RP-HPLC analysis of phenolic antioxidant compound 6-gingerol from in vitro cultures of Zingiber officinale Roscoe

Relation between 6-gingerol content and antioxidant activity in in vitro grown cultures of ginger was studied. Reverse phase HPLC analysis revealed that rhizome derived callus culture and micropropagated plants produced lowest amount of 6-gingerol compare to conventionally grown plants. The antioxidant activity of extracts was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and Ferric Reducing power assay (FRAP) and correlated with the content of total phenolics and total flavonoids in the extracts. Strong correlation was found between antioxidant activity, total phenolics and 6gingerol content.


Introduction
The rhizome of ginger (Zingiber officinale, Rosc.)Zingiberaceae has long served culinary and medicinal uses (Afjal et al., 2001).It has been used as a spice for over 2000 years (Bartley and Jacobs, 2000).Its roots and the obtained extracts contain polyphenol compounds (6-gingerol and its derivatives), which have a high antioxidant activity (Chen et al., 1986;Herrmann, 1994).Two major groups of compounds including gingerol-related compounds and diarylheptanoids have been reported as bioactive components from this plant (Koo et al., 2001;Masuda et al., 2004).Gingerols and shogaols give ginger its pungency and consist of a homologous series of aldols each containing a phenolic group.Its major pungent constituent, [6]-gingerol has been reported to exhibit antioxidative activity against linoleic acid autoxidation and peroxidation of phospholipid liposomes and to scavenge trichloromethylperoxyl-and 1,1diphenyl-2-picrylhydrazyl (DPPH) radicals (Aeschbach et al., 1994;Sekiwa et al., 2000;Pawar et al., 2011).In addition to these antioxidative effects, our recent study (Ippoushi et al., 2003;Ippoushi et al., 2005) revealed that [6]-gingerol inhibits nitric oxide synthesis in activated J774.1 macrophages and prevents oxidation and nitration reactions induced by peroxynitrite (Radi et al., 2001), a strong reactive nitrogen species.The purpose of this study was to assess the antioxidant activities and to determine 6-gingerol content from the different in vitro ginger extract.

Plant material sterilization and inoculation of explant
Rhizome shoot explant was washed with solution of laboline and rinsed thoroughly with distilled water.After the treatment of 0.1% HgCl2 for 6 minutes, the explants were given three to four washes of sterilized double distilled water and blotted well on a sterilized tissue paper.The rhizome explant cut to appropriate size and cultured aseptically on MS medium

Extract Preparation
Rhizome of conventionally grown plant and in vitro grown plant was removed from field and washed with tap water and then rinsed with double distilled water.Rhizomes along with calli were dried under oven for 60 o C and pulverized separately.The one gram of powder of rhizome/calli from each treatment was dissolved in 25 ml methanol and sonicated for 30 minutes.The mixture was centrifuged at 10,000 rpm for 10 minutes and the supernatant was filtered through Whatmans filter paper no.1.All these extracts were kept at 4°C and for assay diluted extracts were used.

Quantification of Total Phenolic Content (TPC)
Total phenolic content was quantified using modified Folin -Ciocalteu method described by (Wolfe, Wu, and Liu, 2003).The assay mixture was prepared using 0.125 ml different concentrations of standard Tannic acid with 0.250 ml of Folin Ciocalteu reagent, 1.25 ml of distilled water and incubated for 10 min in dark.After 10 min, 1 ml 7% aq.sodium carbonate and 1 ml of distilled water is added and the reaction mixture is incubated in dark for 90 min at 37°C.The absorbance of blue colour was read at 760 nm using distilled water instead of std.tannic acid in the reaction mixture as blank on double beam spectrophotometer.Similarly, extracts prepared were also quantified and the results were compared to the standard curve of above standards and expressed as mg/Tannic or equivalent per gram dry powder for the samples under study.

Quantitative determination of total flavonoid contents
Total flavonoid contents in all the above extracts were determined by using a method given by (Luximan -Ramma et al., 2002).1% plant extract (1.5 ml) was taken for the determination of total flavonoids.To this, 1.5 ml of 2% aluminium chloride in methanol was added.The reaction mixture was incubated for 10 minutes at room temperature.The OD was measured at 368 nm against 2% AlCl3, as blank.The OD measurements were compared to standard curve of Quercetin (a standard flavonoid) concentrations and expressed as milligrams of Quercetin equivalent per gram dry weight of ginger.

Antioxidant activity: DPPH (2, 2-Diphenyl-1-picrylhydrazyl) assay
The antioxidant activities were determined as the measure of radical scavenging using DPPH assay as determined by (Brand-Williams et al., 1995).Three ml of a methanolic solution of DPPH (25ppm) was mixed with 20 µl of different concentration of standard Ascorbic acid and the mixture was incubated for 30 min in dark.The absorbance at 515 nm was measured using methanol as blank.The inhibition percentage of DPPH (% DPPH) was calculated and the results were expressed as ascorbic acid equivalent antioxidant capacity (AEAC) as per method described by (Gil et al., 2000).

Antioxidant activity: Ferric Reducing Antioxidant Power (FRAP)
The ferric reducing/antioxidant power (FRAP) assay was used to measure the total antioxidant power ginger extracts.In the FRAP assay, reductants (antioxidants) in the sample reduce Fe 3+ /tripyridyltriazine complex, present in stoichiometric excess, to the blue colored ferrous form, with an increase in absorbance at 593 nm.The ∆A is proportional to the combined (total) ferric reducing/antioxidant power (FRAP value) of the antioxidants in the sample.Antioxidant activity assays were performed by the method described by (Benzie and Strain, 1996).The results were expressed as

Antioxidant activities, total phenolic and total flavonoid content
The highest phenolics content found to be in conventionally grown rhizome sample (1.57gm/100gm of dry weight) compared to callus (0.648gm/100gm of dry weight) and micropropagated grown ginger rhizome (0.79gm/100gm of dry weight).Similar results were obtained for total flavonoid content which was expressed in Quercetin equivalent.Highest content (1.44gm/100gm of dry weight) of flavonoid was in conventionally grown rhizome sample followed by micropropagated rhizome sample (0.77gm/100gm of dry weight) and lowest was in callus sample (0.75gm/100gm of dry weight).The conventionally propagated plants exhibited highest antioxidant activity compared to in vitro cultures.The highest DPPH and FRAP activity was recorded in conventionally grown plant while in micropropagated plant it was 845.34 and 1901.09µM Ascorbic acid eq.respectively and lowest activity was recorded in callus sample.Present results matches with (Grzegorczyk et al., 2007) work on Salvia officinalis L. Correlation between all the traits was above R 2 =0.90.Strong correlation was found between Phenolic and DPPH (R 2 = 0.999) and Gingerol and FRAP (R 2 = 0.999).The trend of increase or decrease of all activities and content was similar for conventionally grown rhizome, micropropagated rhizome and callus.

Conclusion
As expected, the extracts of callus and micropropagated plants of ginger proved to be less active in all methods tested.It is accepted that the dedifferentiation of plant tissues during establishment of callus and cell cultures is often connected with reduction of content of secondary metabolites.According to our knowledge, there are no detailed data regarding the composition of phenolic compounds in ginger cultures were present.So this preliminary study contributes new knowledge of the composition of phenolic compounds by HPLC analysis.Further studies necessary to explain the differences observed.

Fig. 1 .
Fig. 1.Chromatograms: A: Conventional rhizome, B: Micropropagated rhizome and C: Callus in Shoot cultures of Ophiorrhiza rugosa has reported low amount of Camptothecin.The in vitro grown rhizome has lowest 6-gingerol content because, developing normal size of rhizome comparable to that requires three year (Nirmal Babu,

Table 1 .
Nilesh Pawar 1 , Sandeep Pai 2 , Mansingraj Nimbalkar 3 and Ghansham Dixit 3 RP-HPLC analysis of phenolic antioxidant compound 6-gingerol from in vitro cultures of Zingiber officinale Roscoe Showing activities of DPPH and FRAP in mM and content of Phenolic, Flavonoid and 6-gingerol in %