Antioxidant and Antibacterial Activities of Ethanolic Extract of

Therapeutically Important Plant, Anacyclus pyrethrum

Deepak K. Singh

Department of Botany, University of Delhi, Delhi-110007.E mail: singhds3090@gmail.com

This study was performed to evaluate the antioxidant and antibacterial activity of ethanolic extract of Anacyclus pyrethrum. Antioxidant activity of seed, seedling, market sample(root), in vivo and in vitro leaves and root ethanolic extract of different dilution (0.5-10mg/l) were assessed and compared by using DPPH(2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging method. Highest antioxidant activities were 97.88% and 96.01% in market sample of root and in vitro root respectively. Antibacterial activity of in vitro and in vivo root was performed by “Disk diffusion method”. The extracts made of different plant sample of A. pyrethrum showed immense action against selected bacteria Corynebacterium pollutisoli, Pontibacter mucosus and Algoriphagus

2roseus. Maximum area of inhibition was recorded 97.86 mm against Algoriphagus roseus.

Anacyclus pyrethrum, a perennial, procumbent herb of the family Asteraceae, resembling chamomile plant, is indigenous to North Africa (Annalakshmi et al. 2012). It is cultivated on an experimental basis in Jammu and Kashmir from seeds imported from Algeria, Spain, Morocco and other Mediteranean countries (Puri 2003; Kurian and Sankar 2007). In local dialect (Hindi) it is known as ‘Akarkara’, a name also shared by Acmella oleracea (Syn. Spilanthes acmella, S. oleracea). Anacyclus pyrethrum has been extensively used in the traditional systems of medicine for its rejuvenating properties and as a sex stimulant (Puri 2003). Organic extract of its root has been shown to have insecticidal, antimycotic, anti-inflammatory and anti-bacterial properties (Crombie 1955). It is often recommended for treatment of liver disorders (Lamnauer 2005). When mixed with olive oil it is reported to cure rheumatism, cold, epilepsy and

Therapeutically significant plant is any plant which contain bioactive compound synthesized during secondary metabolism, used due to its remedial properties or can be used as a precursor to synthesize useful medicine (Hossain et al. 2012). Impressive number of modern drugs have been successfully isolated from medicinal plants (Owolabi et al. 2007). Worlds 80% inhabitants rely on traditional system of medicine for their primary health care (Farnswort et al. 1985). Diseases curing potential of any plant against chronic alilments may be due to presence of secondary metabolites such as alkaloids, flavonoids, glycosides, phenolics, terpenoids etc. (Cowan 1999).

paralysis (Venkatakrishnabhatt et al. 1988; Annalakshmi et al. 2012). Powdered herbs or root forms a good sniff to treat chronic catarrh of the heads or nostrils by promoting flow of viscid humor, saliva, nasal mucous and tears (Khare 2004; Kurian and Sankar 2007; Annalakshmi et al. 2012). Dried pellitory roots having a pungent taste are used as masticators to allay toothache and headache (Crombie 1952). The diluted essential oil from the roots is used for gargling and in the preparation of tooth powders (Kurian and Sankar 2007; Annalakshmi et al. 2012). Patients are prescribed to chew the roots on a daily basis for several months to get relief from paralyses of the tongue, mouth diseases, rheumatic or neurologic infections of the head and face (Khare 2004; Lamnauer 2005; Kurian and Sankar 2007; Annalakshmi et al. 2012). In India, tobacco is often chewed along with roots of Anacyclus pyrethrum and Acmella calva to reduce the mutagenic action of tobacco by inhibiting nitrosation (Boonen et al. 2012). The powder of whole plant or its roots is mixed with hog's grease to make an ointment, which is an excellent approved remedy in the lethargy (fibromyalgia) and also helps in gout, sciatica and epilepsy (Puri 2003; Khare 2004; Lamnauer 2005). The decoction of the root is useful as a gargle in relaxation of the uvula, odontalgia, tonsilitis and pharyngitis (Annalakshmi et al. 2012). Present reports deals with the screening of therapeutically significant plant, Anacyclus pyrethrum for the evaluation of antioxidant and antibacterial activities in ethanolic extract of different plant samples.

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DPPH free Radical Scavenging Assay:

Plant material : In vivo leaves and roots of Anacyclus pyrethrum were collected from Deoban, Chakrata Uttarakhand (Latitude: 77°52.541ˈE, Longitude: 30°45.149ˈN, Altitude: 2622m) in the month of September 2012, while in vitro leaves and roots were obtained from MS basal medium following the protocol of Singh et al. (2015). The herbarium specimen of one of the collected plant was deposited in the University of Delhi herbarium (Voucher specimen no. DUH 13751).

Test Microorganisms

Leaves and roots of in vitro developed plant maintained on MS medium and those from the native habitat were dried for 48 hr in an oven maintained at

°50 C. Dried plant sample were then pulverized in liquid nitrogen. 100 and 200 mg powder of each sample were added individually to 5ml of ethanol contained in 50 ml Falcon tubes and incubated on a rotary shaker for 12 hr at

o200 rpm and maintained at 25 C. The tubes were then centrifuged at 10,000g for 20 min and supernatant was filtered through Millipore filters (0.22µm). The filtrate containing plant extracts to be analyzed were stored at

°4 C till further use.

MATERIALS AND METHODS

Preparation of ethanolic crude plant extracts

Luria Bertani (LB) agar media was prepared and pH was adjusted to 7.5 with 0.11NaOH or 0.1N HCl. Approximately 25ml of medium was poured into 90mm sterile petridish. Mother plates of each culture were too maintained during subculture.

Bacteria used were Corynebacterium pollutisoli, Pontibacter mucosus and Algoriphagus roseus. These bacterial cultures were obtained from Department of Zoology, University of Delhi, India.

The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging antioxidant potential of seeds, seedling, market sample (roots), in vitro and in vivo roots and leaves ethanolic extract was determined by the method of Brand-Williams et al. (1995) with slight modification. The stock solution of the reagent, DPPH was made by dissolving 24 mg of it into 100mL of methanol and stored

oat -23 C until use for the experiments. 1ml from the stock of DPPH was added to 1ml of extract of different concentrations (0.5, 1, 5 and 10mg/ml). Mixer of 1ml methanol and 1ml DPPH solution was used as control. Resulting mixture was shaken with vortex and incubated in dark for 30min at room temperature. Change in

Preparation of growth media

Antibacterial activities of varying concentration (0, 20 and 40mg/l) of ethanolic extract of in vitro and in vivo roots were tested using "disc diffusion technique"(Doughari et al. 2008). 150 μl of the test cultures of bacterial suspension were spreaded on the top of the solid LB media with the help of L shaped spreader (Hi Media, India) and allowed to dry for 10 min. Different concentration (0, 20, 40mg/ml) of extract loaded sterile discs (6mm sterile disc, Hi media, India) were placed on the surface of the LB medium cultured with individual bacterial species. Antibacterial activities of the plant extract were assessed by measuring clear zone of inhibition around sterile discs.

Screening of plant sample for their antibacterial activities

Antioxidant Activities

absorbance of the reacting mixture was recorded with the spectrophotometer at 593nm. Antioxidant activities of different plant sample were expressed as the percentage of inhibition and evaluated using the following formula:

Experiment design, culture condition and data analysis: Three sterile discs soaked in 0, 10, 20mg/l of ethanolic plant extract were inoculated per petriplate. Bacterial culture plates were incubated at 28 ± 2 °C for 48 hr. Area of inhibition around each disc was measured in milli meters after 48hr of incubation. Each experiment was repeated twice and the results were analyzed using one way ANOVA test and significant difference between each treatment evaluated by the Duncan's multiple range test at P= 0.05 using SPSS (version 22) software package. The values followed by different superscript(s) in each of the tables are significantly different.

RESULTS

Antioxidant activity of extracts from seeds, seedling, market sample (roots), in vitro and in vivo roots, and leaves was compared by DPPH radical scavenging method at four concentrations of each sample. In all the cases, antioxidant activity increased with the increase in concentration of the crude extract. Among all, the roots of the market sample had the highest antioxidant activity, closely followed by roots of the in vitro raised plants. The extracts of the whole seeds had minimum radical scavenging capability (Table 1).

Scavenging activity of plant samples towards DPPH free-radical

Absorbance of the control-Absorbance of the sample% radical scavenging activity = 100

Absorbance of the control´

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Unsaturated fatty acids of membrane lipids are more sensitive to oxidative processes, where arachidonic along with linoleic acid are specially affected by lipid peroxidation (Yu 2001). Free radical scavenging activities of antioxidants can inhibit lipid peroxidation. Because superoxide is the precursor of hydroxyl radical

Effect of different concentration of extract of roots collected from its natural habitat were evaluated against bacteria, Corynebacterium pollutisoli, Pontibacter mucosus and Algoriphagus roseus. Control disc did not show its own effect. Amongst all, maximum area of inhibition was observed against Algoriphagus roseus

2(97.86mm ) followed by Corynebacterium pollutisoli 2(78.76mm ). In vitro root extracts were too assessed for

having antibacterial activities against against same selected bacteria, where most efficient results was

2recorded against Pontibacter mucossus (56.78mm ) but at higher dose(40mg/l). However, extract was less effective against Corynebacterium pollutisoli at both lower and higher concentration (Table 2).

DISCUSSION

Antibacterial activity and singlet oxygen, therefore initiate the process of lipid peroxidation by the elimination of hydrogen atoms from membrane lipids (Gao et al. 2000). In the present study, crude plant sample extract of different concentration (0.5-10mg/l) were tested for its antioxidant activity. Amongst many, higher dose (10mg/ml) of market sample of root generated superlative scoring percentage (97.88%). This result can be considered as complete inhibition of DPPH, as final reacting mixture had some yellowish color, are in conformity with observation of Ordonez et al. 2006, where Sechium edule extracts adsorb DPPH free radicals upto 86% (32 µg/ml). In the present study, higher concentration had higher scoring %, whereas diluted extract have low scavenging activities. These findings are in agreement with the results of Sahu et al. 2013, where leafy extract of various plants showed maximum antioxidant activity in higher dose, while least in lower. Seed extract in the current study had low power for scavenging free radicals, scoring 8.14 to 42.11% only. On the contrary of this, Ordonez et al. (2006) found that decoction of seeds and leaves of Sechium edule had high

Table 1: Antioxidant activity of seeds, seedling, market sample (roots), in vitro and in vivo root and leaves in Anacyclus pyrethrum using DPPH free radical scavenging method.

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Acknowledgements: Financial assistance from Department of Science and Technology, Government of India, New Delhi under DST-INSPIRE program is gratefully acknowledged. The research presented in the paper was financially supported by Research & Development Grant of the University of Delhi.

Earlier as well as recent reports have revealed the power of success of ethnobotanical approach leading to antibiotic drug discoveries (Cox 1994). Medicinally significant plants are supposed the be the backbone of traditional medicine (Farnsworth 1994) In current study, ethanolic extract of micropropagated and in vivo root was tested against different strains of bacteria for its antibacterial activities using disc diffusion method. This highly effective technique has revealed antibacterial effect of different plant's parts of A. pyrethrum. This might be due to presence of simply general metabolic toxins or antibiotic compound in plant extract.

antioxidant activity (80-90%). Seedling, in vitro and in vivo leaf had medium, while in vitro root had optimum (96.01%) scavenging activities in present investigation.

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