The fresh roots of O. sanctum and O. kilimandsacharicum after harvesting were collected from the experimental fields of CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, Uttar Pradesh, India. The voucher specimen samples of both the species were authenticated and deposited in the herbarium unit under reference number 2013-12598 and 2014-12934 respectively in the Botany and Pharmacognosy Department of CSIR-CIMAP, Lucknow, Uttar Pradesh, India.

The fresh roots of both the species of Ocimum were washed in warm water and dried under the controlled conditions in a hot air oven at 40-42°C.The roots were then segregated and grounded in a hammer mill into a coarse powder of 30-40 mesh size. 500 g each of the dried and powdered roots were extracted exhaustively with methanol in a soxhlet type apparatus. The methanolic extract was concentrated under controlled conditions to get a viscous mass. The extracts were re-dissolved in double distilled water and successively partitioned with hexane, chloroform, ethyl acetate and butanol. All the extracts were concentrated under low temperatures and vacuum to obtain the different fractions. The fractions were air dried at room temperature and stored at 4°C till further use.

Hexane, methanol, butanol, ethyl acetate and chloroform (all LR grade) were purchased from Molychem (India) Ltd. Gallic acid, ferrous sulphate, tris-[hydroxymethyl] aminomethane, sodium nitropruside dihydrate, N-(1-naphthyl) ethylenediamine dihydrochloride, sulphanilamide, sodium carbonate, potassium ferricyanide, ferric chloride, tripyridyl-s-triazine (TPTZ), hydrogen phosphate, and m-phosphoric acid aluminum chloride, potassium dihydrogen phosphate, potassium phosphate monobasic, phosphate buffer saline (PBS), ammoniummolybdate, 2, 2-diphenyl-1 picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO), Folin–Ciocalteureagent, sodium acetate quercetin, and ascorbic acid were bought from Sigma Aldrich, Bangalore, India. HPLC grade solvents were procured from Merck Pvt. Ltd, Mumbai, India, Ultrapure water was prepared using a Milli-Q System (Millipore, Milford, MA, USA).

DPPH radical scavenging activity

DPPH radical scavenging assay is a widely used method for the in vitro estimation of antioxidant potential of extracts or phyto-molecules. DPPH radicals produces a dark purple colour in methanol and its characteristic deep purple colour bleaches on accepting hydrogen from a corresponding donor from active extracts Luciana et al., 2001. The antioxidant potential of the different extracts of Ocimum root were assessed according to the method described by Chung et al. (2002) with some variations as reported by Luqman et al. (2009) Chung et al., 2002Luqman et al., 2009. According to the described method, different concentrations of the samples (10, 25, 50, and 100 µg/ml) were added to 100µl of methanol followed by 500µl of Tris HCl buffer (pH 7.4) and 500µl of the DPPH reagent. The reaction mixture was then incubated at 37ºC for 20 min in the dark and the absorbance was measured at 517nm against a blank buffer and percent DPPH inhibition was calculated with respect to reagent control.

Ferric reducing antioxidant power (FRAP) assay

FRAP assay is based on the principle of reduction of ferric tripyridyl triazine (Fe³⁺ TPTZ) complex to ferrous form Benzie and Strain, 1996. The change in absorption is directly proportional to the sum total of reducing capacity of the electron donating antioxidants present in the reaction mixture. A reagent mixture consisting of 20 mM ferric chloride 10 mM TPTZ, and 300 mM acetate buffer (pH 3.6) in ratio of 1:1:10 was prepared. The Ocimum extracts (10, 25, 50 and 10µg/ml) were taken and 50µl of water and 1.5ml of FRAP reagent were added to it. The reaction mixture was then incubated at 37ºC for 5 min and the absorbance was measured at 593nm. The results were expressed in terms of ferrous sulphate equivalents (FSE) which were obtained from the standard curve of ferrous sulphate Luqman et al., 2011.

Reducing power assay

Phytochemicals have the ability to reduce iron like radicals reacts with potassium ferricyanide (Fe³⁺) to form potassium ferrocyanide (Fe²⁺), which further retorts with ferric chloride to form ferric-ferrous complex. The reducing power potential was measured according to the method of Yen and Chen (1995) with some modifications Negi et al., 2011Yen and Chen, 1995. Various concentrations Ocimum root extracts (10, 25, 50 and 100 µg/ml) were pooled with phosphate buffer (200mM, pH6.6) and potassium ferricyanide. The mixture was heated on water bath at 50ºC for 20 min. The supernatant was then transferred to micro plate and to it distilled water and freshly prepared 0.10% FeCl3 was added and optical density was read at 700nm against reagent blank.

Total antioxidant capacity (TAC) estimation

The estimation of the total antioxidant capacity (TAC) is based on method reported by Prieto et al., (1999) where the Mo (VI) is reduced to Mo (V) in suitable acidic medium that lead formation of a greenish complex of phosphate and Mo (V) Prieto et al., 1999. For the experiment different concentration of the Ocimum root extracts (10, 25, 50 and 100 µg/ml) were mixed with 1ml of the TAC reagent (0.605 M ammonium molybdate, 25 mM phosphate mono-basic and 0.60 M concentrated sulphuric acid). The reaction mixture was heated on boiling water bath for 90 min. The optical density of the formed complex was noted at 695nm against a reagent blank and results were expressed as ascorbic acid equivalent.

Total flavonoids (TF) content

The estimation of the TF content in the extract was performed by colorimetric method as described by Meda et al., 2005 Meda et al., 2005. In this phytochemical estimation, various concentrations of the Ocimum roots extract (10, 25, 50 and 100 µg/ml) were dissolved in 50 L of double distilled water with the successive addition of 10 µl of aluminum chloride (10%), 150 µl of methanol, 1 M potassium acetate, and 280 µl of double distilled water. Optical density at 415 nm was measured and compared to blank reagent. The total flavonoid content in the different Ocimum root extracts were articulated as quercetin equivalent (QCE) as mentioned recently Ahmad et al., 2014.

Total phenolic (TP) estimation

Total phenolic compounds present in the plant extracts was estimated by the assay described by Singleton and Rossi, 1965 with some modifications Luqman et al., 2011Singleton and Rossi, 1965. Different concentrations of the Ocimum root extracts (10, 25, 50, and 100 µg/ml) were taken to which folins reagent was added followed with addition of 7.5 % sodium bicarbonate. The reaction cocktail was then incubated at 37ºC for 90 min and the optical density was measured at 765 nm against a reagent blank. The results were expressed as gallic acid equivalent (GAE) which was obtained from a standard curve of gallic acid.

HPLC-UV/PDA fingerprint analysis

As the ethyl acetate extract exhibited the most potent antioxidant activity, so it was taken for further analytical studies. The extraction was carried out in triplicate, filtered, collected and concentrated under vacuum. The uniformity in the three batches of extracts preparation was assured by their reproducible characteristic chemical chromatographic fingerprint. The phytochemical finger printing was done on a chromatographic system from Shimadzu (Shimadzu Corporation, Kyoto, Japan) which consist of model LC-20AD pumps, a rheodyne manual injector, a model CTO-20A oven and a model SDP-M20 diode array detector. For controlling the LC system, LC-MS Solution 3.21 software from Shimadzu was used on a computer system (3000H Series, Lenovo). A model of CBM-20 Shimadzu interface was used to send the signals from the detector to the computer. The separation was achieved by a gradient elution using X select C18 (4.6 × 250 mm, 5µm) at 35°C. The mobile phase consisting, acetic acid (0.5%, v/v) in water (solvent A) and in acetonitrile (solvent B) was used Kumar et al., 2015. Prior to the use, mobile phase was degassed for 15 min using ultrasonication technique (Oscar Ultrasonics, Mumbai, India). The samples and mobile phase was filtered through a 0.45 µm membrane filter in solvent filtration apparatus (Millipore, USA). The gradient elution was started with 5% B with a flow rate of 1.0 ml/min. Detector wavelength was set at 254nm and the injection volume was 20µl. The data acquisition was performed in the range of 200–400 nm to monitor any possible co-elution in plant sample solution. Therefore, considering maximum chromatographic signal response for fingerprint, wavelength 254 nm was selected.

All the data are expressed as mean± SD (n=3) which were computed with the help of MS Office 2007. Comparisons were made between test compounds and reference control for antioxidant activity by one-way analysis of variance (ANOVA) followed by post hoc Dunnet test, all vs control, and a p value of <0.05 was considered statistically significant.

The crude methanolic extract was prepared and fractionated with the solvents of varying polarity to get different extracts containing mixture of compounds. Better yield was obtained in aqueous fraction for both the species of Ocimum which was followed by hexane fraction. The results of extraction procedure are presented in Table 1 .

Various root extracts of two Ocimum species were evaluated for their free radical scavenging activity by DPPH assay. Our finding showed that methanolic root extract of O. kilimandscharicum has the better scavenging potential followed by the methanolic extract of O. sanctum, order of their DPPH radical scavenging potential was 6>5>3>4>1>2 (O. sanctum) and 12>10>8>7>11>9 (O. kilimandscharicum). Overall, it was also observed that methanolic extract of O. kilimandscharicum exhibited low IC50 value (7.02- 24.19 μg/ml) compared to O. sanctum (12.93- 35.46 μg/ml) for DPPH radical scavenging ( Figure 1 ).

The ferric reducing antioxidant power of the Ocimum extracts was expressed as equivalence of ferrous sulphate and it was observed that reducing power of the extracts increases as the concentration increases. The ethyl acetate extract of O.sanctum and O. kilimandscharicum exhibited better ferric reducing activities among all the tested extract (77.05±2.67 and 80.98±3.50 FSE respectively) which was followed by butanolic extracts (75.84±4.85 and 73.91±4.52 FSE respectively). The ethyl acetate extracts of both the Ocimum species showed the maximum potency to donate electrons to the free radical thus terminating the reactive free radical chain reaction. Interestingly, it was also observed that methanolic and butanolic extracts of O. sanctum exhibited similar ferric reducing activity indicated by 75.84±4.85 and 75.61±3.25 FSE respectively. The order of their ferric reducing potential were found as 4>6>5>3>1>2 for O. sanctum and 10>11>7>9>12>8 for O. kilimandscharicum ( Figure 2 ).

The ethyl acetate root extracts of both the species of Ocimum were found most active in iron reducing capacities as compared to other tested extracts, evidenced by a higher optical density recorded at 700 nm (1.64±0.23 and 2.14±0.36 respectively). The reducing power of these extracts rises as the concentration of the extracts increased and was found to follow the similar pattern as that of ferrous reducing power in FRAP assay. The results of iron reducing assays of various extracts are shown in Figure 3 . The optical density of Ocimum extracts ranged from 0.14±0.01 to 2.14±0.36 at 100µg/ml and the order of their iron reducing property was noted as 4>5>6>1~3>2 for O. sanctum and 10>7>11>9>12>8 for O. kilimandscharicum.

The total antioxidant capacity was determined in terms of ascorbic acid equivalent and amongst the tested species of Ocimum, ethyl acetate extract of O. sanctum showed maximum antioxidant capacity. The total antioxidant capacity of chloroform and ethyl acetate extracts of both the species exhibited better total antioxidant potential compared to other which ranges from 7.56±0.47 to 11.31±1.02 µg AsE/mg of dried extracts. The order of antioxidant capacities was observed to be 4>3>6>5>1>2 for O. sanctum 10>9>7>11>8>12 for O. kilimandscharicum ( Figure 4 ).

Flavonoids are the important secondary metabolites reported for various biological activities. Most of the plant extract reported elsewhere for their antioxidant activities are found to contain flavonoids, therefore we have also quantified the total flavonoids content in the extracts. High flavonoid content was reported in ethyl acetate extracts which was followed by hexane extracts of both the Ocimum species. The aqueous and methanolic extracts of O. sanctum and aqueous and butanolic extract of O. kilimandscharicum were found to possess poor amount of total flavonoids. The presence of high amount of flavonoids in ethyl acetate extracts was positively co-related with total antioxidant capacity and the order of flavonoid content were found to be as 4>2>5~3>6>1 for O. sanctumand 10>8>9>12>7>11 O. kilimandscharicum ( Figure 5 ).

The presences of phenolic compound in plant extract also contribute to its antioxidant activity. The total phenolic content was estimated in terms of gallic acid equivalent. The total phenolic content of the two Ocimum species ranges from 3.46±0.49 to 29.88±2.89 µg GAE/mg of dried extracts and the ethyl acetate fraction of both the species showed better phenolic content with respect to other extracts which was positively co-related with the total antioxidant capacity. The order of the total phenolic content of different extracts was found to be as 4>5>3>1>2>6 for O. sanctum and 10>12>11>9>7>8 for O. kilimandscharicum ( Figure 6 ).

Since the ethyl acetate extract of both the species have shown the potent antioxidant activity, the phytochemical profiling of the ethyl acetate extracts with HPLC-PDA and LC-MS was carried out. The presence of various compounds and the hydroxyl cinnamic acid derivatives was observed. It has already been reported that other species of Ocimum contains many phenolics like rosmarinic acid, caffeic acid, etc. The RP-HPLC chromatogram of the ethyl acetate fraction of O. sanctum revealed eight major peaks which were characterized as flavonoids on the basis of UV-spectra matching ( Figure 7 ). The presences of UV active chromophore due to the presence of two aromatic rings provided great support to the flavonoids aglycones which consequently absorb UV absorption peaks. The first maxima found at 240-285 nm range due to the presence of the ring-A benzoyl system and second maximum at 300-400nm range, to the substitution pattern and conjugation of the B-ring cinnamoyl system Mabry et al., 1970Markham, 1982. Our previous studies on roots of O. sanctum have already revealed the presence of ocimol, galactose, arabinose, β-sitosterol, ocimic acid, ursolic acid, trihydroxy ursolic acid, palmityl glucoside, menthylsalicylic glucoside and capryl tetraglycosidic salicylate Ahmad et al., 2012. The UV spectra of eight major peaks showed two absorptions at the max range of 238-240 nm of band-II and another at the range of 307-354 nm of band-I indicated that these compounds tentatively belongs to 3-substituted flavonol or flavones. According to available characteristic, UV spectra of each peak can be used as an indicative tool for the characterization of C-ring, whereas the MS spectra could provide additional, significant information in prediction of active molecules ( Figure 7 ).

DPPH: 2, 2-diphenyl-1-picrylhydrazyl.

FRAP: Ferric reducing antioxidant power

FSE: Ferrous sulphate equivalent.

GAE: Gallic acid equivalent.

LC/MS: Liquid chromatography–mass spectrometry.

QCE: Quercetin equivalent.

RP-HPLC: Reverse phase high performance liquid chromatography.

TAC: Total antioxidant capacity

UV: Ultraviolet

Envisaged and designed the experiments: ST, KS, SL; Root collection and extract preparation: KA, AA; Antioxidant Test: DKS, SL; HPLC/LC-MS Characterization: J, KS; Data Analysis: KA, DKS, AA, KS, ST, SL; Manuscript writing: KA, DKS, J, KS, ST, SL. All authors reviewed and commented on final draft.