Phytochemical Composition and Evaluation of Antimicrobial Activity of Blepharis linariifolia (Pers.) Seeds

Blepharis linariifolia (Pers.) originates from Sudan and grows widely in Africa. The current study has been designed to evaluate the bioactive compounds and antimicrobial activity of the n-hexane extract of Blepharis linariifolia seeds. The extract was analyzed by GC-MS (Model GCMS-QP2010 Ultra, Shimadzu Co., Japan) which revealed the presence of 2-Pentanol, 2-methyl;3-Pentanol, 3methyl; 2-Hexanone; Hexanal; Benzaldehyde, 4-fluoro; 4-Methyl-4-(tetrahydropyran-2yl)oxypentane-2,3-dione; 4-methyl-4-[3',4',5',6'-tetrahydro-2'-H-pyranyl-2'-oxy]-2,3-pentanedione; Hexane, 1,1'-oxybis; 1-Pentanol, 2,2-dimethyl; Butanoic acid, 2-ethyl-2-methyl; 2-Heptene, 5-ethyl2,4-dimethyl; Undeca-4,8-dione; Acetic acid, 4-acetyl-2-isopropyl-5,5-dimethyltetrahydrofuran-2-yl ester; 1-(3,3-dimethyl-bicyclo[2.2.1]hept-2-yl)pentan-2-one 3-cyano-2-oxa-1-ethoxyadamanane; Ethyl 3-methyl-2-oxobutyrate; Heneicosane 4,8,12,16-Tetramethylheptadecan-4-olide; 1,2Benzenedicarboxylic acid, diisooctyl ester; Hexatriacontane and Dotriacontane. All compounds were identified from the spectral libraries of National Institute of Standard and Technology and WILEY. Original Research Article Osama et al.; AJOCS, 2(2): 1-6, 2017; Article no.AJOCS.33182 2 FTIR analysis revealed the presence various functional groups related to different type of organic substances including: Organic halogen, Ester, nitro compound, aromatics, amines, Aldehyde, Alkenes, Phenol, Alcohol and silicon compounds. The in vitro antimicrobial assays showed there is non-significant activity of this extract against five microorganism tested for.


INTRODUCTION
Natural products are a well-known source of new valuable compound for medicinal or industry proposes [1]. Amongst the various natural sources, plants are predominant source of bioactive possess, including anticancer, antifungal and antimicrobial properties. The use of plant compounds as prototypes of new drugs has a historical and economic importance [2][3].
The analysis of plant extracts is extremely valuable to discover the chemical composition and to give better understanding of plant biological activities that may possess. Analysis of small amounts of chemicals has become easier and more and accurate after the discovery and development of chromatographic and spectroscopic techniques such as GC, HPLC, MS, NMR, etc. GC-MS are among the most powerful techniques used for both isolation and detection of samples which can be analyzed sufficiently even in trace amounts, less than 1 ng/ml [4].
Fourier Transform Infrared Spectrophotometer (FTIR) is perhaps the most powerful tool for identifying the types of chemical bonds (functional groups) present in compounds. The wavelength of light absorbed is characteristic of the chemical bond as can be seen in the annotated spectrum. By interpreting the infrared absorption spectrum, the chemical bonds in a molecule can be determined [5].
B. linariifolia (family: Acanthaceae) distributed in Africa especially the areas from Mauritania to Sudan, through Arabia to Northwestern India. B. linariifolia is a low-growing, wiry herb with prickly bracts and blue flowers. Seeds have analgesic activity and are also used in veterinary medicine [6]. However, genus Blepharis have not investigated much [7].

Extraction
B. linariifolia seed samples were collected from River Nile state, Sudan. The fresh seeds were dried in shades for 7 days and then powdered then used for extraction. method was used as per the method described by Osama and Awdelkarim, [8].

Fourier Transform Infrared Spectrophotometer (FTIR) Analysis
The above processed sample was used for FTIR analysis using KBr disk methodology. 1 mg of sample was encapsulated in 100 mg of KBr pellet in order to prepare translucent sample discs. The powdered sample was loaded in FTIR spectroscope (Shimadzu, IR Affinity 1, Japan), with a scan range from 400 to 4000 cm -1 with a resolution of 4 cm -1 . Each analysis was repeated ten times for the spectrum confirmation.

GC-MS Analysis
GC-MS analysis was carried out by using the instrument (Model GCMS-QP2010 Ultra, Shimadzu Co., Japan) equipped with a capillary column Rtx-5 (0.25 m film × 0.25 mm i.d. × 30 m length). The instrument was operated in electron impact mode at ionization voltage (70 eV), injector temperature (250°C), and detector temperature (280°C). The carrier gas used was helium (99.9% purity) at a flow rate of 1.2 mL/min and about 1 L of the sample was injected. The oven temperature was initially programmed at 35°C (3 min) to 240°C at 5°C/min and from 240-280°C at 3°C/min withhold time up to 4 minutes. The identification of compounds from the spectral data was based on the available mass spectral records (NIST and WILEY libraries).

Preparation of bacterial suspensions
One ml aliquots of pure standard bacteria of Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were aseptically distributed onto nutrient agar slopes and incubated at 37°C for 24 hours. The bacterial growth was harvested and washed off with 100 ml sterile normal saline, to produce a suspension containing about 108-109 cfu/ml. The suspension was stored in a refrigerator at 4°C till used. The average number of viable organisms per ml of the stock suspension was determined by means of the surface viable counting technique. Serial dilutions of the stock suspension were made in sterile normal saline solution and 0.02 ml volumes on drop of the appropriate dilutions were transferred by micro pipette onto the surface of dried nutrient agar plates. The plates were allowed to stand for two hours at room temperature for the drops to dry and then incubated at 37°C for 24 hours. After incubation, the number of developed colonies in each drop was counted. The average number of colonies per drop (0.02 ml) was multiplied by 50 and by the dilution factor to give the viable count of the stock suspension, expressed as the number of colony forming units per ml suspension. Each time a fresh stock suspension was prepared. All the above experimental conditions were maintained constant so that suspensions with very close viable counts would be obtained.

Preparation of fungal suspension
The fungal cultures were maintained on Sabouraud dextrose agar, incubated at 25°C for 4 days. The fungal growth was harvested and washed with sterile normal saline and finally suspension in 100 ml of sterile normal saline, and the suspension was stored in the refrigerator until used.

In vitro testing of extracts for antimicrobial activity A) Testing for Antibacterial Activity
The cup-plate agar diffusion method was adopted according to published method of Eltayeb et al. [9] One ml of the standardized bacterial stock suspension 108 -109 C.F.U./ml were thoroughly mixed with 100ml of sterile molten nutrient agar which was maintained at 45°C. 20 ml aliquots of the inoculated nutrient agar were distributed into sterile Petri-dishes. The agars were left to dry and in each of these plates 4 cups (10 mm in diameter) were cut using a sterile cork borer (No. 4) and agar discs were removed. Alternate cups were filled with 0.1 ml seed extract sample using automatic Microliterpipette, and allowed to diffuse at room temperature for two hours. The plates were then incubated in the upright position at 37ºC for 18 hours. Two replicates were carried out for each extracts against each of the test organisms.
Simultaneously addition of extracts was carried out as controls. After incubation, the diameters of the resultants and growth inhibition zones were measured, averaged and the mean values were tabulated.

B) Testing for Antifungal Activity
The same method as for bacteria was adopted. Instead of nutrient agar, Sabouraud dextrose agar was used, which is considered to be more selective for fungal. The inoculated medium was incubated at 25ºC for three days for Aspergillus niger.

RESULTS AND DISCUSSION
The n-hexane dissolved about 2.7% of total sample mass. Hexane is a nonpolar solvent capable to isolate the nonpolar compounds especially fatty acids, terpenes and steroids. This low yield percentage indicates the low amount of oil in this plant. The nature of sample is not the only factor that affect its content of group of compounds. The environmental factors, the part of plant used, methodology of extraction, duration of extraction, solvent used for extraction, etc. all these are factors can effect on percentage yield [10].
In the current study the hexane extract was analyzed using spectrometric techniques (FTIR and GCMS) to determine the functional groups and chemical constituents. The FTIR is the most powerful technique to determine the functional groups according to the response of compounds to the radiation with adjusted wave length. These groups are most likely responsible for the chemical and biological activities of this extract. The functional groups are the active parts of compounds they affect to its reactivity to certain kind of compounds or receptors. The study of the structure and its activities is the base of valuable part of medicinal chemistry which named as structure activity relationship [11]. The results showed the presence of different groups, shown in Table (1).
The functional groups detected by FTIR was mainly belong to aliphatic hydrocarbons, the highest absorption was found in 2958.6 (symmetric C-H stretching) which indicates the high amount of hydrocarbons alkenes. The hydrocarbons are well known for their non-polar properties therefore they can be isolated by hexane, the solvent used for extraction.
However, the alkenes are not active compounds compared to phenols or carboxylic acids. Low absorption was noticed for OH and C=O groups which are known for their antimicrobial activity.
Gas chromatography-mass spectrometry is one of the most updated techniques to isolate and detected the volatile chemical substances. In the present study the GCMS analysis detect different kind of compounds with variable molecular weight. The chromatogram showed the presence of 22 compounds, which they were identified by MS. The MS showed that the molecular weight of these compounds which vary from 100 to 506 amu. These compounds are mainly aliphatic hydrocarbons, shown in Table (2).
The B. linariifolia seed extract was evaluated for its antimicrobial activity and was found to be inactive to five type of bacteria and fungal.