Phytochemical analysis and differential in vitro cytotoxicity assessment of root extracts of Inula racemosa

Biomedicine & Pharmacotherapy 89 (2017) 781–795

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Phytochemical analysis and differential in vitro cytotoxicity assessment of root extracts of Inula racemosa Shikha Mohan, Damodar Gupta* Metabolic Cell Signaling Research, Institute of Nuclear Medicine and Allied Sciences, Defence Research and Development Organization, Timarpur, Delhi, 110 054, India

A R T I C L E I N F O

Article history: Received 2 December 2016 Received in revised form 15 February 2017 Accepted 16 February 2017 Keywords: Inula racemosa Antioxidant capacity Reducing capacity Polyphenols Flavonoids In vitro toxicity

A B S T R A C T

The root of Inula racemosa is known for its antifungal, hypolipdemic and antimicrobial properties in traditional Indian Ayurvedic and Chinese system of medicine. The biological efficacy of Inula species is mainly due to the presence sesquiterpene lactone (Isoalantolactone and Alantolactone), which are reported to be inducers of Nrf2 antioxidant pathway. The investigation of properties and efficacy of root extracts of I. racemosa and their comparison was done with a view to find most efficacious extract for use at cellular level (both normal and transformed). In the present study different extracts of root of I. racemosa (aqueous, ethanolic, and 50% aqueous-ethanolic) were prepared and compared for their antioxidant potential, reducing capacity, polyphenol content and flavonoid content. Our investigations suggested that the aqueous extract possess highest antioxidant capacity and reducing potential. The polyphenol content was found to be highest in aqueous extract in comparison with other two extracts. However, all the three extracts showed less flavonoid content. Further, the preliminary phytochemical screening of all the extracts revealed the presence of terpenoids, phytosterols and glycosides. The TLC profile of ethanolic and 50% aqueous-ethanolic extracts showed the presence of alantolactone while aqueous extracts did not exhibit its strong presence. This warrants the need of more stringent techniques for characterization of aqueous extract in future. The in vitro cell based toxicity assays revealed that the aqueous extract was less toxic to kidneys cells while ethanolic extract was toxic to cells even at low concentrations. Hence, the current investigations showed better efficacy of the aqueous extract with respect to other extracts and found to be promising for its future application at in vitro levels. © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction The Indian trans-Himalayan region of Ladakh (Jammu & Kashmir, India) is rich in ethnobotanical wealth and is known as reservoir of diverse medicinal and aromatic plants. It is one of the global biodiversity hotspots and spans over 186,000 km2 above the natural tree line zone [1]. Some of the known medicinal plants (Achillea milifolium, Artemesia dracunculus, Dracocephalum heterophylum, Gallium pauciflorum, Hippophae rhamnoides, Mentha longifolia, Origanum vulgare, Rhodiola imbricata, Rhodiola heterdonta and Rubia cordifolia etc) of cold desert of Ladakh are being used in

Abbreviations: ARE, anti-oxidant response element; HO-1, heme-oxygenase-1; NQO1, NAD(P)H-quinone oxidoreductase; GCL, glutamate cysteine ligase; GST, glutathione S-transferases; O.D., optical density; SODs, superoxide dismutase; TLC, thin layer chromatogram; TRX, thioredoxin; ROS, reactive oxygen species. * Corresponding author. E-mail addresses: [email protected], [email protected] (D. Gupta). http://dx.doi.org/10.1016/j.biopha.2017.02.053 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

traditional Amchi system of medicine for treatment of ailments related to liver, lungs, heart etc. [2]. These plants are also reported to exhibit anti-viral, antimicrobial, anti-inflammatory, antioxidant properties, which may play vital role in reducing a large number of diseases related to stress-induced oxidative damage [3,4]. Inula racemosa is one of the important medicinal herbs of Western Himalaya’s cold desert area (between 5000–14,000 feet altitude) which finds wide application in Ayurveda, Amchi and Unani system of medicines. I. racemosa is also known as I. royleana (C.B. Clarke), Pushkarmool, Mano (Hindi) and belongs to family Asteraceae. Inula species has been used for treatment of various ailments viz. spasm, hypotension, angina, cancer etc and is also used as expectorant [5,6]. The root of the plants have medicinal value and posses reportedly hypoglycemic, hypocholesterolemic and hypolipidemic properties [7]. The multi herb combination (Lipistat) of I. racemosa with Terminalia a rjuna and Commiphora mukul is reported as cardioprotective and offers protection against isoproterenol induced myocardial ischemia in rats [8]. The extracts

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(aqueous and methanolic) of root have been shown to protect against carbon-tetrachloride, paracetomol and rifamipicin induced hepatotoxicities in rats [9]. The aqueous extract of the roots also shown to exhibit anti-apoptotic efficacy against 4-nitroquinoline1-oxide induced genetic mutations in mice bone marrow cells [10]. Plants from Inula species are known to exhibit anticancer efficacy [11]. Pal et al. reported that the active ingredients in n-hexane fraction of I. racemosa induce apoptosis in HL-60 leukemia cells through generation of ROS intermediates and dysfunctioning of mitochondria [12]. Moreover, I. racemosa has been used in treatment of tuberculosis by native Americans, improving stomach functions, relieving neck and shoulder pain, revitalization of spleen in traditional Chinese medicine [10,13]. The diverse medicinal value of the plant is attributed mainly to the presence of large amount of sesquiterpene lactones, especially eudesmanolides such as alantolactone and isoalantolactone [13,14]. The other sesquiterpene esters viz. dihydro-alantolactone, dihydro-iso alantolactone, dihydroinunolide, isoinunolide, isoallolantolactone, allolantolactone, isoinunal, inunal, isoalantodiene and alantodiene may also be present. Some of the other bioactive compounds which are known to contribute in medicinal efficacy of this plant may include B-sitosterol, daucosterol, D-mannitol, aplotaxene and phenlyacetonitirile. The present study aimed to investigate the free radical scavenging activities, anti-oxidant capacity, reducing capacities, total poly-phenol as well as flavonoid content of aqueous, ethanolic and 50% aqueous-ethanolic extracts of I. racemosa. In addition, preliminary phytochemical screening along with TLC profiling was also performed in order to find the active components of extracts. Cell based in vitro assay was also done on normal kidney epithelial cells (NKE) as wells as on transformed kidney cells (ACHN cells and A498 cells) to assess of differential toxicity of extracts. 2. Materials and methods 2.1. Chemicals Ascorbic acid, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), aluminum chloride (AlCl3), butylated hydroxytoulene (BHT), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), quercetin, gallic acid, methanol, potassium persulfate (K2S2O8) potassium ferricyanide (K3FeCN6), trichloroacetic acid (TCA), 2,4,6tripyridyl-s-triazine (TPTZ), ferric Chloride (FeCl3), Folin-iocalteu phenol reagent, sodium bicarbonate (Na2CO3), glacial acetic acid, ferrous sulphate (FeSO4), iodine, potassium iodide, copper sulphate, sodium hydroxide, nitric acid, hydrochloric acid, lead acetate, acetic anhydride, chloroform, tris base and absolute ethanol were obtained from Sisco Research Laboratories Pvt. Ltd, India. All chemical used during the investigations were of analytical grade. The RPMI 1640 medium, EMEM medium, heat–inactivated fetal bovine serum, penicillin, streptomycin, non-essential amino acids, b-mercaptoethanol, alantolactone, and sulphorhodamine B were obtained from Sigma-Aldrich, St Louis, MO (USA) 2.2. Preparation of root extract The root of the plant were collected (and authentication were done by botanist) in the month of August from Leh, Jammu and Kashmir, India (1000–1100 ft). The roots were washed thoroughly, shade dried (room temperature) and were coarsely powered by using mortar-pestle. The fine powder was prepared with the help of electric mixer and thereafter extracts (aqueous and ethanolic extracts) were prepared by the process of Soxhlet extraction [15]. Briefly, 50 gm of root powder packed in the thimble was kept in Soxhlet apparatus for sequential extraction first with absolute ethanol (10 h) followed by double deionized water (40 h) at 40  C.

The 50% aqueous-ethanolic extract was prepared by the process of Maceration [14] with minor modifications. Briefly, 50 g of the root powder was suspended in 50% ethanol (diluted in 1:10 w/v). The mixture was kept in incubator shaker (MaxQ TM6000, Thermo Fisher Scientific, Massachusetts, USA) for 24 h at 25  C; 250 rpm for extraction. The extracts were concentrated using rotavapour evaporator (BUCHI, R-134, BUCHI Labortechnik, Flawil, Switzerland) and thereafter lyophilized (Lyophilizer (55 C, Sub-Zero Lab Instruments, Chennai, India). The lyophilized extracts were stored in an air-tight container at 20  C. The voucher specimen of extracts has been deposited at institutional library, INMAS [(INM/LIB/ SMKDG01 (a, b, c)] The aqueous and 50% aqueous-ethanolic extract were dissolved in 50% ethanol for all experimental studies, whereas absolute ethanol was used as solvent for ethanolic extract. The percent of ethanol administered to the cells as a vehicle was within non-toxic range (less than 1%). 2.3. Estimation of radical scavenging potential The anti-oxidant capacity of the extracts was determined by ABTS radical scavenging assay as reported by Re et al. [16]. The ABTS radical is a stable free radical and has characteristic absorbance at 734 nm. In the presence of the antioxidant molecule, the reduction of the radical takes place which can be monitored by the decrease in its absorbance. Briefly, the ABTS radical was generated by mixing the two stock solutions [(1:1; v/v, ABTS (7 mM) and potassium persulfate (2.4 mM))] in dark for 12 h followed by preparation of working solution with OD of 0.70  0.01 at 734 nm. Equal volume of working solution and sample (extract/ standard) were mixed, incubated (10 min at room temperature) and absorbance was recorded measured at 734 nm spectrophotometrically using NanoDrop (Implen, Munich, Germany). Ascorbic acid and gallic acid were used as standards for comparison of radical scavenging activity of Inula extracts. The scavenging activity of the extracts was calculated as follows: Radical Scavenging Capacity = [{A0  A1}/A0]  100 Where, A0 = absorbance of ABTS radical; A1 = absorbance of ABTS radical with sample. 2.4. Estimation of reducing potential The reducing potential of extract was determined by method as reported by Oyaizu with minor modifications [17]. The reducing capacity of extracts was examined as transformation of the Fe+3 (Ferric) to Fe+2 (Ferrous) ions, which is mediated by presence of antioxidants in the sample [18]. The presence of Fe+2 is monitored by measuring absorbance of Perl’s Prussion Blue complex at 700 nm. Briefly, the different concentrations of extract was mixed with phosphate buffer (0.2 M, pH 6.6) and 1% potassium ferricyanide (1:25:2.5) and mixture was incubated for 30 min at 50  C. After incubation, 2.5 ml of trichloroacetic acid (TCA; 10%;w/v) was added and the resulting mixture was centrifuged (3000 rpm for 10 min). The supernatant was collected and mixed with ferric chloride (1%) in equal volume and absorbance of final solution was recorded at 700 nm spectrophotometrically. The reducing power of extracts is expressed as difference in optical density with respect to control. The reducing power of extracts is compared with that of ascorbic acid as a standard.

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2.5. Estimation of total antioxidant capacity The anti-oxidant capacity of extracts was measured using ferric reducing antioxidant power (FRAP) assay. A modified method of Benzie and Strain was adopted to measure the antioxidant capacity of herbal extract wherein a ferric salt, Fe+3(TPTZ)2 Cl3 is used as oxidant [19–21]. The antioxidants of the extract provides free electron to ferric-TPTZ complex which get reduced to blue colored ferrous-TPTZ complex and can be monitored at 593 nm spectrophotometrically. The stock solutions of TPTZ (10 mM in HCl), and FeCl3 (20 mM) were prepared fresh at the time of experiment and the working FRAP reagent was prepared fresh too by mixing 25 ml acetate buffer (300 mM acetate buffer (pH 3.6), 2.5 ml TPTZ solution and 2.5 ml FeCl3 (10:1:1 ratio; FRAP reagent). FRAP reagent was mixed with extracts and/or standard and incubated for 30 min in dark. The absorption of colored product (obtained by reduction of Fe+3 TPTZ complex at low pH to blue Fe+2 TPTZ) was recorded at 593 nm. The antioxidant capacity of extracts were estimated by FRAP values expressed in mM Fe(II)/mg of extract. The standard calibration curve of FeSO4 was prepared for determination of FRAP values by using different known concentration of FeSO4 (33.33 mM- 800 mM).

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2.8.2. Detection of carbohydrates For detection of carbohydrate content, extracts were suspended in double deionized water (2 mg/ml) and filtered to obtain filtrate. Carbohydrate content in filtrate was measured by using Fehling’s and Benedict reagents. For the Fehling’s test, the 1 ml of filtrate was boiled with 1 ml of each Fehling A and B solutions. The presence of carbohydrate was indicated by the formation of red precipitate. For the Benedict’s test, the 0.5 ml of Benedict’s reagent was added to 0.5 ml of filtrate and the mixture was heated on water bath for 2 min. The formation of red coloured precipitate indicated the presence of sugar. 2.8.3. Detection of amino acids and protein The amino acid and protein content in the extract (10 mg/ml of extract in double deionized water) were measured using Biuret, Xanthoproteic and Ninhydrin tests. For the Biuret test, an equal volume of filtrate and NaOH (40%) was mixed with few drops of copper sulphate solution (1%) and the appearance of violet colour indicated the presence of amino acid/protein. For the Xanthoproteic test, the filtrate was treated with few drops of nitric acid wherein the formation of yellow colour indicates the presence of proteins. However, presence of amino acids was measured by boiling extract with Ninhydrin reagent (0.25% in acetone) and development of blue colour.

2.6. Estimation of total polyphenol content The Total polyphenol content (TPC) of extracts was measured by Folin-Ciocalteu colorimetric method as described by Gao et al. [22] with minor modifications. Briefly, the reaction mixture containing 100 ml of sample, 2 ml of Follin-Ciocalteu reagent and 2 ml of double deionized water was mixed and incubated for 3 min at room temperature. Following incubation, 1 ml of sodium bicarbonate (20% w/v) was added and again incubated for 1 h at room temperature for formation of coloured products which was measured at 765 nm. Gallic acid was used as standard for comparison of total polyphenol content of extract. The TPC of extract are expressed as Gallic Acid Equivalent or GAE (mg of Gallic acid/mg of extract) and is calculated by linear regression of equation of gallic acid standard curve. 2.7. Estimation of total flavonoid content The total flavonoid content was measured by using method of Quettier-Deleu C et al. with minor modifications [23,24]. Briefly, equal volumes of extract and AlCl3 ethanol solution (2% w/v) were mixed, incubated (at room temperature for 1 h) and absorbance was measured at 420 nm. Quercetin was taken as standard and total flavonoid content of extracts was calculated as quercetin equivalent (mg of quercetin/mg of extract). 2.8. Qualitative phytochemical screening The preliminary phytochemical screening was performed for detection of alkaloids, flavonoids, steroids, terpenoids, anthroquinones, phenols, saponins, tannins, carbohydrates, proteins and amino acids, gums and mucilage as well as oils and resins by methods described earlier with modifications [25,26]. 2.8.1. Detection of alkaloids Extracts mixed with HCl (2:1 w/v) and filtered to obtained filtrate for measurement of presence of alkaloids by Hager’s test and Mayer’s test. Alkaloids forms yellow precipitate with Hager’s reagent (saturated picric acid solution). The Mayer’s reagent (potassium mercuric iodide) forms reddish brown precipitate with alkaloids.

2.8.4. Detection of glycosides The Borntrager’s test was performed to confirm the presence of anthranol glycosides. Briefly, the 5 mg of extracts were hydrolysed with diluted HCl (10%) for 5 min at 37  C. Samples were allowed to cool and equal volume of CHCl3 was added. Thereafter, few drops of 10% NH3 were added to the mixture and heated. The formation of rose-pink colour indicated the presence anthroquinones/anthranol glycosides. The presence of cardiac glycosides was confirmed by Keller Killiani test. Briefly, 0.4 ml of glacial acetic acid and a few drops of 5% ferric chloride solution were added to a little of dry extract. Further, 0.5 ml of conc. H2SO4 was added along the side of the test tube carefully. The formation of blue colour in acetic acid layer confirmed the test. 2.8.5. Detection of terpenoids Salkowski’s test was performed for the detection of terpenoids wherein 5 mg of extract was mixed with 2 ml of CHCl3 and 3 ml of conc. H2SO4 was poured gently to form a layer. The appearance of reddish brown colour in inner phase indicated the presence of terpenoids. 2.8.6. Detection of phytosterols The presence of phytosterols in the extracts was confirmed by Liebermann-Burchard’s Test. Briefly, 2 mg of extract was dissolved in 2 ml of acetic anhydride and boiled. Thereafter, the solution was cooled and 1 ml of conc. H2SO4 was added along the side of the test tube. A brown ring formation at the interphase and the turning of the upper layer to dark green color confirmed the presence of phytosterols. 2.8.7. Detection of phenolic compounds For the detection of phenolic compounds, 10 mg/ml of extract solution (in double deionized water) was mixed with few drops of neutral ferric chloride solution (5%). A dark green colour indicated the presence of phenolic compounds. Lead acetate test was also performed for detection of phenolic compounds. Briefly, a few drops of 10% lead acetate were mixed with 1 ml of the sample and the formation of yellow precipitate confirmed the presence of phenolic compounds. The presence of flavonoids was also confirmed by an alkaline reagent test wherein few drops of

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sodium hydroxide (5%) were added to 1 ml of the test solution. The appearance of yellow colour solution which becomes colourless on addition of a few drops of 2 M hydrochloric acid indicated the presence of flavonoids. 2.8.8. Detection of fixed oils and fats For the detection of fats and fixed oils, 1 ml of copper sulphate solution (1%) was mixed with sample followed by addition of sodium hydroxide solution (10%). The formation of a clear blue solution confirmed the presence of fats and fixed oils. Moreover, appearance of oil stain on the paper, when a small quantity of extract was pressed between two filter papers indicated the presence of fixed oil. 2.8.9. Detection of gum and mucilage 10 mg/ml of extract was prepared (in double deionized water) and incubated with absolute ethanol on constant stirring. The formation of white or cloudy precipitate indicated the presence of gums and mucilages. 2.8.10. Detection of saponins Extracts were dissolved in double deionized water and mixture was vortexed for 15 min. The formation of stable foam confirmed the availability of saponins. Moreover, addition of a few drops of olive oil followed by vortexing formed soluble emulsion indicated the presence of saponins. 2.9. Thin layer chromatography The one dimensional ascending thin layer chromatography was performed on silica gel-66 plate (2.5 cm  6 cm) as described by Pavia et al. [27] with minor modifications. Briefly, the loading spot was marked with help of lead pencil on the TLC plate. The stock of 100 mg/ml of each extract was prepared and approximately 1 ml of ethanolic extract, 5 ml of aqueous extract while 2.5 ml of 50% aqueous-ethanolic was loaded onto plate with the help of glass capillaries. Alantolactone was used as standard for comparison. The spotting of alantolactone was done from 1 mg/ml stock and approx 3 ml was loaded on the plate. The loaded spots were dried before placing it in TLC development chamber. The TLC development chamber was saturated with 50% methanol (mobile phase) and thereafter the plate was introduced in the chamber in such a way that the lower end of plate was inside the mobile phase but loaded samples were not submerged. The chamber was covered and the front of mobile phase was allowed to run till 1 cm from the top. The plate was then taken out of the chamber and the top solvent front was marked. The plate was allowed to air-dry. Later the plate was sprayed with freshly prepared anisaldehyde-sulfuric reagent and kept in hot air oven for 3 min at 100  C. The plate was then taken out and observed for visible color development in daylight. The movement of isolated spots was determined by Retention factor (Rf) calculated as: Rf ¼

Distance travelled by compound Distance travelled by solventfront

2.10. Cell cultures and cytotoxicity screening The immortalized Normal kidney epithelial (NKE) cells was received as a kind gift from Dr Andrei V. Gudknov, Roswell park Cancer Institute, Buffalo, USA, whereas A498 and ACHN cells were obtained from NCCS (National Centre for Cell Science), Pune, India. The NKE cells were maintained and cultured in the RPMI 1640 medium supplemented with 10% heat–inactivated foetal bovine

serum (FBS), 100 units/ml of penicillin, 100 mgm/ml of streptomycin,1% non-essential amino acids, 50 mM b-mercaptoethanol, pH 7.4. The A498 and ACHN cells were maintained in EMEM medium supplemented with 10% heat–inactivated FBS, 100 units/ml of penicillin and 100 mgm/ml of streptomycin, pH 7.4. The cells were maintained in humidified air atmosphere (95–100%) at 37  C in 5% CO2 buffered incubator. The cytotoxic effect of different concentrations (25 mg/ml– 800 mg/ml) of extracts of I. racemosa was determined on NKE cells, and human renal carcinoma cells (ACHN and A498). The cytotoxicity studies of extracts comprised of proliferation assessment and determination of morphological alterations following treatment with different concentrations with extracts. Briefly, logarithmically growing cells were seeded in 96 well plate (5000 cells/well) and incubated in humidified CO2 incubator for proper attachment and growth. The ethanolic extract was dissolved in absolute ethanol while the aqueous and the 50% aqueous-ethanolic extracts were dissolved in 50% ethanol for preparation of stock solutions. The final concentration of ethanol as a vehicle was non-toxic (<1%). Following attachment, cells were treated with increasing concentrations of extracts (25–800 mg/ml) and were observed along with image acquisition at different time intervals (24–96 h) to assess morphological alterations by using Dewinter microscope, Dewinter Optical Inc, Delhi, India. The cell proliferation was assessed by using colorimetric SRB assay at the end of 96 h post treatment with the extract. The SRB assay was performed as described by Voigt [28] with minor modifications. Briefly, the media was removed and the cells were fixed with freshly prepared cold 10% trichloroacetic acid (TCA) for 1 h at 4  C. After fixation, TCA was removed and cells were stained with 0.4% SRB prepared in 1% acetic acid (w/v) for 2 h. Thereafter, the excess dye was washed with distilled water and plates were air dried. The protein-bound dye was extracted with 10 mM tris base, pH 10.5 following which the absorbance was recorded at 565 nm (background wavelength: 670 nm) with BioTek mQuant ELISA microplate reader, USA. The IC50 or inhibitory concentration that kills 50% of the cells for each extract was determined from regression equation between extract concentration and its corresponding absorbance. The cytotoxicity analysis was done through three independent experiment and IC50 values calculated as average of all three experiments. The images of cells were taken from single experiment but were representative of all three experiments. 3. Statistical analysis The three independent experiments were performed for statistical analysis with each experiment conducted with samples in triplicates. The results are average of the findings of the independent experiments with standard deviation. The one way ANOVA (Analysis of Variance) was done to compare the data followed by post hoc analysis using Neuman Keul’s test. The values with p < 0.05 were considered statistically significant. Prism 5, Graphpad Software Inc. was used for statistical analysis of data. 4. Results 4.1. ABTS radical scavenging potential A concentration dependent increase in scavenging of ABTS radical was observed among all the three extracts used in the study (Fig. 1a). The ethanolic extract exhibited 40% radical scavenging potential at 50 mg/ml which increased to be nearly 75% at 150 mg/ ml concentration. In case of the aqueous extract 70% scavenging was observed at concentration of 50 mg/ml which was found to increase with increase of concentrations of extract. ABTS radical

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4.2. Reducing potential All the three extracts of I. racemosa exhibited dose dependent increase in reducing potential (Fig. 2a). The lower concentrations of ethanolic extract (12.5–50 mg/ml) showed marginal increase in their reducing potential. However, a significant increase in reducing potential was observed from 100 to 400 mg/ml concentrations of ethanolic extract. On the other hand, the reducing potential of aqueous extract showed gradual increase from 12.5– 400 mg/ml doses, with a concentration of 400 mg/ml exhibited nearly two fold increase in its reducing potential than that at dose of 200 mg/ml. Similarly, the reducing potential of 200 mg/ml of 50% aqueous-ethanolic was nearly double that the reducing capacity of extract at 100 mg/ml. 50% aqueous-ethanolic extract also showed concentration dependent increase in reducing potential with considerable increase in reducing potential from 50 mg/ml to 400 mg/ml concentrations. Among the extracts, the aqueous extract and 50% aqueousethanolic root extract of I. racemosa exhibited significantly higher (p < 0.0013) reducing capacity as compared to ethanolic extract from 100 mg/ml and above concentration studied. However, the

Fig. 1. The ABTS radical scavenging assay: (a) The ABTS radical scavenging capacity of I. racemosa root extracts (ethanolic, aqueous, 50% aqueous-ethanolic) at different concentration (12.5–200 mg/ml) is represented. The reduction of the ABTS radical is monitored by the decrease in its absorbance at 734 nm. The data is represented as percent scavenging of ABTS radical for each extract. #p = 0.0209 vs 200 mg/ml of ethanolic extract and $$p = 0.0063 vs 150 mg/ml of ethanolic extract. (b) ABTS radical scavenging capacity of ascorbic acid and gallic acid at different concentration (0.039-2.5 mg/ml) as reference compounds is shown. The data is represented as percent scavenging of ABTS radical for each standard. All the data is expressed as mean percent  S.D. (n = 3).

scavenging capacity of 50% aqueous-ethanolic extract was found to be significantly lower with respect to aqueous extract at 50 mgm/ ml concentration (55%). However, no further increase in ABTS radical scavenging capacity was observed at the concentrations higher than 200 mg/ml of all the three extract showed (data not shown). Amongst the three extracts, the aqueous and 50% aqueous-ethanolic extracts exhibited better free radical scavenging ability as compared to corresponding concentrations of ethanolic extract. The concentrations of 150 mg/ml and 200 mg/ ml of ethanolic extract were significantly lower (p = 0.0063 and 0.0209 respectively) in radical scavenging ability than corresponding concentrations of aqueous and 50% aqueous-ethanolic extracts. The ascorbic and gallic acid were used as standards for comparison of scavenging activity with that of extracts. Both of these standards exhibited dose dependent increase in scavenging capacity which reached to saturation by 1.25 mg/ml (Fig. 1b). The concentration of 0.625 mg/ml of gallic acid showed nearly equivalent scavenging (90%) as that of 100 mg/ml of aqueous as well as 50% aqueous-ethanolic extracts. However, percent scavenging of the 100 mg/ml of ethanolic extract was found to be comparable to 0.078 mg/ml of ascorbic acid or 0.312 mg/ml of gallic acid. Hence, the aqueous and 50% aqueous-ethanolic extracts were found to be similar in the free radical scavenging and exhibited better scavenging than the ethanolic extract.

Fig. 2. The Reducing capacity assay: (a) The reduction of Fe+3 to Fe+2 is assessed by measuring absorbance of Perl’s Prussion Blue complex at 700 nm. The reducing capacity of I. racemosa root extracts (ethanolic, aqueous, 50% aqueous-ethanolic) at different concentration (12.5–400 mg/ml) is determined. ***p < 0.0001 vs 400 mg/ ml of ethanolic extract, ###p = 0.0005 vs 200 mg/ml of ethanolic extract and $ $p = 0.0013 vs 100 mg/ml of ethanolic extract. (b) The reducing capacity of quercetin and ascorbic acid at different concentration (2.5–20 mg/ml) as reference compound is shown. The data is represented as absorbance values for each standard. All the data is expressed as mean  S.D. (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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reducing capacity of aqueous and 50% aqueous-ethanolic extracts showed similar increase in their reducing potential across all the concentrations. The reducing capacities of standards, quercetin and ascorbic acid were determined from concentrations of 2.5 mg/ml to 20 mg/ ml (Fig. 2b). Both of these standards showed saturation at concentration of 20 mg/ml beyond which no further increase in reducing potential was observed. Our research findings suggest that the reducing capacities of 400 mg/ml of aqueous and 50% aqueous-ethanolic extracts were comparable to reducing ability of 10 mg/ml of quercetin. Moreover, the reducing capacity 200 mg/ml of these extracts exhibited reducing power equivalent to that of 5 mg/ml of ascorbic acid. 4.3. Antioxidant capacity A calibration curve for determination of FRAP values was found to be linear between 33.33–800 mM concentrations of ferrous sulfate. The equation obtained from the calibration curve was found to be y = 0.001488x + 0.0204; R2 = 0.9964; where x is absorbance and y is FeSO4 concentrations (mM). The assay was performed with four different concentrations of all the three extracts. The FRAP values of ascorbic acid was found to be significantly higher than all the three extracts (Table 1). However, amongst the extracts the aqueous extract exhibited highest FRAP value followed by 50% aqueous-ethanolic and ethanolic extracts. The ethanolic extract had FRAP value of 1.586  0.342 mM Fe(II)/mg of extract which was significantly lower than that of aqueous and 50% aqueous-ethanolic. 4.4. Total polyphenol content In the present study, the calibration equation of gallic acid as standard was obtained for quantification of polyphenol was y = 0.09553x+ 0.1806; R2 = 0.9706, where x is known gallic acid concentration and y is absorbance. The standard curve was found to be linear between 1.25–20 mg/ml concentration of gallic acid. The total polyphenol content of root extract of I. racemosa is observed to be significantly (p = 0.04) higher in aqueous extract and 50% aqueous-ethanolic in comparison to ethanolic extracts as depicted in Table 2. The polyphenol content of the ethanolic extract was found to increase with increase of concentrations, however a considerable increase in polyphenol content was observed at higher Table 1 The total antioxidant capacity assay. Sample

FRAP (mM Fe(II)/mg of extract)

Ethanolic extract Aqueous extract 50% Aqueous-ethanolic Ascorbic Acid

1.586  0.342 2.717  0.32** 2.186  0.215* 64.449  6.848

The total antioxidant capacity assay of I. racemosa root extracts (ethanolic, aqueous, 50% aqueous-ethanolic) is represented as FeSO4 concentration (mM)/mg of extract and ascorbic acid as a standard. All the data is expressed as mean  S.D. (n = 3). * p = 0.0102 vs ethanolic extract. ** p < 0.005 vs ethanolic extract.

concentrations v iz. 200 and 400 mg/ml (Fig. 3). The aqueous and 50% aqueous-ethanolic extract showed gradual increase in polyphenol content with increase of its concentrations. Moreover, the polyphenol content was found to be significantly higher in case of both aqueous and aqueous-ethanolic extracts with respect to the ethanolic extract. 4.5. Total flavonoid content The total flavonoid content of ethanolic extract found to be increased significantly with concentrations (50–400 mg/ml; Fig. 4). However, both aqueous and 50% aqueous-ethanolic extracts exhibited significant increase in flavonoid content from 12.5 mg/ ml till 400 mgm/ml. The flavonoid content assessed by change in O. D. of aqueous extract and 50% aqueous-ethanolic extracts was significantly higher (p = 0.0013) than that of ethanolic extract at concentration of 400 mg/ml. The quantification of flavonoid was done using calibration curve of quercetin y = 1.068x + 0.02212; R2 = 0.9986, where x is known quercetin concentration and y is absorbance. The standard curve of was linear between 0.78 mg/ml and 12.5 mg/ml of quercetin. The 50% aqueous-ethanolic extract and aqueous extract showed significantly higher flavonoid content in comparison with ethanolic extract (Table 3). 4.6. Phytochemical screening The preliminary phytochemical screening of extracts was done in order to find the major class of compounds present in I. racemosa extracts with different solvents. Our preliminary findings suggest that extracts possess less alkaloids (Table 5). All the three extracts showed the slight presence of reducing sugar when tested by Fehling’s reagent. However, the Benedict’s reagent did not show any precipitate formation with aqueous and 50% aqueousethanolic extract while ethanolic extract did exhibit the red precipitate formation. The cardiac glycosides were also present in all the three extract, though in different levels. Ethanolic extract showed higher presence of cardiac glycosides followed by 50% aqueous-ethanolic and aqueous extract. All the extracts were found to be rich in terpenoids and phytosterols as they showed strong positive Salkowski test and Liebermann-Burchard’s test. The aqueous extract exhibited higher phenolic content while ethanolic extract was observed to be deficient in it. The presence of flavonoids in all the three extract extracts was confirmed positive by alkaline reagent test The extracts were found to be deficient in the proteins or free amino acids, anthranol glycosides, fixed oils and fats, gums and mucilage, saponins. 4.7. Thin layer chromatographic profiling The chromatogram showed single intense purple spot for ethanolic extract similar to that of alantolactone with Rf value of  0.59 (Fig. 9). The 50% aqueous-ethanolic extract also showed the presence of a single purple spot with yellow smear. The purple spot appeared with same Rf value of 0.59 suggesting the presence of alantolactone. However, amount of alantolactone extracted in 50% aqueous-ethanolic was observed to be lesser with respect to

Table 2 The Total Polyphenol content.

Total polyphenol content (mg of gallic acid/mg of extract)

Ethanolic extract

Aqueous extract

50% Aqueous ethanolic extract

9.641  6.271

18.693  3.075**

17.729  0.843**

The total polyphenol content of I. racemosa root extracts (ethanolic, aqueous, 50% aqueous-ethanolic) is determined. The data is represented as mg of gallic acid/mg of extract. All the data is expressed as mean  S.D. (n = 3). ** p = 0.04 vs ethanolic extract.

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(800 mg/ml) showed morphological changes after 48 h with respect to untreated (data not shown). The concentration up to 400 mg/ml was found to be non-toxic and no significant morphological changes were observed. The ethanolic extract was found to be toxic to NKE cells at all tested concentrations (25–800 mg/ml; Fig. 5b) measured as morphological alterations and proliferation discussed elsewhere. The 50% aqueous-ethanolic extract showed toxicity to NKE cells from 50 mg/ml. The higher concentrations are found to alter morphology of cells (Fig. 5c). The NKE cells treated with 50 mg/ml of extract were found to be elongated and with increased granularity as observed after 96 h.

Fig. 3. The Total Polyphenol Content: The total polyphenol content of I. racemosa extracts (ethanolic, aqueous, 50% aqueous-ethanolic) at different concentration (12.5–400 mg/ml) is shown. The data is represented as fold change in absorbance values of final colored product measured at 765 nm for each extract. All the data is expressed as mean  S.D. (n = 3). ***p = 0.0002 vs 400 mgm/ml of ethanolic extract, ###p = 0.0003 vs 200 mgm/ml of ethanolic extract and $$p = 0.0023 vs 100 mgm/ml of ethanolic extract.

Fig. 4. The Total Flavonoid Content: The total flavonoid content of I. racemosa root extracts (ethanolic, aqueous, 50% aqueous-ethanolic) at different concentration (12.5–400 mg/ml) was assessed. The data is represented as absorbance values measured at 420 nm for each extract. All the data is expressed as mean  S.D. (n = 3). **p = 0.0013 vs 400 mgm/ml of ethanolic extract.

ethanolic extract as measured by intensity of purple spot. The yellow smear obtained could possibly due the inherent colour of the extract. The aqueous extract showed a yellow smear without significantly visible purple spot of alantolactone. 4.8. Cytotoxicity screening 4.8.1. Morphological assessment of cytotoxicity on normal kidney epithelial (NKE) cells NKE cells treated with aqueous extract showed no morphological changes even after 96 h at up to 600 mg/ml concentration (Fig. 5a). However, further increase in concentrations of extract

4.8.2. Morphological assessment of cytotoxicity on transformed kidney cells (A498) The aqueous extract was found to be non-toxic up to 400 mg/ml. However, further increase in concentration was found to alter morphology (increased granularity, detachment from surface, round shape) with respect to control as observed after 96 h (Fig. 6a). The toxicity pattern of ethanolic extract in A498 cells was observed to be similar to that of NKE cells. The increasing concentration of ethanolic extract (25 mg/ml–800 mg/ml) was found to be toxic to A498 cells (Fig. 6b). The cells were found to be relatively rounded with high granularity in cytoplasm at all tested concentrations. With respect to control treatment of cells with 50% aqueous– ethanolic showed no-toxic effects up to dose of 25 mg/ml. However, further increase in concentrations was found to alter both cell morphology (Fig. 6c) and proliferation discussed elsewhere. 4.8.3. Morphological assessment of cytotoxicity on transformed kidney cells (ACHN) The treatment of ACHN cells up to 200 mg/ml of aqueous extract was found to be non toxic with respect to control. However, further increase in concentrations of extract showed enhanced alterations in concentration dependent manner. However, few cells at a concentration of 800 mg/ml of aqueous extract were observed to be elongated. The ethanolic extract also exhibited toxicity in ACHN at all tested concentrations. The concentrations of 25 mg/ml or higher were observed to be toxic in relation to changes in cell morphology (Fig. 7b). With respect to control, treatment of cells with 50% aqueous– ethanolic showed no-toxic effects up to dose of 25 mg/ml. However, concentration from 50 mg/ml showed morphological alterations (grow singly and elongated). However, higher concentrations were found to be toxic to cells affecting cell viability (Fig. 7c). 4.9. Cytoxicity screening by sulphorhodhamine assay The aqueous extract was found to be less toxic amongst all the three extract as indicated by IC50 values (Table 4). The aqueous extract showed toxicity at higher concentrations (600 mg/ml or

Table 3 The Total Flavonoid Content.

Total flavonoid content (mg of quercetin/mg of extract)

Ethanolic extract

Aqueous extract

50% Aqueous ethanolic extract

31.585  4.93

61.235  13.83*

66.026  35**

The total flavonoid content of I. racemosa root extracts (ethanolic, aqueous, 50% aqueous-ethanolic) is represented as mg of quercetin/mg of extract. All the data is expressed as mean  S.D. (n = 3). * p = 0.01vs ethanolic extract. ** p < 0.005 vs ethanolic extract.

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Fig. 5. Morphological assessment of NKE cells: (a–c) The microscopic images of NKE cells treated with different concentrations of (a) aqueous root extract (b) ethanolic root extract (c) 50% aqueous-ethanolic of I. racemosa is presented. The images presented were from a single experiment and are representative of findings from three independent experiments. The insert in the right hand corner depicts maginified image of cells.

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Fig. 6. Morphological assessment of A498 cells: (a-c) The microscopic images of A498 cells treated with different concentrations of (a) aqueous root extract (b) ethanolic root extract (c) 50% aqueous-ethanolic of I. racemosa is presented. The images presented were from a single experiment and are representative of findings from three independent experiments. The insert in the right hand corner depicts maginified image of cells.

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Fig. 7. Morphological assessment of ACHN cells: (a-c) The microscopic images of ACHN cells treated with different concentrations of (a) aqueous root extract (b) ethanolic root extract (c) 50% aqueous-ethanolic of I. racemosa is presented. The images presented were from a single experiment and are representative of findings from three independent experiments. The insert in the right hand corner depicts maginified image of cells.

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higher) while lower concentrations (<400 mg/ml) showed no significant morphological changes and alterations in proliferation in case of all the three tested cell lines (Fig. 8a–c). The IC50 value of aqueous extract was found to highest in ACHN cells however, the microscopic analysis reveals that concentration of 200–600 mg/ml of aqueous extract bring about physiological changes in the cell indicating stressed cells condition. These vacuolated stressed ACHN cells seem to uptake SRB dye thereby it may be overestimating the IC50 value. The ethanolic extract exhibited least IC50 values with NKE cells, ACHN cells and A498 cells indicating that the extract was most toxic of all the three extracts been tested. The ethanolic extract was more toxic to the renal cancer cells (ACHN and A498) as compared to normal kidney cells (NKE). Similar toxicity trend was noticed with the aqueous-ethanolic extract which was observed to be more tolerant to the NKE cells in comparison with transformed cells. 5. Discussion The trans-Himalayas region of Ladakh has its characteristic vegetation differing from other parts of Himalayas due to the prevailing extreme environmental conditions. The region is marked by the extreme temperatures (average winter and summer temperature of 35  C and 40  C respectively), intensive solar radiations (infra-red and ultra-violet), extreme day and night temperatures fluctuations, low relative humidity and fast blowing winds [29]. Thus, these unique abiotic stresses may lead to upregulated expression of secondary metabolites or intracellular anti-oxidant as an adaptive response in the high altitude vegetation. The secondary metabolites are generally secreted by roots of the plant and are useful to native vegetation for overcoming the natural stress conditions in which they thrive and also for the regulation of their own growth. These plant’s secondary metabolites include families of phyto-chemicals such as

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alkaloids, flavonoids, steroids, terpenoids, phenols, carotenoids, isothiocyanates and others. Most of these phytochemicals combat oxidative stress by acting as a direct natural antioxidants and scavenging reactive species. They may also act as indirect antioxidant and induce the activation of endogenous antioxidant system to neutralize the oxidants [30]. I. racemosa, a stout alpine perennial herb found in the cold dry habitat of North-West Himalayas (2700m–3500 m in the Leh, India) is one of the well known medicinal plant in Ayurvedic system of medicine whose medicinal properties is widely known and studied at rodents as well as human levels [31]. Although the medicinal benefits of Inula species are immense, still much scientific studies needs to be performed in case of many diseases or ailments. The aim of the present study was to find the better solvent system (aqueous or organic/ethanolic) for the preparation of root extract to explore the potential of plant for further in vitro and in vivo applications. Hence, the anti-oxidant/scavenging potential (ABTS and FRAP), reducing capacity, total polyphenol content and total flavonoid content of aqueous extract, ethanolic extract as well as 50% aqueous-ethanolic was evaluated and compared. The anti-oxidant capacity of extracts was assessed by ABTS radical scavenging ability as well as by Fe+3 reducing ability (FRAP assay). The outcome of both the assays highlighted aqueous extract exhibiting highest anti-oxidant potential amongst the three extracts. The ethanolic extract showed least ABTS radical scavenging and Fe+3 reducing capacities. However, the antioxidant capacity of the extracts as assessed by ABTS assay and FRAP assay was found to be comparatively less than that of standards used in the studies (ascorbic acid and gallic acid). This finding may be suggestive that the administration of root extracts may just be a source providing minimal levels of exogenous antioxidants. However, the existing works on Inula species indicates that the plant contains sesquiterpene lactones as active constituents [32]. These sesquiterpene lactones (isoalantolactone,

Table 4 Preliminary Phytochemical screening. S.No

Phytochemical

1

Alkaloids Hager’s test Mayer’s test Carbohydrate Fehling’s test Benedict's test Amino acids & Protein Biuret test Xanthoproteic test Ninhydrin test Glycosides Borntrager test Keller Killiani test Terpenoids Salkowski test Phytosterols/Steroids Liebermann-Burchard’s test Phenolic compounds FeCl3 test Lead acetate test Alkaline reagent test Fixed Oils and Fats CuSO4- NaOH test Filter paper test Gums and Mucilage Saponins Foam test Olive oil test

2

3

4

5 6 7

8

9 10

Aqueous

Ethanolic

50% Aqueous-ethanolic

– –

– –

– –

+ –

+ +

+ –

– – –

– – –

– – –

– +

– +++

– ++

+++

+++

+++

+++

+++

+++

++ ++ +

– – +

+ + +

– – –

– – –

– – –

– –

– –

– –

Preliminary Phytochemical screening indicating presence or absence of different plants metabolite with () indicates absence, (+) indicates positivity, (++) indicates strong positivity in test while (+++) indicates very strong positivity in test.

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Table 5 The IC50 values of root extracts of Inula racemosa. Cell Type

Aqueous extract

Ethanolic extract

50% Aqueous-ethanolic extract

NKE ACHN A498

410.3  1.62 803.3  2.78 666.0  1.01

61.87  2.23 26.00  3.11 50.25  0.98

167.30  1.89 27.42  3.45 105.90  1.22

The IC50 values of I. racemosa root extract (aqueous, ethanolic, 50% aqueous-ethanolic) for each cell lines (NKE, ACHN and A498) were determined from regression equation between extract concentration and its corresponding O.D. of SRB dye uptake.

alantolactone, eupatolide etc) are known to activate of Keap1/Nrf2/ ARE pathway [33–35]. The Nrf2-ARE is a major intrinsic antioxidant pathway that provides cyto-protection against electrophiles and reactive oxygen/nitrogen species by promoting the expression various anti-oxidant enzymes/molecules [36]. The downstream anitioxidant biomolecules may include HO-1, NQO1, GCL, GST, Catalase, SODs, TRX etc. The major sesquiterpene lactones reported in Inula racemosa are alantolactone, isoalantolactone, dihydroalantolactone, dihydroisoalantolactone, daucosterol, eupatolide sitosterol, inunolide, aplotaxene, phenylacetonitrile, isoinunal, isoalloalantolactone, alloalantolacone and iinunal [32,37–39]. Thus, these observations may imply that administration of I. racemosa extracts may prove beneficial in boosting the body’s intrinsic anti-oxidant signaling rather than administration of external source of anti-oxidants. The reducing capacity of extracts as measured by reduction of ferricyanide complex to ferrous form showed similar trend as that of total anti-oxidant capacity with aqueous extract exhibiting statistically higher reducing capacity in comparison with that of ethanolic extract. The polyphenol and flavonoid contents of the plant possess positive correlation to its anti-oxidant capacity [40]. The aqueous and aqueous-ethanolic extracts were found to be better than ethanolic extract in their total polyphenol and total flavonoid contents. However, I. racemosa root extracts showed low flavonoid content which is in accordance with the view that roots of I. racemosa acts by inducing cellular anti-oxidant pathway rather than acting as anti-oxidant itself. Plant metabolites such as alkaloids, carbohydrates, proteins, glycosides, phenols, flavonoids, saponins, phytosterols, terpenoids or their derivatives manifest diverse range of beneficial biological activities. These secondary plant metabolites are harnessed by humans either for the alleviation of diseases or as dietary supplements. These phytochemicals find wide application in cosmetics, medicinal/pharmaceuticals, pesticides and biochemistry industries. Thus, the study on the types of metabolite produced by plant becomes necessary. The present findings suggest that the root extracts of I. racemosa is rich in terpenoids and phytosterols. This observation is in accordance with the fact that plant root has been widely known to consist of mainly sesquiterpenes or its esters [13]. Our study also showed that the extracts are low in their phenolics content. This finding is similar to that of quantitative chemical assay, which states that plant extract are not very rich source of flavonoids. However, among the three extract, aqueous extract was found to be better in total phenolic content. Further, the extracts exhibited low presence of carbohydrates mainly reducing sugars. In general, the plant sugars are stored in root or rhizome and serve either as source of energy or support the endophytic microfungi. The root of Inula species are reported to contain a fructan named Inulin which plant utilizes for storage of energy. Also, the Inula species have been known to contain various types of glycoside namely phenolic glycosides, flavonol glycosides and kaurane glycosides [41–43]. In the present study, the ethanolic extract of I. racemosa showed strong presence of glycoside as well. The 50% aqueous-ethanolic and aqueous extracts exhibited moderate levels of glycoside. However, the extracts did not

showed presence of proteins, oils and fats, alkaloids, saponins, gums and mucilages. Further, thin layer chromatography was performed with the extracts for the presence of terpenes. Since, the roots of I. racemosa have already been known to contain sesquiterpene lactones namely alantolactone, we tested our extracts to find the degree of extraction of alantolactone. The TLC was run with alantolactone as standard. The TLC profiling of extracts depict that alantolactone is the present abundantly in ethanolic extract and comparatively less in abundance in 50% aqueous-ethanolic, while aqueous extract did not contain detectable concentration of alantolactone. These observations imply that aqueous extract either to be deficient in alantolactone or present in very low quantity. Thus, more stringent chromatography techniques like HPLC, GC–MS etc need to be performed later in order to determine the chemical constituents of aqueous extract of I. racemosa as well as other major and minor components. The cyto-toxicity screening of extracts was performed on kidney cells (normal as well as transformed) with the aim to find the extract that exhibits wide range of non-toxic doses for future in vitro and in vivo applications. The images of cells reveal that the aqueous extract did not bring about any morphological changes in the cells up to concentration of 400 mgm/ml with respect to corresponding control. The ethanolic extract was found to be toxic to cells even at low concentrations, thereby limiting its use in in vivo conditions. The 50% aqueous-ethanolic extract however, was less tolerant to cancerous cells than normal cells. The cell proliferation studies corroborated the microscopic observations as indicated by the IC50 values which indicates aqueous extract been well accepted for in vitro application followed by aqueousethanolic extract. Hence, in order to explore the medicinal potential of the plant for human consumption, the water as solvent system may prove to more favorable amongst other. 6. Conclusions The root extracts of I. racemosa was prepared using different solvent systems (water and ethanol). Our aim was to compare antioxidant capacities, reducing potential, total polyphenol and flavonoid content as well as in vitro toxicity of aqueous, ethanolic and 50% aqueous-ethanolic extracts with a view to find the most suitable extract for future in vitro and in vivo application. Our studies clearly demonstrated that in ABTS radical scavenging and FRAP assays the aqueous extract and 50% aqueous-ethanolic extract showed better antioxidant property as compared to ethanolic extract. However, the antioxidant property of I. racemosa root extract was not comparable to standards used (Gallic acid, Ascorbic acid). Hence, Inula species may not be able to act as direct and rich source of antioxidants. However, there are numerous reports that suggests Inula species to be rich sources of sesquiterpene lactones which are reported to be potent Nrf2ARE pathways inducers [33–35]. This may imply that administration of Inula extracts shall improve endogenous antioxidant expression by inducing intrinsic Nrf2-ARE pathway rather than providing exogenous antioxidants. This may useful in field of chemo/radio-therapy where use of antioxidants is not

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Fig. 8. Cell proliferation study: The cell proliferation study of I. racemosa root extracts (ethanolic, aqueous, 50% aqueous-ethanolic) done through Sulphorhodamine dye uptake at different concentration (25–800 mg/ml) on (a)NKE cells, (b)A498 cells and (c) ACHN cells. The data is represented as fold change in absorbance values measured at 565 nm for each extract. All the data is expressed as mean  S.D. (n = 3).

recommended [44]. However, potent Nrf2 inducers may provide survival in such cases by neutralizing the ROS and mitigating the acute radiation syndrome [45]. The aqueous extract was also observed to exhibit statistically better reducing capacity, total

polyphenol content and total flavonoid content as compared to ethanolic extract. 50% aqueous-ethanolic extract was found to be comparable to aqueous extract in its antioxidant capacity, reducing potential, total polyphenol content and total flavonoid content.

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Fig. 9. The TLC profiling of root extracts: The TLC profiling of ethanolic, aqueous and 50% aqueous-ethanolic extracts was done with 50% methanol as mobile phase. Alantolactone was used as standard. The chromatogram was developed with freshly prepared anisaldehyde-sulfuric reagent. The Rf value was calculated by dividing the distance travelled by the purple spot by distance travelled by solvent front.

However, root extracts of the plant was observed not to be rich source of flavonoids. Further, the preliminary phytochemical screening showed that the extracts of I racemosa chiefly contain terpenoids and phytosterols with moderate levels of phenolic compounds, carbohydrates and glycosides. The extracts were observed to be deficient in proteins, oils and fats, alkaloids, saponins, gums and mucilages. This observation was in accordance with the existing fact that root of I. racemosa contains sequiterpenes, namely alantolactone and isoalantolactone. The TLC profiling of the extracts also confirmed the extracts to contain alantolactone. The ethanolic extract exhibited strong presence of alantolactone followed by 50% aqueous-ethanolic. However, TLC profile of aqueous extract did not showed visible detection of alantolactone thereby, indicating the need of sophisticated chromatographic techniques to categorize its chemical constituents. Further, in order to ascertain the performance of extracts on viability of human cell, the in vitro cell proliferation assay and morphological assessment was done with human renal cells which suggest the aqueous extract to be more tolerant in kidney cell (normal vs transformed) with 50% aqueous-ethanolic extract to be less tolerant and ethanolic extract been toxic at most of the tested concentration. The cell proliferation study also is suggestive of 50% aqueous-ethanolic possessing anti-cancer property. However, further studies need to be conducted with in vitro and in vivo systems for validation of its anti- cancer property. Thus, water as a solvent system observed to be more suitable for exploring medicinal potential of I. racemosa. Competing interests The authors have declared that no competing interests exist. Acknowledgments The study was entirely supported by INMAS, DRDO, Ministry of Defense, Government of India. However, the funding body had no

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