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Biochemical potential and screening of bioactive components of Ocimum basilicum

Secondary metabolites and phytochemicals are abundantly found in plants with different remedial properties and pharmaceutical benefits with less or no harmful effects. In the history of mankind, Ocimum basilicum (sweet basil) is reported to be used as antimicrobial, cardio tonic, and as a remedial agent to treat various symptoms of diabetes. As well as it is used as an anticancer agent. Sweet basil has medicinal potential of treating gastric disorders, relaxing blood vessels, healing wounds and different infections in humans. Current study was designed to analyze the biological capacity of essential oil and methanolic extract of Ocimum basilicum leaves to assess the presence of some vital phytochemicals along with its therapeutic and biological potentials. Antioxidant, antifungal, antimicrobial and ant biofilm potential of basil was determined. Fourier transformation infrared spectroscopy (FT-IR) and High Performance Liquid Chromatography (HPLC) techniques were followed to analyze some basic functional groups of crude basil powder and to screen some phenolic compounds. This study can be further helpful for pharmaceutical purpose to treat infectious and non-infectious diseases through the natural drug discovery.

Introduction Plants provide us with many important phytochemicals along with secondary metabolites which serve various benefits for health and different medical properties without or negligible side effects. Since ancient times, different herbs have been used as a remedy to treat most of the human illnesses. Economically it is convenient to obtain the natural extracts or products from plants to treat health disorders and certain infectious illnesses [1]. Due to medicinal capacity of Lamiaceae family plants, they are widely used by mankind. Secondary metabolites of interested research plant (Ocimum basilicum) possess the potential of reducing the risks of certain microbial infections due to the presence of active antimicrobial compounds. Essential oils, alkaloids, tannins, steroids, flavonoids, phenols and resins might be included in these secondary metabolites [2]. Different basil extracts might be consisted of essential oils, vitamins and poly phenolic compounds and many other biologically active compounds which exhibit insecticidal, antipathogenic and stressrelieving properties and treats ailments related to respiratory tract, excretory organs, gastric illnesses, skin and eye problems In drug metabolism, Ocimum basilicum is found to have chemomodulatory effects. Therefore, using natural products of plants is highly beneficial against oxidative damage caused by different degenerative and toxic agents and in preventing from side effects of various chemical products [6]. So, therefore the current study was planned with the biochemical prospective of the Ocimum basilicum (sweet basil) to explore the medicinal prospectives of essential oil & leaves extract of sweet basil. As well as interpretation of chemical analysis of few essential and biologically active phytochemicals that are capable of rendering Ocimum basilicum with many biological activities. Phenolic acids were identified and quantified through High Performance Liquid Chromatography (HPLC). 500 mg dried leaf powder was ground in particle size of 0.125-0.250 mm. immersed in 5 ml of methanol of HPLC grade and left at 4 °C overnight in darkness. Decantation and filtration of supernatants was done through syringe filter [17] and then samples were shifted to HPLC vials. Fourier transformation infrared spectroscopy (FT-IR) FT-IR assay was executed on crude basil powder sample to identify its functional groups. Infrared spectral studies were carried out on FT-IR spectrometer in the presence of dry air, and it was performed at room temperature. In Potassium bromide (KBr) on the FT-IR spectrophotometer using KBr pellet, the spectra from the samples were measured [18].

Statistical analysis
Values of each measurement are expressed as mean ± Standard deviation (SD). Data was checked by using one way ANOVA with the use of Minitab 17 software [19].

Results and Discussion
Current study was planned to investigate the biochemical potential of sweet basil (Ocimum basilicum) along with the screening of some important phytochemicals through biological assays and other analytical techniques. Many biological properties of Ocimum basilicum extracts were identified as; antioxidant activity and antimicrobial potential as well as antibiofilm potential. The most imperative part of our research work was the separation of phenolic acids through HPLC and analyzing crude basil powder by using the FTIR technique. 2, 2-diphenyl-1-1-picrylhydrazylstable radical (DPPH) scavenging assay DPPH is a constant free radical usually used to find out the capability of compounds to scavenge free radicals. So, in this study, DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical was used to assess the In-vitro antioxidant capacity of methanolic extract of Ocimum basilicum and among varying concentrations of essential oil. The results of antioxidant activity of Ocimum basilicum methanolic extract and essential oil extract are shown in (Fig. 1) which depicted that at 100% concentration the essential oil has 93.5% maximum antioxidant activity as compared to methanolic extracts and their radical sifting activity is directly proportional to their concentration. As Shafique and his colleagues in 2011 [20] reported that basil essential oil extracts have higher antioxidant activity at 100% concentration and showed 96.16% activity by DPPH scavenging effect. So, they concluded that the essential oil extract of basil was more effective than Butylated hydroxytoluene (BHT).

Total phenolic content (TPC)
Linear regression gallic acid curve, a standard phenol (y = 0.0055x + 0.0987) was used to determine the Phenolic Content of the methanolic extract of sweet basil leaves and results were shown in mgGAE/g of dried extracted samples weight. TPC results in (Fig. 2) Table 1) shows comparable difference among the minimum inhibitory concentration of the control i.e., Rifampicin and the particular basil essential oil fractions. 100% concentration of basil essential oil fraction was seen with the MIC in the range of 2.46µg/mL to 76.34µg/mL for B. subtilis and E.coli with 5% fraction of basil essential oil respectively. Minimum Inhibitory Concentration (MIC) results of present research work were matched to that of the reported outcome of Hamdan and his co-workers [24]. Both studies were presented with the same results in which the values were lying close to each other and almost within the same ranges.   Similarly, all the three fractions of methanolic extract of sweet basil and 100% concentration of essential oil extracts were checked for antifungal activity against two fungal strains as: Aspergillus Niger and Rhizopus solani. Basil essential oil fractions was observed with the significant antifungal activity against the both Aspergillus niger and Rhizoctonia solani. Whereas fractions of basil methanolic extract did not display antifungal activity as given in (Table 2).

Ocimum basilicum Antibiofilm activity
Methanolic extract of O. basilicum and all of its essential oil divisions were evaluated for their antibiofilm potential through micotitre plate assay describe by Dheepa and his fellows. DMSO (dimethyl sulfoxide) was taken as a negative control. Staphylococcus aureus and Escherichia coli were selected for inhibition of biofilm formation against them. All of the basil essential oil fractions were shown with appreciable intensity of inhibiting biofilm production with the maximum inhibiting activity seen in the concentrated basil essential oil fraction with the supreme value of 55% against S. aureus and 40% against E. coli. Whereas methanolic fractions of sweet basil did not show any antibiofilm potential as shown in ( High Performance Liquid Chromatography (HPLC) was performed to quantify the amount of total phenolic compounds in the crude methanolic extract of Ocimum basilicum. (Table 4) quantifies different phytochemicals and their (%) Quantities like caffeic acid (0.004868), Gallic acid (0.0015), Cinamic acid (0.000896), Quercitin (0.000036) and chromatotropic acid (0.000411). In 2008 Kruma and his colleagues have been observed that basil contain different phenolics and the total phenolic components. They were found to be 12.18mg/100g of phenolic compound through crude methanolic extract [26]. Rosmarinic acid, a derivative of phenolic acid was the only one phenolic compound that was analyzed in that study. Flavonoid was not identified through HPLC whereas present study showed that basil have 0.36ppm of quercitin as we can observed in the HPLC chromatogram of (Fig. 5).

Interpretation of Fourier Transform Infrared Spectroscopy (FTIR) spectral data
Through FTIR wave number, % transmittance, peak values and corresponding presumptive functional groups of the coarse powder of O.basilicum were determined that are presented in (Fig.  6 & Table 5). Characteristic absorption bands of crude sweet basil powder are shown in FTIR spectrum within the range of 1350cm -1 to 3400cm -1 that depict the presence of alkanes and amines or amides. Various functional groups were found between these two peak values which were found in the frequency ranges of 1600cm -1 for C-C stretch, 2100cm -1 for C≡C stretch and 2850cm -1 for C-H stretch at their peculiar peaks.