the aqueous and oil extract of black seed can be considered as antiulcer medication traditionally.