Isolation, Purification of Quercetin from in vitro Cell Suspension Culture of Caesalpinia pulcherrima and its Analysis by HPLC-DAD and NMR

Background: Caesalpinia pulcherrima, belongs to Caesapiniaceae, is a known medicinal plant widely distributed in India and is used in traditional medicine for the treatment of various ailments. Many phytochemicals are reported from the plant as potential source of crude drug. Materials and Methods: An efficient and simple reproducible protocol was developed for callus production using leaf explants of C. pulcherrima. The combination of 2, 4-D, kin and BA, was used for the callus induction. Subsequently, cell suspension culture and quercetin synthesis from in vitro callus was attempted. Role of effect of elicitors (Sucrose, ABA and salicylic acid) in cell suspension culture was carried in MS medium containing 2,4-D + BA + kinetin. Flavonoids was purified, fractionated by HPLC-DAD and NMR. Results: 2, 4-D (2.5 mg/L), BA (2.5 mg/L) + kin (1 mg/mL) was effective for maximum callus induction from leaf explants. Significant cell suspension culture was noticed with liquid MS medium containing 2,4-D (2 mg/L)+ BA (1mg/L)+ kinetin (1.5 mg/L). Sucrose, ABA and salicylic acid (SA) at different concentrations influenced cell biomass and quercetin accumulation. The addition of ABA/SA along with sucrose was found to have no remarkable effect on cell biomass and also quercetin synthesis. However, cells cultured in the medium fortified with 45 g/L sucrose without ABA/ SA showed the highest quercetin content (16.5 mg/g). Flavonoids was purified, fractionated by HPLC-DAD and NMR revealed the presence of 9 components such as quercetin, isoquercetin, quercetrin, rutin, quercetin 3-O-β-D-xyloside, quercetin 3-Oarabinopyranoside, quercetin 3-Oα-arabinopyranosyl (1→2) β-galactopyranoside, isorhamnetin 3-O-rutinoside and an unknown compound. Conclusion: C. pulcherima reveals significant synthesis of quercetin. Quercetin content recorded in cell suspension culture was significantly higher compared with in vivo plants grown in fields and the compounds were identified by NMR.

The biosynthesis of polyphenolic compounds is via shikimate phenylpropanoid pathways.

Caesalpinia pulcherrima Swartz. belonging to
Caesalpiniaceae possess wide folklore medicinal uses.Leaves, flower, bark, and seeds are used in Indian medicine and distributed widely in India.The plant has been considered as a tonic and stimulant.The bark and leaf infusions are used as an abortifacient and cathartic.The plant is rich in active ingredients like caesalpin-type diterpenoids, sitosterol, pulcherrimin, lupeol, lupeol acetate, myricetin, quercetin, rutin, flavonoids, carotenoids, glycosides, peltogynoids, phenols, and steroids. 2 recent decades, there are many well established herbal and plant based medicinal practices like Ayurvedic medicine which are popular in many parts of the world.The World Health Organization reported that 80% of people in the devel-

S45
Effect of sucrose 0.5 g cell biomass obtained from the cell suspension culture which was sub-cultured every 9 th day in liquid MS medium fortified with 2.5 mg/L BA+ 1 mg/L 2, 4-D + 1 mg/L kin and exposed to continuous illumination was inoculated into each of the 100 ml Erlenmeyer flasks containing 20 ml liquid MS medium fortified with different concentrations of sucrose (0, 15, 30, 45, 60, 75 and 90 g/L).Six replicates were used for each sucrose concentration treatment.

Elicitation
Elicitors were employed to the suspension cells to induce the synthesis of quercetin glycosides.Physical elicitors used in this study include light (24 h), dark (24 h) and UV light treatment (45 min/day).Chemical elicitors like gibberellin (0.05-1.5 mg/L), abscisic acid (0.05-1.5 mg/L) and heavy metals like CuSO 4 , and ZnSO 4 (25-100 μM).Organic elicitors such as coconut water (10-50%), yeast extract (0.1-0.5%), phenylalanine (100 -500 µM) and peptone water (0.001-0.1%).Salicylic acid (1-100 μM) and MeJA 100-1000 μM was also trailed.Cultures without elicitor were included as control group.Sampling was performed 6 th day after addition of the elicitors up to 24 th day of culture.Cell aggregates and the liquid medium were separated as per the standard protocol.The fresh cell mass was determined after the cells were harvested.The quercetin content was determined from the harvested cells as stated below.
Effect of sucrose and elicitors combinations 0.5 g cell mass was inoculated into two diverse culture media: (a) liquid MS medium fortified with 1 mg/L BA+ 2 mg/L 2,4-D + 1.5 mg/L kin + 30 g/L sucrose (the normal sucrose content used in MS medium) and (b) liquid MS medium fortified with 1 mg/L BA+ 2 mg/L 2,4-D + 1.5 mg/L kin + 45 g/L sucrose (optimal sucrose concentration that induced the highest quercetin synthesis).SA concentration of 50 µM / 0.1 mg/L ABA was added into the medium at day zero, 3 rd day and 6 th day of culture.The addition of elicitors at the beginning of cell culture (0 day) was used as control.The cultures were maintained under continuous light with intensity of 6.3-11.9µmol photons m -2 sec -1 and the cells were harvested at the end of 9 th day of culture.The cell mass and quercetin content and synthesis were then determined.

Quantification of flavonoids
Total flavonoids were determined by the standard protocol. 8traction, fractionation, and purification of quercetin derivatives Cell suspension cultures were extracted with methanol at room temperature for 48 h and was filtered and concentrated under reduced pressure to yield a crude extract and was diluted in methanol water to a ratio of 9:1, and then extracted successively with n-hexane, dichloromethane, ethyl acetate and n-butanol.Ethyl acetate and n-butanol fractions were separately chromatographed on amberlite XAD-16 columns (2 m × 8 cm).Aqueous methanol solutions (from 0% to 100%, with 10% increments) were used as the mobile phase, and 11 fractions were collected for each extract (fractions EA1-EA11 and B1-B11, for Ethyl acetate and n-butanol, respectively).The fractions were pooled and subjected to Sephadex LH-20 chromatography (30 cm × 45 mm) with methanol water (1:1) as the mobile phase.
Reverse-phase HPLC-DAD analyses HPLC-DAD (High Performance Liquid Chromatography with Diode Array Detector) was used to analyze the chemical composition of ethyl aceate and n-butanol fractions described above.The mobile phase of HPLC-DAD analysis consisted of (A) 1% phosphoric acid in water or (B) 1% phosphoric acid in methanol.Gradients used were: 0-15 min 50-70% of B followed by 15-25 min 70-100% of B. The flow rate was oping countries use herbals for their primary health needs. 3About 40% of compounds used in pharmaceutical industry are directly or indirectly derived from plants because the chemical synthesis of such compounds is either not feasible and/or economically unviable.Therefore, a large number medicinal species are under threat of extinction because of their over exploitation.Tissue culture offers an effective and potential alternative of secondary metabolite production because the amount of secondary metabolites produced in in vitro cultures can be even higher than in parental plants. 4Advances in plant tissue culture have enabled commercial scale production of plant metabolites.Many problems need to be tackled in tissue culture to increase the production of secondary metabolites in plant species.One of the major issues is heterogeneity of plant cells and difference in morphological characteristics of each individual cell from the others, this has a huge effect.Different types of cell cultures (suspension and hairy root) are used to transform the natural molecules like steroid, alkaloid, coumarin, terpenoid, lignan and many others flavouring compounds through biotransformation. 5In this juncture present study is undertaken to analyze flavonoids in Caesalpinia pulcherrima, via cell suspension culture, elicitation of flavonoids, its purification, fractionation and its analyses by HPLC-DAD and NMR.

Plant material
The fresh excised Caesalpinia pulcherrima (L.) Sw. shoot tips and young leaves were collected from their wild habitats.

Sterilization of explants
Explants were washed under running tap water for 20 min to make them free from dirt and foreign particles followed by dipping in 2% tween 20 for 10 to 15 min and then dipped in 1% Bavistin for 30 min, rinsed thrice with deionized distilled water.It was further surface sterilized using 70% ethanol for 3-5 min followed by 0.1% HgCl 2 for a min and then again washed with deionized distilled water thrice to remove the traces of HgCl 2 .

Cell suspension culture
The cell suspension culture of C. pulcherrima was prepared by inoculating 0.5 g (fresh weight) of 9 th day old cells into liquid MS basal medium containing varying concentrations of 0.5 -2 mg/L BA, 0.5-2 mg/L 2,4-D and kin 0.5-2.5 mg/L as the cell proliferation medium. 6,7Sterile stainless steel sieve with 850 μm pore size was used to filter the initial established cell cultures to obtain homogenous cell suspension culture.Homogenous cells or small cell aggregates that has been passed through the sieve (with diameter < 850 μm) were subsequently filtered through What man No. 1 filter paper using an air suction pump to separate the cells from the medium.
The cell cultures were placed on an shaker at 100-120 rpm in a culture room maintained at 24 ± 2 o C under continuous illumination with cool white fluorescent tubes at a light intensity of 6.3-11.9µmol photons m -2 sec -1 .Regularly, the cells were harvested and used as samples for subsequent studies.
1 mL/min and the injection volume 20 μL.The UV-vis spectra were recorded from 254 to 400 nm, with detection at 254 and 365 nm.

Nuclear magnetic resonance (NMR) analysis
13 C-NMR spectra were run on a Bruker AV NMR instrument equipped with 5 mm 13 C operating at 500 MHZ with tetra methyl silane (TMS) as an internal standard.

Statistical analysis
Experiment was repeated three times and triplicates were maintained each treatments.All data are represented as mean ± SD.Data were analyzed by one way analysis of variance (ANOVA) at 5% probability level using SPSS software (version 16).

RESULTS AND DISCUSSION
Total flavonoid content in the flower and leaf extract of C. pulcherrima was 1.9 and 4.2 mg/g tissue respectively.Subsequently, the flavonoids were fractionated by HPLC-DAD.The analysis revealed a pool of fractions displaying varying proportions of compounds having UV absorption typical of flavonoids.In vitro callus initiation was carried out with stem tip and leaf explants of C. pulcherrima.Maximum callus induction (39 -96%) and highest callus proliferation (348±.48-1476 ±0.10mg FW) was observed in half strength MS medium fortified with 2,4-D (2.5 mg/L) + 2.5 mg/L BA and kin (1.5 mg/L) with 30 g/L sucrose for all the leaf explants compared to shoot tip (Figure 1a,b and c) (Table1).Sheeba et al. 9 Studied about the callus induction caused by the effect of phytohormones on in Physalis minima and the best result in terms of % of callus induction (80.67) obtained on NAA (3 mg/L) using leaf as explants after 20 days of inoculation.Ashtari et al. 10 reported efficient callus induction and plant regeneration in Ducrosia anethifolia in stem tips on MS medium (2 mg/L NAA and 1 mg/L BA) and on MS medium containing (3 mg/L NAA and 1 mg/L BA) with stem, leaf and node.In Lilium leucanthum, the highest frequency of plant regeneration (96.7%) and the highest mean number of bulblets per scale (3.07) were obtained on MS medium fortified with 0.5 mg/L BA and 0.1 mg/L NAA. 11Amali et al. 12 trailed MS medium supplemented with 2.0 mg/L of 2,4-D and 0.5 mg/L of kinetin developed embryogenic calli when supplemented with 1.0 mg/L of thidiazuron (TDZ).Lood et al. 13 initiated calli in Lagenaria siceraria with optimum concentration of hormones i.e., 2.0 mg/L 3.3BA + 1.0 mg/L NAA.Bonyanpour and Khosh-Khui 14 induced calli in Punica granatum L. 'Nana' from leaf explants with 1 mg/L BA and 1 mg/L NAA.Hasan et al. 15 induced calli and plant regeneration from shoot tips of Cassia obtusifolia.The highest percentage of callus induction was (96.6%) observed in the medium supplemented with 2.0 mg/L 2,4-D.The highest number of shoots was 5.0 found in the medium having 2.0 mg/L 2.4-D+0.2mg/L Kin.Hasan et al. 16 developed in vitro callus induction and regeneration from nodal explants of Cassia alata.Greenish friable callus was obtained from the excised nodal explants on MS medium supplemented with 1.5 mg/L 2,4-D within 20 days of inoculation.Tan et al. 17 studied the effect of plant growth regulators on callus, cell suspension and cell line selection for flavonoid synthesis from Centella.The highest percentage of callus induction (86.67%) was observed in MS medium fortified with 2.0 mg/L 2,4-D while highest biomass yield (0.27 g dry weight culture −1 ) was obtained in the combination of 2.0 mg/L 2,4-D and 1 mg/L kinetin in MS medium.Aghaei et al. 18 compared different plant growth hormones on callus induction of stem explants in Pistacia atlantica subsp.Kurdica.The high efficient callus formation was observed in the medium containing  19 developed an efficient callus proliferation system for Rheum coreanum.The callus was successfully induced from rhizome explant on MS medium containing 2.4-D (0.2-0.3 mg/L).In the MS medium supplemented with a combination of BA (2 mg/L) and NAA (0.2 mg/L), single NAA (0.5 mg/L), or IBA (0.5 mg/L), a higher number of shoot, root and plantlets was achieved.All these reports confirm that the callus initiation varies among plants with respect to hormonal combinations and explants.Subsequently, the fresh harvested callus was proceeded to initiate cell suspension cultures.
Based on the growth index, the cell culture of C. pulcherrima could be well maintained with an initial inoculum of 0.5 g of fresh cells inoculated into 20 mL of liquid MS medium contain 2 mg/L 2, 4-D + 1 mg/L BA + 1.5 mg/L kin in 100 mL Erlenmeyer flasks and placed on a rotary shaker at 100 -120 rpm.Remarkable cell suspension culture was recorded with these combinations (Figure 2 and table 2).Generally, plant cell suspension cultures fail to detach completely after division, and forms multi-cellular aggregates. 20During subculture, a sterilized stainless steel sieve with 850 μm pore size was employed to separate small cell clumps from the larger clumps.Smaller clumps were selected for the quercetin analysis because as cell aggregates enlarge, cells seated in the interior of a large cell clumps are not properly exposed to nutrients, light, oxygen, and other micro-environmental factors compared to younger peripheral cells.This physico-chemical gradient in turn alters the cell proliferation and phytochemical production in the culture system.The cells cultured in MS medium fortified with 1mg/L BA along with 1 mg/L 2,4-D and 1mg/L Kin exhibited the lowest quercetin content (1.24 ±0.04 mg/g), whereas cells cultured in half MS medium fortified with 2 mg/L 2, 4-D + 1 mg/L BA + 1.5 mg/L kin produced the maximum quercetin content of 6.98 mg/g DW on 24 th day (Table 3).The content of quercetin increased with the growth phase of the suspension culture.
Positive correlation was noticed with biomass and quercetin content in the cell cultures.

Effect of Sucrose
Different sucrose concentration influenced the cell biomass and quercetin synthesis in cell suspension cultures of C. pulcherrima.However, cultures without sucrose (control) showed cell death.Medium fortified with 15 g/L sucrose triggered fresh cell mass (6.8±0.4FW) which was different from the cells cultured in medium fortified with 45 g/L sucrose i.e., optimal dried cell weight was noticed from the cells cultured in the medium fortified with 45 g/L sucrose as compared to medium fortified with 15 g/L (Table 4).Meanwhile, when the level of sucrose was further enhanced (60 g/L or more), there was a reverse trend in cell growth was noticed.
The cells cultured in medium fortified with 15 g/L sucrose yielded the lowest quercetin content (4.4±0.3).However, the cells cultured in medium fortified with high sucrose dose (90 g/L) yielded 2.5±0.04   mg/g quercetin content.Meanwhile, its cell biomass yield was marginal (3.2±0.02g FW) and this resulted in to reduced quercetin.The amount of quercetin produced in the cells cultured in medium fortified with 45 to 75 g/L sucrose achieved remarkable level i.e., 16.5±0.8-3.9±0.24mg/g, each of which was soundly different from the other experimental cell cultures sets.Among these three treatment, culture fortified with 45 g/L sucrose showed the highest growth (10.4 ±0.8 g FW) with optimal quercetin content, hence the addition of 45 g/L sucrose into the cell culture of C. pulcherrima could be used for increasing quercetin level (Table 4).
The relation between quercetin content and sucrose concentration may be due to the osmolytic property of sugar i.e., the increased sucrose content leads to exosmosis in the cell cultures and the supply of 15 g/L sucrose was found to be enough for cellular growth and carbon energy source for metabolism.However, enhanced sucrose level (60 g/L or higher) showed a reverse trend in cell growth of C. pulcherrima (ends in to plasmolysis).Similar trend was reported by Ramirez-Estrada et al. 21by fortifying the culture medium of cell suspension culture with increased sugar content resulted in reduced cell growth.This may be due to the increased osmotic potential in the cell or the high viscosity of the medium which in turn inhibit the normal nutrient uptake.However, Wang et al. 22 stated that if a medium contain all the nutrients in excess, an increase in sugar concentration could result in a proportional increase in cell biomass.Experimenting on Hypericum perforatum cell cultures, they reported that growth rate increased with increase in sucrose level in the medium (15, 30, 45 and 60 g/L) for cultures with initial inocula of 15 and 50 g/wet cells/L.The present results substantiate that higher sucrose level fortified into the culture medium reduce cell biomass due to the increase of osmotic potential which subsequently reduce the nutrient uptake.The cells culture fortified with 15 g/L sucrose exhibited the lowest quercetin content.However, the culture fortified with 90 g/L sucrose yielded decreased cellular biomass and there by the synthesis of quercetin.It is plausible that the high level of sucrose could be related to osmotic stress which results in to cell death as supported by Zhao et al. 23 and hence lower quercetin synthesis in C. pulcherrima cell cultures.Meanwhile, the cell culture medium fortified with 45, 60 and 75 g/L sucrose showed high quercetin synthesis.Therefore, sucrose level in the culture medium should not be exceeded 75 g/L for optimal results.Wang et al. 22 found that for cell suspension culture of Hypericum perforatum, the medium that produced higher cell densities had higher sucrose levels as compared to the medium that produced higher anthocyanin accumulation.This observation was the reverse for cell suspension culture of C. pulcherrima, whereby the medium that enhanced quercetin synthesis had higher sucrose levels and biomass production.A relatively increased concentration of sucrose was also reported to be favorable for flavonoid production in Fagopyrum tataricum cell culture 23 the ajmalicine, serpentine, and quercetin in Citrullus colocynthis cell cultures 24 the phenol production of Calligonum polygonoides 25 the flavonoid accumulation in suspension cultures of Andrographis Paniculata 26 as well as anthocyanin production of grape cells 27 and Camptotheca acuminate cells. 28Phytochemical synthesis could be induced to a certain level of osmotic stress caused by higher sucrose concentration. 23The positive effects of osmotic potential (-0.5 to 0.9 MPa), triggered by increased sucrose concentrations in medium with anthocyanins accumulation was correlated in grapes cell cultures. 29

Elicitation
After standardization of the cell suspension cultures to produce quercetin in optimized MS medium, application of elicitor and precursor were carried to enhance quercetin synthesis.UV-B rays (45 min treatment) increased marginally the yield of quercetin.C. pulcherrima in cell suspension cultures (3.4 ±0.24 mg/g DW) on 18 th day which showed a significant accumulation on 24 th day under physical elicitation by UV light.Meanwhile, light (24 h) or dark (24 h) treatments result no significant changes in the quercetin level in the culture medium.
Similarly, no sound quercetin synthesis was noticed with organic elicitors like peptone water and yeast extract.Meanwhile, among organic elicitors, MS media fortified with coconut water (10 -50%) showed increased quercetin biosynthesis in C. pulcherrima (5.2 ±0.36 mg/g DW) cultures.
The addition of phenylalanine as precursor at lower dose enhanced quercetin content of cells in the plants, which ranged from 3.7 ±0.48 -10.6 ±0.86 mg/g DW.Chemical elicitors, which include GA, ABA and SA at different concentrations, were successful in inducing quercetin biosynthesis in cell suspension cultures of C. pulcherrima (6.2±0.5-14.6 ±1.0 mg/g DW).Impact of heavy metals was also analyzed, zinc sulphate and copper sulphate at various doses showed slight rise in quercetin accumulation (2.4±0.3 -6±0.4 mg/g DW), but the content was lower than that of SA or ABA treatments.MeJ did not showed remarkable increase in quercetin production in cell cultures.Regardless of the parameters, the most promising effect on quercetin synthesis in cell suspension cultures was shown by chemical elicitors, SA (50 μM) or ABA (0.1 mg/L) (14.6±1.0 mg/g DW and 12.8±0.9mg/g DW, respectively) (Table 5).The present results were in consistent with Wang et al. 21who reported a positive correlation between MeJA and flavonoid synthesis in Hypericum perforatum.Similarly, Thanh et al. 30 showed that addition of 200 μM MeJA considerably increased the ginsenoside content in Panax ginseng while, with the increasing concentration (above 200 μM) reduced the ginsenosides content.Bharati and Bansal 31 reviewed that the increased flavonoid caused by MeJA in cell cultures was not always due to increased biomass growth but also by the induction of the enzymes to its synthesis.But in C. pulcherrima cell culture, since the addition of MeJA did not increase the cell biomass and quercetin synthesis.As reported by El-Nabarawy et al. 32 Jasmonic acid and its derivatives play a role in the signal cascade pathway of enzymes catalyzing secondary metabolite synthetic pathways.

Effect of sucrose concentration and day of elicitation by GA/ABA
The results clearly showed that culture medium fortified with 45 g/L sucrose and 0.1 mg/L of ABA or 50 µM GA was not significantly different from the cultures grown in their absence.Cell biomass was also not influenced by the addition of these elicitors at different days into the culture media with 45 g/L sucrose.However, the amount of cell biomass produced was significantly lower than using the culture medium containing 30 g/L sucrose.Cell proliferation medium containing 30 g/L or 45 g/L sucrose with or without the addition of these elicitors did not affect quercetin biosynthesis.However, cells cultured in the medium fortified with 45 g/L sucrose without these showed the maximum quercetin content (Table 4).The reason may be the different feeding time did not influence the synthesis of quercetin.Zhang et al. 33 reported a controversial opinion i.e., addition of jasmonic acid at different feeding time regulates the anthocyanin accumulation in cell suspension cultures of Vitis vinifera.Sajjalaguddam and Paladugu 34 noticed application of phenylalanine enhances quercetin content in in vitro cultures of Abutilon ) .Thus, it can be confirmed that the isolated compound is found to be quercetin.Structural characterization of purified compounds using NMR analysis showed the presence of nine compounds which includes, quercetin, isoquercetin, quercetrin, rutin, quercetin 3-O-β-D-xyloside (Reinutrin), quercetin 3-Oarabinopyranoside (Guajaverin), quercetin 3-O-α-arabinopyranosyl (1→2) β-galactopyranoside, and Isorhamnetin 3-O-rutinoside (Narcissoside) (Figure 3). 38,39,40Besides the expected products one unknown quercetin glycosides from in vitro cultures were noticed by NMR analysis in the plants, of which the biological significance remains to be explored.

CONCLUSION
Quercetin is a naturally occurring flavone found in many medicinal species in the form of quercetin glucosides.Quercetin possesses multiple therapeutic values.In vitro culture offer valuable tool in enhancing the synthesis of secondary metabolites.The present investigation reveals an optimal protocol for the synthesis of quercetin in C. pulcherima.Quercetin content recorded in cell suspension culture was significantly higher compared with in vivo plants grown in fields.Further, the compound was identified by NMR analysis.Future studies are warranted to fractionate the lead molecule, its purification and evaluation of its biological potentialities.

Figure 1a ,
Figure 1a,b,c: In vitro callus induction from leaf explants of C.pulcherima showing green creamy friable type.

Figure 2 :
Figure 2: Viable cells from suspension culture of C.pulcherima

Table 4 : Effect of different concentrations of sucrose on cell biomass and quercetin content (mg/g) of C. pulcherima
37soumian et al.35evaluated the effect of precursors on flavonoid production in Hydrocotyle bonariensis callus tissues.Jedinak et al.36compared flavonoid production in plant tissue cultures using biotic and abiotic factors.Shinde et al.37optimized production of isoflavones in cell cultures of Psoralea corylifolia using elicitation and precursor feeding.