in vitro antibacterial and antioxidant activity of high ...

Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) 914-921 Original Research Article

Microbiology

International Journal of Pharma and Bio Sciences

ISSN 0975-6299

IN VITRO ANTIBACTERIAL AND ANTIOXIDANT ACTIVITY OF HIGH ALTITUDE MEDICINAL PLANT TANACETUM GRACILE MANISHA BHATNAGAR1, ANUPAMA SHARMA AVASTHI2 AND SABARI GHOSAL3* 1,2,3

Amity Institute of Biotechnology, Amity University, Sector 125, Noida-201303, India

ABSTRACT As a part of our ongoing investigation on high altitude medicinal plants, we investigated Tanacetum gracile (Asteraceae) by bioassay guided fractionation for antibacterial and antioxidant activity. The methanolic extract of the plant was fractionated into n-hexane (n-Hex), dichloromethane (DCM), ethylacetate (EtOAc) and aqueous fractions. The antibacterial activity of the fractions was evaluated against three Gram negative viz., Escherichia coli, Acinetobacter sp. and Serratia sp., and two Gram positive including Staphylococcus aureus and Enterococcus sp. MDR clinical isolates. As, oxidative stress is a major cause of cognitive, cardiovascular diseases and cancer, and plant is the main source of antioxidant compounds, we evaluated the antioxidant potential of the fractions by chemical assays including total phenolic and flavonoid content, DPPH scavenging potential and FRAP assays. The results showed that EtOAc fraction possesses significant antibacterial activity against Serratia sp. and also commendable antioxidant activity (DPPH IC50 37.38 ± 0.4 µg/mL) comparable to that of vitamin C. Phytochemical screening of the fractions revealed the presence of phenolics including flavonoids in the fraction. The results may offer the use of the plant fraction as herbal remedy in the therapy of pathological conditions related to multi drug resistant microbes and generation of free radicals. Further phytochemical analysis in this regard could lead to some new drug candidates. Keywords: Tanacetum gracile, Multi drug resistant (MDR), antibacterial activity, antioxidant activity

Dr. SABARI GHOSAL Amity Institute of Biotechnology, AUUP, Noida.

*Corresponding Author

Received on: 10-03-2017 Revised and Accepted on: 24-04-2017 DOI: http://dx.doi.org/10.22376/ijpbs.2017.8.2.b914-921

This article can be downloaded from www.ijpbs.net

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INTRODUCTION Consumption of herbal medicines is widespread and increasing in recent years and approximately 80% of the people in developing countries depend on 1 traditional medicines for primary health care needs . The Himalayan region is a reservoir of variety of medicinal and aromatic plants (MAPs) and designated as one of the global biodiversity hotspots, where ecological, phyto-geographical and evolutionary factors favor high species diversity. Plants growing in extreme climatic conditions like, high altitudinal Ladakh experience abiotic stress in the form of low oxygen and temperature, enhanced UV radiation, short growing 2 seasons etc. . These plants have greater possibilities of having novel molecules and even largest quantities of active compounds which can be exploited for their bioactive properties. Amchi system of Tibetan medicine exploits these plants either as an extract or sometimes in combination with salts or minerals and these concoctions is well acclaimed for their medicinal 3 values . Many such plant extracts are being widely used by local medical practitioners. However, in most of the cases systematic inclusive investigations involving chemists and biologists have not been organized. Tanacetum gracile is a perennial aromatic plant belonging to family Asteraceae. The plant is traditionally used for cold, cough and fever by local 4 Buddhist community, commonly called Botos .The literature survey of the previous phytochemical investigations on the various species of Tanacetum have resulted in the isolation of monoterpenes, sesquiterpenes, triterpenes, flavanoids, essential oils and some other natural products. Essential oil of T. gracile (0.46%) showed the presence of about 40 constituents including lavendulol (21.5%) as a major chemical constituent along with lavandulyl acetate (1.7%), 1,8-cineole (15.2%), cis-β-ocimene (6.4%) borneol (6.1%) and chamazulene (3.7%) as minor 5 components . Three endemic subspecies of T. densum 6 have been explored for antioxidant properties . The oil of T. balsamita L. subsp. Balsamita has also 7 demonstrated cytotoxic and antimicrobial activity . Aerial parts of T. vulgare have also demonstrated 8 antioxidant activities . As a part of our ongoing research to identify bioactive compounds from high altitude plants of Ladakh, we reported two flavonoid compounds 5-hydroxy3,6,7,3’,4’-pentamethoxyflavone (Artemitin) and 5hydroxy-6,7,3',4'-tetramethoxyflavone (chrysosplenetin) from Tanacetum gracile possessing microtubule 9 depolymerization in breast cancer cells . As we know various plant extracts are used by the locals as a remedy for a number of common ailments, we thought of investigating the most promising plant fraction for antibacterial activity against MDR clinical isolates and also antioxidant activity by a number of chemical assays. Since, oxidative stress is a major cause of various diseases including cognitive and cardiovascular disease and cancer, the study could lead to the identification of an active fraction or compounds to combat the adverse effect of the same. Also, the well established fact that antioxidant

MATERIALS AND METHODS Plant material collection and extraction Tanacetum gracile was procured from the natural habitat of Ladakh and was authenticated by Dr. Surinder Kitchloo, Department of Botany, Indian Institute of Intergrative Medicine, Jammu (IIIM). The voucher specimen was deposited in the herbarium of Amity Institute of Biotechnology, Amity University, Noida, India. One kilogram of the whole plant material was extracted with MeOH: H2O (9:1) for five consecutive days. The extract was decanted and the residual plant material was further extracted with 10 water . The organic extract was concentrated under reduced pressure and was fractionated into n-hexane (n-Hex), dichloromethane (DCM), ethylacetate (EtOAc) fractions and aqueous (Aq) residue respectively. Concentrated fractions of the plant were subjected to phytochemical investigations and evaluated for antibacterial activity against five MDR clinical isolates and antioxidant activity. Phytochemical Analysis Detailed phytochemical analysis was performed with nHex, DCM, EtOAc and Aq fractions of methanolic extract of T. gracile to test for the presence of various 11 phytochemicals as described by Rajesh et al., . Flavanoids, steroids, alkaloids and tannins were detected by NaOH/HCl test, Salkowsi’s reaction, Dragendorff’s reaction and ferric chloride test respectively. Additional tests were carried out to check the presence of reducing sugars, cardiac glycosides, anthraquinones, triterpenoids and phlobatannins. Bacterial Strains The five different MDR bacterial clinical isolates including, Escherichia coli (2461), Staphylococcus aureus (2413), Enterococcus sp.(2449), Acinetobacter sp. (2457) and Serratia sp.(2442) were obtained from Dr. Kumardeep Dutta Choudhary, Department of Medical Oncology, Rajiv Gandhi Cancer Research Institute, Delhi, India with their respective antibiotic resistance profiles (Table1). All bacterial strains were revived in nutrient broth for antibacterial assay. Determination of antibacterial activity The antibacterial activity of the plant fractions was determined in accordance with the agar-well diffusion 12 method described by Rojas et al., .In brief the nutrient agar media plates were seeded with the test organism and open wells of 7 mm diameter were bored with a sterile cork borer. The wells were filled with 30 µL of the plant extract prepared to a final concentration of 1mg/mL in DMSO and water having DMSO concentrations not more than 2 %. Sterilised distilled water (MQ) was taken as the negative control. Standard antibiotic disc of gentamicin (30 µg) was used as positive control. The plates were incubated at 37°C for 24 h and observed for development of zones of inhibition around the wells. The diameters of the circular zone of inhibition were measured. The experiments were performed in triplicate and the


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Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) 914-921 antibacterial activity was expressed as mean of inhibition with standard deviation. Determination of antioxidant activity Total phenolic content (TPC) Total phenolics content was determined by modified Folin-Ciocalteau method as described by Celiktas et 13 al., . Briefly 200µL of 1mg/mL of extract prepared in methanol was diluted with 1800 µL of Milli Q water. To this reaction mixture 2.0 mL of Folin-Ciocalteau reagent and 2.0 mL of saturated sodium carbonate was added and mixed well. This was left to stand at room temperature for 30 minutes and the absorbance was determined spectrophotometrically at 760 nm using UV-Visible double beam spectrophotometer. The phenolic content was calculated as gallic acid equivalents (GAE)/g on the basis of standard curve of gallic acid. Total flavonoid content The total flavonoid concentration was measured by the aluminum chloride colorimetric assay as described by 14 Adnan . Briefly, a solution containing NaNO2, AlCl3, NaOH along with plant extract was mixed well and the absorbance was measured at 510 nm. The TFC was calculated in terms of quercetin equivalent (mg/g) by following the calibration curve. DPPH radical scavenging activity The antioxidant activity of the selected extracts was measured in terms of hydrogen donating ability 1, 115 in a diphenyl-2-picrylhydrazyl (DPPH) radical assay reaction mixture containing different concentrations of extracts (2, 10, 20 and 50 µg/mL) and 1 mM methanolic solution of DPPH. Subsequently, the mixture was shaken vigorously and left to stand for 30 min in the dark. Disappearance of the purple colour was monitored at 517 nm using spectrophotometer. Test samples and positive control ascorbic acid were tested in triplicate over the same range of sample concentrations. The radical scavenging activity (RSA) was calculated as

where, AE is the absorbance of solution containing antioxidant extract and AD is the absorbance of the methanolic DPPH solution. The antioxidant activity of each test sample and ascorbic acid was expressed in terms of concentration required to inhibit 50% methanolic DPPH radical formation (IC50 µg/mL) and calculated from the graph of % RSA and plant extract concentrations. Ferric reducing antioxidant power (FRAP) assay FRAP assay was performed according to the modified protocol of Benzie and Strain as adapted by 16 Krishnaraju et al., . The assay was based on the ability of the antioxidant to reduce ferric to ferrous ions in the presence of 2, 4, 6-tri (2-pyridyl)-S-triazine (TPTZ), forming an intense blue ferrous–TPTZ complex with absorption maxima at 593nm. An assay mixture containing 2.5 ml of 30mM acetate buffer (pH 3.6), 0.25 ml of 10 mM TPTZ in HCl, 0.25 ml of 20 mM ferric chloride and different concentration of plant

extract was incubated for 30 min at room temperature and the absorbance of the coloured product (ferrous tripyridyltriazine complex) was read at 593 nm. A standard graph for ferrous sulphate in methanol at different concentrations was prepared. FRAP values of the fractions were expressed as mM of Fe(II)/ g of extract. Cell cytotoxicity assay The cell cytotoxicity was performed by MTT assay as 17 described by Kakad and Dhembare . In brief, fibroblast cells obtained from chick embryo were cultured in DMEM medium supplemented with Fetal Bovine Serum (FBS) and gentamicin. The cells suspension (2 mL) was treated with sample solution at MIC concentration and twice the concentration of MIC. The microtitre plate was incubated aseptically in CO2 0 incubator for 24 hours at 37 C. After incubation, cells were disaggregated using trypsin (0.25%) and cell viability (%) was calculated.

STATISTICAL ANALYSIS All experiments were carried out in triplicates. The mean values of three individual observations were determined and the standard deviation (SD) was calculated and analyzed using Microsoft office excel 2010 and graph pad prism version 5.1.

RESULTS AND DISCUSSION Phytochemical screening The qualitative analysis of the methanolic extract and fractions of the plant revealed the presence of different class of secondary metabolites including flavonoids, saponins, cardiac glycosides, tannins, triterpenes and alkaloids. Specifically, the DCM and EtOAc fractions showed the presence of flavonoids and steroids (Table 2). Tannins were also detected in aqueous residue. Various plant secondary metabolites like flavonoids, saponins, cardiac glycosides, tannins, triterpenes and alkaloids have been reported to possess antimicrobial and antioxidant activities and fractions can be explained by the presence of various groups of potentially active classes of these secondary metabolites. Antibacterial activity The results obtained showed that the extracts of T. gracile have bactericidal effects on MDR clinical isolates (Table 3).The largest zone of inhibition (19.3 ± 0.6 and 18.3 ± 0.3 mm) was demonstrated by the EtOAc and DCM fraction respectively against (2442) Serratia sp. (Figure 1a), while the value dropped to 11.6 ± 0.5 mm and 10 ± 0.1 mm for n-Hex and aqueous fractions respectively when tested against the same organism. However, the aqueous extract was not as effective against any bacterial pathogenas compared to organic solvent fractions even though slight antibacterial activity was observed. The n-Hex fraction also inhibited the growth of (2547) Acinetobacter sp.(Figure 1b) and S. aureus with an inhibition zone 17.6 ± 0.5 mm and 14.6 ± 0.3 mm. The Enterococcus sp. was least inhibited by the plant fractions. Further, standard antibiotic gentamicin


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Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) 914-921 produced significantly larger inhibition zones against the tested bacteria.

antioxidant activities of these plants may be accounted for by their high flavonoids contents.

Total phenolic content EtOAc fraction showed highest phenolic content as determined by the Folin-Ciocalteu method and expressed as mg GAE/g of sample (116.4 ± 2.25 mg GAE/g) followed by DCM fraction (85.5 ± 4.23mg GAE/g respectively) (Table 4 and Figure 2). It is interesting to note that maximum phenolic content was found in the EtOAc fraction as compared to other fractions. Phenolics are widely distributed in the tissuesof plants and play an important role as 18 antioxidants . The results of the present study strongly suggest that the high phenoliccontents of these plants may be responsible for their antioxidant activities.

DPPH scavenging activity DPPH, a highly stable free radical has been widely used to assess antioxidant potential of many natural products. IC50 in µg/mL was calculated for each fraction as amount of antioxidant present in the sample necessary to decrease the initial DPPH concentration by 50%. The lower the IC50 value, the higher is the antioxidant activity. The data for IC50 for each fraction of T. gracile is presented in Table 4 and Figure 4. Lowest IC50 values were found to be for fractions EtOAc and DCM( 37.38 ± 0.4 and 45.82 ± 0.3 µg/mL) respectively which were comparable to the standard antioxidant Vitamin C (35.4 ± 0.2 µg/ mL) signifying the high proton donating and free radical scavenging potential of these fractions. The aqueous fraction did not show any appreciable antioxidant activity.

Total flavonoid content Flavonoids and other polyphenols due to their redox properties play an active role in quenching of free 19 radicals . Flavonoids are polyphenolic substances present in most plants and act as hydrophilic antioxidants, antimicrobials, photoreceptors, visual attractors, feeding repellants, and as UV-light filters and substrate for polyphenol oxidases protecting tissue after physical damage to plants. It is reported that flavonoids might account for atleast part of the health benefits associated with vegetable and fruit 20 consumption . The total flavonoids content of various fractions of the plant under the present study is presented in (Figure 3). Highest flavonoid content was found in DCM fraction (184.6 ± 5.91 mg QE/g)followed by EtOAc fraction (166.8 ± 4.12 mg QE/g) (Table 4). nHexane and Aqueous fractions in general had lower flavonoids content. Since phenolics and flavonoids are important constituents of these plants, the observed

FRAP assay The FRAP assay is a simple and inexpensive procedure that measures the total antioxidant levels in a sample. The method measures the reducing ability of antioxidants against oxidative effects of ROS. Among all the tested fractions, EtOAc fraction of T. gracile exhibited highest FRAP Value (1400 ± 0.95 mM of Fe (II) /g) followed by the DCM fraction (1300 ± 0.82 mM of Fe(II) /g). In contrast, the standard antioxidant Vitamin C had a FRAP value of 1600 ± 0.56mM of 21 Fe(II) /g .The results of FRAP assay are depicted in Table 4 and Figure 5.Flavonoids and other polyphenols due to their redox properties play an active role in quenching of free radicals. The results of the present study indicate that EtOAc and DCM fractions of T. gracile may be useful for treating free radical related pathological damage.

Table 1 Antibiotic resistance profiles of MDR clinical isolates obtained from Rajiv Gandhi Cancer Research Institute, Delhi, India Antibiotics Amikacin Ampicillin Ciprofloxacin Ceftriaxone Chloramphenicol Gentamicin Imepenem Levofloxacin Meropenem Nalidixic acid Nitrofurantoin Norfloxacin Ofloxacin Piperacillin Vancomycin Tobramycin

Gram negative Serratia sp. E. coli (2461) (2442) S S R R R R R R R R S R S S R R S S R S R R R R S R R

Acinetobacter sp. (2457) R R R R R R R

Gram positive S. aureus Enterrococcus sp. (2413) (2449) S R S R S R S R S R S R S R S R S R S R -

(R): Resistant; (S): Sensitive


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Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) 914-921 Table 2 Phytochemical screening of the fractions obtained from the methanolic extract of T. gracile Class of Phytochemicals / Fractions

n-Hex

DCM

EtOAc

Aqueous

Alkaloids ++ + + + Flavonoids ++ +++ +++ Steroids + ++ +++ Tannins + + + + Reducing Sugars ++ Cardiac Glycosides + ++ ++ + Triterpenoids + ++ _ + Anthraquinones Phlobatanins ++ ++++ +++ + “+++” indicates highly present, "++" indicates mode ratelypresent,"+"indicates present in traces, "-" indicates Absence. Flavanoids, steriods, alkaloids and tannins were detected by NaOH-HCl test, Salkowski's reaction, dragondorff reaction and ferric chloride test respectively. Additional tests were carried out to check the presence of reducing sugar, cardiac glycosides, phlobatannins, anthraqinones, saponins and terpenoids (Rajesh et al., 2010).

Table 3 Antibacterial activity of T. gracile plant fractions against five MDR clinical isolates MDR bacterial isolates/ Fractions n-Hex DCM EtOAc Aqueous Positive control (Gentamicin) E. coli (2461) 12.3 ± 0.9 11.3± 0.8 12.6± 0.3 9.3±0.4 21.79±0.58 Acinetobacter sp.(2457) 17.6 ± 0.5 12.3± 0.2 8.0± 0.6 10.6±0.3 25.33±0.5 Serratia sp.(2442) 11.6 ± 0.5 18.3±0.3 19.3±0.6 10±0.1 22.33±0.57 S. aureus (2413) 14.6 ± 0.3 8.3± 0.2 8.3± 0.7 8.0±0.2 25.76±0.58 Enterococcus sp (2449) 12.6 ±0.5 8.0±0.5 18.3±0.5 Inhibition zone in mm includes diameter of the borer (7mm). * 30 µL of 1 mg/mL of the extracts were poured into 7 mm diameter agar wells andzone of inhibition diameter was noted after incubation at 37˚C for 24 hours. (-): No inhibition

Table 4 Antioxidant potential of various fractions of T. gracile Fractions

n-Hexane DCM Ethyl acetate Aqueous Vitamin C

Total phenol content (mg GAE/g dry wt. of sample)* 33.3±1.11 85.5±4.23 116.4±2.25 66.2±0.6

Total Flavonoid content (mg QE/g dry wt. of sample)*

DPPH IC50 (µg/mL)

28.64±2.03 184.66±5.91 166.82±4.21 63.78±1.03

75.83 ± 0.3 45.82 ± 0.3 37.38 ± 0.4 >100 35.4 ± 0.2

FRAP value ± S.D.(µM Fe (II)/g of extract) 1000 ± 0.95 1300 ± 0.82 1400 ± 0.95 100±1 1600 ± 0.56

*Total phenol content expressed as mg GAE/g dry wt. of extract, *Total Flavonoid content expressed as mg QE/g dry wt. of extract. DPPH radical scavenging activity expressed as IC50 in µg/mL extract required to scavenge 50 % of free radicals FRAP value expressed as mM of Fe (II) formed /g of extract

Figure 1 Agar disc diffusion: a) antibacterial activity of T. gracile extracts with significant zone of inhibition against Serratia sp (2442). b) antibacterial activity of T. gracile extracts with moderate zone of inhibition against Acinetobacter sp (2457) *Tg H- n-Hexane extract, Tg D- DCM extract, Tg E- EtOAc extract, Aq- Aqueous extract, Positive control- Gentamycin, MQ- Millipore water as negative control.


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Figure 2 Total Phenolic content of different fractions of T. gracile. The phenolic contents are expressed as mg Gallic Acid Equivalent (GAE)/g dry wt. of extract

Figure 3 Total Flavonoid content of various fractions of T. gracile. The flavonoid content is expressed as mg quercetin equivalent (QE)/g dry wt. of extract

Figure 4 IC50 values for DPPH free radical scavenging of various fractions of T. gracile expressed as µg/mL required to scavenge 50% of free radicals


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Figure 5 FRAP antioxidant capacity of various fractions of T. gracile expressed as mM of Fe (II) formed /g of extract The antibacterial screening was conducted by bioassay guided fractions against two Gram-positive strains including Staphylococcus aureus and Enterococcus sp.; and three Gram-negative strains viz., Serratia sp, Acinetobacter sp and E. coli MDR clinical isolates. The result identified EtOAc fraction followed by DCM and nHex fraction of T. gracile possessing significant activity against Serratia sp. with zone of inhibition (ZI) of 19.3 ± 0.6 mm, 18.3 ± 0.3 mm and 11.6 ± 0.5 mm respectively. Moderate antibacterial activity was also observed with n-Hex and DCM fraction against Acinetobacter sp. The screening result of antioxidant activity conducted by DPPH free radical scavenging and FRAP assay showed that DCM and EtOAc possessed most significant activity. Cell cytotoxicity of the active fractions were evaluated at IC50 concentration against chick embryo culture based fibroblast cells and the result was well within the permissible limit. It is worthy to mention that though, a number of plant extracts exhibit promising biological activity but majority of them suffers from high toxicity on normal cells and could not be considered for drug 22 development . However, our study identified DCM and EtOAc fraction possessing both antibacterial activity and antioxidant activity without any cell cytotoxicity.

CONCLUSION The significant antibacterial potential of EtOAc and DCM fractions of T. gracile has been demonstrated by zone of inhibition assay against MDR Serratia sp. and E. coli and n-Hex fraction showed moderate activity

against S. aureus. The strong antioxidant activity of EtOAc fraction of T. gracile has been demonstrated by high phenolic and flavonoid content and strong free radical scavenging activity in our study. The antioxidant activity may be attributed to chelation of metal ions by phenolics or by quenching of free radicals or a combination of both the mechanisms. In our previous study we reported the presence of artemetin and chrysosplenetin from DCM and EtOAc combined fraction, which showed significant microtubule depolymerization property against MCF-7 and T47D cells. Cell cytotoxicity measured at IC50 (DPPH assay) and twice the concentration of IC50 showed that cytotoxicity was well within the permissible limit i.e., 8 %. The results provide scientific evidence to support the folk medicinal utilization of these plants for the treatment of various ailments.

ACKNOWLEDGEMENTS The authors express their sincere gratitude to Dr. Ashok K Chauhan, Founder Amity Group of Institutions, for his constant guidance and encouragement. We would like to thank Dr. Kumardeep Dutta Choudhary, Department of Medical Oncology, Rajiv Gandhi Cancer Research Institute, Delhi for providing the MDR clinical bacterial isolates.

CONFLICT OF INTEREST Conflict of Interest declared none.

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