Comprehensive Evaluation of Antioxidant Potential of Selected Osbeckia species and their in vitro Culture , Purification and Fractionation

Background: Health-benefit properties of natural pigments have been intensely studied, especially the anthocyanins. In the last few decades, research on anthocyanins has attracted biologists by the increasing evidence of their health beneficial effects. Osbeckia, belongs to Melastomataceae and is well-known for colouring pigments and other bioactive compounds. In the present study, total anthocyanin and antioxidant capacity indicators were evaluated from 8 Osbeckia spp. and anthocyanin was extracted from in vitro cultures of O. aspera and O. reticulata. Materials and Methods: The antioxidant effect was studied using ABTS (2, 2’-azino-bis-3-ethyl benzthiazoline-6-sulphonic acid) radical cation decolourisation assay, the FRAP, the scavenging ability of hydroxyl radicals and the superoxide anion scavenging activity. Anthocyanin extracted from in vitro cultures were purified and fractionated using column chromatography and LC-MS MS analysis. Results: In vitro cultures of O. aspera was obtained in MS medium fortified with various combinations of Benzyl Adenine (BA), Naphthalene acetic acid (NAA) and 2, 4-D. The chromatograms of O. aspera revealed the presence of malvidin-3 -diglucoside, peonidin, delphinidin and cyanindin whereas O. reticulata cultures accumulated large amounts of malvidin, cyanindin and cyanidin aglycone. The purified anthocyanins of these species were evaluated for their antioxidant potential and was found more remarkable than the crude extracts. Conclusion: Osbeckia species are rich in anthocyanin and therefore display potential AOX power. O. aspera and O. reticulata callus was induced in vitro production of anthocyanins. The pool of anthocyanins was purified and fractionated by LCMS/MS and AOX assays were performed with the purified anthocyanin which showed higher level activities.


INTRODUCTION
All over the world, people utilized the herbals for the treatment of various ailments before the advent of allopathic medicine.Medicinal plants have been a major source for ayurvedic drug development.Plant extracts and products are used in the treatment of bacterial, fungal and viral infections.Natural phytochemicals are known to contain lead molecules that can be used for therapeutic purposes or as precursor for the synthesis of novel useful drugs.The natural products play an important role in drug development in pharmaceutical industry.Currently plants are used as a source of medicine and also important in terms of health care system.The genus Osbeckia contains about 12 species.Species of this genus are used in traditional medicine for stress mitigation, detoxicating, hematischesis and also as astringent.Chemical constituents reported from O. crinita, O. aspera and O. chinensis showed the presence of flavonoids, organic acid and steroids.
In experimental studies, the aqueous extract of Osbeckia was shown to protect cells from hepatotoxicity induced by carbon tetrachloride. 1,2D-galactosamine, t-butylhydroperoxide and bromobenzene in vitro. 3The aqueous extract of Osbeckia provided significant protection against paracetamol-induced in vivo liver injury, as assessed by histological changes and liver enzyme levels.In vitro experiments were also performed to test the ability of the aqueous plant extract to protect freshly-isolated rat hepatocytes against damage produced by 2,6-dimethyl N-acetyl p-quinoneimine (2,6-diMeNAPQI), a stable analogue of the toxic metabolite of paracetamol.Thus, there is good evidence that the aqueous extract of Osbeckia has direct protective effects on hepatocytes, and therefore be involved in inducing immune system against viral hepatitis.Antioxidant and immune modulatory effects were investigated with O. aspera and O. wynaadensis. 4

Estimation of Anthocyanin content and antioxidant potential
Protocol by Sutharut and Sudarat was used for the estimation of anthocyanin content in flowers.The absorbance was read at 510 and 700 nm against distilled water as blank. 7ueous acidified methanol and ethanol have been most commonly used in the extraction of anthocyanins.Methanol is not preferred for food use as it is toxic.Ethanol is the most acceptable one for use in food industry.Silica gel, Amberlite IRC 80, Amberlite IR 120, DOWEX 50WX8, Amberlite XAD4 and Amberlite XAD7, were examined for the purification of anthocyanins.
LC-MS/MS analyses were performed on an Agilent 6410A Triple Quad LC-MS/MS system.Briefly, a 100 μL sample was injected onto a Reliasil 4.6 × 250 mm, 5-μm c18 column equilibrated in 95:5 A:B where A was 0.1% Acetic acid and B was acetonitrile.Flow rate was 0.5 mL/min.Anthocyanins were eluted through application of a linear gradient to 80:20 A:B in 25 min.Data dependent MS/MS and MS3 were performed on abundant precursor ions to elucidate chemical structures.Precursor ion scans were obtained in the range m/z 600-1400 and all data manipulation was within Xcalibur software, version 2.0.7.
To estimate the anti-oxidant activity of the plants the flower petal extracts (20 μl) were added to 0.5 ml of methanolic solution of DPPH and 0.48 ml of methanol.The mixture was allowed to react at room temperature for 30 minutes.Methanol served as the blank and DPPH in methanol, without the leaf extracts, served as the positive control.After 30 minutes of incubation, the discolouration of the purple colour was measured at 518nm in a spectrophotometer.The radical scavenging activity was calculated. 8e antioxidant effect of the extracts was also studied using ABTS (2,2'-azino-bis-3-ethyl benzthiazoline-6-sulphonic acid) radical cation decolourisation assay.Radical cations (ABTS+) were produced by reacting ABTS solution (7 mM) with 2.45 mM ammonium persulphate.The mixture was allowed to stand in the dark at room temperature for 12-16 hours before use.Aliquots (0.5 ml) of the different extracts were added to 0.3 ml of ABTS solution and the final volume was made up to 1 ml with ethanol.The absorbance was read at 745 nm in a spectrophotometer and the per cent inhibition was calculated. 9e FRAP assay was performed according to methods described by Benzie and Strain. 10FRAP values, expressed as mmol of Fe(II) equivalent per g plant material, was obtained by comparing the absorption change in the test mixture with doses obtained from Fe(II) standard concentrations curve.
The chelating activity was determined by the method of Dinis et al. 11 The metal chelating capacities of the extracts were expressed as mg EDTA equivalents/100 g extract.A modified thiobarbituric acid-reactive substance (TBARS) assay was used to measure Inhibition (%) of lipid peroxidation. 12Total antioxidant capacity of the plants was evaluated by the phosphomolybdenum method according to the procedure describe by Prieto and his colleagues. 13The ability of extracts to scavenge hydrogen peroxide was estimated according to the method of Ruch et al. 14 The scavenging ability of hydroxyl radicals is measured by the method of Kunchandy and Rao. 15 The superoxide anion scavenging activity was measured as described by Robak and Gryglewski. 16beckia octandra was studied for its hepatoprotective activity.Further O. octandra, O. chinensis and O. nepalensis were showed to exhibit antidiabetic activity. 5espite all these reports, the use of Osbeckia fruits and flowers by ethnic people was highly ignored by investigators.Osbeckia chinensis fruits were used by the Meithi community of Manipur to obtain a medicinally important violet dye and this dye was used in olden days for colouring mouth. 6The crushed fruits and flowers are soaked in local made wine or country liquor for best results.The violet dye being anthocyanins, the brightly coloured flowers and fruits also need to be investigated for medicinal properties.Anthocyanins represent one of the most widely distributed classes of plant flavonoids.Apart from their coloring effects, anthocyanins show ability to prevent lipid oxidation, scavenging activity against various artificially generated free radicals.The common aglycon forms of anthocyanidins are cyanidin, delphinidin, peonidin, petunidin, malvidin, and pelargonidin.The prevalent sugar moieties are glucose, rhamnose, xylose, galactose, arabinose, and fructose.The genus Osbeckia is also facing threats of extinction due to pollution and man-made destructive-activities.No authentic records of micro propagation works were reported except that of O. octandra which was used as a horticultural crop in landscaping.The present work is to evaluate the anti-oxidant activity of the anthocyanins obtained from the selected species of Osbeckia and to standardise protocols for the micro propagation of Osbeckia aspera and Osbeckia reticulata.

The Plant material
Flowers of Osbeckia sps.for the present study were obtained from various parts of Idukki district of Kerala such as Munnar hills, Wagamon and Peerumed.Osbeckia aspera, is a perennial shrub distributed both in tropics and sub tropics.Although the plant is propagated naturally by vegetative means as well as through seeds, seed derived plantlets were found to be less frequent in natural habitat and distribution appear in fragmented patches derived from root stocks.Osbeckia reticulata, is a small tree frequently seen in temperate habitats and is distributed above 3000 feet in Western Ghats.Osbeckia aspera was well established in the green house, but O. reticulata failed to acclimatize with the greenhouse conditions and did not remain alive beyond six months.Identity of the plants were confirmed by referring floras and authenticated by herbaria of Jawaharlal Nehru Tropical Botanical Garden and Research Institute, Palode, Trivandrum.

In vitro culture
The collected plantlets were reared in green house of the college Botanical garden and stem segments and leaf cuttings from healthy, disease free plants were used as explants.The explants were subjected to surface sterilisation using 10% teepol solution followed by rinsing in tap water for 60 min.Then, the stem cuttings were immersed in ethanol 70% (v/v) for 30 s and, finally mercuric chloride or sodium hypochlorate was used.Ethanol treatment was found hazardous in the case of leaves.In the last step, explants were washed in distilled and autoclaved water.After sterilisation nodal segments (2-3 cm) were used for multiple shoot induction and inter nodal fragments and leaf cuttings were used for direct organogenesis and callus induction.The explants were transferred to MS culture medium supplemented with various phytohormones, besides the control group (without growth regulators).During the entire process of in vitro culture, the plantlets were kept in 2.5 × 15 cm test tubes.The MS culture media was supplemented with vitamins, sucrose (30 gm /L), and agar (7 gm /L).The culture media pH was adjusted to 5.7 ± 0.1.Media were sterilized by autoclaving for 15 min at 120°C and 15 lbs of pressure.

Statistical analysis
All the experiments were done in 12 replicates.The results were expressed as mean ±SD (n = 10).SPSS-7.5 version was used for the entire analysis.P<0.05was considered as significant.

RESULTS
Antioxidant-based drug formulations are used for the prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes, Alzheimer's disease and cancer.Anthocyanins are flavonoids, the natural pigments giving wide colour range in plants and showing antioxidant potential.The chemical structure of these molecules seems to be responsible for this potential.Anthocyanins are the most oxidized flavonoids with the C ring fully unsaturated with a hydroxyl group.Anthocyanin was extracted from the tissues using a mixture of ethanol and water in the ratio 70:30 acidified with 1% HCl without any accelerator and energy sources.The solid-liquid separation was done by filtration and centrifugation methods.Anthocyanins were isolated and purified by column chromatography in silica gel and subsequently using amber lite.Amber lite XAD7HP had the highest capacity and desorption ratio.As free sugar was the degradation factor for anthocyanins, the free sugar concentration before and after purification were tested and it was found decreased significantly.There are several studies regarding extraction of anthocyanins by solvents.Extraction of anthocyanin from eggplant peel with acidified alcoholic solvent (ethanol:water:HCl;70:30:1, v/v/v) appeared more efficient than those without ethanol. 17The anthocyanin extraction from black rice using 70% methanol obtained higher anthocyanin concentration than 70% ethanol and it was more effective than water and 70% acetone.However, ethanol has been used in the extraction of anthocyanins from black rice because of low toxicity application in food industries compared to methanol. 18he anthocyanin content of the flowers varied considerably among the species (Table 1).Osbeckia aspera was found to contain the highest anthocyanin content followed by Osbeckia virgata and O. reticulata.Within O. aspera, anthocyanin content varied from 20.5 mg/g to 37.61 mg/g in different varieties.Flowers of the wightiana variety of Osbeckia aspera is found to be the best source of anthocyanin among them.Osbeckia reticulata had 30.3 mg/g and O. virgata was found to contain 32.2 mg/g.LC-MS/MS analysis of purified anthocyanin from Osbeckia aspera and Osbeckia reticulata contain unique fractions of anthocyanins, ie, O. aspera was rich in malvidin-3 -diglucoside, peonidin, delphinidin and cyanindin, whereas, O. reticulata showed large amounts of malvidin, cyanindin and cyanidin aglycone.The two species O. aspera and O. reticulata responded differently in in vitro culture environments.High concentration of cytokinins was found to be sufficient for the rapid sprouting and multiple shoot induction in O. reticulata.3.5 mg/L of BAP produced 18 shoots and 4.2 mg/L of KIN produced 15 shoots.In O. aspera the most suitable medium for shoot induction was the combination of BA and NAA, ie, 2.5 mg/L BA and 0.5 mg/L NAA produced 21.28 shoots, 2.5 mg/L BA alone produced 7.57 shoots and 3 mg/L KIN alone produced 5.57shoots.In O. reticulata, addition of auxins along with BA or NAA resulted in slowing down of shoot proliferation and induced irregular differentiation of tissues.Callusing in both species was normal in equal concentrations auxins and cytokinins.Combinations of BA along with NAA and IBA produced compact tissues with morphogenesis and combination of BA with 2, 4-D produced white friable callus.Combination of either of the cytokinins along with both NAA and IBA and higher concentrations of sucrose in the medium (2 to 2.5 fold) induced synthesis of anthocyanin in friable callus, but darkening of the callus and subsequent death resulted within 5-7 days of the beginning of anthocyanin production (Figure 1a).The highest content of anthocyanin in Osbeckia aspera, (ie, 94.4 mg/g) was obtained in 8% sucrose, 0.5 mg/L BA and 0.5 mg/L 2,4-D (Figure 1b).In O. reticulata the highest content (69.8 mg/g) of anthocyanin producing callus was obtained in half strength MS medium supplemented with 8% sucrose, 1.2% BA and 1.4% 2,4-D (Table 2).Anthocyanin content of the callus was found higher than the flower tissues of both the species.Anthocyanin content and profile of the in vitro raised plants were similar to the naturally grown plants.

Total Antioxidant (AOX) potential
The total antioxidant activities of different Osbeckia species anthocyanin extracts are narrated in Table 3.The values of the total AOX potential of anthocyanin extracts expressed in terms of equivalents of ascorbate.This assay gives an estimate of the overall AOX potential of anthocyanin. 13Anthocyanins reduce Mo (VI) into Mo (V) and forms a green coloured phosphomolybdenum V complex, which shows maximum OD at 700 nm.O. aspera anthocyanin extract showed higher activity in the range of 100 µg/ml in comparison to other Osbeckia species, whereas the O. leschenaultiana showed least activity.Similarly, Baskar et al. showed variable total AOX activity among different banana peel extract under different solvent and incubation conditions. 19Purified anthocyanin of O. aspera and O. reticulate showed similar activity at 25 µg/ml.

DPPH radical scavenging assay
The Table 3 displayed shows DPPH radical scavenging potential of anthocyanin from different Osbeckia species.The mean values across the concentration range, clearly indicates the potential of O. aspera in scavenging DPPH free radicals as 61.8 percentage inhibition at 100 µg/ml when compared to other Osbeckia species.DPPH radicals react with reducing agents, during which the electrons become paired off and the solution loses colour stoichiometrically depending on the number of electrons taken up i.e., the solution gradually reduced to yellowish     -are produced in substantial amounts by diverse biological processes and are toxic to cellular components i.e., a precursor of the most reactive oxygen species (ROS), contributing to tissue damage and various diseases.Anthocyanins of O. aspera exhibited optimal ability in scavenging O 2 .-,when compared to the standards quercetin and ascorbate (Table 2).The results suggest that the O. aspera have superoxide radical scavenging activity which can be of significant interest in health prospects in reducing the level of O 2 -which is elevated during oxidative stress in the body.Potential superoxide anion scavenging was noticed with purified anthocyanin of O. aspera and O. reticulata at 25 µg/ml.

Hydroxyl radical ( -OH) scavenging activity
The .OH is one of the ROSs generated in the body.These radicals are produced through various biological reactions such as Iron (II)-based Fenton reaction.The scavenging capacity of O. aspera may be attributed by their phenolic compounds with the ability to accept electrons, which can combine with free radical competitively to decrease hydroxyl radicals.Umamaheswari and Chatterjee analyzed in vitro varied antioxidant of the different solvent fractions of Coccinia grandis leaf extracts. 21The present AOX potential of Osbeckia species are comparable with the above results.Purified anthocyanin of both the species showed remarkable activity ie 68.5 and 63.4 percentage respectively at 25 µg/ml (Table 4).

Metal chelating activity
Iron is an essential element required for normal physiology, but excess of it may leads in cellular injury i.e., it undergo Fenton reaction, these reduced metals may form reactive hydroxyl radicals and thereby contribute to oxidative stress. 22Ferrozine can quantitively form complexes with Fe 2+ .In the presence of chelating agents, the complex formation is broken, leading to less red coloration of the complex.The metal chelating activity of Osbeckia species was presented in the Table 3.In this assay, Osbeckia aspera registered higher metal chelating activity.The scavenging potential and metal chelating ability of the antioxidants are dependent upon their unique phenolic structure and the number of hydroxyl groups.Raghavan et al.,analyzed free radical scavenging potential of Chlorophytum tuberosum and the values were at par with Osbeckia aspera. 23Purified anthocyanin of O. aspera and O. reticulata showed a twofold higher activity at 25 µg/ml (Table 4).

FRAP assay
The results of the FRAP assay of Osbeckia species are reported in the Table 3.All species showed considerable amounts of antioxidant effects from 15.9 µmol of FeSO 4 /g of anthocyanin equivalent to 27.8 µmol of FeSO 4 / g in DMSO extract.Also, FRAP values, in other species were optimal (P < 0.05) and was comparable to α-tocopherol (10 mg/L) as the reference compound.Purified anthocyanin of O. aspera and O. reticulata displayed optimal activities than crude extracts.

DISCUSSION
Plants display diverse secondary metabolites such as alkaloids, polyphenols, saponins, carotenoids and other molecules.Most of them are proven therapeutic compounds in one way or other.Antioxidant potentials of these phytochemicals are indirectly involved in curing many human diseases including cancer, inflammation etc.For example, Subhasree et al evaluated antioxidant potential in selected green leafy vegetables. 20mamaheswari and Chatterjee analyzed in vitro antioxidant activities of the fractions of Coccinia grandis leaf extract. 21Saito et al. extensively activity is dependent on the chemistry of anthocyanins and not all of them show similar activities for scavenging ROS and RNS.The antioxidant ability of anthocyanins depends on the basic structural orientation of the compound because the ring orientation will decide the way by which a hydrogen atom from a hydroxyl group can be donated to a free radical as well as the potentiality of the anthocyanin to support an unpaired electron. 51In addition, the power to scavenge diverse ROS differs among anthocyanins for instance, delphinidine is active against the (being followed by cyanidin and pelargonidin) and pelargonidin is effective against the OH radical. 52Generally the AOX activity of anthocyanins is connected with the number of free OH around the pyrone ring.More number of OH, the higher is antioxidant activity.Anthocyanins with their 3',4'-dihydroxy groups can chelate metal ions easaily to form stable anthocyanin-metal complexes. 53Anthocyanins at pH 2-4 mostly exist in the form of flavylium cations and because of the charge distribution they are susceptible to nucleophilic attack on positions 2 and 4. Therefore it can be postulated that the hydroxylation of an anthocyanin at these positions increases its chelating capacity, protecting, for example, ascorbic acid from metal-induced oxidation.
Anthocyanins with the ortho-dihydroxyl groups have the potential to scavenge OH radicals through the inhibition of .OH generation by chelating iron. 54In addition to the degree and position of OH groups in the B ring on the AOX activity of anthocyanins, the degree and position of methoxyl groups also influenced the stability and reactivity of these pigments, consequently their AOX activities. 47Kähkönen and Heinonen showed that malvidin-3-glucoside and petunidin-3-glucoside showed lower efficacy compared to cyanidin-3-rutinoside and delphinidin-3-glucoside. 55 Free radical mechanism for the semiquinone stabilization formed from the cyanidin oxidation proposed by Castañeda-Ovando et al. 56 Similarly, the activities are also dependent on type of reactive species.Muselík et al. reported that for the FRAP and TEAC assays the methoxylation of hydroxyl groups in 5´ (petunidin-3-monoglucoside) or 3´ and 5´ positions (malvidin-3-monoglucoside) significantly reduced the antioxidant activity. 47However, the AOX activity showed by malvidin-3monoglucoside in peroxynitrite mediated tyrosine nitration was the same as that of delphinidin-3-monoglucoside and cyanidin-3rutinoside activities.The activity of anthocyanins in inhibiting tyrosine nitration decreased in the following order: cyanidin-3rutinoside > malvidin-3-monoglucoside ≈ delphinidin-3 monoglucoside > petunidin-3-monoglucoside. 47 Tsuda et al. revealed that ONOO-scavenging activity of pelargonidin (anthocyanidin) consists firstly in the break of this pigment by the radical with the formation of p-hydroxybenzoic and secondly the reaction of this acid with ONOO-resulting in the formation of 4-hydroxy-3nitrobenzoic acid. 57epending on the pH, the relative proportions of protonated, deprotonated, hydrated, and isomeric forms of anthocyanins occur.These forms play a role in the AOX activity.In addition, the relative proportions of peroxynitrite anion (ONOO-) and its conjugate acid (HOONO), with different reactivities, are strongly dependent on pH.The peroxynitrite scavenging activity of anthocyanins at pH 7.4 (≈ 80% of peroxynitrite was in the anionic form) decreased in the following order: cyanidin-3-rutinoside > malvidin-3monoglucoside ≈ delphinidin-3-mono-glucoside > petunidin-3monoglucoside. 47Anthocyanins glycisylation in fruits and vegetables decrease the AOX capacity by reducing free hydroxyls and metal chelation sites.However, Kähkönen and Heinonen, 55 reported that depending on the anthocyanidin and lipid oxidation models used for antioxidant analysis, different glycosylation patterns either enhance or diminish the AOX power i.e., activities of the glycosides and the aglycons did not differ remarkably in emulsion, whereas in LDL the aglycons showed in general higher activities than the glycosides.In contrast, in bulk oil, the glycosides were more effective than the aglycons.
screened edible herbal extracts with potent scavenging activity against superoxide anions. 22Raghavan et al., reported free radical scavenging potential of Chlorophytum tuberosum. 23Fernando et al., evaluated antioxidant potential of some species of Bomarea of Alstroemeriaceae. 24enugopalan and Revathy proved antioxidant activity of Cassia fistula flower extracts. 25Hadi Al-Anbari and Azeez Hasan compared antioxidant activity in some Citrus leaves and seeds ethanolic extracts. 26icel et al. correlated phenolic profile and antioxidant potential of leaves from selected Cotoneaster species. 27Angel et al. correlated phenolics with antioxidant activity in five underutilized starchy Curcuma species. 28hanavi et al. compared antioxidant activity and total phenols of some date varieties. 29Rathi and Rajput reviewed anthocyanins antioxidant potential of grapes. 30Oliveira et al., quantified total phenolic content and antioxidant activity of Malvaceae species. 31Goodarzi et al., evaluated antioxidant potential and reduction capacity of some plant extracts in silver nanoparticles synthesis. 32Camatari analyzed antioxidant potential of flours from cereals, tubers, beans and seeds and also chemical profile of Curcuma longa flour. 33Vadlapudi and Naidu analyzed in vitro antioxidant capacity of few selected medicinal and mangrove species. 34igueiredo and Lima, accessed antioxidant activity of anthocyanins from Sideroxylon obtusifolium fruits. 35Vaidya et al. compared antioxidant capacity of fresh and dry leaf extracts of sixteen Scutellaria species. 36andhiappan and Rengasamy compared antioxidant activity of different species of Solanaceae. 37Medina-Medrano et al. fractionated phenolic constituents and antioxidant properties of five wild species of Physalis. 38ulniute et al comprehensively evaluated antioxidant potential of selected Salvia species using high pressure methods for the isolation of lipophilic and hydrophilic plant fractions. 39In the present study, anthocyanins from Osbeckia species also showed optimal varied AOX potentials as revealed by the results.
The mode of activity of anthocyanins as antioxidants may be a) decreasing localized oxygen concentrations; (b) preventing chain initiation by scavenging reactive oxygen and/or nitrogen species (ROS/RNS), e.g., superoxide anion, hydrogen peroxide, hydroxyl radical, peroxynitrite; (c) binding metal ions in such a manner that they will not generate species such as HO , ferryl or Fe2+/Fe3+/O2, and/or decompose lipid peroxides to peroxyl and alkoxyl radicals; (d) decomposing peroxides by converting them to non-radical products, such as alcohols; (e) chain-breaking, i.e. scavenging intermediate radicals, such as peroxyl and alkoxyl radicals, to prevent continued hydrogen abstraction. 40,41ntioxidant assays in foods and biological systems can be classified into analysis of lipid peroxidation, or measuring the free radical scavenging ability. 41In assessing lipid peroxidation, several lipid substrates can be used and the AOX activity in these systems can be analysed by measuring the substrate and the oxidant consumption, and the intermediates or the end products formation.For evaluation of free radical scavenging potential, there are methods such as chemical reactions involved: electron transferand hydrogen transfer-based assays or evaluating effectiveness against several ROS and RNS (O 2 , OH, ONOO−, NO, H 2 O 2 ). 427][48][49][50] Fundamentally, this Therefore, the in vitro effect of glycosylation on antioxidant activity also depends on the environment in which oxidations accounts. 51Different number of sugar residues and their position in the anthocyanidin may also have different roles on the AOX activity of an anthocyanin. 52,55The number of sugar residues at the C3 position seems to be unique for AOX activity.The smaller the number of sugar units at C3, the greater the AOX activity i.e., delphinidin and cyanidin-3-rutinoside are less active in the DPPH scavenging activity than their corresponding monoglucosides. 47For example, the glycosylation of malvidin-3-monoglucoside to malvidin-3, 5 diglucoside significantly reduces the AOX ability when measured through the TEAC method, but without significant effect on the inhibition of tyrosine nitration (ONOO-scavenging), or being better as scavenging free radicals when measured through the FRAP assay when compared to malvidin-3-monoglucoside. 47 The presence of acyl groups also shows influence on the AOX activity, nevertheless and as reported for sugar moieties, the scavenging activity is highly dependent on the type of free radicals.Similarly, pyranoanthocyanins of cyanidin, petunidin, malvidin and pelargonidin showed significant capacity to scavenge superoxide anion radicals but did not scavenge OH radicals. 58ncorporation of pyruvic acid into delphinidin-3-monoglucoside and malvidin-3monoglucoside caused significant decrease in AOX activity in aqueous phase assays. 47In the present study the purified anthocyanin contain malvidin 3 glucosides, peonydine, delphynidine and cyanidin.The significant AOX activity showed by the species may be considered as a culmination of activities attributed by these compounds.

CONCLUSION
Osbeckia species contain optimal anthocyanin and therefore display potential AOX power.O. aspera and O. reticulata were subjected to in vitro culture using optimal concentrations of hormones in the MS medium.Callus was further induced by altering the hormones for in vitro production of anthocyanins.Further, the pool anthocyanins were purified and fractionated by LC-MS/MS.As the last phase, AOX assays were performed with the purified anthocyanin which showed higher level activities.Further studies are planned to analyse the antiinflammatory potential of the purified anthocyanin from these two species.
Inhibition of lipid peroxidation by anthocyanin at various concentrations from different Osbeckia species is represented in the Table3.Lipid peroxides are unstable and decompose to form reactive carbonyl molecules responsible for damage of DNA, protein, unsaturated fatty acids, generation of life style diseases.Most abundant among them is malondialdehyde (MDA), which reacts with thiobarbituric acid (TBA) to form a pinkish chromogen.The decrease in the MDA levels in the presence of increased dose of anthocyanin suggests the role of anthocyanins as antioxidants.TBARS assay was used to determine the anti-lipid peroxidation properties of the anthocyanins.The result values obtained indicate a moderate percentage inhibition with O. aspera exhibiting highest inhibition among other species at 100 µg/ml.Remarkable lipid peroxidation inhibition, ie, 74 and 62.3 was showed by the purified anthocyanin of O. aspera and O. reticulata at 25 µg/ml (

Figure 1 :
Figure 1: a -Anthocyanin rich callus produced in 1.4 mg/l 2,4D and 1.2 mg/l BA in O. reticulata; b-friable callus produced in 0.5 mg/l of 2, 4-D and 0.5 mg/l BA in O.aspera; c -Multiple shooting in O. reticulata; d-shoots produced in 2.5 mg/L BA and 0.5 mg/l NAA in O. aspera.

Table 1 : Anthocyanin content of Osbeckia species
product viz., diphenylpicryl hydrazine, with the addition of the anthocyanin.Purified anthocyanin of O. aspera and O. reticulata revealed more remarkable DPPH scavenging activity at 25 µg/ml.

Table 3
20dicates the ABTS radical scavenging capacity of anthocyanin at various concentrations from different Osbeckia species.From the results, it may be interpreted that O. aspera and O.reticulata, among all species, inhibit or scavenge the radical in a dose dependent manner with highest percentage inhibition (47.6% and 40.2% respectively) noticed at 100 µg/ml.Studies by Subhasree et al.20determined the scavenging activity on ABTS radical from selected green leafy vegetables which were at par with the present results.Purified anthocyanin of O. aspera and O. reticulata displayed more activity at 25 µg/ml concentration.

Table 4
). Anthocyanins are polyphenols can donate electrons to H 2 O 2 and converted to H 2 O. H 2 O 2 is a signaling molecule that inactivates a few enzymes directly, usually by oxidation of essential thiol group (-SH) leads to toxic effects that can cross membranes and reacts with Fe 2+ and Cu 2+ ions to form hydroxyl radical.Osbeckia species showed insignificant AOX activity i.e., ranged from 14.5 to 25.4%.Synthetic antioxidants such as quercetin and ascorbate standards yielded 66.1 % and 75.5 % H 2 O 2 scavenging activity.Purified anthocyanin of O. aspera and O. reticulata showed increased activity ie, 49.6 and 41.2 percentage respectively at 25 µg/ml.

Table 4 : AOX Potential of purified anthocyanin of Osbeckia aspera and Osbeckia reticulata from in vitro callus cultures at 25 µg/ml concentration Species of Osbeckia studied
Pharmacognosy Journal, Vol 9, Issue 5, Sep-Oct, 2017