Antioxidant activity of essential oil of three cultivars of Amomum subulatum and standardization of high performance thin layer chromatography ( HPTLC ) method for the estimation of 1 , 8-cineole

Essential oils of the fruits of three cultivars of Amomum subulatum (Family-Zingiberaceae), such as varlangy, seremna and sawney were isolated. Antioxidant potential was determined using 1,1-diphenyl2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenging and FeCl3-reducing models and the presence of 1,8-Cineole was quantified using the developed high performance thin layer chromatography (HPTLC) method. In the DPPH scavenging model, the IC50 value of the essential oil of the seremna, varlangy and sawney were found to be 172.3, 216.9 and 274.3 μg/ml, respectively, while in the ABTS scavenging model, the IC50 value of the seremna, varlangy and sawney were found to be 27.96, 31.34 and 32.49 μg/ml respectively. The antioxidant power of the FeCl3 reducing model, the absorbance value of the essential oil of the seremna was found comparatively higher than the essential oils of varlangy and sawney. The content of 1,8-cineole was determined by the developed HPTLC densitometric method, and the value of the percentage (mean ± SD, % w/w) content of the essential oils of the seremna, varlangy and sawney were found to be 69.59 ± 1.45%, 48.78 ± 3.21 and 47.84 ± 1.76 respectively. The highest antioxidant value of seremna cultivar may be due to the presence of high content of 1, 8-cineole. The HPTLC method, therefore confirms that monoterpene 1,8-cineole is the main antioxidant compound present in the fruits of A. subulatum.


INTRODUCTION
The fruits of Amomum subulatum or greater cardamom is one of the most expensive fruits of the large family, Zingiberaceae of the Kingdom Plantae (Kumar et al., 2013).It is a perennial herb consisting of subterranean rhizomes and leafy aerial shoots which stands erect at 1.7 to 2.6 m depending on the cultivars.Inflorescence of this plant has a condensed spike, commonly found, and has 10-40 fruits in each spike depending on the cultivars.Flowering season starts during March-April at lower altitudes, and in May, at higher altitudes.Harvesting starts during August-September at lower altitudes and October-December at higher altitudes (Gupta and John, 1987).It is also known by other common names such as, English name: black cardamom, hil cardamom, Bengal cardamom, Nepal cardamom; Sanskrit: Aindri, Sthula ela, Brihatupakunchika, Hindi, Punjabi and Gujarati kali ilaichi; Malayalam: karutta elakka; Marathi: Moto-eldori, Moteveldode and Thorveldoda.There are a number of cultivars of A. subulatum species grown worldwide, with some of the cultivars such as ramsey, sawney, golsey, varlangy, seremna, etc. popularly cultivated in the Sikkim State and North Bengal, Darjeeling (West Bengal State) of India.Sikkim is one of the leading states in India, which produce huge quantity of the fruits of A. subulatum (Dubey and Yadav, 2001).In the area, Sikkim ranked 28 th ; situated in the north eastern side of India and contributes approximately 53% of the world production of the greater cardamom (Sharma et al., 2000;Agnihotri and Wakode, 2010;Joshi et al., 2013).In India, this perennial herb is commonly planted at an elevation of 700 to 1650 m above sea level and is widely used for the condiments, homemade remedies, and Indian and Chinese systems of medicine (Mukherjee, 1972;Madhusoodhanan and Rao, 2001).In addition to India, the other cultivars of A. subulatum is also cultivated in the several other countries such as Nepal, Bhutan, China and Indonesia.
Nowadays, research is concerned with finding the safe and natural antioxidant for the commercial food products to avoid the carcinogenicity of the food products linked with synthetic antioxidants, but in the most of the investigations, the natural antioxidants showed comparatively lower potency than synthetic antioxidants (Ramalho and Jorge, 2006).There is need to investigate the antioxidant potential of the natural sources in the whole extract, because in addition to the principal compounds several other agonist and antagonist compounds present in it, which may together show synergistic potential or other compounds may revert the foremost side effects (Jimenez-Andrade et al., 2003).The oxidative stress is an imbalance between free radical production and antioxidant defences, due to overproduction of free radical through metabolic process and caused several diseases (Uttara et al., 2009;Lobo et al., 2010).Numerous research suggests that consumption of oils, fruits and vegetables or other natural products are important for decreasing the ROS oxidative stress by a variety of mechanisms (Rahal et al., 2014).HPTLC is one of the most advanced form of chromatography, highly applicable for the quantification of active compounds in the extracts, oils and other natural products and it is widely accepted in all over the world (Attimarad et al., 2011;Kathirvel et al., 2012).The main purpose of this study was to select a highly antioxidant varieties of fruits of A. subulatum species following a new approach, that is, comparing the essential oil of three popular varieties of A. subulatum fruits, using DPPH, ABTS and ferric reducing methods along with quantifying the main active constituents 1-8 Cineole using newly developed, reproducible and accurate HPTLC method.

Separation of essential oil
The fruits of sawney, seremna and varlangy cultivars of A. subulatum were coarsely powdered in the mixer grinder and passed through sieve No. 60.About 100 g of each powder was hydrodistilled for 4 h using Clevenger apparatus (lighter than water).Further, distilled essential oil was collected and dried over anhydrous sodium sulphate; the obtained essential oils were transferred using a glass vial, and stored in the deep freezer for further study.

Free radical scavenging activity (DPPH model)
The DPPH scavenging activity of the essential oil was assessed by using reported method (Asnaashari et al., 2016), with slight modification.In short, essential oils and standard (BHT) were separately diluted in methanol to obtain 1000 µg/ml stock solution.80 µg/ml of DPPH solution was prepared.Six dilutions (500 to15.6 μg/ml) of samples and standard were prepared separately.Accurately, 5 ml of DPPH was mixed with 5 ml of samples and standard solution and the mixtures were kept in the dark at 25°C for 30 min.The absorbance was recorded immediately at 517 nm by UV spectrophotometer.Percentage inhibition of free radical was calculated by the following formula: Where, A was the absorbance of sample and standard solution and B was the means absorbance of blank (without essential oil or standard, BHT).Further, a plot between percentage inhibition and concentration of different dilutions of samples and standards was plotted for the estimation of IC50 (50% inhibition).

Free radical scavenging activity (ABTS model)
The ABTS antiradical activity of essential oils and the standard was assessed using reported methods (Yang et al., 2009) that was slightly modified.In short, 1000 µg/ml stock solution was used to prepare the serial dilutions (200 -12.5 μg/ml) of samples and standard.The ABTS solution was prepared by reacting equal amount of ABTS (7 mM) with potassium persulfate (2.45 mM).The reaction mixture was kept in the dark for at least 4 h at 25°C.Further, it was diluted with 0.1 M sodium phosphate buffer (pH 7.4) to acquire an absorbance of 0.70 ± 0.02 at 734 nm.Accurately, 2.99 ml of ABTS solution was mixed with 10 µl of serial diluted solution of samples and standard and kept in the dark at 25°C for 30 min.An absorbance was recorded immediately at 734 nm by UV spectrophotometer.Percent inhibition and IC50 were calculated by equation mentioned in DPPH assay.

Reducing power assay (Ferric chloride model)
The reducing power assay of the essential oils was assessed using reported methods (Olugbami et al., 2015) with slight modification.In short, a stock solution of samples and standard were used for the serial dilutions (200 -25 μg /ml).About 1 ml of solution from each dilution was mixed with 2.5 ml of phosphate buffer of 0.2 mol/l (pH 6.6) and 2.5 ml of 1% C6N6FeK3 solution and were incubated at 50°C for 30 min.About 2.5 ml of 10% trichloroacetic acid (TCA) solution was added into it and was then centrifuged for 10 min at 3000 g.Further, about 2.5 ml of the supernatant was pooled with 2.5 ml of distilled water, shaken with 0.5 ml of 0.1% FeCl3 solution (freshly prepared) and incubated at room temperature for 10 min.The absorbance was recorded at 700 nm against blank (n=3).A graph was plotted between the average of absorbance and concentration for estimation of antioxidant activity.

Chromatographic conditions
The high performance thin layer chromatography (HPTLC) method was developed on a system consisting; TLC applicator Linomat V, automatic development chamber 2 (ADC2), derivatization chamber, TLC plate heater, TLC Scanner equipped with WinCATS software (version 1.4.6) and TLC Reprostar 3 (all from Camag, Muttenz, Switzerland).Separation, derivatization and identification were done with TLC Silica Gel 60 F254 Glass plates, 20 × 20 cm and derivatization of plates was developed by automatic TLC sprayer using Vanillin-H2SO4 reagents.

Preparation of standard and sample solutions
1000 μg/ml w/v of the standard solution of 1,8-cineole, and the Alam and Majumdar 1131 sample solution of the essential oils were prepared separately in the solvent methanol.

Chromatographic conditions
Estimation of 1,8 cineole was performed on a TLC plate without pre-washing.Standard and samples of essential oils were applied to the silica gel plates as 8 mm bands with Linomat V.The plates were developed in automatic development chamber 2 to a distance of 80 mm, relative humidity 50-60%, at 25 ± 5°C, using mobile phase composed of hexane: ethyl acetate (8:2), saturation for 25 min.After development, the plates were air-dried and observed in a visualizer at white light, 254 nm and 366 nm.The reagent Vanillin-H2SO4 was used for the derivatization and then the plates were heated at 100°C using a TLC plate heater.The plate was then cooled and scanned in the scanner at UV 665 nm for the quantification.

Calibration curve for standard
The calibration curve for the standard was prepared after application of 1 to 7 μg on a TLC plate.It was then developed and scanned as per the conditions of chromatography.The peak areas were recorded and the calibration curve of standard 1,8-cineole was made by plotting peak area verses concentration.

Method validation
The intended method was validated, referring to the International Conference on Harmonization guidelines (ICH, 2005).The linearity of the current method for the 1,8-cineole was checked (100 to 700 ng/spot) and concentration was plotted against peak area.LOD (Limit of detection) and LOQ (limit of quantification) were determined from the slope of the calibration curve.The equation 3.3×SD×S ˉ1 and 10×SD×S ˉ1 were used for the determination LOD and LOQ respectively.The accuracy of the present method was tested by the pre examined samples spiked with 1,8-cineole (0, 50, 100, and 150%); the mixtures were re-examined; and the percentage of recovery and RSD (% Relative standard deviation) were calculated.The precision of standard was determined by study of repeatability and intermediate precision.Repeatability precision was measured in 6 replicates of a standard solution at three different concentration levels 300, 400 and 500 ng/spot on the same day (intra-day precision).Intermediate precision was also measured in 6 replicates of a standard solution at three different concentration levels on different days (inter-day precision).Robustness of the method was determined by estimating the effect on small changes in the polarity of the mobile phase.

Statistical data
The mean ± standard deviation (SD), relative to percentage of scavenging activity by means of non-linear regression, followed by log (inhibitor) vs. response -Variable slope (four parameters) was used to estimate the IC50 using software GraphPad Prism, version 7.03 (San Diego, CA).

DPPH free radical scavenging activity
Figure 1 shows the percentage DPPH radical scavenging Table 1.Antioxidant activity of essential oils obtained from the fruits of seremna, varlangy and sawney determined using three different methods such as DPPH, ABTS and FeCl3-reducing models.

Essential oils DPPH method ABTS method
FeCl3-reducing method IC50 (μg/ml)  84.54, 172.3, 216.9 and 274.3 µg/ml of BHT, seremna, varlangy and sawney respectively.The previous study confirmed that the in vitro DPPH free radical scavenging assay is a reliable and stable method for the investigation of antioxidant activity (Amiri, 2012).Pathogenesis of various diseases were caused by free radicals, which are produced during metabolic reaction.Free radicals can oxidize all types of biomolecules, spread in all the body parts and damage cells.The antioxidant compounds that are present in extracts or essential oil of spices and herbs scavenge the free radical and prevent the various diseases.The DPPH antioxidant study of essential oil of fruits of A. subulatum has been previously reported by some of the investigators (Bisht et al., 2011;Dhuley, 1999;Kapoor et al., 2008), but as per our knowledge, this is the first report pertaining to the comparative antioxidant activity of essential of fruits of three cultivars.DPPH and ABTS methods have been widely used for the study of the essential oils (Ballester-Costa et al., 2017;Afoulous et al., 2013).The most powerful scavenging constituent by DPPH was found in the essential oils of seremna cultivar.

ABTS free radical scavenging activity
Figure 2 shows the different levels of ABTS radical scavenging activity in the essential oil of each selected cultivar.The percentage scavenging of essential oil of fruits of seremna was 84.1% comparatively higher than for varlangy (52.6%) and sawney (44.7%).Table 1 shows the IC 50 value of essential oils and standard such as  27.96, 31.34 and 32.49 µg/ml of seremna, varlangy and sawney respectively.Like DPPH, the ABTS radical scavenging activity also reproduces hydrogen-donating ability.ABTS method is commonly used for the antioxidant profiling of the essential oils; it is a method that is generally superior to DPPH especially for the essential oil activity, due to the complexity and polarity of compounds (Okoh et al., 2016;Kaviarasan et al., 2007).
The most powerful scavenging using the ABTS method was found in the essential oils of seremna cultivar, showing IC 50 , 27.96 µg/ml.

Ferric chloride reducing assay
Figure 3 shows the reductive capabilities of the essential oils of fruits of cultivars of A. subulatum cultivars compared with BHT and the results showed different reducing levels.The reducing power of the essential oil of seremna fruit, 1.453 was found to be higher than the varlangy, 1.011 and sawney, 1.001 at 200 g/ml, which increased gradually with a rise in the concentration.The method of reducing power assay based on the reaction between antioxidant compounds and potassium ferric cyanide to form potassium ferrous cyanide, which then reacts with ferric chloride to form complex mixture of ferric-ferrous that has an absorption maximum at 700 nm.The reducing power of the essential oils and BHT increases with the increase in the concentration of antioxidant compounds (Bhalodia et al., 2013).

Selection of mobile phase
Figure 4 shows the HPTLC densitogram of the developed method for the standard 1,8-cineole.For the optimization of mobile phase, different trials were made using several solvents alone and in different proportions.Solvent system consisting of hexane and ethyl acetate was used in the ratio of 8:2, v/v, where a spot was observed at the R f value 0.28±0.12for the standard.

Specificity
The specificity of the method was determined by analysing samples and standard spectra at the peak apex, peak start and end positions of the peak.When excess standards were added to the essential oil of fruits of cultivars to check the specificity of the method, the spectro-densitogram of standard 1,8-cineole and essential oil at λ 665 nm was observed.A satisfactory peak purity was obtained and it was observed that the other essential oil present in the essential oil did not interfere with the standard peak and it was found that chromatogram was stabled in solution and on the TLC plate at room temperature.

Linearity
Table 2 shows the linearity of the developed method, for which a calibration curve of five dilution was prepared by plotting peak area against concentrations.It was evaluated by applying different concentrations of 100 to 700 ng/spot for standard solution 1,8-cineole.A good

LOD and LOQ
Table 2 also shows the limit of detection (LOD) and limit of quantification (LOQ) of the developed method for the identification of 1,8-cineole.The LOD 5.14 ng/b and LOQ, 14.66 ng/b respectively, were calculated for standard solution and validate the sensitivity of the present method.

Accuracy
Table 3 shows the accuracy of the present method; for this the pre-analysed samples were spiked with 50, 100 and 150% of the standard 1,8-cineole and the mixtures were re-analysed using the proposed method.The range of percentage recovery (98 to 99.33%) and percentage relative standard deviation, (0.26 to 0.99 % RSD) for standard 1,8-cineole endorse the accuracy of the present method.

Precision
Table 4 shows the precision data on the intraday and inter-day variation for three different concentration levels.The results of repeatability were stated in terms of the relative standard deviation (% RSD).The low % RSD showed the method is precise for the analysis of 1,8cineole.

Robustness
Table 5 shows the robustness of the present method of the introduction of small changes in the composition of the mobile phase and the effects on the results were observed.Low % RSD indicated that the developed HPTLC method is robust.

Quantification of 1,8-cineole in the essential of A. subulatum cultivars
Table 6 shows the percentage contents of 1,8-cineole in the essential oils of fruits of seremna, varlangy and sawney and these were analysed using developed HPTLC methods.The contents of 1,8-cineole were 69.59 ± 1.45%, 48.78 ± 3.21% and 47.84 ± 1.76% of the essential oils of fruits of seremna, varlangy and sawney, respectively.

Conclusion
The developed HPTLC method was found to be accurate, specific, precise and easy to use for the quantitative analysis of 1,8-cineole.Quantification of the 1,8-cineole using developed method confirmed that the monoterpenes, 1,8-cineole is a principal ingredient found in the essential oil of fruits of A. subulatum cultivars.1,8-Cineole was found to be higher in seremna cultivar fruit in comparing to varlangy and sawney fruits.So, the seremna fruits obtained essential oil is a good choice of antioxidant cultivar in comparison to the other two cultivars fruit.The comparative antioxidant activity of fruit oil further confirmed the antioxidant nature of monoterpene, 1,8-cineole and the study concluded that the higher the concentration of 1,8-cineole, the greater the value of antioxidant.This method can be adopted for the quality control of essential oils and formulations that contains 1,8-ineole as active compounds as well as for the selection of good antioxidant cultivars that contain 1,8-cineole as an active marker.

Figure 1 .
Figure 1.DPPH scavenging activity of essential oils obtained from the fruits of seremna, varlangy and sawney.Values are expressed as mean ± SEM (n=3).

Figure 2 .
Figure 2. ABTS scavenging activity of essential oils obtained from the fruits of seremna, varlangy and sawney.Values are expressed as mean ± SEM (n=3).

Figure 3 .
Figure 3. Ferric chloride reducing assay of essential oils obtained from the fruits of seremna, varlangy and sawney.Values are expressed as mean ± SEM (n=3).

Table 2 .
Linear regression data for the calibration curve (n=6).

Table 5 .
Robustness of the proposed HPTLC method.

Table 6 .
The contents of 1-8 Cineole in the essential oils obtained from the fruits of Seremna, Varlangy and Sawney determined using developed HPTLC method.