Antioxidant and antibacterial activity of Xanthuim strumarium L

N. B. Sridharamurthy¹* and R. Yogananda²

¹Department of Pharmacology, Dayanandsagar College of Pharmacy, Bangalore, India.

²Department of Pharmaceutics, SJM college of Pharmacy, Chitradurga, India.

ABSTRACT:

The main objective of this study is to evaluate the antibacterial and antioxidant activity of ethanol extract of Xanthium strumarium L. Disc diffusion and broth serial dilution tests were used to determine the antibacterial activity of the ethanol extract on two Gram-positive strains (Bacillus subtilus NCIM 2718, Staphylococcus aureus ATCC25923) and three Gram-negative bacterial strains (Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniea ATCC 70063 and Eschrichia coli ATCC 25922). The ethanol extract of the root of plant was subjected to preliminary phytochemical analysis. Free radical scavenging activity of the ethanol extract at different concentration was determined with 1, 1-diphenyl-2 picrylhydrazyl (DPPH), superoxide free radical free radical scavenging activity and lipid peroxide inhibition activity. Ethanol extract showed inhibition in all the organisms under test. The result from the present study shows that the ethanol extract of X. strumarium has antibacterial activity. The antibacterial activity may be attributed to the presence of tannins, flavonoids and triterpinoids and phenolic acids in the ethanol extract. The antibacterial and antioxidant activity exhibited by the ethanol extract can be corroborated to the usage of this plant in Indian folk medicine.

KEYWORDS: Antibacterial, DPPH, Superoxide, Lipid peroxide and Xanthium strumarium

INTRODUCTION:

There are so many plants on the earth are still under investigation one such plant belongs to the genus Xanthium which consists of more than 25 species which are distributed in the several parts of the world and they represent untapped natural resources for man to exploit. Traditionally, many species of this genus have been exploited for various ailments from decades.

The plant X. strumarium has been widely reported to have several medicinal properties in traditional form of medicine. The beneficial properties are diuretic, astringent, sedative, demulcent, diaphoretic, analgesic, sialagogue, styptic, sudorific, anodyne, antibacterial, antifungal, antispasmodic, bactericide, bitter, depressant, hemostat, laxative, refrigerant, and antirheumatic¹.

There are few reports on the systematic studies on the plant Xanthium strumariumL. Most of the studies carried out by earlier workers were on the phytochemical characterization of X. strumanium and evaluation of few pharmacological properties.

In 1993 Aguta et al have reported 1, 3, 5-tri-0-caffeoylquinicacid from X. strumarium². Malik et al., (1993) reported xanthanolides from X. strumarium³. Alberto et al., (1993) reported xanthanolide from Xanthium: Absolute configuration of xanthanol, isoxanthanol and their C-4 epimers⁴.

Ma et al., (1998) isolated a new thiazinedione from X. strumarium⁵. Han et al., (2006) reported new thiazinediones and other components from X. strumarium⁶.

There are very few reports on the pharmacological activities of the plant. Jawad et al., (1998) reported anti microbial activity of the extracts while studying the antimicrobial activity of some of the medicinal plants⁷. Murillo-Alvarez et al., (2001) reported the positive response with reference to antimicrobial and cytotoxic activity of X. strumarium⁸. Very recent reports of Sherer et al., (2009) suggest the antimicrobial activity⁹.

Talakal et al., (1995) reported in-vitro and in-vivo anti-trypanosomal activity of X. strumarium leaves¹⁰. The nematocidal activity of extracts of X. strumarium has been observed by Malik et al., (1987)¹¹. The anti-malarial activity of the same plant has been shown by Joshi et al., (1997)¹².

MATERIALS AND METHODS:

Collection and authentication of plant material:

The roots of Xanthium strumanium L. were collected from uncultivated lands of Chitradurga District, Karnataka state, India. The taxonomic authentication of the plant was done

Preparation of extracts:

Various extracts of the plant material were prepared by successive solvent extraction method as described below.

The powdered material of roots of Xanthium strumanium L was refluxed successively with the solvents petroleum ether (40⁰-60⁰, E- Merck Mumbai, India), Chloroform (50⁰– 70⁰, E-Merck Mumbai, India) and Ethanol (E- Merck Mumbai, India) in a soxhlet extractor for 48 hrs in batches of 350g each. Every time, before extracting with the next solvent the marc was dried. After extraction with ethanol lastly, marc was kept in closed jar in distilled water for 48 hrs with occasional shaking, then distilled water obtained by pressing the marc with tincture press.

All the extracts were concentrated in vacuum using rotary flash evaporator (Buchi-Flawil, Switzerland). The solvents were removed completely over the water bath and finally desiccator dried. The extracts so obtained from each of the solvents were labeled, weighed and the yield was calculated in terms of grams percent of the weight of the powdered roots.

Microorganisms:

The antibacterial activity of ethanol extract was determined by individually on Gram-positive and Gram-negative bacterial strains obtained from NCIM (National Collection of Industrial Microorganisms, National Chemical Laboratory), Pune. The Gram-positive organisms used were Bacillus subtilus (NCIM 2341) and Staphylococcus aureus (ATCC 25924).Gram-negative strains used was Pseudomonas aeruginosa (ATCC27867), Klebsiella pneuminiae (ATCC 70068) and Escherichia coli (ATCC25929). All the strains were maintained on nutrient agar at 4⁰C and were sub cultured every month.

Preliminary phytochemical tests:

To detect the presence of possible phytochemicals in ethanol extract, preliminary phytochemical tests^8,13 were performed. (1) Test for alkaloids (1ml extract+ 1% hydrochloric acid on steam bath, 1ml filtrate + 6drops of Mayer’s reagent, appearance of cream white precipitate indicated the presence of alkaloids. (2) Test for tannins (1ml extract + few drops of 10% lead acetate), appearance of precipitate indicated the presence of tannins. (3) Test for saponins (1ml of extract + 9ml distilled water, shaken vigorously), appearance of stable froth indicated the presence of saponins. (4) Test for steroids and triterpinoids (Liebermann-Burchard) (2ml extract + 1ml chloroform+ few drops of acetic anhydride + conc. sulfuric acid added along the side of test tube), appearance of blue or green color indicated the presence steroids, and appearance of red, brown color indicates the presence of triterpinoids, (5) Test for cardiac glycosides (1ml of extract + few drops of ferric chloride + 3-4 drops of conc. Sulfuric acid), appearance of blue-green color indicated the presence of glycosides. Test for flavonoids (2ml extract + conc. Hydrochloric acid+ magnesium ribbon), appearance of pink-red color indicated the presence of flavonoids.

Antibacterial assay:

The antibacterial activity of ethanol extract was determined by disc diffusion and broth dilution method^9,14. Nutrient Agar (NA) and Muller Hinton broth (MHB) were used for the tests. Overnight cultures were grown at 37⁰C in MHB. Bacterial suspensions of 1.0% X 10⁸ colony-forming units (CFU) per ml were obtained (OD₆₀₀=0.08nm). Petri plates containing 20ml of (NA) were used for the disc diffusion assay. A total of 200μl of the bacterial culture (1CFU) was spread over the surface of the plate and was allowed to dry for 10 min. The filter paper discs (6mm in diameter) were loaded with ethanol extract (5mg/disc) and were allowed to dry completely. Disc with 10μl DMSO and gentamicin (10μg/disc) were placed as controls. The plates were incubated overnight at 37⁰C. The antibacterial activity against each test organism was qualified by determining by average diameter of the zone of inhibition around the paper discs in millimeters. The tests were performed twice and average diameters of zones were calculated.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC):

MIC of ethanol extract was determined by serial dilution method. A total of 500μl of MHB was added to tubes. Stock solution of 50mg/ml of ethanol extract was subjected to two fold dilutions such that concentration ranged between 50 mg/ml and 0.0241mg/ml. Again, 10μl of 10⁶ CFU bacterial suspensions were added to the tubes. The tubes were incubated at 37⁰C for 24hrs. MIC was taken as the highest dilution of the extract that inhibited the growth of the bacteria. Lowest concentration of the ethanol extract, which inhibited the bacterial growth after a period of 24hrs of incubation at 37⁰C, was recorded as MIC. Minimum bactericidal concentration (MBC) was determined by sub culturing 10μl of the MIC tube solution (showing no visible growth) on fresh drug free MHA plate and incubating for 24hrs at 37⁰C. The highest dilution that yielded no bacterial growth was taken as MBC¹⁴.

Evaluation of in vitro antioxidant activities of the extracts of Xanthium strumarium L.

Free-radical scavenging activity of all extracts at different concentrations was tested in three in vitro models.

· 1, 1-Diphenyl 2-Picryl Hydrazyl (DPPH) radical scavenging activity

· Super oxide anion scavenging activity

· Lipid peroxidation inhibition activity

Preparation of Stock Solutions of extracts:

The stock solutions of extracts prepared by dissolving 100 mg of dried extracts in 100 ml of methanol to make a stock solution of 1 mg/ml. Aliquots from this stock solution were further diluted with methanol to get the final concentrations viz. 20, 40, 60, 80, 100 and 120 mg/ml.

Estimation of 1, 1-Diphenyl 2-Picryl Hydrazyl (DPPH) radical scavenging activity:

DPPH:

Stock solution of DPPH was prepared by dissolving 32.5 mg in 10 ml of methanol and then the total volume was made up to 25 ml with methanol in volumetric flask.

Test solutions:

Samples of various concentrations were prepared by dissolving in methanol as described above.

Preparation of Ascorbic Acid Stock Solution:

Ascorbic acid used as a standard for the study. Ascorbic acid stock solution was prepared in the concentration of 1000 mg/ml in water. It was prepared freshly and used immediately for the study to avoid the spontaneous decomposition of ascorbic acid in water solution. From the stock solution different concentrations viz. 10, 20, 40, 60, 80, 100 and 120 mg/ml were prepared in water and used for antioxidant studies.

DPPH free radical scavenging activity

Free scavenging activity was measured by a decrease in absorbance at 516 nm of a methanol solution of colored DPPH brought about by the sample¹⁵. A stock solution of DPPH (1.3 mg/ml in methanol) was prepared such that 75 ml of it in 3 ml methanol gave an initial absorbance of 0.9. Decrease in the absorbance in the presence of sample extract at different concentrations was noted after 15 min. EC₅₀ (i.e. the concentration of the test solution required to give a 50% decrease in the absorbance compared to that of blank solution) was calculated from percent inhibition. A blank reading was obtained using methanol instead of the extract. Ascorbic acid was used as standard. The percentage inhibition of antiradical activity was calculated using the formula,

Absorbance of blank – Absorbance of test sample

% inhibition = _____ X 100

Absorbance of blank

Estimation of Superoxide Anion scavenging activity in the NADH/PMS/NBT System:

The superoxide anion scavenging activity of extracts were determined by the method described¹⁶ by Nishimiki et al., slightly modified. About 1 ml NBT solution containing 156 μM NBT dissolved in 1.0 ml 100 mM phosphate buffer, pH 7.4, 1 ml NADH solution containing 468 μM NADH dissolved in 1 ml 100 mM phosphate buffer, pH 7.4, and 0.1 ml of various concentration of test samples and reference compound (20, 40, 60, 80, 100 and 120 μg) were mixed and the reaction was started by adding 100 μl phenazine methosulfate solution containing 60 μM phenazine methosulfate in 100 mM phosphate buffer, pH 7.4. The reaction mixture was incubated at 25ºC for 5 min and absorbance at 560 nm was measured against control samples. BHT was used as reference compound. Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. All tests were performed in triplicate. The capability of scavenging the superoxide anion radicals was calculated using the following equation,

Where, A₀ is the absorbance of the control (without test samples); A₁ is the absorbance of test samples.

Estimation of lipid peroxidation:

Stock TBA-TCA-HCl reagent: 15% w/v trichloroacetic acid, 0.375% w/v thiobarbituric acid and 0.25N hydrochloric acid. This solution was mildly heated to assist the dissolution of TBA.

1.15% KCl: 1.15 g KCl was dissolved in 20 ml of distilled water and volume was made up to 100 ml with distilled water in volumetric flask. This solution is kept in refrigerator.

Preparation of rat brain homogenate: Albino rats (180-200 g) of either sex were used for the study. After decapitation, the brain was removed carefully. The tissue was immediately weighed and homogenate with cold 1.15% KCl to make 10% homogenate. This homogenate was immediately used foe the in vitro lipid peroxidation study.

Lipid Peroxidation Assay:

Lipid peroxidation was quantified by the method of determination of thiobarbituric acid-reactive substances (TBARS). TBARS were determined by a method described by John and Steven¹⁷.

The 0.5 ml of brain homogenate was added to 1 ml of various concentrations of the test compound. The mixture was incubated for 30 min. Peroxidation was terminated by the addition of 2 ml TBA–TCA–HCl reagent. The solution was heated for 15 min in a boiling water bath and then cooled. The flocculent precipitate obtained after cooling was removed by centrifugation at 1000 rpm for 10 min. The intensity of the pink colored complex was measured at 535 nm in a UV-vis Spectrophotometer (Shimadzu UV-2450). The experiment was performed in triplicate. The capability of scavenging the free radicals was calculated using the following equation,

RESULTS:

The phytochemical analysis of the ethanol extract performed in the present study showed the presence of alkaloids, triterpinoids, tannins, cardiac glycosides and flavonoids. The antibacterial activity of ethanol extract of X. strumarium assayed by disc diffusion method is almost near to the standard antibiotic gentamycin (Fig-1). The MIC values for E. coli and B. subtilus were found to be 6.25mg/ml and 1.36mg/ml, respectively. The ethanol MBC values for E. coli and B. subtilus were found to be 15mg/ml and 22.5mg/ml, respectively. The ethanol extract of the root of the plant exhibited the increasing scavenging activity at different concentrations. The scavenging activity was seen to increase gradually with increase in concentration. The scavenging activity was almost nearer to scavenging activity of ascorbic acid as shown in Table-1, Fig-2; Table-2, Fig-3; Table-3, Fig-4.

Figure 1: Comparison of antibacterial activities of ethanol extracts of X. strumarium with the standard antibiotic Gentamicin. Zone of inhibition was measured after 24h of incubation for ethanol extract and plotted against bacterial strains

In-vitro antioxidant property:

Phytochemical investigations conducted both by quantitative and qualitative methods showed that all the extracts of the root of X. strumarium contain variable concentrations of polyphenols. Hence it is necessary to carry out the antioxidant property for all the four extracts of X. strumarium.

All the extracts of the root of X. strumarium were subjected for free radical scavenging activity by the following three methods.

a) 1,1-Diphenyl 2 picryl Hydrazyl (DPPH) radical scavenging activity

b) Superoxide anion scavenging activity

c) Lipid peroxidation inhibition activity

1, 1-Diphenyl 2 picryl Hydrazyl (DPPH) radical scavenging activity:

The free radical scavenging activity of various extracts is expressed in terms of percentage inhibition. The decrease in percentage of inhibition shows increased absorbance. The decrease in optical absorbance at 517nm after addition of the test compounds is measured. The percentage of DPPH radical scavenged for various extracts ranges from 22.13% (Aqueous extracts) to the maximum of 85.67% as in ethanol extract. The ethanol extract exhibited a significant dose dependent inhibition of DPPH activity, with a 50% inhibition (IC 50) at a concentration of 50µg. The results of other extracts are given in the Table-4. The IC 50 values of ethanol extract was found to be far more than that of pet-ether extract and aqueous extracts. The IC 50 value of ethanol extract was found to be nearer to the IC50 value of standard Ascorbic acid. The free radical scavenging is maximum with ascorbic acid >alcohol>chloroform>pet-ether>aqueous which is given in Table-1, Fig -2 .

Fig-2: DPPH free radical scavenging activity

Superoxide anion scavenging activity:

The super oxide anion scavenging activity of the crude extracts of the root of X. strumarium were determined by NBT system. The ethanol extract showed the significant scavenging of the super oxide anion when compare to other extracts. The ethanol extract was found to possess good scavenging activity on super oxide anion at all concentrations under test. Ethanol extract at concentrations range from 10-100µg/ml inhibited the production of super oxide anion radical by 24.43% to 86.53% Table-4. On the other hand the standard Butylated hydroxyl toluene showed significant scavenging activity in a dose dependent manner. The greatest scavenging activity was observed with BHT which effectively depressed the formation of super oxide anion. The maximum inhibition of super oxide anion was observed at 100µg/ml concentration and is 94.57% (Table-2). The super oxide scavenging activity is least with aqueous extract which is 19.36% and that of pet-ether and chloroform extract is 29.78% and 41.20% respectively. The data is graphically represented in Fig-3.

Fig-3: Superoxide anion free radical scavenging activity

Lipid peroxidation inhibition activity:

The four extracts of the root of X. strumarium were subjected to lipid peroxidation inhibition activity against non-enzymatic in vitro lipid peroxidation in rat brain by method of determination of thiobarbituric acid reactive substances (TBARS). The malondialdehyde formed as a result of lipid peroxidation induced by ferric chloride which reacts with thiobarbituric acid releasing pink chromogen that indicates the extent of lipid peroxidation. Inhibition of pink chromogen formed indicates inhibition of lipid peroxidation. The ethanol extract under test showed the low absorbance values which indicates the highest level of antioxidant activity. It showed the ability of free radical inhibition activity in a dose dependent manner and maximum being 84.30% at 100µg/ml concentration. The absorbance is lesser than the other extracts (Table-3 and Fig-4). The inhibitory activity of other extracts such as pet-ether, chloroform, aqueous and BHT are 24.75%, 42.17%, 21.37% and 91.90% respectively. Interestingly, the activity exhibited by ethanol extract is almost very close to the standard BHT (Fig-4).

Fig-4: Lipid peroxidation inhibition activity

DISCUSSION:

In the present study, crude ethanol extract isolated from X. strumarium exhibited both antibacterial and antioxidant activities. The study revealed that ethanol extract of X. strumarium is bactericidal against all pathogens under test which may be due to the presence of phenolic compounds and tannins in the extract which is shown in the phytochemical investigation of the extract. The antioxidant property of the extracts may due their hydrogen donating ability¹⁸. This reveals that the extract has antiradical action and serves as free radical inhibitor or scavenger. Fenglina and co-workers reported that 56 plant extracts has antiradical activity out of 300 selected Chinese medicinal plants¹⁹. These extracts contained tannins, flavonoids and these plants have been used traditionally in the treatment of bleeding, dysentery, wounds and skin infections. The selected plant X. strumarium is used for the treatment of dysentery, wound healing and antibacterial in traditional practice as given in the introduction. As per the review of literature in 1993 Aguta et al have reported 1, 3, 5-tri-0-caffeoylquinicacid from X. strumarium², Jawad et al., reported anti microbial activity of the extracts⁷, Very recent reports of Scherer et al., suggest the antimicrobial activity⁹. Based on the presence of phytochemicals, free radical scavenging property of the extract and the reports from the review of literature clearly indicates that the ethanol extract X. strumarium definitely has antibacterial property and can be used in the treatment of wound healing and burns as described in traditional medicinal practice.

Tannins are known for their astringent property and antimicrobial activity²⁰. It is explained that in the wound healing process, the tannins bind to proteins of exposed tissues, thus precipitating proteins and forms antiseptic protective coat enabling the regeneration of new tissues to take place^{21, 22}. It is well known that tannins and flavonoids are also responsible for the strong free radical scavenging activity and anti-inflammatory property^{23, 24}. Free radical scavengers can inhibit the process of inflammatory response ²³. In a recent review article, cellular mechanisms for anti-inflammatory activity of flavonoids have been explained²⁵. Flavonoids possess anti-oxidative, free radical scavenging activities and regulate cellular activities of the inflammatory cells like- Mast cells, macrophages, lymphocytes and neutrophils. For instance, some flavonoids inhibit histamine release from mast cells and others inhibit-cell proliferation. In addition, certain flavonoids modulate metabolizing enzymes such as phospholipase A₂ (PLA2), cycloxygenase (COX), lipoxygense (LOX) and the nitric oxide (NOS). An inhibition of these enzymes by flavonoids reduces the production of inflammatory mediators like prostaglandins (PG), leukotrienes (LT), and NO. Thus, the inhibition of these enzymes by flavonoids is definitely one of the important cellular mechanisms of anti-inflammation²⁵. Triterpinoids also have anti-inflammatory, anticancer and antioxidant activities²⁶. They are well known to promote the wound healing process mainly due to their astringent and antimicrobial property, which seems to be responsible for wound contraction and increased rate of epithelialization²⁷. Thus, the outcome the present work definitely supports traditional use of the plant.

CONCLUSION:

In the present study, crude ethanol extract isolated from X. strumarium exhibited both antibacterial and antioxidant activities. The study revealed that ethanol extract of X. strumarium is bactericidal against all pathogens under test which may be due to the presence of phenolic compounds and tannins in the extract. The antioxidant property of the extracts may due to their hydrogen donating ability. It is well known that tannins and flavonoids are also responsible for the strong free radical scavenging activity and anti-inflammatory property. Free radical scavengers can inhibit the process of inflammatory response. In a recent review article, cellular mechanisms for anti-inflammatory activity of flavonoids have been explained²⁵. Flavonoids possess anti-oxidative, free radical scavenging activities and regulate cellular activities of the inflammatory cells like- Mast cells, macrophages, lymphocytes and neutrophils. For instance, some flavonoids inhibit histamine release from mast cells and others inhibit-cell proliferation. In addition, certain flavonoids modulate metabolizing enzymes such as phospholipase A₂ (PLA2), cycloxygenase (COX), lipoxygense (LOX) and the nitric oxide (NOS). An inhibition of these enzymes by flavonoids reduces the production of inflammatory mediators like prostaglandins (PG), leukotrienes (LT), and NO. Thus, the inhibition of these enzymes by flavonoids is definitely one of the important cellular mechanisms of anti-inflammation²⁵. Triterpinoids also have anti-inflammatory, anticancer and antioxidant activities²⁶. They are well known to promote the wound healing process mainly due to their astringent and antimicrobial property, which seems to be responsible for wound contraction and increased rate of epithelialization²⁷. Thus, the outcome the present work definitely supports traditional use of the plant.

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Received on 17.08.2010

Accepted on 11.09.2010

Research J. Pharmacology and Pharmacodynamics. 2(6): Nov. –Dec. 2010, 407-413

Conc of extract/std mcg/mL	Ascorbic acid	Ether extract	Chloroform extract	Ethanol extract	Aqueous extract
10	24.95±0.27	9.10±0.43	14.33±0.35	18.13±0.31	8.27±0.32
20	41.17±0.36	11.27±0.44	17.17±0.29	35.10±0.16	12.93±0.17
40	61.27±0.45	16.98±0.55	21.02±0.42	45.05±0.45	14.10±0.16
60	80.35±0.78	20.88±0.40	29.12±0.47	68.70±0.23	17.27±0.27
80	90.25±0.38	22.75±0.19	36.65±0.22	78.98±0.25	19.58±0.28
100	93.67±0.30	25.70±0.33	40.88±0.38	85.67±0.29	22.13±0.7

Conc of extract/std mcg/mL	BHT	Ether extract	Chloroform extract	Ethanol extract	Aqueous extract
10	26.98±0.12	10.17±0.25	14.65±0.25	24.43±0.28	6.19±0.21
20	42.08±0.18	13.62±0.29	16.55±0.29	34.85±0.37	12.11±0.32
40	65.32±0.23	16.38±0.33	23.68±0.32	52.43±0.49	15.31±0.25
60	81.05±0.15	24.63±0.17	29.25±0.73	69.87±0.11	17.43±0.11
80	92.18±0.09	26.85±0.25	37.07±0.33	82.00±0.45	18.17±0.21
100	94.57±0.15	29.78±0.19	41.20±0.25	86.53±0.31	19.36±0.29

Conc of extract/std mcg/mL	BHT	Ether extract	Chloroform extract	Ethanolic extract	Aqueous extract
10	25.00±0.34	12.31±0.15	14.21±0.25	22.33±0.46	9.22±0.36
20	41.83±0.6	14.48±0.11	16.32±0.28	37.17±0.38	12.78±0.32
40	59.83±0.19	17.39±0.12	21.21±0.24	49.78±0.44	15.35±0.31
60	81.22±0.26	19.13±0.35	25.17±0.21	62.88±0.27	17.21±024
80	87.40±0.10	22.92±0.29	31.28±0.36	75.77±0.22	19.45±0.27
100	91.90±0.33	24.75±0.20	42.17±0.42	84.30±0.28	21.37±0.36