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Sunita Khatak et al. / Journal of Pharmacy Research 2014,8(9),1279-1284 Research Article ISSN: 0974-6943 Available online through http://jprsolution...
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Sunita Khatak et al. / Journal of Pharmacy Research 2014,8(9),1279-1284

Research Article ISSN: 0974-6943

Available online through http://jprsolutions.info

Antimicrobial, antioxidant and phytochemical property of Cassia tora against pathogenic microorganisms Sunita Khatak*, Preeti Sharma, Shikha Laller and Deepak Kumar Malik Department of Biotechnology Engineering, Kurukshetra University Kurukshetra, India Received on:14-08-2014; Revised on: 05-09-2014; Accepted on:19-09-2014 ABSTRACT The methanol and ethyl acetate extracts of seeds and pods along with seeds showed the highest activity in comparison to leaves and bark with regard to the inhibition of microbial growth while distilled water showed ineffective antimicrobial activity against different plant extracts. All plant parts exhibited antimicrobial activity but the highest activity is observed in the pods taken along with seeds. The methanolic extracts were considered for antioxidant analysis.All extracts showed significant antioxidant scavenging activities against DPPH radical as observed from IC50 values. The methanolic extracts of seeds proved to be most effective antioxidant as compared to methanolic extracts prepared using bark, leaves and pods.The Cassia tora is very easily available medicinal plant. The role of medicinal plants in disease prevention has been attributed to antimicrobial, phytochemical and antioxidant properties of their constituents. The antimicrobial activity of Cassia tora was analyzed by agar well diffusion assay. The antioxidant and phytochemical properties was analyzed by standard protocols. KEYWORDS:Cassia tora, agar well diffusion assay, antimicrobial, plant extract INTRODUCTION Use of plants for the treatment of many diseases dated back to prehistory and people of all continents have this old tradition. Every culture on earth has relied on the vast variety of natural chemistries’ found in plants for their therapeutic properties [1]. Beyond this pharmaceutical approach to plants, there is a wide tendency to utilize herbal product to supplement the diet, mainly with the intention of improving the quality of life and preventing the diseases of elderly people [2]. Despite the remarkable progress in the preparation of synthetic drugs, over 25% of prescribed medicines in industrialized countries are derived directly from plants [3]. Plant synthesizes a wide variety of chemical compounds, which can be sorted by their chemical class, biosynthetic origin and functional groups into primary and secondary metabolites. The steadily increasing microbial resistance to existing drugs is a serious problem in antimicrobial therapy and necessitates continuing research into new classes of antimicrobials [4]. One way to prevent antibiotic resistance of pathogenic species is to use new compounds that are not based on existing synthetic antimicrobial agents [5]. Plant and plant derived agents have long history to clinical relevance as a source of potential chemotherapeutic agents [6]. Many studies have been undertaken with the aim of determining the antimicrobial and phytochemical constituents of medicinal plants and using them for the treatment of both topical and systemic microbial

infections as possible alternatives to chemical synthetic drugs to which many infectious microorganisms have become resistant [7]. The antimicrobial activity of plant extracts is due to different chemical agents in the extract, which function to attract beneficial and repel harmful organisms, serve as phytoprotectants and respond to environmental changes in plants. In humans, however the compounds have beneficial effects [8].

The role of medicinal plants in disease prevention or control has been attributed to antioxidant properties of their constituents [9]. Besides playing an important role in physiological systems, antioxidants have been used in the food industry to prolong the shelf life of foods, especially those rich in polyunsaturated fats [10]. Therefore, researchers have focused on natural antioxidants and numerous crude extracts and pure natural compounds have been recognized to have beneficial effects against free radicals in biological systems as antioxidants [11]. The Cassia tora L. syn. Cassia obtusifolia belongs to the Leguminosae family. It is an annual herb, 30–90 cm high which occurs as wasteland rainy season wild plant in India. Cassia tora is a wild crop that grows in most parts of India as a weed. The most popular English names of Cassia tora are Foetid Cassia, The Sickle Senna and Wild Senna. Common Hindi names are Puwad, Charota, Chakvad, Chakavat. The main useful parts of Cassia tora are leaves, *Corresponding author. roots and seeds. The chemical component of Cassia tora are anSunita Khatak thraquinones, chrysophanol, emodin, obtusifolin, obtusin, chrysoDepartment of Biotechnology Engineering, Kurukshetra University, obtusin, aurantio-obtusin, and their glycosides. Naphthopyrones, Kurukshetra, India rubrofusarin, norrubro fusarin, rubrofusaring, entiobioside. Journal of Pharmacy Research Vol.8 Issue 9. September 2014 1279-1284

Sunita Khatak et al. / Journal of Pharmacy Research 2014,8(9),1279-1284 Toralactone,torachrysone. Roots contains 1, 3, 5-trihydroxy-6-7dimethoxy-2-methylanth- roquinone and beta-sitosterol. While seeds contains chrysophanol, physcion, emodin, rubrofusarin, chrysophonic acid-9-anthrone. Emodin, tricontan-1-0l, stigmasterol, beta-sitosteral-beta- Dglucoside, freindlen, palmitic, stearic, succinic and dtartaricacids, uridine, quercitrin and isoquercitrin are isolated from leaves [12,13]. According to Ayurveda the leaves and seeds are acrid, laxative, antiperiodic, anthelmintic, ophthalmic, liver tonic, cardiotonic and expectorant [14]. The leaves and seeds are useful in leprosy, ringworm, flatulence, colic, dyspepsia, constipation, cough, bronchitis, cardiac disorders [15]. Therefore the objective of present work was to explore the antibacterial and antioxidant aspects of Cassia tora. MATERIAL AND METHODS Plant materials, chemicals, reagents and pathogenic cultures The bark, leaves and fruit of Cassia tora used in present investigation for the analysis of antimicrobial, antioxidant and phytochemicals were collected from roadside of NH-65 highway, near by Kurukshetra University, Kurukshetra, Haryana, India. All the chemicals and reagents used in the present investigation were of high purity and analytical grade. The different solvents were used (methanol, dichloromethane, distilled water, ethyl acetate, petroleum ether, chloroform and hexane). Pathogenic microbial cultures were procured from Institute of Microbiology and Technology, Chandigarh. Escherichia coli (MTCC No.40) Pseudomonas fluorescence (MTCC No.1748), Staphylococcus aureus (MTCC No.7443), Bacillus megaterium (MTCC No.428-BM) and Bacillus subtilis (MTCC No.121) were used in present study. Preparation of the plant extracts and antimicrobial activity The collected different plants parts were shade dried and pulverized and powdered to fine powder. From this 5 g of powder was soaked in 20 ml of each solvent. After 72 hours it was filtered and the residue was discarded. The solvent present in filtrate was evaporated at 4550º C in water bath. The residual powder after solvent extraction was dissolved in DMSO and stored at 4º C. The antimicrobial activity of plants/part was evaluated by agar well diffusion assay. The microbial inoculums was inoculated aseptically and spread uniformly on surface of pre solidified Mueller Hinton Agar (MHA) plates with the help of sterile glass spreader. A well of about 6.0 mm diameter was aseptically punctured using a sterile cork borer. The cut agar was carefully removed by the use of sterile forceps. To each well 50µl plant extracts were poured. DMSO was used as negative control whereas Ciprofloxacin and Fuconaozole were used as positive controls. The petriplates were kept in laminar for 30 minutes for pre-diffusion to

occur then petriplates were incubated overnight at 37 ºC for 24 hours. The antimicrobial spectrum of the extract was determined in term of zone sizes (inhibition zone diameters) around each well. Zones were measured by Hi-media Zone scale. The experiment was repeated thrice and the average values were recorded for antibacterial activity. Antioxidant activity The DPPH solution (0.004%) was prepared by dissolving the DPPH in methanol. Amber color bottles were used to avoid the effect of light. An equal amount of methanol and DPPH served as control. Standard ascorbic acid solution (0.1 ml) of different concentrations viz. 10 to 100ìg/ml was added to 3 ml of a DPPH solution. After 30 minutes incubation in the dark, absorbance was recorded at 517 nm, and the percentage inhibition activity was calculated. The antioxidant activity of the extract was expressed as IC50. The IC50 value was defined as the concentration (in ìg/ml) of extracts that inhibits the formation of DPPH radicals by 50%. The antioxidant activity of the extract was expressed as IC50. The IC50 value was defined as the concentration (in ìg/ml) of extracts that inhibits the formation of DPPH radicals by 50%. % Scavenging = [(A0–A1)/A0] ×100. A0 = the absorbance of the control, A1 = the absorbance in the presence of the extract. The effective concentration of sample required to scavenge DPPH radical by 50% (IC50 value) was obtained by linear regression analysis of dose response curve plotting between % inhibition and different concentrations of extracts. Phytochemical Analysis Freshly prepared extracts were subjected to standard phytochemical analysis to find the presence of flavanoids, tannins, saponins and steroids in the sample [16]. The 5 ml of extract was shaken vigorously with 10 ml of distilled water for 2 min. The appearance of foam that persisted for at least 15 min, confirmed the presence of saponins. A few drops of 5% FeCl3 solution were added to 2 to 3 ml of ethanolic extract. Appearance of deep blue color indicated the presence of tannins. The chloroform was added in equal amount to 2ml of extract followed by addition of 2 ml of conc.H2SO4 and shaken well. Chloroform layers appearing red and acid layer showing greenish yellow fluorescence, indicated the presence of steroid in the test extract. A few drops of lead acetate solution were added to 2 to 3 ml of extract. Formation of yellow color indicated the presence of flavonoid. RESULTS Antimicrobial activity of different solvent extracts using bark The Bark extract of Cassia tora were prepared in various solvents and were subjected to agar well diffusion assay. The methanolic, dichloromethane and ethyl acetate extracts prepared using bark part

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Sunita Khatak et al. / Journal of Pharmacy Research 2014,8(9),1279-1284 gave better results as compared to other solvents. The antimicrobial effect of Cassia tora by using different solvents is shown in Table (1, 2,3,4, 5). The dichloromethane extract of bark showed maximum inhibition zone diameters of 18mm for Bacillus megaterium, 17mm for Escherichia coli, 17mm for Staphylococcus aureus, 16mm for Bacillus subtilis, and 13mm for pseudomonas fluorescene in Agar well diffusion assay. The chloroform was not effective against any pathogen used. DMSO was served as negative control in which, no inhibition zone was observed against Escherichia coli. Amoxicillin (1mg/ ml) antibiotic served as a positive control for bacterial cultures respectively. Amoxicillin showed inhibition zones of 21mm against Escherichia coli, 17mm against Pseudomonas fluorescens, 24mm against Staphylococcus aureus, 22mm against bacillus subtilis, 23mm against bacillus megaterium. Inhibition zone diameters of Cassia tora extracts were comparable to antibiotics used in positive control. Antimicrobial activity of different solvent extract using leaves Various solvent S1-S7 (Table 3.1) extracts of Cassia tora leaves were subjected to agar well diffusion assay. Inhibition zone diameters (in mm) of leaves extract of Cassia tora in various solvents against various pathogenic strains were tested as shown in Table 4.2. Petroleum ether and chloroform proves to be better solvents then other. Chloroform extract of leaves detected maximum inhibition zone diameters of 17mm for Escherichia coli (MTCC No.40), 17mm for Bacillus megaterium(MTCC No.428), 16.5mm for Staphylococcus aureus (MTCC No.7443), 16mm for Bacillus subtilis (MTCC No.121) in agar well diffusion assay but no activity was observed against Pseudomonas fluorescens (MTCC No. 1748). Distilled water showed no activity against any pathogen used in present investigation. Antimicrobial activity of different solvent extracts using seeds The seed extract of Cassia tora in various solvents against various pathogenic strains were tested. The methanol and chloroform extracts prepared using seeds gave better results as compared to other solvent used. Methanol extract of seeds showed maximum inhibition zone diameters of 18mm for Staphylococcus aureus, 17mm in Bacillus subtilis, 16mm for Bacillus megaterium, 14mm for Escherichia coli and 10mm for Pseudomonas fluorescens in agar well diffusion assay. Chloroform, petroleum ether and dichloromethane extract showed no activity against Pseudomonas fluorescens. Antimicrobial activity of different solvent extract of pods along with seeds The pods along with seeds extract of Cassia tora in various solvents against various pathogenic strains were tested. The ethyl acetate and dichloromethane extracts prepared using pods along with seeds gave better results as compared to other solvents. Thee ethyl acetate

extract of pods along with seeds showed maximum inhibition zone diameters of 26 mm against Bacillus megaterium, 21mm for Pseudomonas fluorescens, 21mm for Staphylococcus aureus, 19mm for Escherichia coli, 19mm for Bacillus subtilis. Petroleum ether showed minimum zone of inhibition against various pathogenic strains used. Antimicrobial activity of different solvent extract of only pods The distilled water and ethyl acetate extracts prepared using pods when taken alone gave better results as compared to other solvents. Distilled water extract of only pods showed maximum inhibition zone diameters of 18mm for Escherichia coli, 13mm for Pseudomonas fluorescens, 15mm for Staphylococcus aureus, 16mm against Bacillus subtilis, 16mm for Bacillus megaterium. Dichloromethane showed no activity against any used pathogen. Phytochemical analysis of Cassia tora Phytochemical analysis of bark extract of different solvents gave both positive and negative test for saponin, tannins, steroids and flavonoids tested. Hexane and methanol showed presence of all phytochemical constituents. The ethyl acetate showed absence of saponins, flavonoids and steroids. In phytochemical analysis of leaves extract, ethyl acetate showed all positive tests revealing presence of all active compounds. Hexane extract showed mainly presence of steroids and absence of other phytochemical constituents. Different solvent extract of leaves showed both presence and absence of saponins, tannins, steroids and flavonoids. The different solvent extract of seeds, pods along with seeds and only pods of Cassia tora showed presence and absence of saponins, tannins, steroids and flavonoids. The methanolic extract of seeds showed presence of all active compounds. The different solvents extract of seeds showed maximum presence of saponins, tannins, flavonoids and steroids. Chloroform extract of pods along with seeds showed all positive tests in phytochemical analysis. Some results may be not determined as in dichloromethane and ethyl acetate. Chloroform and methanol showed all positive tests for the presence of saponins, tannins, flavonoids and steroids. Some negative results showed in hexane only presence of tannins. Antioxidant activity of Cassia tora All the tests were performed in triplicate and the graph was plotted with the average of three observations. A significant decrease in the concentration of DPPH radicals due to the scavenging ability of various methanolic extracts was observed. This activity was dose dependent. Maximum scavenging activity of ascorbic acid (98.059) was observed at 100µg/ml and the IC50 value of 38.28µg/ml. DPPH radicals due to the scavenging activity of methanolic solvent extract of

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Sunita Khatak et al. / Journal of Pharmacy Research 2014,8(9),1279-1284 different parts of Cassia tora. The maximum scavenging activity of Table 4. Inhibition Zone diameters (mm) pods along with seeds exbark extract (62.008%), leaves extract (87.474%) and pods extract tract of Cassia tora in different solvent extracts against various (82.064%) was observed at 100 µg/ml concentrations, Maximum scav- pathogenic stains. enging activity of seeds extract (91.628%) and pods along with seeds Solvent Pathogens (66.400%) was observed at 100 µg/ml concentrations. The IC50 value P. fluorescens B. subtils E. coli B. megaterium S. aureus of bark, leaves, seeds, pods along with seeds and only pods extracts Hexane 16 14 15 12.5 17 were found to be 79.75µg/ml, 57.48µg/ml, 18.85µg/ml, 59.26µg/ml and Chloroform 14 15 16 17 17.24µg/ml respectively. IC50 values were recorded for methanol exDistilled water 10 13 12 Petroleum Ether 10 11 tracts of seeds was higher than all other methanol extract of different Methanol 10 17 15 16 12 plants used. Dichloromethane 1 3 17 18 18 16 Table 1. Inhibition Zone diameter (mm) bark extracts of Cassia tora in different solvent extracts against various pathogenic stains Solvent

Pathogens P. fluorescens B. subtils E. coli B. megaterium S. aureus

Hexane 9 Chloroform Distilled water 1 0 Petroleum Ether Methanol 12 Dichloromethane 17 Ethyl acetate 11 Positive control 1 7 Negative control -

13 15 14 16 13

13 15 14 16 17

13 7 10.5 14 17

13 7 15 13 15 18

15 21 -

15 21 -

11 23 -

15 24 -

Table 2. Inhibition Zone diameters (mm) leaf extract of Cassia tora in different solvent extracts against various pathogenic stains. Solvent

Pathogens P. fluorescens B. subtils E. coli B. megaterium S. aureus

Hexane Chloroform Distilled water Petroleum Ether Methanol Dichloromethane Ethyl acetate Positive control

11 12 10 12 17

14 16 18 16 11 16 22

16 17 16 16 15 15 21

10.5 17 16 14 18 9 23

13 16.5 18 14 15 15 24

Negative control

-

-

-

-

-

Table 3. Inhibition Zone diameters (mm) seed extract of Cassia tora in different solvent extracts against various pathogenic stains. Solvent

Pathogens P. fluorescens B. subtils E. coli B. megaterium S. aureus

Hexane 13 Chloroform Distilled water 11 Petroleum Ether Methanol 10 Dichloromethane Ethyl acetate 12 Positive control 17 Negative control -

13 14 14 14 17 11 14 22 -

15 15 13 13 14 8 10 21 -

15 16 12 15 16 10 10 23 -

14 17 15 15 18 14 14 24 -

Ethyl acetate Positive control

21 17

19 22

19 22

26 23

21 24

Negative control

-

-

-

-

-

Table 5. Inhibition Zone diameters (mm) pods extract of Cassia tora in different solvent extracts against various pathogenic stains. Solvent

Hexane Chloroform Distilled water Petroleum Ether Methanol Dichloromethane Ethyl acetate Positive control Negative control

Pathogens P. fluorescens B. subtils E. coli B. megaterium 16 13 11 9 17 -

14 14 16 14 15 18 22 -

13 15 18 15 14 19 21 -

13 16 16 11 23 -

S. aureus 14 13 15 14 12 15 24 -

DISCUSSION Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is antibacterial activity assay [17]. The use of Ayurveda medicines has increased now days [18]. The bio active compounds obtained from medicinal plants have been used to treat various ailments caused by microorganisms. The most important of these bioactive principles are alkaloids, phenolic compounds, flavanoids and tannins that may be evolved in plants as self defense against pests and pathogens [19]. In present study the activity of different solvent extracts of bark, leaves, seeds, pods along with seeds and pods of Cassia tora screened for antimicrobial activity against Escherichia coli, Pseudomonas fluorescence, Staphylococcus aureus, Bacillus subtilis and Bacillus megaterium Plant based products have been effectively proven for their utilization as source for antimicrobial compounds. Our results well correlated that ethyl acetate extracts of various plants exhibited good antimicrobial activity than other solvents [20]. Ethyl acetate extracts of pods along with seeds and pods alone, methanolic extracts of seeds were found effective against Bacillus megaterium and Escherichia coli. The antimicrobial activity of dichloromethane extracts of pods were found comparatively lower. The ethanol extract

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Sunita Khatak et al. / Journal of Pharmacy Research 2014,8(9),1279-1284 inhibited only B. subtilis, where as it was not effective against the other bacteria tested [21]. Methanol extract was effective against two of the tested organisms i.e., S.aureus and E.coli both at the concentration of 64mg/ml. Petroleum ether did not inhibit the growth of any of the bacteria. [22]. In present study methanolic extracts were effective against all the bacterial strains i.e. Escherichia coli, Pseudomonas fluorescens, Staphylococcus aureus, Bacillus subtilis and Bacillus megaterium while petroleum ether extracts of any plant part did not showed any antimicrobial activity against Pseudomonas fluorescens. Results of chloroform extracts of plant parts were contradictory [23] finding that chloroform was the best solvent for extracting the effective antimicrobial sub-stances from the medicinal plant C. auriculata than the other three solvents i.e. petroleum ether, ethyl acetate and methanol. Therefore, the chloroform extract has been selected for investigating antimicrobial activity. But our results revealed that ethyl acetate and methanol extracts of bark, leaves, seeds, pods along with seeds and pods showed best antimicrobial activity against all bacterial strains. Our findings were supported by investigations of [24] that aqueous extracts of various plant exhibited negligible antimicrobial potential. In present study aqueous extracts did not showed any antimicrobial activity in leaves extracts of Cassia tora. The results of present investigation clearly indicate that the antimicrobial activity vary with thesolvent used for extraction of antimicrobial metabolite. Thus, the study ascertains the value of plants used in ayurveda, which could be of considerable interest to the development of new drugs. The extracts did not show any activity against the tested strains, they may be active against other microbial species which were not tested [25]. Antioxidants can inhibit or delay the oxidation of oxidizable substrates and this appears to be very important in the prevention of oxidative stress which is suggested as the leading cause of many oxidation related diseases. Recently, and mainly due to undesirable side effects such as toxicity and carcinogenicity of synthetic additives, interest has considerably increased for finding naturally occurring antioxidant and antimicrobial compounds suitable for use in food and/or medicine. The crude methanolic extracts of bark, leaves, seeds, pods along with seeds and pods of Cassia tora tested for the in vitro antioxidant activity using the DPPH method. Among selected solvents extracts seeds and pods were good antioxidants and showed IC50 value 18.85µg/ml and 17.24µg/ml and respectively. Results showed that all plant parts have significant antioxidant activity but less than standard ascorbic acid. The methanolic extract of all plant parts of Cassia tora showed good antioxidant activity. Phytochemical constituents such as alkaloids, flavonoids, tannins, phenols, saponins, and several other aromatic compounds are secondary metabolites of plants that serve a defense mechanism against prediction by many microorganisms, insects and other herbi-

vores [26]. The present study carried out on the plant samples revealed the presence of medicinally active constituents. Analysis of different solvent extracts of bark, leaves, seeds, pods along with seeds and pods revealed the presence of flavonoids, saponins, steroids and tannins in most of the selected plants which could be responsible for the observed antimicrobial property. CONCLUSION The activity of different solvent extracts of bark, leaves, seeds, pods along with seeds and pods against E, coli, P. fluorescens, S. aureus, B. subtilis and B. megaterium was analyzed. Pathogenic microbes confirmed that the plant contains compounds that exhibit measurable antimicrobial activity against bacteria and fungi used in this study. Among the solvents used methanol and ethyl acetate extracts of seeds and pods along with seeds showed the highest activity in comparison to leaves and bark with regard to the inhibition of microbial growth while distilled water showed ineffective antimicrobial activity against different plant extracts. All plant parts exhibited antimicrobial activity but the highest activity is observed in the pods taken along with seeds. Ethyl acetate extract of pods along with seeds and pods were found to be highly active against the growth of bacterial strains such as Escherichia coli, Pseudomonas fluorescens, Staphylococcus aureus, Bacillus subtilis and Bacillus megaterium. Methanolic extract of seeds showed highest antimicrobial activity against Staphylococcus aureus and petroleum ether extract of leaves showed highest antimicrobial activity against Staphylococcus aureus, Bacillus subtilis but distilled water extract of leaves ineffective against all pathogen tested. Dichloromethane extract of bark were found to be highly active against the growth of bacterial strains such as Bacillus megaterium. Antioxidant assays were carried out using bark, leaves, seeds, pods along with seeds and pods of Cassia tora. Only methanolic extracts were considered for antioxidant analysis. All extracts showed significant antioxidant scavenging activities against DPPH radical as observed from IC50 values. Methanolic extracts of seeds proved to be most effective antioxidant as compared to methanolic extracts prepared using bark, leaves and pods. Plants posses a wide spectrum of various phytochemical and can be exploited for the infectious diseases therapy. Phytochemical analysis of different extract of Cassia tora gave positive test for saponins, tannins, flavonoids and steroids. Methanol fraction was rich in flavonoids while chloroform extract mainly showed the presence of steroids. Our findings confirm the traditional therapeutic claim of consumption of Cassia tora as a leafy vegetable especially for stomach related ailments. The outcome of the present study has opened the way for addressing several other research problems in the current scenario.

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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.8 Issue 9. September 2014

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