A 1H NMR Spectroscopic Method for the Quantification Of

Food Chemistry 331 (2020) 127278

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Food Chemistry

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A 1H NMR spectroscopic method for the quantification of propenylbenzenes T in the essential oils: Evaluation of key odorants, antioxidants and post- harvest drying techniques for Piper betle L. Phirose Kempraia,b, Bhaskar Protim Mahantaa,b, Pranjit Kumar Boraa, Deep Jyoti Dasb,c, ⁎ Jyoti Lakshmi Hati Boruahc, Siddhartha Proteem Saikiaa, Saikat Haldara, a Medicinal, Aromatic and Economic Plants Group, Biological Sciences and Technology Division, CSIR-North East Institute of Science and Technology (NEIST), Jorhat, Assam 785006, India b AcSIR-Academy of Scientific and Innovative Research, CSIR-North East Institute of Science and Technology, Jorhat, Assam 785006,India c Natural Products Chemistry Group, Chemical Sciences and Technology Division, CSIR-North East Institute of Science and Technology (NEIST), Jorhat, Assam 785006, India

ARTICLE INFO ABSTRACT

Keywords: 1H quantitative Nuclear Magnetic Resonance (qNMR) spectroscopy technique has certain advantages such as Essential oil low-temperature operation, authentic structural prediction and short data acquisition time. In this study, a 1H qNMR qNMR method was developed for the analysis of propenylbenzenes (eugenol and seven analogues) in the es- Betel sential oils, a broadly distributed class of natural flavours. It was validated in terms of specificity (methoxy/ Sensory analysis acetate signal), linearity (range 0.05–5.00 mg per assay), sensitivity (limit of detection and quantification 4.4 Post-harvest drying and 14.9 µg/mL respectively), accuracy and precision. The qNMR technique was utilized during the sensory or Antioxidant activity-guided identification of chavibetol as the key odorant and antioxidant in thebetel(Piper betle L., Bangla cultivar) oil, a widely consumed chewing stimulant and valuable flavouring agent. The method was also applied for the evaluation of six different post-harvest drying techniques for betel leaves through the quantitative analysis of unambiguously identified propenylbenzene markers (chavibetol, chavibetol acetate and 4-allyl-1,2- phenylene diacetate).

1. Introduction Raymond, Davies, & Larkman, 2017). Although GC–MS provides a high resolution and sensitivity, it is generally time-consuming (25–60 min The global market size of the essential oils was estimated to be 7.51 for a single run), reports a relative quantification and library based billion USD in 2018 and it is growing with impressive annual growth tentative identification of the constituents. Thermolability of the oleo- (Global Market Insights Inc., 2019). Eugenol (1) and its analogues in- chemicals also may restrict the application of GC (Turek & Stintzing, cluding eugenyl acetate (2), chavibetol (3), methyleugenol (6) are 2013). Few studies have reported liquid chromatography (LC) such as chemically classified as propenylbenzenes and they are the major high performance thin layer chromatography (HPTLC) (Dhalwal, constituents of many essential oils (Supplementary Fig. S1)(Kamatou, Shinde, Mahadik, & Namdeo, 2007; Gopu, Aher, Mehta, Paradkar, & Vermaak, & Viljoen, 2012; Tan & Nishida, 2011). These oleochemicals Mahadik, 2008) or high performance liquid chromatography (HPLC) are extensively used in flavour, fragrance, cosmetics and pharmaceu- (Yun et al., 2010) based analysis of the propenylbenzenes. However, LC tical industries (Kamatou et al., 2012; Tan et al., 2011). Quantitative methods generally require a long run time, reference compound for analysis of these essential oils possessing a large share by the prope- peak identity and often suffer from low solubility or specificity fora nylbenzenes is important when authentication, quality control and complex mixture of non-polar analytes like essential oils. On the other safety are concerned. hand, 1H quantitative Nuclear Magnetic Resonance (qNMR) spectro- Gas chromatography-mass spectrometry (GC–MS) has been used scopy based technique is fast, reproducible and provides higher relia- extensively for the analysis of propenylbenzenes (Ferreira, Lopez, & bility on the structural identification of the constituents. Moreover, it Cacho, 2000; Li, Liu, Wang, Yang, & Han, 2018; Li, Zhang, & Liu, 2015; operates at ambient temperature; thus prevents the degradation of

⁎ Corresponding author. E-mail address: [email protected] (S. Haldar). https://doi.org/10.1016/j.foodchem.2020.127278 Received 28 January 2020; Received in revised form 22 May 2020; Accepted 6 June 2020 Available online 10 June 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved. P. Kemprai, et al. Food Chemistry 331 (2020) 127278 thermolabile analytes. Quantitative analysis of the major chemical were individually subjected to different drying conditions as follows: markers from essential oils through 1H qNMR technique possesses a (a) air-drying at the shade (25–30 °C) for 3 days, (b) sun-drying for great significance in rapid screening and quality control (Pauli, 3 days, (c) oven-drying at 40 °C for 20 h, (d) oven-drying at 60 °C for Godecke, Jaki, & Lankin, 2012; Pauli, Jaki, & Lankin, 2005). However, 6 h, (e) microwave-drying at 520 W for 4.0 min (1.0 min × 4) and (f) very limited information is known in the literature on qNMR methods freeze-drying for 24 h. The remaining batch was further processed dealing with the analysis of propenylbenzene-rich essential oils (Freitas without drying and considered as the control. All the experiments were et al., 2018; Grosch et al., 2013). performed in triplicate using three individual sets of samples within a Betel (Piper betle L.) leaf is consumed as the chewing stimulant single batch. The moisture content in the leaf samples was calculated as majorly in the Asian countries with more than two billion regular [(sample weight – dry weight) / sample weight] × 100%. consumers (Das, Parida, Sandeep, Nayak, & Mohanty, 2016; Guha, 2006). The peppery and spicy essential oil of betel is rich in prope- 2.4. Extraction of the essential oil nylbenzenes. It is mainly used by the flavour industries and in the herbal medicines (Balasubrahmanyam & Rawat, 1992; Das et al., 2016). Fresh or the dried leaves (30 g or equivalent) was subjected to the Betel (Paan) is cultivated in India as an important cash crop in > hydrodistillation in a Clevenger-type apparatus for 10 h. Then, the 55,000 ha land with an annual production of about 9 billion Indian extracted oil was separated and the residual oil in the apparatus was rupees (Das et al., 2016; Guha, 2006). The current price of betel es- taken together through the dichloromethane wash. Further, the col- sential oil in the Indian market is in the range of 28–35,000 Rs per Kg lected oil was dried over anhydrous sodium sulphate, dichloromethane (equivalent to ~ 389–486 USD per Kg) (Dubey, 2019). ‘Bangla’ is one of was removed under reduced pressure and the oil was weighed. Each the popular cultivars of betel in India and it is cultivated in many states experiment was performed in three independent replicates. The oil including Assam, West Bengal, Bihar, Orissa, Madhya Pradesh, Uttar yield was expressed as the percentage with respect to the fresh weight Pradesh and Maharashtra (Preethy, Aswathi, Mannambeth, & Pillai, of the sample. It was stored at −20 °C before the analysis. 2016). The aim of this study was to develop a rapid, sensitive and reliable 2.5. GC–MS analysis 1H qNMR spectroscopy based technique for the quantification of propenylbenzenes (1–8) in the essential oils (Supplementary Fig. S1). GC–MS analysis was performed on Thermo Fisher TRACE GC Ultra Further, the credibility of the developed 1H qNMR method was estab- chromatograph coupled to DSQ mass detector. TR‐5MS capillary column lished through the sensory or bioactivity-guided fractionation and (5% phenyl polysilphenylene‐siloxane, 30 m × 0.25 mm × 0.25 μm) was identification of key odorants and antioxidants in the betel oilfrom used for the separation. Samples were dissolved in ethyl acetate at a Bangla cultivar. The method was also used for the analysis of essential concentration of ca. 0.3 mg/mL and 1.0 μL was injected with a split ratio oil samples obtained from the betel leaves (Bangla cultivar) subjected to of 1:10. The GC runtime was 29.75 min with a column temperature pro- different post-harvest drying conditions. gramme as follows: (a) initial temperature 50 °C for 1.0 min, (b) an in- crement with a ramp of 8 °C/min up to 200 °C and hold for 2.0 min, (c) 2. Experimental section increase up to 300 °C with a gradient of 20 °C/min and a hold for 3.0 min. Helium was used as the carrier gas with a flow rate of 1.0 mL/min. MS 2.1. Chemicals and general experimental procedures parameters were maintained as follows: ion source temperature 200 °C, mass range m/z 50–400 with full scan in positive ion mode. Xcalibur Deuterated chloroform (99.8 atom %D), anhydrous benzene 3.0.63 software integrated with NIST Mass Spectral library (Version 2.0f, (99.8%), dipropylene glycol (DPG, 99.0%) and 2-allylphenol (98%) build 11 Aug 2008) was used for data processing and structural prediction. were procured from Sigma-Aldrich, St. Louis, MO, USA. 2,2-Diphenyl-1- The quantity of individual constituents (mg/g of oil) was determined by picrylhydrazyl (DPPH, > 85.0%) and ascorbic acid (> 99.0%) were injecting a solution (1.0 μL) containing betel oil (0.3 mg) and 2-allylphenol purchased from HiMedia, Mumbai, India. For column chromatographic as the internal standard (0.05 mg) in ethyl acetate (total volume 1.2 mL). purification, 60–120 mesh silica gel (Avra Synthesis, Hyderabad, India) was used and the solvents of purity ≥ 99.0% (Merck Life Sciences, 2.6. NMR instrumentation and optimization Mumbai, India) were employed as the mobile phase. Thin-layer chromatography (TLC) was performed on silica gel G-coated 0.25 mm alu- 1H NMR data were recorded in a Bruker (Karlsruhe, Germany) minium plates (Merck, Darmstadt, Germany). Clove, anise, bay leaf and AVANCE III FT-NMR spectrometer of 500 MHz using 5 mm probe at basil oil were obtained through hydrodistillation in a Clevenger-type 296 K. A conventional pulse program for proton NMR (i.e. zg30) was apparatus for 10 h. Eugenol (1) was purified (> 99.0%) from hydro- used for the acquisition of data with acquisition parameters as follow: distilled clove oil through silica gel column chromatography technique acquisition mode DQD, pre-scan delay 6.50 µs, spectral width using 4:1n-hexane : dichloromethane (v/v) as the eluent. 10,000 Hz, FID resolution ~0.3 Hz, acquisition time 3.277 s, number of scans 8 and relaxation delay 1.0 s. The samples were prepared and data 2.2. Collection and authentication of the plant materials were recorded in deuterated chloroform. The recorded data were analyzed through Bruker TopSpin 4.0.7 software. Baseline and phase cor- The fresh leaves of betel (P. betle) were collected in October 2018 rection was made before further analysis. The residual solvent signal at from Teok, Jorhat (India) located at the geographical coordinate of δH 7.26 ppm was designated as the reference. The stock solution of the 26°50′00.6″N 94°25′50.7″E; altitude 94 m. Identity of the plant species internal standard (IS) (10.0 mg/mL) was prepared by dissolving an- was confirmed by the taxonomist Dr. Dipanwita Banik and the voucher hydrous benzene in CDCl3 and stored at −20 °C. To optimize the re- specimen was deposited at the herbarium of CSIR-North East Institute laxation delay (D1), a solution of 1 (4.0 mg) and IS (0.5 mg) was pre- 1 of Science and Technology, Jorhat, India (specimen no. P. Kemprai pared in CDCl3 (total volume 600 µL). H NMR spectra of the solution NEIST 1897). The collected plant sample was stored at the refrigerator were recorded with varying D1 (1.0, 5.0, 10.0, 20.0 and 30.0 s). The (-20 °C) before further use. peak area ratio of 1 versus IS was evaluated with varying D1.

2.3. Drying techniques for the betel leaves 2.7. Method validation

Collected fresh betel leaves (630 g) were segregated into seven The specificity of the developed method was tested through the batches each consisting of three sets of 30 g each. Six of the batches following three individual sets of experiments. (a) 1H NMR spectra were

2 P. Kemprai, et al. Food Chemistry 331 (2020) 127278 recorded for five selected essential oil samples (clove bud, bay leaf, follows: (A) eluent: n-hexane, 0.44 g; (B) eluent: 4:1n-hexane : di- betel leaf, anise seed and basil leaf) which are known to be rich in chloromethane (v/v), 1.98 g; (C) eluent: 1:1n-hexane : dichloromethane propenylbenzenes. Major propenylbenzenes and their methoxy or (v/v), 0.36 g; (D) eluent: dichloromethane, 0.19 g. TLC analysis was acetate signals were identified in the individual tested oils. Further, the carried out for the fractions A-D in reference to the crude oil using 1:1 possibility of peak over-lapping for those key methyl signals was in- v/v dichloromethane : n-hexane as the mobile phase. Anisaldehyde vestigated. (b) Spiking studies of 1 and 2 were performed with clove staining agent was employed for developing the spots. Further pur- bud oil. A known quantity of 1 or 2 (2.0 mg) was added to clove bud oil ification of the individual fractions B (0.50 g) and C (0.30 g) yielded (2.0 mg) maintaining an assay volume of 600 µL. Specificity was pure chavibetol (3, 0.35 g) and chavibetol acetate (4, 0.11 g) respec- checked through enhancement in the signal intensity exclusively for the tively. Silica gel column (60–120 mesh, L × ID 15.0 × 1.5 cm) chro- added marker (1 or 2). Similarly, the spiking study of 3 and 4 was matography with an eluent of 5:1 n-hexane: dichloromethane (v/v) (1.0 performed with betel leaf oil. (c) Specificity was also tested through the L) and flow rate of 20.0 mL/min was used for this purpose. Purified 3 analysis of samples from multiple locations. Betel leaves were collected and 4 were structurally characterized by 1H, 13C NMR spectroscopic from five different locations of Assam and the essential oil washydro- and mass spectrometric data which agreed well with the previous re- distilled. 1H NMR spectra were recorded for those samples and the ports (Eliasen, Thedford, Claussen, Yuan, & Siegel, 2014; Rathee, Patro, possibility of peak over-lapping was evaluated for the selected methoxy Mula, Gamre, & Chattopadhyay, 2006). The purity of 3 and 4 was de- or acetate signals of the major propenylbenzenes (3, 4 and 5). termined to be > 99% with no detectable impurity as analyzed through To test the linearity of the response, an increasing amount of 1 GC–MS technique. Fractions A-D and purified 3, 4 were stored at 4 °C (0.05, 0.10, 0.25, 0.50, 0.75, 1.00, 2.00, 3.00, 4.00 and 5.00 mg) was until further experiment. added to a fixed quantity of IS (0.5 mg) in a total sample volume of 600 µL. The peak area ratio of 1 versus IS was plotted against their ratio 2.10. Difference from control (DFC) test of molar concentrations and the data points were fit into a linear trend. The linearity was expressed through the linear regression equation and The difference from control (DFC) test was performed in orderto its coefficient of determination2 (R ). evaluate the difference in sensory (odour) perception of the oil-fractions The sensitivity of the method was tested through LOD (limit of A-D, purified chavibetol (3) and chavibetol acetate (4) in reference to detection) and LOQ (limit of quantification) values. An increasing the crude betel oil (Whelan, 2017). A total of 36 panelists were selected concentration of 1 (0.05–1.00 mg) was added to a fixed quantity of IS for this sensory analysis. The panelists with a nasal disorder were ex- 1 (0.5 mg) in CDCl3 (600 µL of assay volume) and H NMR data were cluded. The test included eight coded samples (i) control - crude oil, (ii) recorded. S/N (signal-to-noise) values were determined and they were fraction A, (iii) fraction B, (iv) fraction C, (v) fraction D, (vi) chavibetol, plotted against the concentration. Analyte (1) concentrations producing (vii) chavibetol acetate and (viii) blind control. The samples were S/N values 3:1 and 10:1 were considered as LOD and LOQ respectively. prepared in dipropylene glycol (DPG) at 5.0% (v/v) concentration. The To check the accuracy and precision of the developed method, three panelists were requested to access the odour difference in the individual known concentrations of 1 covering a wide range (0.25, 1.00 and test samples (ii-viii) with respect to the control (i). The degree of dif- 4.00 mg) were added to a fixed quantity of IS (0.5 mg) in 600 µLof ference was scored in a five-point structured ‘difference scale’ (differ- assay volume. The concentration of 1 in each of the samples was de- ence scores: 0 no difference, +1 slightly different, +2 moderately termined using the developed method. Accuracy of the quantification different, +3 largely different and + 4 no similarity). All the assessors was represented by ‘% recovery’ which was calculated as (observed were made familiar with the test instructions and meaning of the dif- value / true value × 100%). To test the repeatability, NMR data were ference scale. The mean values of the difference scores for the in- recorded in triplicate. For checking the intermediate precision (rug- dividual test samples were plotted. Difference score for the blind con- gedness), data for each sample was recorded in three consecutive days trol signified the ‘placebo effect’. (total 9 determinations for 3 samples). The precision of the measurements was represented through the percent relative standard deviation 2.11. Determination of the odour threshold value (OTV) and odour activity (% RSD). value (OAV)

2.8. qNMR analysis of the betel oil samples Odour threshold value (OTV) was determined using the single as- cending method for crude betel oil, chavibetol (3) and chavibetol For the quantitative NMR analysis of the betel oil samples (crude oil acetate (4) in 1:1 (v/v) DPG : water mixture (Tsukatani, Miwa, or oil-fraction), a known amount of the oil (ca. 10 mg) was added to a Furukawa, & Costanzo, 2003). Five dilutions (0.01, 0.05, 0.10, 0.50, fixed quantity of IS (0.5 mg) in a total sample volume of 600 µL.The and 1.00%) were prepared for the individual samples. Each set of the absolute quantity of the selected oleochemical makers (3, 4 and 5) and dilutions was presented to the panelists with an increasing concentra- tentative total propenylbenzene (PB) was determined using the linear tion gradient and asked to detect the odour. The dilution at which the regression equation obtained for eugenol (1) i.e. Manalyte /MIS = [slope odorant stimulus was detected first was considered as OTV. The odour × (Aanalyte /AIS) - intercept]; where (Manalyte /MIS) and (Aanalyte /AIS) activity value (OAV) for 3 and 4 was determined by dividing the in- represented the mole ratio and peak area ratio of the analyte versus IS dividual concentration in the crude oil with their OTV. respectively. The quantity of 3, 4, 5 and PB was expressed in milligrams per gram (mg/g) of the oil sample. 2.12. Triangle test

2.9. Chromatographic fractionation and purification of the crude betel oil The triangle test was conducted with 48 panelists and two samples (crude betel oil and chavibetol) to evaluate their similarity (Sinkinson, Betel oil from multiple batches of hydrodistillation was pooled and 2017). The samples were prepared at 2.0% (v/v) concentration in 1:1 subjected to fractionation through silica gel column chromatography. (v/v) DPG: water mixture. The level of statistical significance was set at The silica gel column (60–120 mesh, L × ID 26.5 × 3.0 cm) was loaded β risk 0.10 and the maximum proportion of discriminators (Pd) 30%. A with crude oil (3.30 g) and eluted with an increasing percentage of set of three samples were presented to the panelists in a randomized dichloromethane in n-hexane (0.5 L of n-hexane, 2.5 L of 4:1n-hexane : manner among which two were identical (crude oil) and the remaining dichloromethane, 0.5 L of 1:1n-hexane : dichloromethane and 0.5 L of one was chavibetol (3). The panelists were requested to identify the odd dichloromethane) with a flow rate of ~ 25.0 mL/min. The elutes were sample in terms of sensory (odour) perception and the total number of individually dried under reduced pressure to produce four fractions as correct identification was counted.

3 P. Kemprai, et al. Food Chemistry 331 (2020) 127278

2.13. DPPH radical scavenging assay delay) were optimized. Optimization and method validation was performed on eugenol (1). Deuterated chloroform was selected for the Free radical-scavenging activity for the crude oil, fractions (A-D) sample preparation due to the high solubility of these oleochemicals and the purified oleochemicals (3 and 4) was determined using DPPH and a residual solvent signal that was not overlapped by the oil con- assay (Paul et al., 2015). The oil sample solution (1.0 mL) of varying stituents. Anhydrous benzene of high purity (99.8%) was used as the concentrations (1–500 µg/mL in methanol) was mixed with 0.003% internal standard (IS) for developing the method. Single, sharp and non- DPPH (0.076 mM) in methanol (1.2 mL). After an incubation of 30 min overlapping NMR signal (equivalent to six hydrogens), sufficient solu- in dark, the absorbance was measured at 517 nm. The percentage of bility and stability in the analytical condition made benzene suitable as inhibition was calculated using the equation: [(ADPPH-AS)/ the IS for the intended purpose. Considering the low-boiling point of the ADPPH] × 100, where ADPPH was the absorbance of the control (DPPH solvent and IS, low-temperature storage was preferred. Storage of the solution without any oil sample) and AS was the absorbance for the test sample at −20 °C in a well-capped NMR tube at least for a week neither sample (DPPH solution with oil sample). The half maximal inhibitory showed a noticeable alteration in the sample volume nor a significant concentration (IC50) was graphically determined by plotting the per- deviation from the desired precision of quantification. The influence of centage of inhibition versus the concentration of the sample (µg/mL). the varying relaxation delay (D1) on the quantification was tested in the Ascorbic acid was used as the standard antioxidant in the assay. range 1.0–30.0 s for 1, in specific for the methoxy and benzylic signals at δH 3.88 and 3.32 ppm respectively (Fig. 1B). It did not alter the peak 2.14. Hierarchically clustered heat map analysis area ratio of 1 (4.0 mg) versus IS (0.5 mg) significantly for both the signals. Therefore, 1.0 s was considered and further employed as the RStudio version 1.2.1335 (Rstudio Inc, Boston, MA, USA) was used optimized D1 for the developed method. for generating a hierarchically clustered heatmap. It was created with The developed 1H qNMR method was validated in terms of speci- the quantified values of 3, 4, 5 and PB. In the output, the quantity of ficity, linearity, sensitivity, accuracy and precision. ICH guidelines Q2 chemical markers (3, 4, 5 and PB) represented the individual columns (R1) was followed for the validation purpose (ICH, 2005). The selection whereas the rows corresponded to individual oil samples. An associated of a well-defined, sharp and non-overlapping characteristic peak forthe dendrogram clustered the drying techniques according to their com- quantification of individual propenylbenzenes (1–8) ensured the spe- positional similarity. cificity of the developed method. Sharp singlet for the methoxy groupof the studied propenylbenzenes (1–4, 6–8) was chosen as the key signal 2.15. Statistical analysis for the quantification. In the absence of any methoxy group(5), the

signal for acetate was opted. In specific, the signals atδH 3.88 (s, 3H) 1 The determination of oil yield and H qNMR experiments were for 1, δH 3.82 (s, 3H) for 2, δH 3.87 (s, 3H) for 3, δH 3.81 (s, 3H) for 4, carried out in triplicate and the absolute quantity was expressed as δH 2.28 (2 × s, 6H) for 5, δH 3.86 (s, 3H) for 6, δH 3.79 (s, 3H) for 7 and mean ± standard deviation. DPPH radical scavenging assays were δH 3.78 (s, 3H) for 8 were selected (Fig. 1A). The specificity of the 1 performed in triplicate and repeated twice. Mean values of IC50 were selected peaks was tested in three different ways. (a) Firstly, H NMR plotted for the test samples and the standard deviations were re- spectra for five different essential oils that are rich in propenylbenzenes presented as the error bars. Microsoft Office Excel 2007 (Microsoft including clove bud, bay leaf, betel leaf, anise seed and basil leaf were Corporation, Redmond, WA, USA) was used for statistical analysis. investigated (Fig. 1A). The specificity of the selected methoxy or acetate signals was evaluated for 1 and 2 in clove bud oil, 1 in bay leaf oil, 3, 4 3. Results and discussion and 5 in betel leaf oil, 7 and 8 in anise seed oil and 6 in basil oil. Selected signals were found to be free from any interference by the 3.1. Selection of the propenylbenzenes other oleochemicals, IS or solvent in the corresponding tested oils (Fig. 1A). (b) Secondly, the spiking study individually for 1 and 2 in Identification of the phytochemical markers and their analysis play clove bud oil showed a relative enhancement in the signal intensity at a critical role in the authentication and quality control of the plant δH 3.88 and 3.82 ppm respectively indicating the specificity of the se- materials and plant-produce. Eugenol (1) and seven of its structural lected peaks (Supplementary Fig. S4). Similarly, the spiking study for 3 analogues namely eugenyl acetate (2), chavibetol (3), chavibetol and 4 with betel leaf oil confirmed the specificity of its key signal atδH acetate (4), 4-allyl-1,2-phenylene diacetate (5) (synonymous with 4- 3.87 and 3.81 ppm respectively (Supplementary Fig. S4). (c) To check allyl-1,2-diacetoxybenzene), methyleugenol (6), trans-anethole (7) and the sample dependent variation, specificity was evaluated on a set of estragole (8) were chosen for the study. They make a major or sig- five different betel oil samples. Betel leaves from five different locations nificant contribution in many essential oils, broadly belonging tothe (Assam, India) were collected, hydrodistilled and the specificity of spices, leafy herb and floral categories (Supplementary Fig. S1). These methoxy or acetate signals for three major propenylbenzenes (3, 4 and oleochemicals can be the representative chemical markers for those 5) was evaluated. It was observed that the selected key signals were free propenylbenzene-rich aromatic plants. For example, commonly known from any overlap by the other peaks in all the samples (Supplementary Indian aromatic plant materials such as clove bud, anise seed, basil, bay Fig. S3). and betel leaf are rich in these propenylbenzenes (Fig. 1A). Secondly, Linearity of the method was tested in the concentration range of they were selected for the current study based on their structural var- 0.05–5.00 mg (in 600 µL of the sample volume) of eugenol (1) added to iation on a common framework of propenylbenzene. Acetylation (2, 4, a fixed quantity (0.5 mg) of IS(Fig. 1C). The plot of the mole ratio 5), O-methylation (1–4, 6–8) and propenyl isomerization (7) are versus peak area ratio (1/IS) showed a linear trend. The linear regres- commonly occurred downstream modifications during the biosynthesis sion equation was expressed as y = 0.784x − 0.005; where ‘y’ and ‘x’ of propenylbenzenes (Koeduka et al., 2006; Vogt, 2010). Keeping it in represents the mole ratio [M1/MIS] and peak area ratio [A1/AIS] re- mind, these naturally occurring molecules (1–8) were selected for the spectively. The coefficient of determination2 (R ) was calculated to be development of the quantification method. 0.999. The equation for the quantification of 1 in the oil sample was derived from the linear regression equation as follows: C (mg/ 1 3.2. Development and validation of the H qNMR method g) = [0.784 × (A1/AIS) – 0.005] × MIS × MW1 × (1000/Woil); where C represents the quantity (mg) of 1 per gram of the oil, (A1/AIS) is the 1 To develop a H NMR based quantification technique for the se- peak area ratio of 1 versus IS, MIS is the moles of IS added in the sample, lected propenylbenzenes, sample preparation (e.g. solvent, internal MW1 is the molecular weight of 1,Woil is the known added quantity of standard) and critical data acquisition parameters (e.g. relaxation oil in the sample. This equation was utilized further for the

4 P. Kemprai, et al. Food Chemistry 331 (2020) 127278

Fig. 1. Development and validation of the 1H qNMR method: (A) Specificity of the selected methoxy and acetate signals of the studied propenylbenzenes (1–8) in five different essential oil samples. Two relevant segments ofthe 1H NMR spectra i.e. 3.2–4.2 ppm (methoxy and benzylic protons) and 2.2–2.4 ppm (acetate protons) have been represented; (B) Optimization of the relaxation delay (D1) in the range 1.0–30.0 s for the signals of 1 at δH 3.88 (-OMe) and 3.32 (–CH2) ppm, (C) Linearity test for the signal (δH 3.88 ppm) of 1 in the range 0.05–5.0 mg per assay added with a fixed quantity (0.5 mg) of IS; (D) Sensitivity test for 1 in the range

0.05–1.00 mg per assay for the signals at δH 3.88 (-OMe) and 3.32 (–CH2) ppm; (E) Accuracy (% recovery) and precision (% RSD) test for the quantification of 1 at three individual concentrations 0.25, 1.00 and 4.00 mg per assay.

5 P. Kemprai, et al. Food Chemistry 331 (2020) 127278 quantification of other propenylbenzenes considering their molecular 2015; Tissot, Rochat, Debonneville, & Chaintreau, 2012)(Fig. 2A). On weights. the other hand, 1H NMR signal is proportional to the molar con- The sensitivity parameters (LOD and LOQ) were determined from centration resulting in absolute quantification of the targeted analytes. the S/N of 1 in the lower concentration range (0.05–1.00 mg per 600 µL assay volume) (Fig. 1D). The LOD and LOQ values were determined as 3.3.2. Sensory-guided identification of major key odorant(s) in the betel oil

4.4 and 14.9 µg/mL respectively for the methoxy signal at δH 3.88 ppm. Despite the extensive use of the betel oil as the flavouring agent, no For the methylene protons (δH 3.32 ppm) at the benzylic position, the systematic sensory study has been demonstrated to date to find out the LOD and LOQ values were found to be 37.0 and 123.5 µg/mL respec- key odorants. Sensory-guided fractionation followed by compositional tively. Considering the higher sensitivity and sharp singlet for the analysis through 1H qNMR technique was adopted to identify the key methoxy protons, it was preferred over the benzylic signals for the odorant molecules from the betel oil. The crude oil was subjected to quantification. The sensitivity of the developed method was well ade- fractionation through silica gel column chromatography with in- quate considering the working concentration range of the targeted creasing polarity of the eluent. The chromatographic elution furnished analytes. four fractions A-D containing 13.21, 59.97, 10.85 and 5.82% (w/w) of Accuracy and precision of the developed method were tested on the crude oil mass (Fig. 3A-C). Sensory analysis of the individual frac- three different concentrations (0.25, 1.00 and 4.00 mg in 600 µLsample tions A-D through the difference from control (DFC) test evaluated their volume) of 1 covering the linear response range (Fig. 1E). Accuracy (% odour difference in reference to the crude oil(Fig. 3G). The results recovery) of the quantification was found to be 99.33, 97.74 and depicted fraction B to possess the highest odour similarity to the crude 98.03% respectively. Percent relative standard deviations (% RSD) of which was evident from its low difference score (0.84). The fractions A, the measurements were calculated as 1.68, 0.51 and 0.48% respec- C and D showed much higher difference scores which were found tobe tively. Therefore, the observed accuracy and precision over the studied 3.38, 2.02 and 2.93 respectively. The placebo effect was assessed concentration range were within the acceptable limit. through a blind control experiment showing the difference score as 0.24. Quantification of the major chemical markers (3–5) through the 3.3. Applicability of the developed 1H qNMR method in betel (P. betle, developed qNMR technique revealed that chavibetol (3) content was Bangla cultivar) essential oil very high (88.0% w/w) in the fraction B (Fig. 3D-E). Highly non-polar fraction A contained terpene hydrocarbons with no detectable 3, 4, 5 or 3.3.1. Identification of the chemical markers and the advantages of 1H any other propenylbenzenes. In the fraction C, a rich abundance of qNMR technique chavibetol acetate (4) (47.1%) was observed with a low percentage of 3 Identification of the major metabolites (or chemical markers) inthe (3.0%) and 5 (7.2%). The fraction D was a complex mixture of prope- plant extracts forms the basis of qualitative or quantitative phyto- nylbenzenes (including 3, 4 and 5) and oxygenated terpenes. These chemical analysis. Eugenol (1) and eugenyl acetate (2) were predicted results were supported by the TLC analysis (Fig. 3C). Hydrocarbon-rich as the major oleochemicals in the betel oil through GC–MS analysis (Rt non-polar fraction A appeared as a blue spot on the TLC plate near to 13.8 and 16.2 min respectively). Library-based search predicted their the solvent front (retention factor or Rf 0.86). Fraction B, highly rich in structure with well acceptable SI (Similarity Index) and RSI (Reverse 3 appeared as a green spot after staining at Rf 0.44. Fraction C was a Similarity Index) values (Fig. 2A). A similar observation was also made mixture of mainly 3 and 4 (blue, Rf 0.30) whereas fraction D was ob- in our earlier report where 1 and 2 were predicted as the major oleo- served to be a complex mixture of relatively polar metabolites. These chemicals through GC–MS analysis in the betel oil samples from observations were also substantiated by the GC–MS analysis. Further northeast India (Kemprai et al., 2020). However, purification followed purification of the fraction B and C furnished purified chavibetol (3) by NMR based structural elucidation of these two oleochemicals in the and chavibetol acetate (4) respectively (Fig. 3A). The DFC test clearly current study revealed an erroneous GC–MS prediction. 1H and 13C showed a low difference score for the purified 3 (0.76) and very high NMR studies unambiguously confirmed their structural identity as for 4 (2.93) (Fig. 3G). It indicated that chavibetol (3) possessed a high chavibetol (3) and chavibetol acetate (4) respectively (Supplementary degree of odour similarity to the crude betel oil. When the odour Data). This result was in agreement with the previous investigation in threshold value (OTV) was determined individually for the crude oil, which 3 and 4 were isolated and characterized systematically as the chavibetol (3) and chavibetol acetate (4) in 1:1 (v/v) dipropylene glycol major constituents in the essential oil from Philippine betel leaves (DPG) and water mixture, the average values were found to be 0.07, (Rimando, Han, Park, & Cantoria, 1986). Isomeric relationship (posi- 0.09 and 0.37% respectively (Fig. 3H-J). The odour activity value tional) between eugenol (1) and chavibetol (3) produced no significant (OAV) for 3 and 4 in 1.0% solution (1:1 DPG : water) of betel oil was mass spectral difference which was the probable source of inaccuracy in determined to be 5.61 and 0.33 respectively (Fig. 4K). A comparable GC–MS based structural prediction (Supplementary Fig. S2)(Babushok OTV with the betel oil and a high OAV for chavibetol (3) further sup- & Andriamaharavo, 2012). When 1H NMR spectra of the crude betel oil ported its strong influence on the overall odour of crude oil. Onthe (Bangla cultivar) samples were carefully analyzed, three prope- other hand, a large difference score in the DFC test, higher OTV andlow nylbenzenes namely chavibetol (3), chavibetol acetate (4) and 4-allyl- OAV (less than1.0) for chavibetol acetate (4) confirmed its contribution 1,2-phenylene diacetate (5) were identified as the major oleochemicals to be insignificant towards the betel essence. A triangle test (forced- (Fig. 2B). Therefore, these three propenylbenzenes (3, 4 and 5) were choice discrimination technique) between the crude betel oil and cha- selected as the chemical markers for the quantitative analysis of betel vibetol (3) (5.0% in DPG) was conducted to confirm the odour simi- oil. Besides, a tentative estimation of total propenylbenzenes (PB) in the larity (β risk 0.10 and maximum proportion of discriminators or Pd as oil was made by utilizing benzylic methylene signals which were 30%). Fifteen panelists out of forty eight could respond correctly which commonly present in 3, 4 and 5. This quantification considered the was less than the maximum number of correct answers (i.e. 19) to gross peak area in the range δH 3.25–3.40 ppm and an average of conclude similarity. Therefore, the triangle test further substantiated molecular weights (3, 4 and 5). chavibetol (3) as the major key odorant molecule in the betel essential In this study, 1H NMR based technique provided not only an un- oil. ambiguous structural identification but also rapidity in the analysis. Data acquisition for single sample required less than 3.0 min with the 3.3.3. Activity-guided identification of major antioxidants in the betel oil current acquisition parameters (in comparison to 29.75 min for a single The potential of betel as the rich source of natural antioxidants has GC–MS run). Secondly, the use of internal standard during gas chro- been demonstrated through in vitro and in vivo experiments with solvent matography based analysis provides a semi-quantification of the ana- extracts and essential oil (Arambewela, Arawwawala, & Rajapaksa, lytes considering all response factors to be unity (de Saint Laumer et al., 2006; Rathee et al., 2006). In the current study, activity-guided

6 P. Kemprai, et al. Food Chemistry 331 (2020) 127278

Fig. 2. (A) GC–MS analysis of betel oil from Bangla cultivar (Abbreviations: SI, Similarity Index; RSI, Reverse Similarity Index) added with 2-allylphenol as the 1 internal standard, showing an erroneous identification (eugenol and eugenyl acetate) of major constituents (3 and 4); (B) H NMR spectrum (500 MHz, CDCl3) of betel essential oil from the fresh leaves (δ 0.5–8.0 ppm). The signals for IS, residual solvent and individual chemical markers (3, 4 and 5) have been assigned. The inset is showing a representative picture of betel leaf of Bangla cultivar (scale bar 2.3 cm).

7 P. Kemprai, et al. Food Chemistry 331 (2020) 127278

Fig. 3. (A) Column chromatographic fractionation and purification scheme for the crude betel oil; (B) Isolated yield (%w/w) of the individual fractions A-D from the crude oil; (C) Image of the TLC plate for the fractions A-D in reference to the crude oil; (D) Stacked 1H NMR spectra for the crude oil and the fractions A-D added with a fixed quantity of IS; (E) Quantification (mg/g) of 3, 4 and 5 in the crude oil and fractions A-D through the developed 1H qNMR method; (F) Half-maximal inhibitory concentration (IC50 in µg/mL) for the crude oil, fractions A-D, 3, 4 and ascorbic acid (positive control) in DPPH radical scavenging assay; (G) Difference from control (DFC) test result (difference score) for the fractions A-D, 3, 4 and blind control in reference to the crude oil; (H-J) Evaluation of the odour threshold value (OTV) for the crude oil, 3 and 4 respectively in 1:1 v/v DPG : water mixture; (K) Odour activity values (OAV) for 3 and 4 in 1.0% betel oil taken in 1:1 v/v DPG : water mixture.

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DPPH assay (Arambewela et al., 2006). In this study, activity-guided fractionation demonstrated chavibetol (3) as the major antioxidant in the betel oil.

3.3.4. Evaluation of the post-harvest drying techniques for betel (Bangla cultivar) 3.3.4.1. Post-harvest drying of the betel leaves. Betel leaf is a perishable commodity and undergoes microbial spoilage during storage and transportation (estimated 35–70% in India), causing a huge economic loss (Guha, 2006). Post-harvest drying of the leaves leading to an enhanced shelf-life can be a feasible approach to retain the value of this cultivar in the flavour industries (Balasubramanian, Sharma, Gupta, & Patil, 2011; Pin et al., 2009). But, post-harvest processes including storage, drying or extraction may greatly influence the oil yield and composition of the aromatic crops. It is very important to develop a reliable and rapid analytical technique for the quality control or optimization of the post-harvest processing for these aromatic oil- bearing plants. The developed 1H qNMR method was utilized for the evaluation of different drying techniques for P. betle (Bangla cultivar) leaves. The leaves of Bangla cultivar were subjected to six different drying methods including (i) air-drying, (ii) sun-drying, (iii) oven drying at 40 °C, (iv) oven drying at 60 °C, (v) microwave drying and (vi) freeze drying. The moisture content in the dried betel leaf samples was determined to be 11.11, 13.89, 11.40, 8.82, 15.00 and 11.85% respectively, in reference to 79.33% in the fresh leaves. Further, the hydrodistilled oil from differently dried leaves was subjected to qNMR analysis using the developed method.

3.3.4.2. Effect of the drying techniques on the oil-yield. Drying of the aromatic plant material generally influences the yield of distilled oil depending on the method, temperature and duration of the drying. In this study, different drying methods reduced the oil yield inawide extent (Fig. 4A). The oil-yield was calculated with respect to the fresh Fig. 4. (A) Variation in the yield (%w/w) of hydrodistilled oil from betel leaves weight of the sample. The yield of hydrodistilled oil in the case of fresh subjected to six different post-harvest drying techniques (in reference tothe leaves was 0.55%. Out of six drying techniques, microwave and freeze- fresh leaves); (B) Hierarchically clustered heat map for the quantity of chemical drying produced comparatively higher oil yield (0.49% and 0.47% markers (3, 4, 5 and PB) in the betel oil samples from the fresh and differently dried leaves. Each column represents the individual chemical marker whereas respectively). Sun-drying, oven-drying (40 and 60 °C) and air-drying the rows represent different oil samples. significantly lowered the oil-yield, producing 0.32, 0.24, 0.11 and 0.19% of oil respectively (Fig. 4A). These results were similar to the earlier observations with leafy aromatic herbs. Rahimmalek and co- fractionation followed by 1H qNMR based quantitative analysis of the workers reported that freeze-drying furnished the highest oil yield fractions facilitated the identification of major antioxidants in the betel (1.7%) in the case of Thymus daenensis among six different tested oil (Fig. 3F). When the chromatographic fractions A-D were evaluated drying-techniques (Rahimmalek & Goli, 2013). According to the for antioxidant activity through DPPH radical scavenging assay, highest observation by Sellami et al, the leaves of bay laurel (Laurus nobilis) activity was observed in the chavibetol-rich fraction B. The observed when dried at 45 and 65 °C, the oil yield reduced to 0.32 and 0.22% activity in the fractions was in the following order: B (IC 4.22 µg/ 50 respectively in comparison to the fresh leaves (0.58%) (Sellami et al., mL) > D (IC 34.87 µg/mL) > C (IC 41.58 µg/mL) > A 50 50 2011). (IC50 > 100 µg/mL) (Fig. 3F). For the crude oil and positive control (ascorbic acid), IC values were found to be 9.00 and 12.81 µg/mL 50 3.3.4.3. qNMR analysis: comparative evaluation of the post-harvest drying respectively. Further purification of chavibetol (3) from the active techniques. Different post-harvest drying techniques influenced the fraction B, indicated it as the major antioxidant in the betel oil (50.5% chemical composition of betel essential oil as well. Quantitative w/w) with an IC of 4.60 µg/mL (28.01 µM) (Fig. 3F). In comparison, 50 variation was determined for 3, 4, 5 and PB through the developed chavibetol acetate (4) isolated from fraction C exhibited much lower 1 H qNMR method (Table 1, Fig. 2B). In the oil sample from the fresh activity with an IC of 37.77 µg/mL (183.20 µM). Phenolic hydroxy 50 leaves, the quantity of 3, 4, 5 and PB was estimated to be 223, 293, 177 plays a critical role in the antioxidant activity of natural phenolics due and 771 mg/g respectively and it was considered as a reference for to its ability to donate hydrogen that acts as the free radical scavenger evaluating the tested drying techniques. Significant quantitative (de Pinedo, Peñalver, & Morales, 2007). In this case, a lower activity variation was observed in the oleochemical profile during air-drying, exhibited by chavibetol acetate (4) was plausibly due to the masking of sun-drying and oven-drying (40 and 60 °C) in comparison to the fresh phenolic hydroxy through acetylation. plant material. An elevation in the level of chavibetol (3) was observed Previously the antioxidant activity of ethanolic extract from betel in the range of 344–568 mg/g. On the other hand, the quantity of leaf has been attributed majorly to the low abundant (1.23%) allyl- chavibetol acetate (4) and 4-allyl-1,2-phenylene diacetate (5) reduced pyrocatechol. However, the contribution of chavibetol (0.21% of the significantly to 55–140 and trace to 37 mg/g respectively inthese extract) was concluded to be insignificant towards the activity of the samples. Also, a temperature dependent trend was observed in the extract (Rathee et al., 2006). To the best of our knowledge, no sys- elevation of chavibetol (3) level. Quantity of 3 increased in the tematic investigation has been made to date to identify the antioxidants following order: air drying (344 mg/g) < sun drying (408 mg/ in the betel oil for which EC was reported as 12.66 µg/mL in the 50 g) < oven drying at 40 °C (435 mg/g) < oven-drying at 60 °C

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Table 1 CRediT authorship contribution statement Absolute quantification (mg/g of the oil sample) of the selected oleochemical markers (3, 4 and 5) and total propenylbenzenes in the betel leaf oil through Phirose Kemprai: Methodology, Investigation, Formal analysis, 1 the developed H qNMR method. The samples included hydrodistilled oil from Writing - original draft. Bhaskar Protim Mahanta: Investigation, the fresh and dried (six different post-harvest drying conditions) leaves of betel. Formal analysis. Pranjit Kumar Bora: Investigation, Formal analysis. Abbreviations: PB, propenylbenzenes; nd, not detected. Deep Jyoti Das: Investigation, Formal analysis. Jyoti Lakshmi Hati Betel oil sample details Quantity (mg/g of the oil) Boruah: Methodology. Siddhartha Proteem Saikia: Supervision, Visualization, Writing - original draft. Saikat Haldar: 3 4 5 PB Conceptualization, Supervision, Visualization, Writing - original draft. Fresh leaves 223 ± 4 293 ± 4 177 ± 3 771 ± 11 Air dried 344 ± 19 55 ± 3 nd 480 ± 11 Declaration of Competing Interest Sun dried 408 ± 7 140 ± 6 37 ± 2 693 ± 5 Oven dried (40 °C) 435 ± 5 102 ± 2 33 ± 1 689 ± 17 Oven dried (60 °C) 568 ± 15 56 ± 2 nd 756 ± 14 The authors declare that they have no known competing financial Freeze dried 301 ± 8 228 ± 8 149 ± 4 788 ± 11 interests or personal relationships that could have appeared to influ- Microwave dried 210 ± 2 308 ± 3 142 ± 2 734 ± 15 ence the work reported in this paper.

Acknowledgements

(568 mg/g) while lowering the level of 4 and 5. To note, the quantity of P.K. acknowledges UGC, New Delhi for the fellowship. The current chavibetol (3) in the sun dried, 40 and 60 °C dried samples was 1.83, work is supported by the CSIR-AROMA Mission (HCP007) project and 1.95 and 2.55 times higher than the fresh one respectively. It happened the Director, CSIR-NEIST. plausibly due to the evaporation of low-boiling terpene hydrocarbons at higher drying temperature which was further reflected through the Appendix A. Supplementary data reduced oil-yield obtained in these drying techniques (Fig. 4A). Besides, to assess the possibility of thermal degradation of chavibetol acetate (4) Supplementary data to this article can be found online at https:// to chavibetol (3), crude betel oil and purified 4 were incubated at 60 °C doi.org/10.1016/j.foodchem.2020.127278. for 6 h separately. However, no significant change in the quantity of 3 and 4 was observed in the crude oil sample. Also, the formation of References chavibetol (3) was not detected in the thermally treated chavibetol acetate (4). However, in the case of freeze drying and microwave Arambewela, L., Arawwawala, M., & Rajapaksa, D. (2006). Piper betle: A potential natural drying the oleochemical composition was much closer to the oil from antioxidant. International Journal of Food Science & Technology, 41(s1), 10–14. the fresh leaves. Freeze drying resulted in 301, 228 and 149 mg/g of 3, Babushok, V. I., & Andriamaharavo, N. R. (2012). Use of large retention index database for filtering of GC-MS false positive identifications of compounds. Chromatographia, 4 and 5 respectively in the oil. Microwave drying best retained the 75(11–12), 685–692. oleochemical composition among all the tested conditions. In this case, Balasubrahmanyam, V. R., & Rawat, A. K. S. (1992). Flavour characteristics of Piper betle the quantity of 3, 4 and 5 was found to be 210, 308 and 142 mg/g L. 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