A 1H NMR Spectroscopic Method for the Quantification Of
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Food Chemistry 331 (2020) 127278 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem 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 pro- penylbenzenes (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 chro- matography (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.