Attributes of bioactive compounds isolated from commercial brands of fenugreek ( Trigonella foneum-graecum ) in relation to organic solvent systems and their potential as antioxidants and biological activity

The present study is about potential variations of bioactive compounds from different commercial brands of fenugreek; in relation to multiple organic solvent systems, antioxidants and antimicrobial assays. Total phenolic contents, total flavonoid contents, individual phenolic acids, and DPPH assays were checked whereas phenolics analysis was performed by HPLC spectrophotometrically. Antimicrobial activity of different extracts was ascertained by disc diffusion method. The crude extract yields for different commercial brands of fenugreek ranged from 35.53-62.77 g/100g dry weight. Maximum yield was obtained from 100% Local Fenugreek with methanol while minimum was obtained from 100% local fenugreek chloroform. A considerable amount of TPC and TFC 24.442.60 mg of Gallic acid equivalents per g of sample dry weight (mg GAE/g DW) and 0.75-1.87 as mg of catechin equivalents per gram of sample dry weight (mg CE/g DW) respectively was observed. The amount of DPPH and reducing power essay was 0.927-3.78 mg/mL and 4.20-7.76 mg/mL respectively. Different brands of fenugreek had significant amount of phenolics acids ranging from 14.36-1035.44 mg/kg, where Gallic acid was found in all extracts. Antimicrobial activity was tested against microorganisms and were found to be considerable significant. These finding indicates that fenugreek has strong potential to be used as pharmaceuticals, antioxidants and preservatives.


Introduction
Lots of changes have been observed in modern civilization which, no doubt provide many significant benefits but on other hand, has brought a greater risk to human life, for instance use of chemicals, pesticides, environmental pollutants, and others cause increased level of free radical production in the body.In living organisms oxidation reactions produced by free radicals cause the oxidation of bio-molecules like lipid, DNA, RNA and protein etc [1, 2].Whereas the food issues are concerned, the shelf life of the fresh and processed food decreases by oxidative reactions.Thus, the human health is affected due to toxicity from the environmental factors and also from the preserved food stuff [3].Antioxidants have the ability to retard the damage by free radicals in biological systems as they capture or quench those free radicals that are involved in causing damage to human body and food.They are also very important in increasing the shelf life of food which is affected by oxidative reaction.Synthetic antioxidants are mostly used in food processing and pharmaceuticals industries, but they have many side effects such as liver damage and carcinogenic effect [4].So, there is need to explore and isolate natural antioxidants in high amount, which have very fewer or no side effects and can be used in medicine instead of the synthetic antioxidants [5].Plants and fruits are the most important, more safer and compatible sources of natural antioxidants.Epidemiological evidence in the literature have shown that the intake of natural products have been responsible for the lower prevalence of chronic diseases, for example, cancer and heart diseases.Natural products, in addition to the vitamins and minerals, contain phytochemicals such as flavonoids and other phenolics, which may exert therapeutic effects in human diseases [6].Many phytochemicals possessed the antioxidant activity which helps to protect the cells against the oxidative damage caused by free radicals [7].Despite, there is great interest in determining the role of phytonutrients, as the synthetic molecules have some side effects, and there is increasing trend for natural active compounds.In this scenario, beside nutritional value, functional foods have a potentially positive effect on the health [8,9].Trigonella foenum-graecum also called Fenugreek (locally known as Methi) is widely grown in Pakistan, India, Egypt, and Middle Eastern countries [10].It belongs to the family Fabaceae.It often used as a condiment in Mediterranean countries and Indian sub-continental location.Fenugreek contains a gum or mucilage which is called galactomannan which acts as a thickener or stabilizer or additive in food [11].Fenugreek (Trigonella foneum-graecum) is considered to be the oldest medicinal plant, and had been used as a tonic for treatment of edema of legs, lactation stimulant, in treatment of baldness, anti-diabetes and cholesterol reducing properties, as an antibacterial, antiinflammatory and antiulcerogenic [12].Recent research has also shown that fenugreek helps in lowering the blood glucose level and plays a significant role for treatment of diabetes.Diabetic rats treated by supplementation of fenugreek seeds showed elevation in their antioxidant potential [13].Fenugreek has a great role in pharmacological aspect that may include anti-inflammatory, antiviral, antitumor, hypertensive, anti-microbial and antioxidant activity [14].The functional constituents in natural sources are affected by varieties, cultivation, maturation, storage, and processing.The nature of extracting solvent also plays an important role in resulting extracts yields for their antioxidant activities and other important bioactive compounds [15, 16].Multiple solvents system plays a vital role in extraction of maximum bioactive compounds from selected plants it also avoids fruitless effort in future by avoiding solvent that extract minimum compound [17].As fenugreek is reported to be rich source of valuable metabolites and antioxidants such as phenolic acid and flavonoids [18], it would be interesting to evaluate the extract, by using different solvents systems, from different commercial brands of fenugreek that includes shan, national and local verity for their different antioxidant attributes.

Sample collection
Three different samples of fenugreek were collected.Two samples were marketed by shan and national while the third sample of fenugreek was bought from the local market.

Preparation of extracts
The samples were dried after cleaning, crushed into fine powder, placed separately in polythene bags and marked individually and then placed in refrigerator.Extraction of all samples was done by different solvents having different ratios e.g.100%, 80% and 50% methanol and chloroform.Ratio of sample plus solvent are kept as 1/10 (w/v).Extraction was done for 12 hours thrice by using orbital shakers.Extract was separated by the process of filtration by keeping residue left behind.These extracts were concentrated by using vacuum rotary evaporator at reduced temperature under vacuum.After that extract was kept in refrigerator at a temperature of 4 o C and then used for further analysis [19].Assessment of total phenolic contents (TPCs) Quantity of TPC was calculated using FC (Folin-Ciocalteu) reagent as described by Kalpna et al. [20].The result expressed as GAE (gallic acid equivalents) mg/g of dry weight of sample.Readings were taken in triplicate and the results were averaged.Results were reported on the basis of dry matter.

Determination of total flavonoid contents (TFCs)
TFCs were determined by the procedure followed by Zhishen et al. [21].The results were expressed as catechin equivalent (10-500 ppm) (R 2 =0.9913)CE mg/g of dry weight.Assay was performed in triplicate.

Determination of reducing power (RP)
The reducing power of CFPE was determined as reported earlier by Zhuang et al. [22] with slight modifications.In this assay, different concentrations of extracts (2-10mg/mL) were mixed with 3.5 mL phosphate buffer (0.2 M, pH 6.6) and 2 mL of 1 % potassium ferricyanide.For 20 min, the mixture was incubated at room temperature.Following the addition of 2.5 mL of 10 % TCA (trichloroacetic acid) the mixture and the mixture was centrifuged for 10 min at 3000 rpm.2.5 mL of supernatant was collected by pipette and mixed with 2.5 mL of distilled water.To this mixture 0.5 mL of 0.1 % ferric chloride was added and then absorbance of the mixture calculated at 700 nm with a spectrophotometer.

DPPH radical scavenging assay
The antioxidant activity of Trigonella foenum-graecum was assessed by DPPH (1, 1 diphenyl-2-picrylhydrazyl) radical scavenging assay described by Zhuang et al. [22].BHA that is synthetic antioxidant was used as a positive control.Calculation was made at IC50, concentration of extract that is scavenged/ neutralized 50% of DPPH free radicals.Acontrol = the absorbance of control which contain all reagents except sample Aextract = the absorbance of extracts % scavenging activity = (Acontrol -Aextract)/Acontrol * 100 The % scavenging activity at 50% is IC50 High performance liquid chromatography (HPLC) analysis Individual phenolics profile was analyzed by using HPLC.Separation of phenolic acids was carried out on HPLC using ODS (C18) reversed phase column (250 × 4.6 mm) [23].The mobile phase used was acidified acetonitrile (99.5%) at a constant flow rate of 1 mL/min in isocratic mode.With the help of a microsyringe 20-μL sample was injected into the column.The detection was carried out at 280 nm.Phenolics compounds were identified by matching their relative retention times with those of the pure standards of targeted compounds.On the basis of peak area measurement, the contents of an individual compounds were calculated.

Antibacterial activity
The Antibacterial activity of the extracts was determined by disc diffusion method.Nutrient agar medium was prepared and autoclaved.100 μL inoculums was added in medium while it was liquid and cool, mixed and then poured into petri plates.After that 6 mm wicks paper discs were laid flat on medium and 100 uL extract was put on each disc.For 24h, the petri plates were then incubated at 37C° to check the growth of bacteria.The extracts showing the antibacterial activity inhibited the bacterial growth by forming clear zone of inhibition, measured in millimeters [24].The bacterial strains that used were Bacilus Substilis, Streptococcus aureus, Salmonella typahe and E.coli.Ciprofloxacin was used as standard.[28] also reported the highest TPC content in methanol followed by chloroform.The values were observed comparatively higher as compare to the present trend.Other factors that contribute to the recovery of phenolic contents may include polarity and solubility of compounds and solvents as well.TPC that has been observed from different fenugreek brands was found to be in order of local < national < shan (Table 2).

Reducing power
The EC50 for different commercial brands of fenugreek is given in (

Phenolic acids profile
With the help of HPLC, qualitative and quantitative analysis of phenolic acid is performed.Comparison has been made between commercial brands of fenugreek and standard phenolic acids.Ten phenolic acids have been used that include gallic acid, caffic acid, chlorogenic acid, protocatechuic acid, 3-hydroxy benzoic acid, syringic acid, 4-hydroxyl benzoic acid, p-coumeric acid, 3,5 dihydroxyl benzoic acid and vanillic acid.Total phenolic acids were ranged between 18.36-1035.99.The range of gallic acid and protocatechuic acid is 10.90-96.89mg/kg and 2441-277.70mg/kg of extract respectively.Gallic acid was present in almost all the samples except few ones.The highest value of gallic acid was found in 100% methanol (shan 96.89) and lowest value was found in 50% chloroform (national 10.90).Higher amount of all present phenolic acids was found in shan 80% (275.70 mg/kg) as shown in Table 4.
These phenolic acids play a great role as antioxidant and has a great medicinal value.Gallic acid is found in a frequent amount that can easily be absorbed by human body and it has a positive influence on the canserious cell and prevent their growth.Pcoumaric acid plays a significant role in prevention of stomach cancer by inhibiting the activity of carcinogenic nitrosamines

Table 1 . Extract yields (%) of fenugreek
eValues are means ± SD (n=3) of three separate experiments.Different small letter in subscript within the same column show significant (p < 0.05) differences of means among Citrus species.Different superscript letters within the same row show significant (p < 0.05) differences of means among extraction solvents

Table 2 . Total phenolic and flavonoid contents of extracts from fenugreek
BValues are means ± SD (n=3) of three separate experiments.Different small letter in subscript within the same column show significant (p < 0.05) differences of means among Citrus species.Different superscript letters within the same row show significant (p < 0.05) differences of means among extraction solvents.

Table 3 . DPPH radical scavenging activity and reducing power
AValues are means ± SD (n=3) of three separate experiments.Different small letter in subscript within the same column show significant (p < 0.05) differences of means among Citrus species.Different superscript letters within the same row show significant (p < 0.05) differences of means among extraction solvents

Table 5 . Antibacterial activity of different commercial brands of fenugreek (Trigonella foenum graecum) against Escherichia coli and Bacillus subtilis Solvents Zone of inhibition (mm) against Escherichia coli Zone of inhibition (mm) against Bacillus subtilis
Values are means ± SD (n=3) of three separate experiments.Different small letter in subscript within the same column show significant (p < 0.05) differences of means among Citrus species.Different superscript letters within the same row show significant (p < 0.05) differences of means among extraction solvents

Table 6 . Antibacterial activity of different commercial brands of fenugreek (Trigonella foenum-graecum) against Salmonella tyaphimurium and Staphlococus aureus Solvents Zone of inhibition (mm) against Salmonella tyaphimurium Zone of inhibition (mm) against
Values are means ± SD (n=3) of three separate experiments.Different small letter in subscript within the same column show significant (p < 0.05) differences of means among Citrus species.Different superscript letters within the same row show significant (p < 0.05) differences of means among extraction solvents Conceived and designed the experiments: T Mehmood, Performed the Experiments: M Ahmed & Z Jabeen, Analyzed the Data: T Mehmood, A Karim, MA Shaheen & F Siddique, Contributed reagents/ materials/ analysis tools: T Mehmood .Wrote the paper: T Mehmood & F Siddique.