L. Rhizomes and (+)-Nootkatone Quantitatio - ACS Publications

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Tissue-Specific Metabolite Profiling of Cyperus rotundus L. Rhizomes and (+)-Nootkatone Quantitation by Laser Microdissection, Ultra-High-Performance Liquid Chromatography−Quadrupole Time-of-Flight Mass Spectrometry, and Gas Chromatography−Mass Spectrometry Techniques Yogini Jaiswal, Zhitao Liang, Ping Guo, Hing-Man Ho, Hubiao Chen, and Zhongzhen Zhao* School of Chinese Medicine, Hong Kong Baptist University, Kowloon, Hong Kong Special Administrative Region, People’s Republic of China S Supporting Information *

ABSTRACT: Cyperus rotundus L. is a plant species commonly found in both India and China. The caused destruction of this plant is of critical concern for agricultural produce. Nevertheless, it can serve as a potential source of the commercially important sesquiterpenoid (+)-nootkatone. The present work describes comparative metabolite profiling and (+)-nootkatone content determination in rhizome samples collected from these two countries. Laser dissected tissues, namely, the cortex, hypodermal fiber bundles, endodermis, amphivasal vascular bundles, and whole rhizomes were analyzed by ultra-high-performance liquid chromatography−quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). Gas chromatography−mass spectrometry (GC-MS) analysis was used for profiling of essential oil constituents and quantitation of (+)-nootkatone. The content of (+)-nootkatone was found to be higher in samples from India (30.47 μg/10 g) compared to samples from China (21.72 μg/10 g). The method was validated as per International Conference on Harmonisation (ICH) guidelines (Q2 R1). The results from this study can be applied for quality control and efficient utilization of this terpenoid-rich plant for several applications in food-based industries. KEYWORDS: Cyperus rotundus L. (purple nut sedge), laser micro dissection, metabolite analysis, (+)-nootkatone, UHPLC-QTOF MS, GC-MS

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INTRODUCTION Cyperus rotundus L. (purple nut sedge), belonging to the family Cyperaceae, is a perennial herb, indigenous to India and found in tropical and subtropical regions throughout the world.1−6 It is a notorious weed and has a destructive effect on agricultural yields after it invades the crop fields. (+)-Nootkatone is also an important entity for industries that manufacture perfumes and insecticides.7−12 It is also derived from peels of various fruits like kabusu (Citrus aurantium L.), bergamot (Citrus bergamia Risso), and pummelo (Citrus grandis L. Osbeck).12 Thus, finding natural and economic sources of (+)-nootkatone from a weed like C. rotundus has significant importance for the industrial and agricultural sector. Purple nut sedge is also considered as a traditional food and an important medicinal herb in many countries, including China and India.13−17 The rhizomes of C. rotundus are a good source of carbohydrates, fiber, oil, and minerals.17,18 It is used in manufacture of candies, bakery products, and flavored beverages in Middle east and Southeast Asia.18−20 It is used as a staple and functional food and as a flavoring agent in preparation of curries and pickles.21,22 The starch obtained from its rhizomes has physiochemical properties similar to potato, and it has been exploited for its application in manufacture of noodles.23 The rhizomes have also been used as a substitute for maize as a feed in poultry farms.24 For other species belonging to Cyperaceae family, similar applications in manufacture of food products have also been reported.25 © 2014 American Chemical Society

Recently, some reports have suggested the protective effect of the rhizome extracts of C. rotundus and its constituent (+)-nootkatone in neurodegeneration and arterial thrombosis complications.15,16 The Ayurvedic Pharmacopoeia records the use of C. rotundus (commonly known as Mustã) in Ayurveda, for treatment of various pathological conditions including neurodegenerative diseases. It is an important constituent of several Ayurvedic formulations.26 The Chinese pharmacopoeia recommends the use of C. rotundus L. (Xiangfu) for treatment of many disorders including depression and pain.27,28 Several reports have stated the presence of sesquiterpenes, furochromones, sterols, flavonoids, and triterpenes in the rhizomes of this plant.29,30 Some studies report the occurrence of four chemotypes of purple nut sedge found in Asian countries (H, K, M, and O types).31 The rhizomes and its herbal products are reported to have antifatigue, antibacterial, antidiabetic, antiapoptotic, and anti-inflammatory properties.31−34 Some plant species are exhaustively exploited from their natural habitats to meet consumer demands. Thus, finding representative substitutes and evaluating the quality of herbs of the same species from different geographical locations becomes Received: Revised: Accepted: Published: 7302

February 12, 2014 June 17, 2014 June 17, 2014 June 17, 2014 dx.doi.org/10.1021/jf502494z | J. Agric. Food Chem. 2014, 62, 7302−7316

Journal of Agricultural and Food Chemistry

Article

cryotome and then placed on poly(ethylene terephthalate) (PET) slides at working temperature of −15 °C. Laser Microdissection and Tissue Extraction. Sections of samples were prepared at −15 °C by use of a Thermo Shandon As620 cryotome. A Leica LMD 7000 system (Leica, Benshein, Germany) was used for laser dissection and investigation of microscopic features. At magnifications of 6.3× and 10×, the target tissues were dissected under a Leica LMD-BGR (blue−green−red) fluorescence filter system. A diode-pumped solid-state (DPSS) laser beam was used with an exposure time of 116.9 ms, aperture size of 1, gain of 2.3×, and speed of 3. The target tissues for investigation were excised by LMD, to obtain tissues with a total area of about 1 × 106 μm2. The tissues were collected in Eppendorf tube caps, prefixed in the LMD collecting device, and used for further analysis. For preparation of samples for UHPLC-QTOF MS analysis, the dissected rhizome tissues were centrifuged at 12 000 rpm for 5 min in a 5415Rcentrifuge (Eppendorf, Hamburg, Germany). Following centrifugation, 100 μL of HPLC-grade methanol was added into each sample tube, and the tubes were sonicated for 30 min (Crest 1875HTAG ultrasonic processor). After sonication, the tubes were subjected to centrifugation at 12 000 rpm for 10 min. The resultant supernatants were collected into amber-colored glass HPLC vials containing glass inserts with bottom springs (400 μL, Grace, HK) and stored at 4 °C until analysis. For analysis of whole rhizome sections, each presoaked sample was sectioned with the cryotome. The sections were collected in Eppendorf tubes and subjected to extraction with 500 μL of methanol by the procedure described above for isolated tissues. The dissected whole sections were used for whole-rhizome metabolite profile analysis. For analysis of raw material, the rhizome samples were coarsely powdered (mesh size 2−8) and extracted as per the procedure used for whole sections. For GC-MS analysis, the rhizomes were coarsely powdered (mesh size 2−8) and extracted for 1 h with hexane/ethyl acetate mixture (1:1).39 The obtained extracts were then concentrated by use of a B-480 rotary evaporator from Buchi (Flawil, Switzerland). Metabolite Profiling by UHPLC-QTOF MS. For ultra high performance liquid chromatography, an Agilent 6540 accurate−mass Q-TOF LC-MS system (Agilent Technologies, Santa Clara, CA) was used. A UPLC C18 analytical column [dimensions 2.1 mm × 100 mm, i.d. 1.7 μm, Acquity UPLC ethylene-bridged hybrid (BEH); Waters, Milford, MA] with a C18 precolumn (2.1 mm × 5 mm, i.d. 1.7 μm, VanGuard BEH, Waters), was used for separation. The analysis was carried out at room temperature of 20 °C. The operation parameters applied for UHPLC-ESI-MS analysis of adduct ion species generated by electrospray ionization (ESI) were as follows: dry gas temperature 300 °C, fragmentor voltage 150 V, dry gas (N2) flow rate 6 L/min, Vcap 4500, nozzle voltage 500 V, and nebulizer pressure 40 psi. Mass spectra were acquired in both negative and positive modes. Scanning was carried out for adduct ions of mass to charge ratio (m/z) from 110 to 1700. A mixture of water (A) and acetonitrile (B), both containing 0.1% formic acid, was used as the mobile phase with an optimized linear gradient elution as follows: 0−8 min, 2−15% B; 8−18 min, 15−55% B; 18−23 min, 55−100% B; 23−26 min, 100% B; 26−26.1 min, 100−2% B, 26.1−30 min, 2% B. The injection volume was 2.0 μL. The flow rate was set at 0.4 mL/min. An ESI−low concentration tuning mix solution (Agilent Technologies) was used for accurate molecular mass analysis calibration. The accuracy error threshold of 10 ppm was applied for analysis. GC-MS-Based Metabolite Profiling and (+)-Nootkatone Quantitation. GC-MS analysis was performed on a Shimadzu GCMS-QP2010 gas chromatograph−mass spectrometer, equipped with a DB-5 capillary column (dimensions 30 m length, 0.25 mm i.d., and film thickness 0.25 μm). An autoinjector (AOC-20i) was employed for sample injection. The oven temperature was programmed from 80 to 280 °C at the rate of 5 °C/min. A splitless injection mode was applied for analysis. The oven temperature was held at 80 °C at the start of the run, then increased to 110 °C at 8 °C/min and 180 °C at 3 °C/min, before being held at 280 °C for 10 min. Helium with a flow rate of 0.95 mL/min was used as a carrier gas. The ion source and interface temperatures were

significantly important to ascertain the validity of their use as substitutes for each other. In our previously published and unpublished work, we have found similarities and differences in metabolite profiles of plant species from same genus grown in China and India.35−37 Furthermore, study of tissue distribution of metabolites paves a way to unravel the specific tissue sites for metabolic pathways of the plants.38 Plants of the same species growing in different geographical regions exhibit varying morphological and genetic features, and also differences in the contents of their secondary metabolites are often observed. This is due to the effect of environmental conditions on their metabolic pathways and type of evolution in particular taxa. Evidences to these facts have been provided by various studies. For example, in a study involving broccoli, a variance in the glucosinolate profiles was observed in accessions grown in different environments.38 In a report including Brassica oleracea, dissimilarities in glucosinolate−myrosinase system were found due to different climatic conditions.39 Genetic diversity was reported in a study involving Phyllanthus amarus species grown in different geographical locations.40,41 Recently, laser microdissection (LMD) has been recognized as an important tool in isolation of specific plant and animal tissues due to its contamination-free tissue isolation procedures. Combined with hyphenated chromatography techniques like ultra-high-performance liquid chromatography−quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS) and gas chromatography−mass spectrometry (GC-MS), the LMD method can be exploited for application in tissue-specific metabolite analysis.35,36 In consideration of the significance of (+)-nootkatone as a biomarker for important human pathological conditions (like neurodegeneration) and its high demand in various industrial segments, it was selected as a marker for evaluation of the pharmaceutical quality of C. rotundus L. in the studied samples. The aim of the current study was to compare the tissue-specific metabolite profiles of C. rotundus L. rhizomes collected from various regions of China and India and evaluate the pharmaceutical quality of rhizomes based upon the content of the bioactive sesquiterpenoid (+)-nootkatone.

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MATERIALS AND METHODS

Chemicals. For LC-MS analysis, acetonitrile (HPLC-grade) was obtained from E. Merck (Darmstadt, Germany). Formic acid (HPLCgrade, purity 96.0%) was used as a mobile phase modifier, and it was purchased from Tedia Company Inc. Ultrapure water used in various steps of the experiments was obtained from a Milli-Q water purification system (Millipore, Bedford, MA). Standard (+)-nootkatone (purity >95%) used as a reference compound, was procured from Extrasynthese, Genay cedex, France. For GC-MS analysis, n-hexane and ethyl acetate used in extraction were procured from ACI Lab Scan Ltd., Thailand. Plant Material. Dried rhizome samples of C. rotundus L., comprising five sample sets from different regions of China and five sample sets from various locations in India, were used for analysis. The samples were authenticated by Professor ZhongZhen Zhao, and voucher specimens (voucher CM/005) were deposited in the Chinese Medicines Centre of Hong Kong Baptist University. Microscopy Sample Preparation. The sampling of specific microscopic tissues from the C. rotundus rhizome samples was carried out as detailed in our previously published reports.36 Prior to sectioning, the rhizome samples were wrapped in water-soaked noncellulose paper and kept under vacuum at 25 inHg pressure at room temperature for 24 h. After softening by application of vacuum, small sections of the rhizomes were cut and embedded in a cryogel matrix (Leica Microsystems, Germany). Rhizome sections of 25 μm thicknesses were cut with a 7303

dx.doi.org/10.1021/jf502494z | J. Agric. Food Chem. 2014, 62, 7302−7316


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Figure 1. Pictorial representation of C. rotundus rhizome samples collected from (A) India and (B) China.

Figure 2. Microscopic characteristics of rhizomes of C. rotundus. Leica LMD 7000 system microscope was used with 10× magnification and a 200 μm scale bar. Images were observed under (A) normal light and (B) blue fluorescence. Tissues are denoted as (a) hypodermal fiber bundles, (b) epidermis, (c) hypodermis, (d) cortex, (e) leaf trace-type vascular bundles, (f) secretory cells, (g) endodermis, and (h) vascular bundles (amphivasal type). held at 200 and 280 °C, respectively. The retention times and characteristic ions for the samples were studied by recording the

electron ionization mass spectra of analytes in scan mode (range of m/z 35−425). For quantitative analysis, selected-ion monitoring (SIM) 7304



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Figure 3. Representative LC-MS base peak chromatograms of C. rotundus rhizomes: (A) whole-rhizome sections of samples collected from India; (B) whole-rhizome sections of samples collected from China; (C) methanol used as blank; (D) cryogenic gel. Peak numbers indicating the identified constituents are listed in Table 1. mode was used and the method was validated.42 The characteristic ions selected for the analysis had m/z values 190 and 203 for (+)-nootkatone. Working solutions of standard (+)-nootkatone were prepared by appropriate dilutions of stock of concentration 1.0 mg/mL. Hexane/ ethyl acetate mixture (1:1) was used as the solvent for preparation of sample solutions.

Data Analysis. For analysis of LC-MS data, Agilent Mass Hunter Workstation software for qualitative analysis (version B 4.00, Build 4.0.479.5, Service Pack 3, Agilent Technologies, Inc., 2011) was used. Both positive and negative ion modes were applied for analysis with the following settings: peaks with height ≥2000 counts to be used, extraction restricted retention time of 1.0−30.0 min, and charge state of 7305


266.1517 176.0834 170.1307 252.1722 234.1681 220.1823 202.1716 234.1618 134.1093 234.1618

C15H24O3 C15H22O2 C15H24O C15H22 C15H22O2 C10H14 C15H22O2

C15H24O2 C15H24O2 C29H50O C15H10O8 C15H22O C15H22O

C15H24O C14H28O2

sugetriol ligucyperonol (−)-epoxycaryophyllene calamenene mandassidione 4-cymene sugeonol

cyperusol B1 cyperusol B2 sitosterol myrcetin cyperotundone valerenal

isolongifolanone myristic acid

1 2 3 3 4 5 6 7 8 9 10 10 11 12 13 14 15 16 16 17 18 18

7306

19.42 22.32

17.52 17.77 18.02 18.44 19.14 19.29

11.67 12.07 13.09 15.45 16.08 16.55 16.94

10.20 10.36 11.11

TR (min) calcd 249.1485 177.091 171.1378 193.1206 253.1798 217.1587 203.1794 203.1794 235.1693 135.1168 235.1693 257.1510 237.1849 237.1849 437.3754 319.2843 219.1743 219.1743 241.1563 221.1900 229.2162 251.1982

species (M+H)+ [−H2O] (M+H)+ (M+H)+ (M+Na)+ (M+H)+ (M+H)+ [−H2O] (M+H)+ [−H2O] (M+H)+ (M+H)+ (M+H)+ (M+H)+ (M+Na)+ (M+H)+ (M+H)+ (M+Na)+ (M+H)+ (M+H)+ (M+H)+ (M+Na)+ (M+H)+ (M+H)+ (M+Na)+

obsd 249.1483 177.0906 171.138 193.1199 253.1796 217.1583 203.1791 203.1788 235.1691 135.1165 235.1691 257.1512 237.1854 237.1855 437.3756 319.2856 219.1745 219.1747 241.1559 221.1912 229.2173 251.1988

0.80 2.33 0.91 −3.71 1.05 1.75 1.41 2.98 0.72 2.06 0.72 0.79 −1.98 −2.60 −0.38 4.15 −0.87 −1.77 1.64 −5.25 − 4.79 −2.75

Δ ppm

+ + + + + + + + + + + + + +

+

CW

+

+ + + + + + +

CA

+

+ + + + + + +

CB

+

+ +

+ + +

CC

+

+ + +

+ + + + + +

CD + + + + + + + + + + + + + + + + + + + + +

IW

samplesa

+

+ + +

+ + + +

IA

+

+ + +

+ + + +

IB

+

+ + + + + + + +

IC

+

+ + +

+ + + +

ID

a CW, extract of sections of whole rhizomes collected from China; CA, CB, CC, and CD represent cortex, hypodermal fiber bundle, endodermis, and amphivisal-type vascular bundle tissues isolated from C. rotundus rhizomes collected from China; IW, extract of sections of whole rhizomes collected from India; IA, IB, IC, and ID represent cortex, hypodermal fiber bundle, endodermis, and amphivisal-type vascular bundle tissues isolated from C. rotundus rhizomes collected from India. (+) denotes presence of constituents in respective samples.

220.1839 228.2099

236.1781 236.1782 414.3861 318.2350 218.1673 218.1673

calcd mass

formula

C15H22O4 C11H12O2 C10H18O2

compd

cyperanic acid 7-methoxy-2-tetralone sobrerol

peak

m/z

Table 1. Data for LC-MS-Based Identification of Various Constituents in C. rotundus Rhizomes

Journal of Agricultural and Food Chemistry Article



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Figure 4. LC-MS base peak chromatograms of various laser-dissected tissues of rhizomes collected from India: (A) cortex, (B) hypodermal fiber bundles, (C) endodermis, and (D) amphivasal-type vascular bundles. Peak numbers indicating the identified constituents are listed in Table 1. 1 and −1 for positive and negative modes, respectively. The compound relative height was ≥2.5%, and absolute height was set to ≥5000 counts with elements of C, H, O, and N from 3−60, 0−120, 0−30, and 0−30, respectively, to generate formulas. The ranges 3−60, 0−120, 0−30, and 0−30 indicate the minimum and maximum limits for the number of elements C, H, O, and N for generation formulas. Peak spacing tolerance was set to 0.0025 m/z plus 7.0 ppm for analysis. The obtained results are depicted as base peak chromatograms (BPC with m/z range 150−950) for each dissected tissue and whole-rhizome tissue analysis. The resultant peak list for LC-MS analysis was processed through Mass

Hunter Workstation software and Microsoft Excel software (Microsoft, Redmond, WA). GC-MS analysis data was processed by using the postrun analysis utility of GC-MS solution software (version 2.50 SU3). The results shown in the paper are average values of determinations performed in five sample sets collected from each of the selected countries.

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RESULTS AND DISCUSSION Macroscopic and Microscopic Studies. Pictorial representations of dried C. rotundus rhizomes collected from India and 7307



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Figure 5. LC-MS base peak chromatograms of various laser-dissected tissues of rhizomes collected from China: (A) cortex, (B) hypodermal fiber bundles, (C) endodermis, and (D) amphivasal-type vascular bundles. Peak numbers indicating the identified constituents are listed in Table 1.

China are shown in Figure 1. The rhizomes of C. rotundus are usually spindle-shaped, 2.0−3.0 cm in length and 0.5−1.0 cm in diameter. The outer surface is dark brown or dull black in color. Longitudinal wrinkles are observed on the outer surface along with scars of fibrous roots. The internal surface appears pale cream or yellowish brown in color and has a pleasant odor.

Laser microdissection (LMD) was used to study the microscopic features of rhizomes; a UV laser beam was used to dissect tissues. In the present study, the cortex, hypodermal fiber bundles, endodermis, and amphivasal-type vascular bundles were isolated for analysis of constituents. The microscopic features of transverse sections of rhizomes observed under white light and blue fluorescence for samples from India and China were found 7308



Article

Figure 6. Representative GC-MS chromatograms of C. rotundus whole rhizomes: (A) extract of rhizomes collected from India; (B) extract of rhizomes collected from China; (C) (+)-nootkatone standard; (D) blank solvent (hexane/ethyl acetate); (E) cryogenic gel. The peak numbers indicating the identified constituents are listed in Table 2.

Figure 2A). Under blue fluorescence, the epidermis appears blackish-brown (see Figure 2B). Followed by the epidermis, the hypodermis consists of 2−3 layers of fibers filled with dark brownish contents. Under fluorescence, the fibers appear reddish

to be similar. A representative image indicating various tissues of rhizomes is shown in Figure 2. A single layer of yellowish-brown epidermis is found to occur with scattered hypodermal fiber bundles (as indicated in 7309



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Table 2. Data for GC-MS-Based Identification of Various Constituents in C. rotundus Rhizome Samples samplesa b

peak

compd

obsd m/z

formula

TR (min)

RI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

pinocarveol pinocarvone (1R)-(−)-myrtenal (+)-verbenone ionone α-cubebene longipinocarveol copaene β-cadinene β-vatirenene methylisoeugenol α-fenchone (+)-cycloisosativene germacrene D eudesma-4(14),11-diene (+)-aromadendrene α-guaiene cycloisolongifolene alloaromadendrene oxide-(1) (−)-spathulenol γ-gurjunenepoxide-(1) trans-Z-α-bisabolene epoxide caryophyllene oxide 9H-cycloisolongifolene, 8-oxo(−)-neoclovene-(II) epiglobulol longiverbenone aristolone longipinocarvone isopulegol α-cyperone isoaromadendrene epoxide (+)-nootkatone menthol corymbolone trans-9-octadecen-1-ol erucic acid

152.25 150.20 150.20 150.00 192.30 204.35 220.35 204.35 204.35 300.45 178.25 152.25 204.35 204.35 204.35 204.35 204.35 202.35 220.35 220.35 220.35 220.35 220.35 218.35 206.35 222.35 218.35 218.35 218.35 154.25 218.35 220.35 242.30 156.25 236.35 268.50 338.55

C10H16O C10H14O C10H14O C10H14O C13H20O C15H24 C15H24O C15H24 C15H24 C20H28O2 C11H14O2 C10H16O C15H24 C15H24 C15H24 C15H24 C15H24 C15H22 C15H24O C15H24O C15H24O C15H24O C15H24O C15H22O C15H26 C15H26O C15H22O C15H22O C15H22O C10H18O C15H22O C15H24O C16H18O2 C10H20O C15H24O2 C18H36O C22H42O2

5.92 6.33 7.01 7.31 10.39 11.00 11.65 11.95 12.91 14.00 14.48 14.82 15.24 15.24 15.81 15.91 16.36 16.80 17.45 18.55 18.61 19.12 20.11 20.52 21.61 21.82 22.40 23.22 23.66 24.10 24.92 25.64 26.99 27.79 29.33 37.76 40.00

1118 1161 1194 1203 1493 1348 1169 1376 1538 1179 1453 1087 1381 1481 1487 1438 1446 1319 1641 1577 1419 1671 1573 1197 1454 1564 1204 1763 1559 1144 1706 1475 1810 1171 1853 2062 2473

CW

CA

CB

CC

CD

IW

IA

IB

IC

ID

− − − + + − + − + + + + + + + + + + + − + + + + + + + + + + + + + − + + −

− − − − + − − − + − + + − − − − − + + − − + − + + − − + − − + − + − − − −

− − − − + − − − + − + − − − − − − + + − − + − + + − − + − − + − + − − − −

− − − − + − − − + − + − − − − − − + + − − + − + + − − + − − + − − − − − −

− − − − + − − − + − + − − − − − − + + − − + − + + − − + − − + − + − − − −

+ + + + + + + + + + + + + + + + − + + + + + − + + + + + + − + + + + − − +

− − − − + − − − + − + − − − − − − + + − − + − + − − − − − − − + − − − − −

− − − − + − − − + − + + − − − − − + + − − + − + − − − − − − − + + − − − −

− − − − + − − − + − + + − − − − − + + − − + − + − − − − − − − + − − − − −

− − − − + − − − + − + + − − − − − + + − − + − + − − − − + − − + + − − − −

CW, extract of sections of whole rhizomes collected from China; CA, CB, CC, and CD represent cortex, hypodermal fiber bundle, endodermis, and amphivisal-type vascular bundle tissues isolated from C. rotundus rhizomes collected from China; IW, extract of sections of whole rhizomes collected from India; IA, IB, IC, and ID represent cortex, hypodermal fiber bundle, endodermis, and amphivisal-type vascular bundle tissues isolated from C. rotundus rhizomes collected from India. (+) denotes presence of constituents in respective samples. bCalculated Kovats retention indices. a

sections and the target isolated tissues of C. rotundus rhizomes, a database of 86 phytoconstituents reported for Cyperus species in scientific publications and mass banks was prepared.43−48 The constituents found in LC-MS spectra were deduced on the basis of extracted fragment groups and losses in molecular masses of fragment ions. Representative LC-MS base peak chromatograms of the rhizomes of both Indian and Chinese samples and controls of cryogenic gel and methanol are shown in Figure 3. Retention times, molecular fragment ions, m/z values of measured adduct ions, and calculated molecular masses of all the secondary metabolites detected in samples of C. rotundus rhizomes by LCMS analysis are listed in Table 1. The database of 86 reported constituents was prepared through literature survey and used for identification of components (see Table S1 of Supporting Information). The cryogel used in preparation of tissue sections

brown. The cortex consists of numerous layers of parenchymatous cells with intercellular spaces. In the cortex, there are numerous secretory cells scattered, and a few leaf trace vascular bundles are also found. When observed under blue fluorescent light, the parenchymatous cells of cortex appear pale blue and secretory cells and endodermis appear reddish brown. An endodermis made of subrectangular cells forming a distinct ring is observed. The stele shows the occurrence of several amphivasal vascular bundles and secretory cells. The samples collected from both India and China belong to the same species, and in the microscopic and morphological investigations carried out in this study, these features were found to be similar between them. Metabolite Profiling by UHPLC-QTOF MS. Cyperus rhizomes are well-known for their terpenoidal constituents.43 The sesquiterpenoids are a particular class of terpenoids found in this plant. For metabolite profiling of the whole-rhizome tissue 7310



Article

Figure 7. GC-MS mass spectra of some abundant constituents found in whole rhizome samples collected from India: (A) copaene, (B) caryophyllene oxide, (C) trans-Z-α-bisabolene epoxide, and (D) longiverbenone.

Figure 8. GC-MS mass spectra of some abundant constituents found in whole rhizome samples collected from China: (A) β-cadinene, (B) α-fenchone, (C) aristolone, and (D) α-cyperone. 7311



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Figure 9. GC-MS base peak chromatograms of various laser-dissected tissues of rhizomes collected from India: (A) cortex, (B) hypodermal fiber bundles, (C) endodermis, and (D) amphivasal-type vascular bundles. Peak numbers indicating the identified constituents are listed in Table 2.

exhibits sharp peaks of glycols and poly(vinyl alcohol) polymers between 11 and 14 min of retention time. Methanol, used as solvent, exhibits few peaks from 18 to 21 min. The positive mode in comparison to negative mode exhibited a better response for profiling of constituents; hence the positive mode was used for analysis. As seen in the chromatograms of Figure 3, there are several constituents that occur in common between the samples collected from India and China. There were also some constituents that occurred exclusively in each of the samples from India and China. The components cyperanic acid, 7-methoxy-2-tetralone, sobrerol, sugetriol, and (−)-epoxycaryophyllene were found only in samples from India. The common constituents for samples from India and China were ligucyperonol, calamenene,

mandassidione, 4-cymene, sugeonol, cyperusol B1 and B2, sitosterol, myrcetin, cyperotundone, isolongifolanone, and valerenal. Tissue-Specific Metabolite Analysis by UHPLC-QTOF MS. The metabolites present in the selected target tissues were identified by LC-MS analysis. The tissues isolated for analysis were cortex, hypodermal fiber bundles, endodermis, and amphivasal-type vascular bundles. The base peak chromatograms obtained after LC-MS analysis of these tissues of samples from India and China are shown in Figures 4 and 5 respectively. The peak numbers assigned to all the identified secondary metabolites and their occurrence in various tissues is indicated in Table 1, along with the details of their molecular characteristics in LC-MS analysis. Table S1 of Supporting Information lists all the constituents used for profiling. 7312



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Figure 10. GC-MS base peak chromatograms of various laser-dissected tissues of rhizomes collected from China: (A) cortex, (B) hypodermal fiber bundles, (C) endodermis, and (D) amphivasal-type vascular bundles. Peak numbers indicating the identified constituents are listed in Table 2.

constituents listed above. The amphivasal vascular bundles, in addition to the common constituents, also had calamenene, mandassidione, 4-cymene, and cyperotundone. On the basis of comparative profiling of tissue metabolites, we infer that the C. rotundus rhizome samples collected from both India and China can be used as qualitative substitutes for each other. GC-MS-Based Metabolite Profiling. As mentioned earlier, several reports indicate the presence of terpenoids, alkaloids, flavonoids, and steroids in rhizomes of C. rotundus. In this study, various essential oil constituents present in C. rotundus were characterized and the content of the sesquiterpenoid constituent (+)-nootkatone was quantified by GC-MS analysis. Representative chromatograms of extracts of whole rhizome samples from India and China, with hexane/ethyl acetate (1:1) used as blank

As indicated in Figure 4 and Table 1, the tissues of rhizomes collected from India contained 4-cymene, sugeonol, cyperusol B1, sitosterol, myrcetin, cyperotundone, and myristic acid. The cortex, hypodermal fiber bundles, endodermis, and amphivasal vascular bundles did not differ significantly in constituents except for the endodermis, which contained cyperusol B2 in addition to the common constituents. With regard to samples collected from China, some differences were observed in the type of constituents in the selected tissues (see Figure 5). The common constituents in all tissues were sugeonol, cyperusol B1, sitosterol, myrcetin, and myristic acid. The cortex and hypodermal fiber bundles also contained cyperusol B2 and cyperotundone in addition to the common constituents. The endodermis contained all the common 7313



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solvent and (+)-nootkatone standard, are presented in Figure 6. The 37 constituents identified in the study samples through GC-MS analysis are listed in Table 2, along with their retention times and calculated Kovats retention indices. (+)-Nootkatone was found to have a retention time of 26.99 min. The constituents found in common between the samples from India and China are trans-Z-α-bisabolene epoxide, longiverbenone, and (+)-nootkatone. From percentage areas it was found that the most abundant constituents in samples from India were copaene (6.96%), trans-Z-α-bisabolene epoxide (9.75%), caryophyllene oxide (9.01%), and longiverbenone (8.50%). In samples collected from China, the abundant constituents found were β-cadinene (33.45%), α-fenchone (7.44%), aristolone (23.31%), and α-cyperone (23.31%). Overall, in the optimized experimental conditions used for this study, 23 constituents were detected in samples from India and 20 constituents were detected in samples from China. Representative mass spectra of some major constituents in samples from India and China are indicated in Figures 7 and 8, respectively, and their occurrence or absence is indicated in Table 2. GC-MS-Based Tissue-Specific Metabolite Profiling. The presence of nonpolar metabolites in cortex, hypodermal fiber bundle, endodermis, and amphivasal-type vascular bundle tissues of rhizomes was estimated by GC-MS analysis. The base peak chromatograms for tissues of rhizome samples from India and China are shown in Figures 9 and 10, respectively. The peak numbers for all the identified constituents are indicated in the chromatograms are listed in Table 2. In the rhizome samples collected from India, all the tissues contained ionone, β-cadinene, methyl isoeugenol, cycloisolongifolene, alloaromadendrene oxide-(1), trans-Z-α-bisabolene epoxide, 9H-cycloisolongifolene, and isoaromadendrene epoxide. All these metabolites were found to occur in the cortex. In addition to these common metabolites, the hypodermal fiber bundles and endodermis indicated the presence of α-fenchone. The amphivasal-type vascular bundle tissues contained longipinocarvone in addition to α-fenchone and the above-mentioned common constituents. In the rhizome samples collected from China, the common metabolites among all the target tissues were ionone, β-cadinene, methyl isoeugenol, cycloisolongifolene, (−)-neoclovene-(II), aristolone, and α-cyperone. The cortex, hypodermal fiber bundles and amphivasal-type vascular bundle tissues in addition to the common metabolites contained (+)-nootkatone; however, it was not detected in the endodermis tissues. Quantitation of (+)-Nootkatone and Method Validation. The content of (+)-nootkatone was determined in the rhizome samples of C. rotundus through GC-MS analysis. SIM mode was used for quantitative analysis and the peak areas obtained for the standard analyte in the calibration curve were used for calculation of (+)-nootkatone content in the samples. The content of (+)-nootkatone in samples from India was found to be higher than in samples from China (see Table 3). The method was validated as per International Conference on Harmonisation (ICH) guidelines (Q2 R1).42 The results obtained for recovery studies and other parameters of validation are reported in Table 4. The sesquiterpenoid component (+)-nootkatone has significant insecticidal, anticancer, and antiplatelet activity, and it is an important flavoring agent used in biotechnology, pesticide, and perfume industries.46−50 Finding sources of rich (+)-nootkatone content like C. rotundus, which is an easily available weed and an

Table 3. Content of (+)-Nootkatone in C. rotundus Rhizome Samples samplea

(+)-nootkatoneb (μg/10 g)

CW IW

21.72 ± 0.572 30.47 ± 0.716

a

CW, extract of whole rhizome samples collected from China; IW, extract of whole rhizome samples collected from India. bContents were calculated on the basis of dry weight of the rhizomes (n = 3) and are presented as mean ± relative standard deviation (RSD).

Table 4. Results of Various Parameters of Validation Studies parameter calibration curve equation correlation coefficient (r2) LOD (ng/mL) LOQ (ng/mL) calibration range (ng) intraday precision (% RSD, n = 5) interday precision (% RSD, n = 3) repeatability (RSD, n = 5) recovery (n = 3) low-level spike (50% ± RSD) intermediate-level spike (100% ± RSD) high-level spike (150% ± RSD)

value for (+)-nootkatone y = 109 851x + 56 174 0.9887 0.303 1.0 1.0−100.0 0.239 0.645 0.423 56.15 ± 0.203 99.78 ± 0.019 125.73 ± 0.372

agricultural waste product, would be of significant importance for the relevant industries. In conclusion, our study is the first to compare tissuespecific metabolites and (+)-nootkatone content in C. rotundus, which is a common plant species found in China and India. Quantitatively, the samples from both these geographical regions have a difference in their percentage contents of (+)-nootkatone. However, qualitatively they have considerable similarities, which suggests their validity of being used as substitutes for each other. The plant C. rotundus is important not only due to its therapeutic utility in traditional medicine and its applications in food industry but also due to its use as an economic and easily available source of (+)-nootkatone. Thus, its (+)-nootkatonerich content adds to its beneficial values despite its reputation of being a notorious weed in the agricultural sector. The experimental approach was designed with a perspective of evaluating the quality of C. rotundus rhizome from two different countries in terms of differences in their metabolites and (+)-nootkatone content. Such studies can help in identification of geographical locations for obtaining herbal material of superior quality. Our study provides information that can be utilized for quality control and standardization of C. rotundus rhizomes and its products. Furthermore, our study provides a perspective for advantageous utilization of the notorious weed C. rotundus, which has been a major concern for the agricultural sector.

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ASSOCIATED CONTENT

S Supporting Information *

One table listing constituents identified in C. rotundus by UHPLC-QTOF MS analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; phone +852 34112424; fax +852 34112461. 7314



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Funding

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This work was supported by the Hong Kong Research Grants Council (GRF-HKBU-263412). Notes

The authors declare no competing financial interest.

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