Value addition of khus (Vetiveria zizanioides L. Nash) root extracts by elicitor and heat treatment

Industrial Crops & Products 144 (2020) 112037

Contents lists available at ScienceDirect

Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop

Value addition of khus (Vetiveria zizanioides L. Nash) root extracts by elicitor and heat treatment

T

Utkarsh Ravindra Moona,*, Arpana Ashokrao Durgeb, Mahesh Kumarc a

Department of Microbiology, Mahatma Gandhi College of Science, Gadchandur, 442908, India Department of Biochemistry, Guru Nanak College of Science, Ballarpur, 442701, India c Agricultural and Food Engineering Department, Natural Product Biotechnology Group, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Vetiveria zizanioides Elicitor Heat-treatment Khus sharbat Antioxidant Acetylcholinesterase inhibitory activity

Phenolic acids are known to possess many health beneficial effects. Presence of phenolic acids in root extracts of khus (Vetiveria zizanioides L. Nash) established the need to enhance these compounds in khus roots which are used in khus sharbat preparation. Among all the treatments, dual strategy of elicitor (roots treated with chitosan at 200 mg/l for 18 h) followed by heat treatment (100 °C for 20 min) showed 5.3, 27.5, 14 and 31.3 folds increase in p-hydroxybenzoic acid, vanillin, p-coumaric acid and ferulic acid, respectively. This treatment also showed the highest antioxidant activities measured in terms of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging (IC50 66.71 ± 2.02 mg fresh weight/ml), ferric reducing antioxidant potential (FRAP) value (4670 ± 4.09 μM of Fe2+ equivalent/g fresh weight) and 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) value (28.11 ± 0.23 mM/g fresh weight ascorbic acid equivalent) as well as highest total phenolic content (TPC) (10.07 ± 0.02 mg gallic acid equivalent/g fresh weight. Relative acetylcholinesterase (AChE) inhibitory activity was also found to be highest after chitosan elicitor treatment. Principal component analyses of khus root extracts after elicitor and heat treatment confirmed the major role of vanillin and ferulic acid in the enhancement of antioxidant capacities and AChE inhibitory capacities of khus root extracts. Thus, contributing in the value-addition of khus extracts used in sharbat preparation.

1. Introduction Khus (Vetiveria zizanioides L. Nash) is a densely tufted plant, mostly located in swamp regions of subcontinent and belongs to the grass family (Sreenath et al., 1994). Khus root extract is used for preparation of khus sharbat. It is used as a refreshing drink in summer and serves as additive to milk, kulfi, ice-creams and food salads. Since, the sharbat is known for relieving stress, anxiety, insomnia and has cooling effect to brain (Skerman and Riveros, 1990), it is essential to study the metabolites present in the root extract and their potential health benefits. Studies on khus roots are mainly focused on aromatic khus oil, however, very less information is available on the status of phenolic acids in root extracts. Presence of phenolic acids has only been reported from aerial parts of khus (Jha et al., 2013). Phenolic compounds are the secondary metabolites produced in plant as a response to defense against pathogens (Pandey and Rizvi, 2009; Limwachiranon et al., 2019). Epidemiological studies suggest that by the end of 20th century, population having phenolic rich diet will be less susceptible to cardiovascular diseases, cancer, neurodegenerative diseases and diabetes

⁎

mellitus (Arts and Hollman, 2005). Phenolic compounds are reported to show antioxidant activities which are involved in scavenging the free radicals formed in the body, thereby preventing the harmful physiological effects in human body (Huang et al., 2019). Phenolics can be divided in two groups as hydroxycinnamic acids and hydroxybenzoic acid (Shahidi et al., 1992). Hydroxycinnamic acids such as ferulic acid (Graf, 1992), caffeic acid, coumaric acid, chlorogenic acid along with p-hydroxybenzoic acid (Shahidi et al., 1992) and vanillin (Tai et al., 2011) are known for their role as potent antioxidants. Ferulic acid is known for its role as food preservative and skin protection (Graf, 1992). Coumaric acid is known to have antimicrobial and anticancer activity (Mussatto et al., 2007). p-Hydroxybenzoic acid has its role in food industry (Tomás-Barberán and Clifford, 2000). It acts as antimicrobial (Vosmann et al., 2008) and anti-cancerous agent (Tanaka et al., 1995). Role of vanillin as flavoring agent is widely known (Walton et al., 2000). Recent studies on malt (Szwajgier and Borowiec, 2012; Szwajgier, 2013) and various medicinal plants (Santos et al., 2018) established the role of phenolic acids as neuroprotective agents. Phenolic acids are potential inhibitors of acetylcholinesterase (AChE) and

Corresponding author. E-mail address: [email protected] (U.R. Moon).

https://doi.org/10.1016/j.indcrop.2019.112037 Received 12 September 2019; Received in revised form 18 November 2019; Accepted 9 December 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

Industrial Crops & Products 144 (2020) 112037

U.R. Moon, et al.

2.3. Antioxidant capacities

butyrylcholinesterase (BChE) enzymes (Szwajgier and Borowiec, 2012). AChE enzyme is linked with Alzheimer’s disease (AD) which is characterized by memory loss and defective cognitive function (Urbain et al., 2004). AChE hydrolyses acetylcholine, a neurotransmitter, which aggravates the symptoms of AD. Availability of acetylcholine can be increased by the inhibition of AChE (Houghton and Howes, 2005). Since, phenolic contents of the khus roots, their antioxidant and AChE inhibitory activities have been poorly examined; it becomes important to study phenolic contents of the roots and their involvement in AChE inhibition and free radical scavenging processes. This manuscript also addresses the enhancement of metabolites involved in these activities using different elicitors and heat treatment. Elicitors are the microbial derived small molecules which trigger similar response in plants as it would have triggered in microbe itself (Keen, 1975). They are responsible for the production of secondary metabolites by the activation of phenylpropanoid pathway (Moon and Mitra, 2016). Plant derived foods which contains phenolic compounds have about one-third as phenolic acids. They are either free or linked to esters or ethers in cell wall (Robbins, 2003). It is assumed that antioxidant activity shown by phenolic compounds is mainly contributed by the covalently bound phenolic acids. Heat treatment in citrus peel has shown the release of low molecular weight phenolic acids along with the enhancement of antioxidant activities (Seok-Moon et al., 2004). In totality, this manuscript deals with study of phenolic acids in khus aqueous root extracts, effect of elicitor and heat treatment on phenolic acids, antioxidant capacities and AChE inhibitory activities.

2.3.1. DPPH free radical scavenging assay DPPH assay was determined according to previously described method (Brand-Williams et al., 1995). The free radical scavenging activity of aqueous extracts was tested using DPPH solution which consisted of DPPH free radical. Crude extracts of root (0.05 ml) were mixed with the 0.95 ml ethanolic solution of DPPH (0.1 M). The mixture was vortexed and kept in dark for 30 min. The decrement in O.D was noted at 517 nm. The percentage inhibition (% I) was calculated using formula given below: % I = (Acontrol – Asample) / (Acontrol) Where, Acontrol and Asample stand for absorbance of control at 0 min and absorbance of sample after 30 min of incubation. IC50 value is calculated as, the concentration of sample required to scavenge 50 % of DPPH radicals. IC50 values are expressed in mg fresh weight/ml. 2.3.2. ABTS free radical scavenging assay ABTS assay was performed according to Re et al., 1999 with some modifications. ABTS solution was prepared by dissolving 7 mM ABTS and 2.45 mM potassium persulfate in deionized water and incubation in dark for 12−16 h. For working solution, absorbance of ABTS solution was adjusted 0.7 ( ± 0.02) by diluting it with ethanol at 734 nm. The reaction mixture consisted of working solution (0.997 ml) and crude extract (0.003 ml). The reaction mixture was vortexed and incubated for 6 min at 30 °C. Decrease in absorbance was monitored at 734 nm. The ABTS free radical scavenging activity of the extract was expressed in ascorbic acid equivalent antioxidant capacity (AEAC). This value was calculated by comparing the change in absorbance at 734 nm in a test sample to that of calibration plot of ascorbic acid for ABTS assay.

2. Materials and methods 2.1. Chemicals AChE from Electrophorus electricus (eel), acetylthiocholine iodide (ATChI), 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), methyl-jasmonate (MeJa) and p-coumaric acid were purchased from Sigma-Aldrint was adopted with excised roots dipped in different elicitor concentrations (Moon and Mitra, 2016). Roots were cut approximately in 5−6 cm of length and treated with chitosan (100, 200 and 300 mg/l), methyl jasmonate (25, 50 and 75 μM) and yeast extract (5, 10 and 15 mg/ml). After elicitor treatment roots were dipped in water for 12 h followed by maceration, ultra-sonication and heated at 100 °C for 20 min (heat treated). Unheated roots served as control. Chitosan (1 g) was dissolved in 2 ml glacial acetic acid at 60 °C; volume was adjusted to 100 ml and autoclaved at 121 °C for 20 min before use. Stock solution of methyl jasmonate (1 mM) was prepared by dissolving it in 95 % ethanol. Yeast extract was prepared freshly in deionized water and autoclaved at 121 °C for 20 min before use.

2.3.3. FRAP assay The FRAP assay was performed according to previously published procedure (Benzie and Strain, 1996) with slight modifications. Stock solutions included TPTZ (2,4,6-tripyridyl-s-triazine) solution (10 mM) which was prepared in HCl (40 mM), FeCl3.6H2O (20 mM) and acetate buffer (300 mM, pH 3.6). The working solution was prepared as 25 ml acetate buffer, 5 ml TPTZ and 5 ml FeCl3.6H2O. The extracts (0.003 ml) were allowed to react with FRAP working solution (0.997 ml). The mixture was then incubated for 6 min at 37 °C and absorbance was recorded at 593 nm. The FRAP value was expressed in μM of Fe2+ equivalent/g fresh weight. 2.3.4. Determination of total phenolic content Estimation of total phenolic content was done as described previously (Moon et al., 2015). Aqueous extracts (0.1 ml) were mixed with 10 % Folin-Ciocalteu reagent (0.2 ml) and 0.8 ml of Na2CO3 (700 mM). After incubation for 2 h at room temperature, increase in absorbance was then measured at 765 nm. Total phenolic content was expressed as mg gallic acid equivalent/g fresh weight.

2.2. Plant material, elicitor and heat treatment Fresh nature-grown khus (Vetiveria zizanioides L. Nash) plants were collected in the month of September from Palasgaon-Jat from the area near water pond. This place is situated in Chandrapur district in Maharashtra state (20°34′36′′ North 79° 65′86″ East), India. In vivo method of elicitor treatment was adopted with excised roots dipped in different elicitor concentrations (Moon and Mitra, 2016). Roots were cut approximately in 5-−6 cm of length and treated with chitosan (100, 200 and 300 mg/l), methyl jasmonate (25, 50 and 75 μM) and yeast extract (5, 10 and 15 mg/ml). After elicitor treatment roots were dipped in water for 12 h followed by maceration, ultra-sonication and heated at 100 °C for 20 min (heat treated). Unheated roots served as control. Chitosan (1 g) was dissolved in 2 ml glacial acetic acid at 60 °C; volume was adjusted to 100 ml and autoclaved at 121 °C for 20 min before use. Stock solution of methyl jasmonate (1 mM) was prepared by dissolving it in 95 % ethanol. Yeast extract was prepared freshly in deionized water and autoclaved at 121 °C for 20 min before use.

2.4. Acetylcholinesterase (AChE) inhibiting capacity AChE inhibition capacity of different extracts was studied as described previously (Moon et al., 2014). The reaction mixture consists of 0.5 ml of 3 mM DTNB, 46 μl of 15 mM, 0.356 ml of 50 mM Tris-HCl buffer (pH 8.0), extract (as required), 0.1 ml 0.28 U/ml AChE. The absorbance was recorded after incubating reaction mixtures for 10 min at 405 nm. The rate of reaction before addition of enzyme was subtracted from reaction rate after addition of enzyme in order to nullify the absorbance changes due to spontaneous hydrolysis of substrate (Ellman et al., 1961). The velocities of the reaction are calculated by subtracting the absorbance at the end of reaction with that of beginning of the reaction. Enzyme activity was calculated as a percentage reaction 2

Industrial Crops & Products 144 (2020) 112037

U.R. Moon, et al.

Fig. 1. HPLC chromatograms showing detection of phenolic acids from aqueous extract of V. zizanioides roots at 254 nm and 310 nm. (1) p-Hydroxybenzoic acid, (2) vanillin, (3) p-coumaric acid and (4) ferulic acid.

explanatory variables.

velocities compared with an assay using a buffer without any inhibitor. Data were represented as relative AChE activity, which were calculated in respect to untreated control (100 %). The untreated control is the sample without any treatment neither elicitor nor heat.

3. Results and discussion 3.1. Identification and quantification of soluble phenolics from Khus (V. Zizanioides L. Nash) aqueous root extracts

2.5. Extraction and identification of soluble phenolics Nature-grown roots (1 g) were thoroughly washed and treated with different elicitors (20 ml each) for 18 h. Control roots were dipped in the water only. Afterwards, Rroots were dipped 12 h in water (50 ml). The extract was prepared by following methods, first by simply straining the water (untreated) and secondly, by maceration followed by ultra-sonication and boiling of the roots at 100 °C for 20 min (heat treated) in water. The extracts were cooled and centrifuged at 8000 g for 10 min (MiniSpinplus, Eppendorf, Germany). After drying the supernatant in vacuum concentrator (Concentrator plus, Eppendorf, Germany), the final extraction yields (%) were calculated by the formula,

Khus aqueous root extract serves as the main ingredient for “khus” sharbat. After dipping roots in water, the scented extracts are often mixed with sugar syrup which constitutes sharbat preparations. Above study only focused on the analyses of aqueous root extracts because of its main role in sharbat preparations. Khus roots were treated with different elicitors (chitosan, methyl jasmonate and yeast extract) and dipped in distilled water for 12 h. The extracts were then prepared in two ways, either by boiling roots at 100 °C for 20 min (heat treated) or without boiling (untreated). The final extraction yields of the aqueous root extracts were 1.32 % and 2 % for untreated extract and heat treated extracts, respectively. The HPLC separation of extracts resulted in the identification of four major phenolic acids based on their comparison with UV–vis spectrum and HPLC retention time (Rt) (Fig. 1, Fig. S1). The major compounds identified were p-hydroxybenzoic acid (Rt 6.48 min), vanillin (Rt - 12.31 min), p-coumaric acid (Rt - 15.82 min) and ferulic acid (Rt - 18.47 min). Presence of phenolic acids was also reported earlier in khus particularly from leaves (Jha et al., 2013). Concentrations of phenolic acids varied in different treatments. Detailed chart is shown in Table 1. Untreated and heat treated extracts showed concentrations in following order: p-hydroxybenzoic acid < pcoumaric acid < ferulic acid < vanillin and p-hydroxybenzoic acid < pcoumaric acid < vanillin < ferulic acid, respectively. Heat treatment showed notable increase in phenolic acids concentrations. Khus roots treated with chitosan (200 mg/l) for 18 h followed by heat treatment (100 °C for 20 min) showed 5.3, 27.5, 14, and 31.3 folds increase in phydroxybenzoic acid, vanillin, p-coumaric acid and ferulic acid, respectively. These findings are in agreement with the earlier studies in citrus peel extract which showed increase in phenolics after heat treatment at 121 °C for 30 min. The release of phenolic acids after cleavage of ester and glycosidic bond in plant material contributed the increase in total phenolics (Xu et al., 2007). Plant cell walls of monocots are usually rich in hydroxycinnamic acids like p-coumaric acid, ferulic acid are linked with lignin via ester-bonds (Faulds and Williamson, 1996). Thus, heating of roots may release these phenolics in sharbat extract. Chitosan is known to induce systemic acquired resistance to plant tissue by elevating the defense related enzymes such as superoxide dismutase, catalase, NADPH oxidase and phenylalanine ammonia lyase activities which contribute increase in total phenolic content. These phenolic compounds play an important role plant defense and act as antioxidants, antimicrobials and anti-inflammatory agent against plant infection (Tuan et al., 2010). Industrial use of phenolic acids (phydroxybenzoic acid, vanillin, ferulic acid) is widely known in food based industries (Tomás-Barberán and Clifford, 2000; Renger and Steinhart, 2000; Natella et al., 1999). This provides an opportunity to

Extraction yield (%) = [W2-W1/W0] Where, W0, W1 and W2 were the weights of initial plant material, container without extract and container with extract, respectively. The dried aqueous extract was re-suspended in 0.5 ml deionized water, filtered through 0.45 syringe filter and analyzed by Breeze™ HPLC system (Waters, Milford, MA, USA). A Synergi™ Hydro-RP (4 μ) C18 reversephase column (250 × 4.6 mm) was used and the detection of compounds was simultaneously monitored at 254 nm and 310 nm using isocratic solvent system comprising 1 mM trifluoroacetic acid (TFA) in deionized water and methanol (MeOH) (70:30, v/v) with a flow rate of 1 ml/min. The compounds were eluted and confirmed by comparing the retention time and UV–vis spectrum Quantification of isolated compounds was done by running the samples in triplicate. 2.6. Statistical analyses All the analyses were performed in triplicates and represented as means ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA) followed by multiple comparison of samples by Duncan’s New Multiple Range Test using statistical package for windows SPSS (Version 16.0; SPSS Inc. Chicago, IL, USA). Results were considered statistically significant with p < 0.05. The correlation coefficients between the characteristics were calculated as Pearson’s correlation coefficient using Microsoft excel 2007. Principal component analysis of all the variables were performed using, Multi-Variate Statistical Package (MVSP Version 3.22) (Maiti et al., 2014). Two principal components with highest eigen-values were selected. All the antioxidant activities (DPPH, FRAP, ABTS), TPC, AChE inhibition properties and phenolic acid concentrations (p-hydroxybenzoic acid, vanillin, coumaric acid, ferulic acid) served as dependent variables whereas; different elicitor treatments (chitosan, methyl jasmonate, yeast extract) and heat-treated and non-treated samples served as 3

Industrial Crops & Products 144 (2020) 112037

U.R. Moon, et al.

Table 1 Variations of phenolic acid composition in V. zizanioides roots before and after treatment. Sample

Control CH 1 CH 2 CH 3 MJ 1 MJ 2 MJ 3 YE 1 YE 2 YE 3

p-hydroxybenzoic acid

vanillin

Untreated*

Heat treated*

Untreated*

Heat treated*

Untreated*

Heat treated*

Untreated*

Heat treated*

95 ± 5.1a,b 102 ± 4.2a,b 383 ± 8.8f 108 ± 4.7b,c 102 ± 4.1a,b 180 ± 4.6e 160 ± 2.6d 92 ± 1.5a 120 ± 2.1c 108 ± 1.9b,c

139 173 509 127 271 330 210 105 161 140

893 ± 8.8a 7230 ± 25.2 g 10160 ± 17.6 h 6330 ± 35.3f 1200 ± 5.8d 1330 ± 20.8e 1156 ± 29.6c,d 1138 ± 9.3c 1190 ± 20.8d 973 ± 14.5b

6410 ± 15.3c 9077 ± 39.3 g 24636 ± 320 h 7110 ± 58.6d 8087 ± 44.9e 8600 ± 28.9f 7320 ± 15.3d 5609 ± 21.3b 7070 ± 35.1d 2297 ± 20.3a

991 ± 10.8a 1423 ± 14.5d 2123 ± 14.5e 1033 ± 38.4a,b 1233 ± 24.1c 1273 ± 8.8c 1100 ± 57.7b 1056 ± 28.5a,b 1216 ± 23.3c 1070 ± 35.1a,b

5647 ± 29.1b 8082 ± 41.5 g 13840 ± 50.2 h 6420 ± 15.3d 7220 ± 32.1e 7550 ± 28.9f 6127 ± 17.6c 6113 ± 8.8c 7223 ± 23.2e 1953 ± 29.1a

74.66 ± 2.44a 2801 ± 52.1 g 5617 ± 44.1i 2906 ± 34 h 1604 ± 21e 2859 ± 30.1 g,h 1413 ± 18.6d 1313 ± 18.6c 1700 ± 29.1f 701 ± 12.1b

445 ± 10a 16800 ± 57.7 g 33700 ± 115i 17367 ± 88 h 9608 ± 58.3e 17151 ± 28.9 h 8559 ± 70.9d 7793 ± 96.6c 10096 ± 60.6f 4166 ± 88.1b

± ± ± ± ± ± ± ± ± ±

4.6b 4.9c 8.1 g 3.5b 6.4e 6.2f 4.3d 5.2a 2.9c 3.1b

p-coumaric acid

ferulic acid

CH 1, CH 2 and CH 3 stands for chitosan concentrations (100 mg/l, 200 mg/l and 300 mg/l, respectively), MJ 1, MJ 2 and MJ 3 stands for methyl jasmonate concentrations (25 μM, 50 μM and 75 μM, respectively), YE 1, YE 2 and YE 3 stands for yeast extract concentrations (5 mg/ml, 10 mg/ml and 15 mg/ml, respectively). Concentration of phenolic acids (μg/g FW). Each experiment was performed in triplicate and the values are expressed as mean ± SD (n = 3). Mean value followed by different alphabets differ significantly from each other at p ≤ 0.05. * Significant differences between untreated and heat treated samples at p ≤ 0.05.

vanillin and ferulic acid followed by coumaric acid and p-hydroxybenzoic acid ferulic acid, vanillin and p-coumaric acid. This may be due to the higher concentration of vanillin and ferulic acid ferulic acid, vanillin and p-coumaric acid as compared with coumaric acid and to phydroxybenzoic acid. In FRAP assay, the antioxidants in the sample act as reducing agent in a redox-linked colorimetric reaction (Guo et al., 2003). At low pH, antioxidants favored the conversion of colorless ferric-tripyridyltriazine complex to blue coloured ferrous form. This can be detected at 593 nm (Benzie and Strain, 1996). The results are shown in Table 2. Among all the treatments the sample from roots treated with chitosan (200 mg/l) for 18 h followed by maceration, ultra-sonication and heating at 100 °C for 20 min resulted highest blue coloration. It showed FRAP value of 4670 ± 4.09 μM of Fe2+ equivalent/g FW. Correlation coefficients of untreated samples (without heat treatment) between FRAP and phenolic acids are 0.68, 0.95, 0.79 and 0.94 for p-hydroxybenzoic acid, vanillin, p-coumaric acid and ferulic acid, respectively. Correlation coefficients of heat-treated samples between FRAP and phenolic acids are 0.78, 0.67, 0.74 and 0.82 for p-hydroxybenzoic acid, vanillin, pcoumaric acid and ferulic acid, respectively. Thus, FRAP activity is contributed by all the phenolic acids identified.Generation of blue/ green ABTS radical cation by reaction of ABTS and potassium persulfate forms the basis of ABTS assay. The radical cation formed has absorption maxima at wavelengths 645 nm, 734 nm and 415 nm which got reduced to ABTS with the reduction of blue/green colour. This can be monitored at 734 nm and antioxidant potential of extracts can be evaluated. The results are shown in Table 2. Among all the treatments the sample from roots treated with chitosan (200 mg/l) for 18 h followed by maceration, ultra-sonication and heating at 100 °C for 20 min resulted highest discoloration with ABTS value of 28.11 ± 0.23 mM/g FW ascorbic acid equivalents. Correlation coefficients of untreated samples (without heat treatment) between ABTS and phenolic acids are 0.55, 0.80, 0.64 and 0.90 for p-hydroxybenzoic acid, vanillin, p-coumaric acid and ferulic acid, respectively. Correlation coefficients of heat-treated samples between ABTS and phenolic acids are 0.60, 0.67, 0.76 and 0.90 for phydroxybenzoic acid, vanillin, p-coumaric acid and ferulic acid, respectively. Thus, ABTS value is mainly contributed by ferulic acid, vanillin and p-coumaric acid followed by p-hydroxybenzoic acid. The order of effectiveness of elicitors followed the trend as methyl jasmonate < yeast extract yeast extract < methyl jasmonate < chitosan for all antioxidant assays. The values with different alphabets differ significantly from each other at p < 0.05 (Table 2).

explore this dual approach for the isolation of individual phenolic acids. Role of phenolic acids as antioxidants is widely known (Szwajgier and Borowiec, 2012). Thus, it becomes important to test the qualities of V. zizanioides roots extracts for their antioxidant potential after various elicitor and heat treatments.

3.2. Enhanced antioxidant capacities of khus aqueous root extracts after elicitor and heat treatments Free radicals (O2−, H2O2 etc.) are formed in human body in two ways, either by endogenous sources such as mitochondria leak, enzymatic reaction, respiratory burst, auto-oxidation reactions or exogenous sources such as pollutant, ultraviolet radiations and xenobiotics. These radicals are involved in lipid peroxidation, modification of DNA bases or protein degradation and thus, cause tissue damage (Young and Woodside, 2001). Therefore, any food material with good antioxidant properties are considered boon for human consumption. Owing to its cooling effects, khus sharbat is widely consumed in subtropical countries especially in summer (Skerman and Riveros, 1990). Khus leaves and roots extracts have been tested for their antioxidant potentials earlier (Luqman et al., 2009). Till date, no approach was carried out to enhance the antioxidant capacities of field grown vetiver roots. A dual approach of elicitor and heat treatment has been adopted in this study to establish any positive changes in antioxidant capacities of roots. Antioxidant capacities of treated roots are determined by performing various antioxidant assays such as DPPH, FRAP and ABTS. Antioxidant capacities of all the extracts are shown in Table 2 . DPPH free radical was used to study the free radical scavenging capacity of the different elicitor treated khus root aqueous extracts. The free radical solution shows a deep blue colour at 517 nm. Lightening of the blue colour is noticed after the donation of protons to DPPH free radicals. Thus lighter the blue more effective is the extract in scavenging the free radicals. The results are shown in Table 2. Among all the treatments the sample obtained by treating roots with chitosan (200 mg/l) for 18 h followed by maceration, ultra-sonication and heating at 100 °C for 20 min resulted in the highest discoloration of DPPH free radical solution. It showed IC50 value of 66.71 ± 2.02 mg fresh weight/ml. Correlation coefficients of untreated samples (without heat treatment) between DPPH and phenolic acids are 0.51, 0.94, 0.66 and 0.88 for p-hydroxybenzoic acid, vanillin, p-coumaric acid and ferulic acid, respectively. Correlation coefficients of heat-treated samples between DPPH and phenolic acids are 0.45, 0.71, 0.71 and 0.86 for p-hydroxybenzoic acid, vanillin, p-coumaric acid and ferulic acid, respectively. Thus, DPPH scavenging activity is mainly contributed by 4

Industrial Crops & Products 144 (2020) 112037

U.R. Moon, et al.

Table 2 Antioxidant capacities of V. zizanioides roots before and after treatment. Sample

Control CH 1 CH 2 CH 3 MJ 1 MJ 2 MJ 3 YE 1 YE 2 YE 3

DPPH1

ABTS2

FRAP3

TPC4

Untreated*

Heat treated*

Untreated*

Heat treated*

Untreated*

Heat treated*

Untreated*

Heat treated*

223.66 ± 6.33e 121.66 ± 3.33b 108.00 ± 2.12a 117.66 ± 1.46b 193.67 ± 2.41d 177.01 ± 4.93c 201.13 ± 1.51d,e 193.83 ± 4.16d 181.43 ± 3.5c 213.78 ± 1.52f

163.33 ± 2.72f 92.03 ± 1.73c 66.71 ± 2.02a 79.33 ± 2.96b 149.32 ± 3.21e 136.81 ± 3.18d 163.64 ± 6.56f 151.09 ± 8.02e 136.72 ± 6.11d 166.67 ± 4.02f

4.20 ± 0.09a 12.42 ± 0.29e 14.04 ± 0.01 g 13.03 ± 0.28f 8.66 ± 0.17d 12.44 ± 0.24e 7.00 ± 0.12c 6.68 ± 0.07c 7.01 ± 0.06c 5.10 ± 0.11b

8.32 ± 0.06a 24.02 ± 0.05 g 28.11 ± 0.23e 25.30 ± 0.15 h 18.85 ± 0.07f 24.34 ± 0.67 g 13.34 ± 0.04b 14.22 ± 0.12c 17.10 ± 0.15d 8.42 ± 0.08a

40.33 ± 0.89a 90.33 ± 3.18d 120.61 ± 1.79e 90.11 ± 1.72d 49.71 ± 2.11b 70.32 ± 1.09c 39.51 ± 1.52a 43.32 ± 1.49a 50.62 ± 1.24b 40.29 ± 2.01a

500.67 ± 6.98a 2800 ± 11.15e 4670 ± 4.09 h 2730 ± 2.88e 3820 ± 7.26f 4250 ± 11.05 g 1510 ± 24.68c 1910 ± 37.56d 2710 ± 42.27e 1320 ± 87.75b

1.39 1.83 2.27 1.94 1.50 1.72 1.60 1.46 1.59 1.38

1.80 ± 0.03a 5.49 ± 0.13e 10.07 ± 0.02 g 5.80 ± 0.03f 2.25 ± 0.07b,c 3.44 ± 0.08d 2.28 ± 0.02b,c 2. 13 ± 0.02a,b,c 2.5 ± 0.03c 1.91 ± 0.01a,b

± ± ± ± ± ± ± ± ± ±

0.03a 0.02d 0.08f 0.04e 0.08a,b 0.03d 0.03b,c 0.01a,b 0.06c 0.02a

1 IC50 value (mg FW/ml), 2ABTS value (mM/g FW ascorbic acid equivalent), 3FRAP value (μM of Fe2+ equivalent/g FW), 4mg gallic acid equivalent/g FW. CH 1, CH 2 and CH 3 stands for chitosan concentrations (100 mg/l, 200 mg/l and 300 mg/l, respectively), MJ 1, MJ 2 and MJ 3 stands for methyl jasmonate concentrations (25 μM, 50 μM and 75 μM, respectively), YE 1, YE 2 and YE 3 stands for yeast extract concentrations (5 mg/ml, 10 mg/ml and 15 mg/ml, respectively). Each experiment was performed in triplicate and the values are expressed as mean ± SD (n = 3). Mean value followed by different alphabets differ significantly from each other at p ≤ 0.05. *Significant differences between untreated and heat treated samples at p ≤ 0.05.

3.3. Enhanced total phenolic content of khus aqueous root extracts after elicitor and heat treatments Total phenolic content (TPC) in plant material plays a major role by acting as a scavenger of free radicals (Wan et al., 2011). Thus, TPC was also determined from all the extracts. Among all the treatments, chitosan treatment at 200 mg/l for 18 h followed by heat treatment at 100 °C for 20 min showed highest TPC (10.07 ± 0.02 mg GAE/g FW), ABTS value (28.11 ± 0.23 mM/g FW ascorbic acid equivalent), FRAP value of 4670 ± 4.09 μM of Fe2+ equivalent/g FW and lowest DPPH IC50 value (66.71 ± 2.02 mg FW/ml). All heat-treated aqueous extracts showed highest TPC and compared with untreated (without heattreatment) extracts with statistical difference from each other at p < 0.05. Positive correlation was obtained between TPC and antioxidant assays (Pearson’s correlation coefficients for TPC- DPPH, TPCFRAP, TPC-ABTS in untreated samples (without heat treatment) are 0.92, 0.96 and 0.89, respectively, and in heat-treated samples are 0.93, 0.64 and 0.80 respectively). A positive correlation between TPC and antioxidant capacities was also established in the flowers of Uvaria hamiltonii (Barman et al., 2019). Thus, total phenolics which also included p-hydroxybenzoic acid, vanillin, p-coumaric acid and ferulic acid, synthesized after elicitor treatment are responsible for high antioxidant activities. All the results indicated that chitosan followed by heat-treatment is the best alternative for improving antioxidant capacities of khus aqueous root extracts. Thus, a plant-derived enhanced antioxidant extract can be a good source of antioxidant rich khus sharbat.

Fig. 2. Effect of heat and elicitor treatments on relative AChE activity of aqueous root extracts of V. zizanioides. CH 1, CH 2 and CH 3 stands for chitosan concentrations (100 mg/l, 200 mg/l and 300 mg/l, respectively), MJ 1, MJ 2 and MJ 3 stands for methyl jasmonate concentrations (25 μM, 50 μM and 75 μM, respectively), YE 1, YE 2 and YE 3 stands for yeast extract concentrations (5 mg/ml, 10 mg/ml and 15 mg/ml, respectively). Each experiment was performed thrice and the values are expressed as mean ± SD (n = 3). Comparisons of elicitor-untreated sample mean values followed by different alphabets differ significantly from each other at p ≤ 0.05. Comparisons of elicitor-heat treated sample mean values followed by different alphabets differ significantly from each other at p ≤ 0.05. *** p < 0.001, ** p < 0.004.

with TPC (correlation coefficients are 0.97 and 0.87 for untreated and heat-treated samples, respectively) and all the four major phenolic acids. The correlation coefficients for AChE inhibition and phenolic acid concentration in untreated (non-heated) samples are 0.79, 0.86, 0.85 and 0.99 for p-hydroxybenzoic acid, vanillin, p-coumaric acid, ferulic acid, respectively and for heat-treated samples are 0.62, 0.99, 0.80 and 0.94 for p-hydroxybenzoic acid, vanillin, p-coumaric acid, ferulic acid, respectively. These values suggest lesser role of p-hydroxybenzoic acid and coumaric acid in AChE inhibition as compared with vanillin and ferulic acid. Phenolic acids such ferulic acid, p-coumaric acid and sinapic acid are proven to be active AChE and butyrylcholinesterase (BChE) inhibitors (Szwajgier and Borowiec, 2012). Vanillin is known as a most demanding flavoring agent in food industry (Lomascolo et al., 1999). In recent past, vanillin has been demonstrated to possess AChE inhibitory properties (Kundu and Mitra, 2013). AChE is involved in the hydrolysis of acetylcholine neurotransmitter which conducts signals through synapses. AChE also forms three-dimensional complex with β-amyloid fibrils (Inestrosa et al., 1996). AChE inhibitor

3.4. Enhanced acetylcholinesterase (AChE) inhibitory capacities of khus aqueous root extracts Khus roots extracts showed promising AChE inhibition as shown in Fig. 2. Since, khus sharbat is consumed as refreshment drink, it is important to establish any link between increase in phenolic acids and antioxidant capacities of khus roots and the inhibition of AChE enzyme. Among all the treatments, chitosan treatment at 200 mg/l for 18 h followed by heat treatment at 100 °C for 20 min showed maximum inhibition of AChE (Fig. 2). As discussed earlier, chitosan is involved in activation of defense related genes. These genes are involved in the synthesis of secondary metabolites (Moon and Mitra, 2016) which may act as AChE inhibitors when tested in vitro. Heat-treated aqueous extracts showed highest AChE inhibition and compared with untreated (without heat treatment) extracts and are statistically different from each other at p < 0.05. AChE inhibition showed positive correlation 5

Industrial Crops & Products 144 (2020) 112037

U.R. Moon, et al.

Fig. 3. a Score plot of phenolic acids, antioxidant activities and AChE activities from elicitor treated, non-heated (untreated) aqueous extracts of V. zizanioides after PCA analysis. Where, AE, TP, HBA, VA, CM and FR represents AChE inhibition, total phenolic content, p -hydroxybenzoic acid, vanillin, coumaric acid and ferulic acid, respectively. b Score plot of phenolic acids, antioxidant activities and AChE activities from elicitor treated, heated (treated) aqueous extracts of V. zizanioides after PCA analysis. Where, AE, TP, HBA, VA, CM and FR represents AChE inhibition, total phenolic content, p-hydroxybenzoic acid, vanillin, coumaric acid and ferulic acid, respectively.

by principal component analysis (PCA). From the scatter plot of untreated (non-heated) samples (Fig. 3a), two principal components (PC1 - 86.39 % and PC2 - 8.95 %) were sufficient to explain total variance i.e. 95.35 %. Group I on right hand side of the graph showed all the variables (DPPH, FRAP, ABTS, TPC, vanillin, ferulic acid, AChE) together which suggests a close relationship between these activities. Phenolic acids, such as p-hydroxybenzoic and p-coumaric acid are excluded from the group which may indicate their lesser role in contribution of antioxidant and AChE inhibitory capacities. From the scatter plot of treated (heat-treated) samples (Fig. 3b), two principal components (PC1 - 82.98 % and PC2 - 9.14 %) were sufficient to explain total variance i.e. 92.12 %. Two groups can be plotted on right hand side of the graph. Group I at bottom right showed all the variables (vanillin, ferulic acid, TPC, DPPH, ABTS and AChE) together which suggest a close relationship between these activities. Vanillin and ferulic acid are well antioxidant phenolic acids which are known to scavenge free radicals (Kundu and Mitra, 2013; Graf, 1992). Their role in AChE inhibition is also studied in

plays a major role by blocking the AChE and preventing acetylcholine hydrolysis and progression of this complex and thereby, the progression of disease. Positive correlation between AChE inhibition and phenolic acids is indicative of the major contribution of phenolic acids in AChE inhibitory properties. Moreover, literature citing the correlation of phenolic acids as neuroprotective agents is very less. This dual treatment approach is novel in the way as it establishes the strategy for value addition of root extracts with the increase in phenolic acids concentrations and antioxidant capacities which finally enhances AChE inhibition capacity of the khus extract and would be beneficial for the patients of Alzheimer’s disease. 3.5. Statistical analyses Relationship between all the antioxidant assays (DPPH, FRAP and ABTS), TPC, phenolic acids (p-hydroxybenzoic acid, vanillin, p-coumaric acid and ferulic acid) and AChE inhibitory activities were studied 6

Industrial Crops & Products 144 (2020) 112037

U.R. Moon, et al.

in Uvaria hamiltonii flowers. Nat. Prod. Res. 1, 1–4. https://doi.org/10.1080/ 14786419.2019.1610959. Benzie, I.E.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem. 239, 70–76. https://doi.org/ 10.1006/abio.1996.0292. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci. Technol. 28, 25–30. https://doi.org/10. 1016/S0023-6438(95)80008-5. Ellman, G.L., Courtney, K.D., Andes, V., Featherstone, R.M., 1961. A new rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. https://doi.org/10.1016/0006-2952(61)90145-9. Faulds, C., Williamson, G., 1996. A major bioactive component of plant cell walls, ferulic acid influences feruloyl esterase production in Aspergillus niger. Biochem. Soc. Trans. 24, 386S. https://doi.org/10.1042/bst024386s. Graf, E., 1992. Antioxidant potential of ferulic acid. Free Rad. Biol. Med. 13, 435–448. https://doi.org/10.1016/0891-5849(92)90184-i. Guo, C.J., Yang, C.J., Wei, J., Li, Y., Xu, J., Jiang, Y., 2003. Antioxidant activities of peel, pulp and seed fractions of common fruits as determined by FRAP assay. Nutr. Res. 23, 1719–1726. https://doi.org/10.1016/j.nutres.2003.08.005. Houghton, P.J., Howes, M.J., 2005. Natural products and derivatives affecting neurotransmission relevant to Alzheimer?"s and Parkinson?"s disease. Neurosignals. 14, 6–22. https://doi.org/10.1159/000085382. Huang, H., Xu, Q., Belwal, T., Li, L., Aalim, H., Wu, Q., Duan, Z., Zhang, X., Luo, Z., 2019. Ultrasonic impact on the viscosity and extraction efficiency of polyethylene glycol: A greener approach for anthocyanins recovery from purple sweet potato. Food Chem. 283, 59–67 10.10.1016/j.foodchem.2019.01.017. Inestrosa, N.C., Alvarez, A., Pérez, C.A., Moreno, R.D., Vicente, M., Linker, C., Casanueva, O.I., Soto, C., Garrido, J., 1996. Acetylcholinesterase accelerates assembly of amyloid-beta-peptides into Alzheimer?"s fibrils: possible role of the peripheral site of the enzyme. Neuron 16, 881–891. https://doi.org/10.1016/s0896-6273(00)80108-7. Jha, P., Jindal, R., Chakraborty, D., 2013. HPLC quantification of phenolic acids from Vetiveria zizanioides (L.) Nash and its antioxidant and antimicrobial activity. J. Pharm. (Cairo) 270342, 1–6. https://doi.org/10.1155/2013/270472. Keen, N.T., 1975. Specific elicitors of plant phytoalexin production: determinants of race specificity in pathogens. Science. 187, 74–75. https://doi.org/10.1126/science.187. 4171.74. Kundu, A., Mitra, A., 2013. Flavoring extracts of Hemidesmus indicus roots and Vanilla planifolia pods exibit in vitro acetylcholinesterase inhibitory activities. Plant Foods Hum. Nutr. 68, 247–253. https://doi.org/10.1007/s11130-013-0363-z. Limwachiranon, J., Jiang, L., Huang, H., Sun, J., Luo, Z., 2019. Improvement of phenolic compounds extraction from high-starch lotus (Nelumbo nucifera G.) seed kernels using glycerol: new insights to amylase/amylopectin ? phenolic relationships. Food Chem. 274, 933–941. https://doi.org/10.1016/j.foodchem.2018.09.022. Lomascolo, A., Stentelaire, C., Asther, M., Lesage-Meessen, L., 1999. Basidiomycetes as new biotechnological tools to generate natural aromatic flavours for the food industry. Trends Biotechnol. 17, 282–289. https://doi.org/10.1016/S0167-7799(99) 01313-X. Luqman, S., Kumar, R., Kaushik, S., Srivastava, S., Darokar, M.P., Khanuja, S.P., 2009. Antioxidant potential of the root of Vetiveria zizanioides (L.) Nash. Indian J. Biochem. Biophys. 46, 122–125. http://nopr.niscair.res.in/handle/123456789/3331. Maiti, S., Moon, U.R., Bera, P., Samanta, T., Mitra, A., 2014. The in vitro antioxidant capacities of Polianthes tuberose flower extract. Acta Physiol. Plant. 36, 2597–2605. https://doi.org/10.1007/s11738-014-1630-9. Moon, U.R., Mitra, A., 2016. A mechanistic insight into hydrogen peroxide-mediated elicitation of bioactive xanthones in Hoppea fastigiata shoot cultures. Planta. 244, 259–274. https://doi.org/10.1007/s00425-016-2506-6. Moon, U.R., Sen, S.K., Mitra, A., 2014. Antioxidant and acetylcholinesterase-inhibitory activity of Hoppea fastigiata. J. Herbs Spices Med. Plants 20, 115–123. https://doi. org/10.1080/10496475.2013.840711. Moon, U.R., Sircar, D., Barthwal, R., Sen, S.K., Beuerle, T., Beerhues, L., Mitra, A., 2015. Shoot cultures of Hoppea fastigiata (Griseb.) C.B. Clarke as potential source of neuroprotective xanthones. J. Nat. Med. 69, 375–386. https://doi.org/10.1007/s11418015-0904-x. Mussatto, S.I., Dragone, G., Roberto, I.C., 2007. Ferulic and p-coumaric acids extraction by alkaline hydrolysis of brewer?"s spent grain. Ind. Crop Prod. 25, 231–237. https:// doi.org/10.1016/j.indcrop.2006.11.001. Natella, F., Nardini, M., di Felice, M., Scaccini, C., 1999. Benzoic and cinnamic acid derivatives as antioxidants: structure-activity relation. J. Agric. Food Chem. 47, 1453–1459. https://doi.org/10.1021/jf980737w. Pandey, K.B., Rizvi, S.I., 2009. Plant polyphenols as dietary antioxidant in human health and disease. Oxid. Med. Cell. Longev. 2, 270–278. https://doi.org/10.4161/oxim.2.5. 9498. Re, R., Pellegrinni, N., Proteggente, A., Pannala, A., Yang, M., RiceEvans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231–1237. https://doi.org/10.1016/s0891-5849(98) 00315-3. Renger, A., Steinhart, H., 2000. Ferulic acid dehydrodimers as structural elements in cereal dietary fibre. Eur. Food Res. Technol. 211, 422–428. https://doi.org/10.1007/ s002170000. Robbins, R.J., 2003. Phenolic acids in foods: an overview of analytical methodology. J. Agric. Food Chem. 51, 2866–2887. https://doi.org/10.1021/jf026182t. Santos, T.C.D., Gomes, T.M., Pinto, B.A.S., Camara, A.L., Paes, A.M.D.A., 2018. Naturally occurring acetylcholinesterase inhibitors and their potential use for Alzheimer?"s disease therapy. Front. Pharmacol. 9, 1. https://doi.org/10.3389/fphar.2018.01192. Seok-Moon, J., So-Young, K., Dong-Ryul, K., Seong-Chun, J., Nam, K.C., Ahn, D.U., SeungCheol, L., 2004. Effect of heat treatment on the antioxidant activity of extracts from

malt and Vanilla planifolia (Szwajgier and Borowiec, 2012; Kundu and Mitra, 2013). Phenolic acids, such as p-hydroxybenzoic and p-coumaric acid are out of group, which indicates a lesser role of these phenolics in contribution to antioxidant activities such as DPPH and ABTS. These phenolics play a greater role in reduction of Fe3+ to Fe2+ which is justified by close proximity of FRAP assay with p-coumaric acid and phydroxybenzoic acid in Fig. 3a. Thus from PCA analyses, it can be concluded that the elicitor treatment by chitosan and heat treatment is followed by the enhancement of phenolic acids especially, vanillin and ferulic acid which played a major role in the enhancement of antioxidant capacities and AChE inhibitory capacities of khus root extracts. 4. Conclusions Phenolic compounds are known to possess pleiotropic health beneficial effects against cancer, diabetes, infections and cardiovascular diseases. They are considered excellent antioxidants that can neutralize the activity of reactive oxygen and nitrogen species produced as a byproduct of metabolic processes. Detection of phenolic acids in khus roots provided an opportunity for the enhancement of phenolics and value-addition of the khus sharbat. Dual treatment with chitosan elicitor and heat enhanced phenolic acids, p-hydroxybenzoic acid (5.3 fold), vanillin (27.5 fold), p-coumaric acid (14 fold) and ferulic acid (31.3 fold). Enhancement of these compounds also contributed in enhanced antioxidant activities, total phenolic contents and acetylcholinesterase inhibitory activities which are also indicated by their positive correlation with phenolic acids. Principal component analyses (PCA) of khus root extracts after elicitor and heat treatment also established and confirmed the major role of vanillin and ferulic acid in the enhancement of antioxidant capacities and AChE inhibitory capacities of khus root extracts. Thus, the dual treatment strategy to enhance phenolic acids in khus aqueous extracts will be commercially beneficial in terms of value-addition of khus extracts used in khus sharbat preparation. Declaration of Competing Interest The authors declare that they have no conflict of interest. CRediT authorship contribution statement Utkarsh Ravindra Moon: Conceptualization, Investigation, Methodology, Formal analysis, Writing - original draft, Writing - review & editing. Arpana Ashokrao Durge: Writing - review & editing, Formal analysis, Visualization. Mahesh Kumar: Methodology, Validation, Writing - review & editing. Acknowledgement Utkarsh Ravindra Moon would like to thank Prof. Adinpunya Mitra, Natural Product Biotechnology Group, Indian Institute of Technology Kharagpur, Kharagpur, India, for extending support for this work at his laboratory. It is a pleasure to thank Mahendra Moon and Ravindra Moon for collecting khus plants for this work. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.112037. References Arts, I.C.W., Hollman, P.C.H., 2005. Polyhenols and disease risk in epidemiologic studies. Am. J. Clin. Nutr. 81, 317–325. https://doi.org/10.1093/ajcn/81.1.317S. Barman, M., Ghissing, U., Dey, P.K., Agarwal, A., Bera, B., Kotamreddy, J.N.R., Karmakar, P., Mitra, A., 2019. Specialized metabolites contributing to colour and scent volatiles

7

Industrial Crops & Products 144 (2020) 112037

U.R. Moon, et al.

nature, occurrence and dietary burden. J. Sci. Food Agric. 80, 1024–1032 https:// doi.org/10.1002/(SICI)1097-0010(20000515)80:7 < 1024::AIDJSFA567 > 3.0.CO;2-S. Tuan, P.A., Park, N.I., Li, X., Xu, H., Kim, H.H., Park, S.U., 2010. Molecular cloning and characterization of phenylalanine ammonia lyase and cinnamate 4-hydroxylase in the phenylpropanoid biosynthesis pathway in garlic (Allium sativum). J. Agric. Food Chem. 58, 10911–10917. https://doi.org/10.1021/jf1021384. Urbain, A., Marston, A., Queiroz, E.F., Ndjoko, K., Hostettmann, K., 2004. Xanthones from Gentiana campestris as new acetylcholinesterase inbhibitors. Planta Med. 70, 1011–1014. https://doi.org/10.1055/s-2004-832632. Vosmann, K., Wiege, B., Weitkamp, P., Weber, N., 2008. Preparation of lipophilic alkyl (hydroxy) benzoate by solvent-free lipase-catalyzed esterification and transesterification. Appl. Microbiol. Biotechnol. 80, 29–36. https://doi.org/10.1007/s00253008-1534-y. Walton, N.J., Narbad, A., Faulds, C.B., Williamson, G., 2000. Novel approaches to the biosynthesis of vanillin. Curr. Opin. Biotech. 11, 490–496. https://doi.org/10.1016/ s0958-1669(00)00125-7. Wan, C., Yu, Y., Zhou, S., Tian, S., Cao, S., 2011. Isolation and identification of phenolic compounds from Gynura divaricata leaves. Pharmacogn. Mag. 7, 101–108. https:// doi.org/10.4103/0973-1296.80666. Xu, G., Ye, X., Chen, J., Liu, D., 2007. Effect of heat treatment on the phenolic compounds and antioxidant capacity of citrus peel extract. J. Agric. Food Chem. 55, 330–335. https://doi.org/10.1021/jf062517l. Young, I.S., Woodside, J.V., 2001. Antioxidants in health and disease. J. Clin. Pathol. 54, 176–186. https://doi.org/10.1136/jcp.54.3.176.

citrus peels. J. Agric. Food Chem. 52, 3389–3393. https://doi.org/10.1021/ jf049899k. Shahidi, F., Janitha, P.K., Wanasundara, P.D., 1992. Phenolic antioxidants. Crit. Rev. Food Sci. Nutr. 32, 67–103 10.10408399209527581. Skerman, P.J., Riveros, F., 1990. Tropical Grasses, first ed. Rome, Italy. Sreenath, H.L., Jagdishchandra, K.S., Bajaj, Y.P.S., 1994. Vetiveria zizanioides (L.) Nash (Vetiver Grass): In vitro culture, regeneration, and the production of essential oils, Vetiveria zizanioides (L.) Nash (Vetiver Grass): In vitro culture, regeneration, and the production of essential oils. In: Bajaj, Y.P.S. (Ed.), Medicinal and Aromatic Plants VI. Biotechnology in Agriculture and Forestry. Springer, Berlin, Heidelberg, pp. 403–421. https://doi.org/10.1007/978-3-642-57970-7_27. Szwajgier, D., 2013. Inhibition of cholinesterase by phenolic acids detected in beer: a dose-response model approach. Afr. J. Biotechnol. 12, 1675–1681. https://doi.org/ 10.5897/AJB12.2699. Szwajgier, D., Borowiec, K., 2012. Phenolic acids from malt are efficient acetylcholinesterase and butyrylcholinesterase inhibitors. J. Inst. Brew. 118, 40–48. https://doi.org/10.1002/jib.5. Tai, A., Sawano, T., Yazama, F., Ito, H., 2011. Evaluation of antioxidant activity of vanillin by using multiple antioxidant assays. Biochim. Biophys. Acta 1810, 170–177. https://doi.org/10.1016/j.bbagen.2010.11.004. Tanaka, T., Kojima, T., Kawamori, T., Mori, H., 1995. Chemoprevention of digestive organs carcinogenesis by natural product protocatechuic acid. Cancer. 75, 1433–1439 https://doi.org/10.1002/1097-0142(19950315)75:6+ < 1433::aidcncr2820751507 > 3.0.co;2-4. Tomás-Barberán, F.A., Clifford, M.N., 2000. Dietary hydroxybenzoic acid derivatives-

8