Metabolomics and Marker Based Accelerated Stability of Kanji and Ethanol Extract of its Main Ingredient, Daucus carota L. Roots

A self-fermented probiotic beverage Kanji-prepared from roots of Daucus carota L. subsp. sativus (Hoffm.) Arcang. var. vavilovii Mazk. (Apiaceae)-is widely consumed in many Asian countries due to a number of therapeutic claims. However, this beverage is only available for 2-3 months due to instability and availability of raw material. Therefore, to make this remedy available for longer period, the present study describes accelerated stability and different kinetic parameters of Kanji and ethanol extract of its major ingredient using metabolomics comparison and marker-based HPLC method. The samples were stored at three different conditions of temperature and relative humidity for a period of six months. UV-visible metabolomics fingerprints of both the samples taken at different time intervals compared to assess the stability under stress conditions. The same samples were then analyzed using HPLC method with florescent detection for the determination of ferulic acid contents which were used to determine kinetic parameters and predict shelf life at 25°C. Metabolomics fingerprints comparison showed decrease in peak intensities and appearance of entirely different profiles in samples stored at high temperature and relative humidity. Based on ferulic acid contents, both the products followed zero order degradation. Ethanol extract was found to be having higher shelf life, activation energy and pre-exponential factor than that of the Kanji. The results of the present study indicate that Kanji manufacturers may use UV-visible metabolomics profiles for the assessment of stability and ethanol extract of roots of Daucus carota L. to make this beverage available throughout the year. Page 2 of 8 Citation: Latif A, Hussain K, Bukhari NI, Shafi H, Mazhar M (2018) Metabolomics and Marker Based Accelerated Stability of Kanji and Ethanol Extract of its Main Ingredient, Daucus carota L. Roots. Pharm Anal Acta 9: 589. doi: 10.4172/2153-2435.1000589 Volume 9 • Issue 7 • 1000589 Pharm Anal Acta, an open access journal ISSN: 2153-2435 classified into eight categories which can be used to maintain chemical constancy in products [3]. A marker(s) which is characteristic to plant and has pharmacological activity is better choice to determine kinetic parameters and predict shelf life like modern medicine. The authors of this study isolated two components from black carrot root extract, ferulic acid and 6,4’-dihydroxy 3’-propen chalcone with %age yield of 0.1% and 0.005% respectively. Therefore, ferulic acid was considered as major component for further study. Current study described two approaches-UV/Visible metabolomics fingerprint profiling and HPLC method-for the stability studies of Kanji and ethanol extract of roots of Daucus carota L. Materials and Methods Plant material The roots of the plant-Daucus carota L. subsp. sativus (Hoffm.) Arcang. var. vavilovii Mazk.-also called black carrots were purchased from local vegetable market in the month of March. The material was authenticated by Professor Dr. Zaheer ud Din Khan, Department of Botany, Government College University, Lahore, Pakistan; wherein a voucher specimen was deposited vide reference No. G. C. Bot. Herb. 958. The roots were rinsed in tap water to remove extraneous matter and the residual water was evaporated. About 1 kg of dried roots was crushed and 500 g of this material was macerated in 1.0 L ethanol at room temperature for 8 h with occasional shaking. Then the extract was collected and the residue was again extracted with 1.0 L of ethanol. Both the extracts were pooled and dried in vacuum at 40°C. Preparation of fermented beverage (Kanji) The Kanji was prepared using the most commonly used traditional recipe as 113 g vertically sliced thin-long pieces of roots and 5 g each of red chilies, mustard seeds and table salt (sodium chloride) were added in a glass jar containing 1.50 L water. The jar was covered and the contents were allowed to ferment spontaneously at room temperature for 4 days [7,8]. Afterwards, the Kanji was stored at 10°C-15°C until used. The red chillies, mustard seeds and salt are used to impart good taste and color to the Kanji. Ultraviolet/visible spectroscopy: The samples were scanned in ultraviolet and visible region using UV/Visible spectrophotometer equipped with UV-Probe operating system (Shimadzu Scientific Instruments, USA). High performance liquid chromatography (HPLC): Chromatographic analysis was performed using liquid chromatography system 1200 series (Agilent Technologies, Waldronn, Germany) equipped with isocratic pump (G1310 A), auto sampler (G1329 A), column oven (G1316 A), DAD (G 1315 B) and florescent detector (G1321 A). The data acquisition was carried out using ChemStation, version A. 08.03. Stability study protocol The study was performed using protocol described by International Conference on Harmonization (ICH) as suggested by Working Party of Herbal Medicinal Products of the European Agency for the Evaluation of Medicinal Products [4,9]. Kanji and ethanol extract were kept in screw caped transparent glass bottles and exposed for a period of 6 months to three different conditions of temperatures and relative humidity (Table 1). The humidity was controlled by saturated salt solution [10-13]. The samples were withdrawn and analyzed at 0 month (before starting experiment), 1, 2, 4 and 6 months. Preparation of samples for metabolomics fingerprints Kanji having concentration 1.0 mg/ml of ferulic acid was taken as a stock solution. The working sample solution having concentration 0.1 mg/ml of ferulic acid was prepared by diluting the stock solution in ethanol. The stock solution of ethanol extract having concentration 1.0 mg/ ml of ferulic acid was prepared in ethanol. The working sample solution having concentration 0.1 mg/ml of ferulic acid was prepared by diluting the stock solution with ethanol. UV-Visible spectroscopy The working sample solutions of Kanji and ethanol extract were scanned in UV-visible range (200-800 nm) using ethanol as a blank. The UV-visible profiles of samples taken at different time intervals from different storage conditions were compared to observe metabolomics changes. Preparation of samples for HPLC Samples of Kanji taken from different storage conditions at different time intervals were freeze-dried in order to avoid deterioration. The dried Kanji was then dissolved in HPLC grade methanol to get sample solution having concentration 5 mg/ml. Ethanol extract taken from various storage conditions was also dissolved in in HPLC grade methanol to get sample solution having concentration 5 mg/ml. Preparation of standard solutions for HPLC A stock solution of ferulic acid having concentration 1.0 mg/ml was prepared in HPLC grade methanol. Then a range of working standard solutions (10.0, 20.0, 40.0, 60.0, 80.0, 100.0 and 120.0 μg/ml) was prepared by diluting the stock solution with methanol. Chromatographic conditions All the samples and standard solutions for were filtered using 0.45 μm PTFE syringe filters (Whatman, Maidstone, England). Each sample/ standard solution (20 μL) was eluted through column-Eclipse X DB-C18 (5 μm, 4.6X 1500 mm)-using isocratic mobile phase comprising water: methanol: phosphoric acid (300: 200: 2.5, v/v/v) at flow rate of 1.0 ml/ min. The column temperature was maintained at 25°C and fluorescent detection was performed at 250 nm excitation and 410 nm emission wavelengths. The peaks were identified by comparing retention time to that of the standard. Calibration curve was constructed between concentration and peak area for the quantification of the ferulic acid in samples. Determination of kinetic parameters The order of the reaction was determined using graphical method [14,15]. For each storage temperature, graphs were plotted between % concentration and time, lnK and time, and reciprocal of concentration and time for zero order, first order and second order, respectively. The Temperature (oC) Relative humidity (RH %) Saturated salt solution 30 ± 2 65 ± 5 NaCl 40 ± 2 75 ± 5 NaCl 60 ± 2 85 ± 5 KCl Table 1: Storage conditions for stability studies of Kanji and ethanol extract of roots of Daucus carota. Page 3 of 8 Citation: Latif A, Hussain K, Bukhari NI, Shafi H, Mazhar M (2018) Metabolomics and Marker Based Accelerated Stability of Kanji and Ethanol Extract of its Main Ingredient, Daucus carota L. Roots. Pharm Anal Acta 9: 589. doi: 10.4172/2153-2435.1000589 Volume 9 • Issue 7 • 1000589 Pharm Anal Acta, an open access journal ISSN: 2153-2435 correlation coefficient of each of the graphs was evaluated and the plot with the best linearity was taken as order of the chemical reaction. The slope of the plot having the best linearity was taken a rate constant (K) at each storage temperature. Then the rate constants corresponding to three storage temperatures were converted in natural logarithm and the storage temperature in Kelvin is converted into reciprocal (1/T). Then a plot was constructed between lnK and 1/T to get linear regression equation for the determination of K at 25°C, activation energy (Ea) and frequency constant (A). The slope of this plot corresponds to Ea/R (universal gas constant, 8.314 J. mole-1. K-1). Shelf life (t90) depends on the order of the reaction and can be calculated using different equations. For compounds following zero order degradation, a factor 0.105 is divided by rate constant to calculate the shelf life. Data analysis The samples were analyzed in triplicate and the results were averaged and expressed as mean ± SD. Results and Discussion The accelerated stability of Kanji and ethanol extract of roots of its main ingredient, Daucus carota L. was determined using two methods such as metabolomics fingerprint profiling and active marker quantification. Both the samples were kept at different storage conditions such as 30°C/65% RH, 40°C/75% RH and 60°C/85% RH [4]. The samples drawn at different time intervals were analyzed to get metabolomics fingerprints usi