Evaluation of antioxidant, antimicrobial activity and GC-MS analysis of Phlomis stewartii

The myths of curing several ailments related to Phlomis in different countries since centuries brought our attention to unveil one of its potent specie. The main target was to explore biological activities of this species and to investigate presence of chemical constituents. The present work consists of preliminary phytochemical analysis, Gas Chromatography-Mass Spectrometry (GC-MS) analysis, antioxidant, antibacterial and antifungal activities of the Phlomis stewartii . Qualitative phytochemical analysis deduced the presence of the alkaloids, terpenes, coumarins


Introduction
Natural products either obtained from plants, animals or microbes are considered as important tools in medicinal field.A large number of natural products are used as starting material for drug development.Additionally, they are allowed to be marketed after several modifications to overcome side effects [1].Natural products are categorized as primary and secondary metabolites, the later one are actually responsible for making them unique with respect to several characteristic benefits provided to different life forms [2,3].Medicinal plants are potent with respect to ailments cure and famous since ancient history.The plants are used for curing several ailments from simple fever to major diseases caused by pathogens in different parts of the world [4].The hidden chemistry for such curing potential lies in their phytochemicals which brings some uniqueness in plant and these substances are actually the center of attraction for several activities.These reported substances are up to 4500 and 350substances are completely characterized by using different techniques.These phytochemicals make the plant attractive by imparting color, flavor, and taste as well as protect them against foreign attack of living as well as non-living like exposure of ultraviolet rays, polluting agents etc. [5].These substances when isolated and applied on other living organisms then they impart mixture of two behaviors that is detrimental as well as benign [6].The most potent phytochemicals found in most of the plants are flavonoids, carotenoids, polyphenols, lignins, anthocyanins, indole-3-carbinol and glycosides [7].Phlomis  In present work, Phlomis stewartii was studied for the GC-MS analysis of n-hexane fraction and preliminary phytochemical analysis of the plant along with evaluation of antibacterial, antifungal and antioxidant activities.

Plant material
The plant sample was collected from Ziarat, Balochistan in the month of April, 2017 and identified by Dr. Rasool Bakhsh Tareen, Professor Department of Botany, University of Balochistan, and Quetta.The plant (7 kg) was extracted thrice with methanol in order to obtain maximum chemical constituents in the extract [10].The obtained methanolic extract was fractionated into n-hexane and ethyl acetate fractions.

GC-MS analysis
Gas Chromatography (GC) was used to determine the retention time and Mass spectrometry (MS) was used to determine the molecular ion peak and fragment ion peaks of compounds.
The column (30m×250µm×0.25µm)was packed with 5% phenyl methyl siloxane as stationary phase.The helium gas used as mobile phase which allowed the sample to move across the column.The oven temperature was initially set at 50 0 C for at least 3 minutes and then it was increased from 10-180 0 C /15 minutes.In the second step, temperature was raised up to a maximum of 300 0 C, the maximum temperature and pressure of oven recorded as 360 0 C and 9.05psi respectively.The complete run up time was 68 minutes [11] and the mass spectra thus obtained were matched with already available data in Replib and Mainlib [12].The electron impact (EI) used as a source for ionization at 250 0 C.

Qualitative analysis
The methanolic extract and n-hexane fraction were evaluated for the presence of alkaloids, terpenes, coumarins, saponins, cardiac glycosides, phlobatannins, flavonoids, quinone, steroids and tannins by adopting methods described in previous reported literature [12].The test methodsare included in (Table 1).

Quantitative analysis
The quantitative analysis of n-hexane and methanolic extract were determined for total alkaloid contents, total phenolic contents, total saponins and total flavonoids contents.The methodology thus adopted was already reported [13,14].

Antioxidant activity
The n-hexane fraction and methanolic extract were evaluated for antioxidant activity.The 2,2-diphenyl-1-picrylhydrazyl(DPPH) was used as free radical while BHT as a standard antioxidant.The plant sample and DPPH were dissolved in methanol, so methanol was used as reference.The DPPH (0.004 g) was dissolved in 100 mL of methanol.The dilutions (250, 125, 50, 10) mg/L were prepared from the 5mg/L stock solution for plant sample and BHT standard.All the prepared samples were kept in dark for a period of 20-25 minutes in dark for providing

Antifungal activity
The antifungal activity of sample was tested by using disc diffusion method and agar well diffusion method.The Potato Dextrose Agar (PDA) was used as nutrient and fungi were Alternaria alternate, Rhizoctonia solani and Fusarium oxysporum.

Disc diffusion method
The fungi were placed at center of the discs and samples applied at peripheral region.The diameter of disc was 6mm and allowed for incubation for 3 days at 37 0 C in dark.This method was used to assess the samples on the basis of qualitative analysis [16].

Agar well diffusion method
This method adopted for the quantitative analysis of the plant samples against fungi.

Results and discussion GC-MS analysis
The n-hexane fraction of Phlomis stewartii was investigated via GC-MS technique.The list of compounds, their molecular formula (MF), molecular weight (MW), and retention time (RT), area sum % peaks are incorporated in (Table 2).The GC chromatogram showed relative concentration of each compound verses retention time.The chromatograms included as (Figure 1 & 2).The mass spectra of most abundant compounds are added as (Figure 3-6).
Among them, stigmasterol, β-sitosterol and phytol have antioxidant potential.The ester based compounds have the potential of scavenging free radicals [18].The long chain fatty acids including eicosanoic acid, linoleic acid etc. are potent agent for antibacterial activities.The presence of above compounds in nhexane fraction revealed its medicinal importance [19-21].3).

Quantitative analysis
The methanolic extract and n-hexane fraction were evaluated for quantitative analysis.It was revealed that highest %age of phenolic compounds were present in methanolic extract that was 48% while 42% in n-hexane fraction.The results are incorporated in (Table 4).The compounds present in methanolic extract and n-hexane fraction are reportedly biologically important such as flavonoids are antioxidant and antiviral agents [27].

Antioxidant activity
The antioxidant activity of Phlomis stewartii was tested and IC50 obtained from different dilutions showed the potency of sample as an antioxidant.The decoloration of DPPH from purple color indicated the scavenging of it by sample and BHT.The results showed that methanolic fraction (28.42±0.1) is more potent antioxidant than n-hexane fraction (37.16±0.007).The results are shown in (Table 5).The DPPH scavenging effect was calculated by absorbance of control and sample difference, the samples were tested in triplicate and then IC50 was obtained from the DPPH scavenging activity graphs at different dilutions.The graphical representation is included in (Figure 7 & 8).The scavenging activity indicating the presence of some antioxidant in plant sample such as polyphenols and quinines can be reason for antioxidant activity.The antioxidant activity was found to be dependent upon hydrophilic and hydrophobic nature of components, larger electron donor groups like -OH in phenylpropanoids favours more activity [10].BHT has such phenolic group which resembled with phenolic skeleton of antioxidants present in plants.The stable free radical (DPPH) was chosen and absorbance was measured at 517nm and reduction in absorbance was observed due to engulfing of free radicals.It was due to provision of hydrogen by antioxidant [15].Flavonoids and several glycosides which contain hydroxyl groups are much effective agents of such activities [26].

Antifungal activity
The n-hexane and methanolic extract were assessed for antifungal activity and Alternaria alternate, Rhizoctonia solani and Fusarium oxysporum were used as fungal agents.The potency of n-hexane fraction and methanolic extract was tested by means of qualitative as well as quantitative analysis.The results revealed that n-hexane fraction inhibited the R. solani (40+0.94), A. alternate (25+0.81)and F. oxysporum (12+0.67)whereas methanolic extract inhibited only A. alternate (20+0.58).The results are shown in (Table 6 & Figure 9).The use of medicinal plants as antifungal agents is in practice since ancient times and their use in development of drugs against fungal pathogens has minimized the chances of adverse side effects on human health.The demand for appropriate anti-fungal agents also get importance after finding of adverse action of many fungi, especially in destroying the quality, quantity, mortality and shelf life of useful crops.10).The previous findings related to this activity supported present results, antibacterial activity depends upon synergism of components in a mixture rather than a single isolated compounds, while for some bacteria one compound can be effective than extract or fraction [16].The presence of these phytochemicals can be reason behind the biological activities.The methanolic extract showed the more antioxidant potential, it may be due to combined effect of many compounds such sterols, particularly esters have the potential of scavenging free radicals.n-hexane fraction showed significant antibacterial activity.The presence of long chain fatty acids including eicosanoic acid and linoleic acid etc. are potent agent for antibacterial activity.n-hexane fraction also exhibited the higher antifungal activity.The Phlomis stewartii is a potential plant which may further be explored and studied for the bioassay guided isolation of pure compounds.

Author's contributions
them the time to complete the reaction.The UV Spectrophotometer (Shimadzu, Japan) was used and all the prepared dilutions absorbance measured at 517 nm.The decrease in absorbance confirmed the scavenging activity of sample[10,15].The following formula was used to assayed antioxidant activity of plant sample.%age scavenging of DPPH = Abs control-Abs sample/Abs control The effectiveness of sample was further checked by IC50 value obtained from slope by using formula as; IC50 = 50-b/a (a and b are obtained from slope)

Figure 7 .Figure 8 .Figure 9 .Figure 10 .
Figure 7.Comparison of scavenging activity of n-hexane fraction, methanolic extract and BHT standard Conceived and designed the experiments: S Ali, Performed the experiments: A Farooq, Analyzed the data: S Ali & H Ullah, Contributed reagents/materials/ analysis tools: A Khan, N Jahan, I Agha & RB Tareen, Wrote the paper: A Farooq & S Ali.
stewartii belongs to Lamiaceae family.It consists of rosy pink flowers; located in different areas of Pakistan especially in Suleiman and salt range.It is perennial plant with erect stems having 30-45 cm height.It consists of dense hairs and much branching at lower side.The leaves are thicktextured, aromatic, elliptic or narrow oblong, crenulate, acute at apex,length of calyx is 12mm.The corolla are rose to dusky pink with length of 2 cm, tube exerted beyond calyx, calyx is surrounded by thin bracts nearly 18 in number [8, 9].

Table 1 . Qualitative analysis for checking strength of components in n-hexane fraction and methanolic extract
+++ indicated the more abundance of respective phytochemicals, while ++ revealed moderate level of phytochemicals and + showed the lowest concentration of that phytochemical in particular sample.indicated the absence of that substance in sample