Nutritional investigation and biological activities of Parthenium hysterophorus

Parthenium hysterophorus is used as a vegetable and in the treatment of various human ailments traditionally in Pakistan. The current study was arranged to investigate the nutritional importance, microbial inhibition and its potency against free radicals. The results revealed that extract possesses macro elements Ca, K, Na, Mg, Fe, Zn, as well as micro elements including Ni, Li, Pb, Co, Mn, Cu, Cr, and Cd. Scavenging of free radicals revealed that methanolic extract of P. hysterophorus (PM) possesses lowest IC50 (2.5 ± 0.05 μg/ml) comparatively to ascorbic acid (3.7 ± 0.03) and n-hexane extract of P. hysterophorus (PH) (5.1 ± 0.07 μg/ml). The extracts also showed maximum antibacterial potential in various concentrations of n-hexane and methanolic extract of P. hysterophorus. The results revealed that P. hysterophorus contain a remarkable fungal inhibition due to the presence of bioactive constituents. Based on this screening various fractions of P. hysterophorus are recommended for future bioassay guided isolation of bioactive constituents.


INTRODUCTION
Plants possess a number of bioactive compounds which are used as medicine or in preparation of new drugs.Nowadays 30% of worldwide drugs are based on natural products isolated from medicinal plants (Grabley and Thiericke, 1999;Sahreen et al., 2010;Sahreen et al., 2011a, b).Many efforts have been made to extract new antimicrobial bioactive compounds from various kinds of sources.One of such resource is fractions of folk medicinal plants.In ancient times people used spices and herbs in their food as flavoring agents (Beuchat, 1994;Cutler, 1995;Khan et al., 2009;Khan et al., 2011).These can be used locally as food preservatives and in folk medicine also.Many herbs and spices also possessed free radical scavenging, antioxidant and antimicrobial activities like bactericidal and bacteriostatic (Khan et al., *Corresponding author.E-mail: Rahmatgul_81@yahoo.com.Tel: +92 51 90643086.Fax: +92 51 9205753. 2010a, b).Today, worldwide, many medicinal plants, spices and herbs, exert antimicrobial activities.A large number of these spices and herbs are in regularly used as alternative medicines (Lis-Balchin et al., 1996).Reactive oxygen species (ROS) are major free radicals generated in many redox processes, which may induce oxidative damage to biomolecules, including carbohydrates, proteins, lipids and DNA.Reactive oxygen species affect living cells, which mediate the pathogenesis of many chronic diseases, such as atherosclerosis, Parkinson's disease, Alzheimer's disease, stroke, arthritis, chronic inflammatory diseases, cancers, and other degenerative diseases (Halliwell and Grootveld, 1987;McDermott, 2000).The action of ROS is opposed by a balanced system of antioxidant compounds produced in vivo (Halliwell and Gutteridge, 1999;Khan et al., 2011a, b).Endogenous antioxidants are insufficient, and dietary antioxidants are required to countermeasure excess ROS (Lim and Murtijaya, 2007).Significant attention has been paid to antioxidants in food as well as additives to cosmetic and pharmaceutical products.
Parthenium hysterophorus L. (Asteraceae) is a common weed distributed worldwide.It´s decoction has been used in traditional medicine to treat fever, diarrhoea, neurologic disorders, urinary infections, dysentery and malaria and as emmenagogue (Ramos et al., 2001;Kalsi et al., 1995;Surib-Fakim et al., 1996).The present study was therefore undertaken to investigate the free radical scavenging and antimicrobial efficacy of P. hysterophorus.

Biochemical analysis of plants
Pharmacological activities of plants are rendered by the occurrence of bioactive compounds.P. hysterophorus is medicinally used in the treatment of different disorders as folk medicines and also as pot herb.On account of ethno pharmacological use of these plants, evaluation for the biochemical composition is utmost important.

Plant collection
Collection of P. hysterophorus was carried out from Bannu (Pakistan) in June and September 2010.The identification of the plants was carried out at Herbarium of botany Department University of Science and Technology Bannu, KPK, Pakistan and a specimen was submitted vide voucher R-97 (P.hysterophorus).P. hysterophorus were shade dried at 25°C for two weeks, chopped and powdered.

Extract preparation
2 kg powder of P. hysterophorus was extracted two times in 5 L of methanol, after a week of socking, filtration were conducted through Whatmann filter paper # 45 and evaporated via rotary evaporator to obtain methanolic crude extract (PM).The extract was further fractionated with n-hexane (PH).Both fractions were stored further in phytochemical and in vitro investigations at 4°C.

Elemental analysis
100 ppm stock solution of the magnesium (Mg), calcium (Ca), potassium (K), ferrous (Fe), sodium (Na), cobalt (Co), manganese (Mn), copper (Cu), chromium (Cr), zinc (Zn), nickel (Ni), lithium (Li), lead (Pb), and cadmium (Cd) were formed by mixing required quantity of salts in distilled water for elemental analysis of P. hysterophorus in whole plant powders.Perchloric acid digestion method was used for elemental analysis (Allen, 1974).0.25 g powder was immersed in 6.5 ml of mixed acid solution, that is nitric acid, sulfuric acid and perchloric acid (5:1:0.1)and digested in a flask (50 ml) in fume hood on hot plate till the digestion was completed which was indicated by white fumes coming out from the flasks.Digested samples were allowed to cool and transferred in 50 ml volumetric flask, by rising volume with distilled water.Filtrate (Whatmann No. 42) was collected and concentration of each element was determined on Shimadzu AA-670 atomic absorption spectrophotometer.Quantity of each element was calculated by using the formula as follows: Nutrient cation in plants = (ppm in extract -blank) × A/W × dilution factor where A is the total volume of extract (ml) and W is the weight of dry plant.

Nutritional investigation of P. hysterophorus
Estimation of leaf protein Lowry et al. (1951) method was used for determination of leaf protein content.Monobasic sodium phosphate (16 ml) and dibasic sodium phosphate (84 ml) was mixed to get the desired pH (7.5) of phosphate buffer.More so, the following reagents were used: 1. Reagent A: 2 g sodium carbonate (Na2CO3), 0.4 g NaOH (0.1 N) and 1 g Na-K tartrate was dissolved in100 ml of distilled water.2. Reagent B: CuSO4.5H2O(0.5 g) was dissolved in 100 ml of distilled water.3. Reagent C: Solution A (50 ml) and Solution B (1 ml) were mixed.4. Reagent D: Folin phenol reagent was diluted with distilled water in 1:1 ratio.Fresh leaves 0.1 g was homogenized in 1 ml of buffer solution (phosphate buffer pH 7.5).The homogenate was centrifuged for 10 min (3000 rpm).The supernatant (0.1 ml) was poured in the test tubes and finally 1 ml volume was made by distilled water.Reagent C (1 ml) was added to it.0.1 ml of reagent D was added after shaking for 10 min.The absorbance of each sample was recorded at 650 nm after 30 min of incubation and protein content was calculated with help of standard bovine serum albumin (BSA).

Sugar estimation
Sugar was estimated using fresh plant materials, homogenized and treated with concentrated sulphuric acid.The incubation of sample was carried out for 4 h at 25°C.Optical density (OD) of each sample was measured at 420 nm.The concentration of glucose in both fractions was calculated using standard curve of glucose.

DPPH radical scavenging activity
Procedure of Gyamfi et al. (1999) was used for determination of DPPH scavenging capacity of various fractions.2.4 mg DPPH were dissolved in 100 ml methanol to prepare stock solution.The stock solution was further diluted with methanol until attaining an absorbance less than 1.00 using the spectrophotometer at 517 nm. 3 ml solution was mixed with 100 µl sample solution (1 to 100 µg/ml) and measured absorbance at 517 nm.% inhibition was calculated as: Scavenging effect (%) = [(OD of control -OD of sample) / (OD of control)] ×100 While IC50 value was obtained by using graph prism pad software.

Antibacterial assay
The antibacterial potency of various fractions of P. hysterophorus was carried out through protocol (Bagamboula et al., 2003).
Preparation of samples: 100 mg of each fraction was dissolved in DMSO (10 ml) to prepare stock solution.Media for bacteria: It was composed of peptone (5 g/L) and meat extract (3 g/L), while nutrient agar medium (MERCK) was composed of peptone (5 g/L), meat extract (3 g/L) and agar-agar (12 g/L).Nutrient broth medium was prepared by dissolving 0.8 g/100 ml nutrient broth in distilled water, while nutrient agar medium by dissolving 2 g of nutrient agar in 100 ml of distilled water (pH 7.0) and was autoclaved.
procedure (agar diffusion method): 2 g nutrient agar was dissolved in 100 ml distilled water (pH 7.0) and autoclaved.It was allowed to cool up to 45°C.Then, it was seeded with 1 ml of prepared inoculums to have 10 6 CFU/ml.14 cm Petri plate was used.Eleven wells per plate were made with sterile cork borer (8 mm).Using micropipette, 100 µl of test solutions were poured in to respective wells.Eight samples of two solutions for positive control (Roxithromycin (Rox) 1 mg/ml and Cefixime-USP (Cefix) 1 mg/ml, one for each) and one for negative control (DMSO) were applied to each petri plate.Finally, the petri plates were incubated for 24 h at 37°C.Antibacterial activities were measured as a mean of 3 replicates.
Determination of total chlorophylls: Methanolic solutions of plant extracts of the appropriate concentration (1.0 to 4.0 mg/ml) were analyzed through UV/VIS spectrophotometer at 470, 653 and 666 nm.The concentrations of chlorophylls α and b were determined according to the equations reported by Lichtenthaler and Wellburn (1985)

Statistical analysis
Statistical Processor System Support (SPSS) 13 was used for determination of Mean ± SE using replicate data of various experiments.

Elemental and nutritional analysis
Mineral analysis of P. hysterophorus showed that potassium, calcium, magnesium, sodium, iron, zinc, copper, molybdenum, lead, lithium, nickel, cadmium, chromium and manganese contents were found as presented in Table 1.The digestion method revealed that P. hysterophorus possessed highest concentration of macro elements in µg/g of Ca (75.14 ± 0.45), K (29.09 ± 0.21), Na (11.37 ± 0.14), Mg (6.161 ± 0.31), Fe (7.36 ± 0.01) and Zn (4.63 ± 0.009), as well as micro elements including Ni (0.056 ± 0.0005), Li (0.081 ± 0.004), Pb (0.45 ± 0.002), Co (0.69 ± 0.003), Mn (0.089 ± 0.003), Cu (0.096 ± 0.002), Cr (0.098 ± 0.002) and Cd (0.54 ± 0.005) dry powder.The nutritional analysis of P. hysterophorus revealed the presence of protein (5.15 ± 0.32 mg/g weight) and sugar (2.07 ± 0.04 mg/g weight), respectively (Figure 1).Wild growing leafy vegetables play crucial role as an integral component in the diet of inhabitants of different parts of the world.The proximate analysis of P. hysterophorus shows that their leaves are a good source of metallic elements, protein and sugar.Due to the presence of these constituents, consumers are more benefited when they use them as a substitute of leafy vegetable in various food preparations.These inorganic elemental constituents have profound effects in the maintenance of normal glucose-tolerance and in the release of insulin from β-cells islets of Langerhans through amelioration of oxidative stress (Choudhary and Bandyopadhyay, 1999;Khan et al., 2010c).Similar investigation was reported by Tadhani and Subhash (2006) during the preliminary studies on Stevia rebaudiana leaves for phytochemical and mineral screening.Study of Garg et al. (2009) reported the presence of the same metallic elements in brahmi (Bacopa monnieri) extract comparable to our results.

DPPH free radical scavenging antioxidant assay
Oxidation is a necessary process of living organisms for energy production; however, during normal metabolism oxygen consumption, through many enzymatic systems, reactive free radicals (RFR) are produced.In small amounts, these ROS are beneficial in signal transduction and growth regulation.However, large amount of ROS produce oxidative stress, attack many molecules, such as protein, DNA and lipids (Halliwell and Gutteridge, 1999;Ahmad et al., 2011).1,1-diphenyl 1-2-picryl-hydrazyl (DPPH) a free radical possess the ability to steal electron from antioxidant, therefore, it is widely used for estimation of in vitro antioxidant scavenging activities of medicinal plants.Figure 2 shows the scavenging activity of different fractions of P. hysterophorus for free radicals of DPPH.
Lowest IC 50 has been recorded with PM (2.5 ± 0.05 µg/ml) and compared to ascorbic acid (3.7 ± 0.03) and PH (5.1 ± 0.07 µg/ml).Our result shows similarity with the investigation of Hagerman et al. (1998) andFalleh et al. (2008) who have reported that medicinal plants markedly scavenge free radicals.The antioxidant potential of various fractions of both fractions could be due to the presence of plant bioactive phenolic and polyphenolic compounds which significantly reduce the free radicals which cause oxidative stress.

Antibacterial assay (MIC) and antifungal assay
Due to emergence of antibiotic resistant strains as well as side effects of synthetic drugs, investigation of potent antimicrobial drugs obtained from natural resources has been an objective of researchers and investigators.The minimum inhibitory concentrations (MIC) of both fractions of P. hysterophorus were evaluated for antibacterial activity and are tabulated in Table 2. Growth of the Gram negative bacteria, such as S. aureus was inhibited with a MIC value of 1.5 mg/ml for PM and 3.8 mg/ml for PH.MIC for E. coli was PM (2.5 mg/ml), PH (5 mg/ml); however, MIC for K. pneumoniae growth was 1 mg/ml for PM, 5 mg/ml for PH.In case of Gram positive bacteria the observed MIC was PM (5 mg/ml), PH (2.5 mg/ml) against M. luteus, while B. subtilis was inhibited by 3 mg/ml of PM, 4.5 mg/ml PH.PM and PH inhibited the growth of E. aerogenes with MIC is 2.3 and 3.6 mg/ml, respectively.Data of the antifungal assay are shown in Table 3.Growth of A. niger was inhibited more than 50% by the PM (56.0 ± 2.0%), while, PH showed less than 25% inhibition (12.6 ± 0.04%).In case of F. solani PM (70.7 ± 8.0%) and PH showed no inhibition of the growth of the said fungus. A. flavus was inhibited by liquid-phase

Conclusion
Methanolic plant extracts of P. hysterophorus showed significant antimicrobial and antioxidant activity.This might be due to the presence of bioactive elements and polyphenolic constituents present in the extract.Based on the significant results obtained in this study, further work is in progress towards isolation and purification.

Table 2 .
Effects of various fractions of P. hysterophorus on MIC.