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Variation in the essential oil composition of Angelica archangelica from three different altitudes in Western Himalaya, India
Industrial Crops and Products 94 (2016) 401–404
Contents lists available at ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Variation in the essential oil composition of Angelica archangelica from three different altitudes in Western Himalaya, India Rajendra S. Chauhan a,∗ , Mohan C. Nautiyal a , Roberto Cecotti b , Mariella Mella c , Aldo Tava b a
High Altitude Plant Physiology Research Center, H.N.B. Garhwal University, Srinagar, Uttarakhand, India Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie, CREA-FLC, Viale Piacenza, Lodi, Italy c Dipartimento di Chimica Organica Università di Pavia, v.le Taramelli 10, 27100 Pavia, Italy b
a r t i c l e
i n f o
Article history: Received 6 May 2016 Received in revised form 24 August 2016 Accepted 25 August 2016 Keywords: Adaptation Angelica archangelica Chemotypes Dillapiole Essential oil MAPs Nothoapiole
a b s t r a c t Angelica archangelica L. (Apiaceae) is one of the important perennial medicinal and aromatic plant, distributed in mid hill region of Europe and Asia. The essential oil composition of the rhizomes of Angelica archangelica from three different altitudes of Western Himalaya was investigated. Essential oils were predominantly composed of dillapiole and nothoapiole quantified by GC and identified based on their GC/MS spectra and NMR data. Composition of the essential oil varied greatly with altitude of collection as well as previous studies. Dillapiole was quantified between 35.–91.5% of the total essential oil, while nothoapiole was quantified between 0.1–62.8% of the total essential oil, which is not reported so far. Such great variations in the composition of the essential oil may be attributed to existence of chemotype and adaptation of the species to particular habitat. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Angelica archangelica L. (Apiaceae) is a perennial medicinal and aromatic plant species (MAPs), 60–230 cm tall, commonly known as European angelica. It is native to Europe and northern Asia, from where it was later introduced to other parts of the globe as important MAPs, as well as for ornamental purposes (Anon, 1985; Gaur, 1999). More southerly, it is common in the Western Himalayan region of India encompassing Kashmir, Himachal Pradesh, Uttarakhand and Sikkim Himalaya, between the altitudes 2600–3900 m asl. This species grows on poor soils, on steep slopes or near the river banks, preferably in moist environments (Alonso, 1998). A. archangelica has long been associated to magic of protection and healing when tried as a remedy against the Black Plague epidemics (Alonso, 1998). Tea made from roots of A. archangelica has been used as a folk remedy for stomach cancer (Duke, 1987). This plant used as a carminative, a gastric stimulant, rheumatic and skin dis-
∗ Corresponding author. Present address: College of Horticulture, VCSG Uttarakhand University of Horticulture & Forestry, Bharsar, Pauri Garhwal, Uttarakhand, 246 123, India. E-mail addresses: [email protected], [email protected] (R.S. Chauhan). http://dx.doi.org/10.1016/j.indcrop.2016.08.044 0926-6690/© 2016 Elsevier B.V. All rights reserved.
orders (Louis, 2002), treat respiratory problems as well as a tonic to improve disease recovery (Hutchens, 1992). Roots used internally in the treatment of gastrointestinal disorders, including gastric ulcers, as well as anorexia, migraine, bronchitis, chronic fatigue and menstrual complaints. It had been stimulated gastric and pancreatic secretions (Alonso, 1998), antiseptic and antidepressant properties (Anon, 1985; Gaur, 1999; Alonso, 1998; Louis, 2002; Hutchens, 1992; Sarker and Nahar, 2004). Recent sources have further substantiated it as an antiseptic, expectorant, emmenagogue and diuretic (Murray, 1995; Duke, 2002). A. archangelica exert an antifeedant activity against the larvae of two major plant pathogens such as Leptinotarsa decemlineata Say. and Spodoptera littoralis Bois (Pavela, 2010). A. archangelica oil showed good antimicrobial activity against Clostridium difficile, Clostridium perfringens, Enterococcus faecalis, Eubacterium limosum, Peptostreptococcus anaerobius and Candida albicans (Fraternale et al., 2014) and in vitro antifungal activity against Fusarium sp., Botrytis cinerea and Alternaria solani (Fraternale et al., 2016). Besides the described therapeutic uses, all parts of A. archangelica have been extensively employed as food flavorings, spices and condiments. The essential oil from the root is also an ingredient in liquors and in high-grade perfumery, notably to impart a musky note as well as a fixative (Stanchev et al., 1993). In general, essen-
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tial oils, flavonoids, tannin, resins, silica, carbohydrates, coumarins, organic acids and terpenes are reported from A. archangelica. This species is mainly cultivated for the production of root essential oil; content of which varies between 0.25–1.16% (Hornok, 1992; Holm et al., 2012). Root essential oils are mainly composed of ␣-phellandrene, -phellandrene, ␣-pinene, ␦-3-carene, limonene, etc. Fraternale et al. (2014) reported that root oil mainly contains ␣-pinene (21.3%), ␦-3-carene (16.5%), limonene (16.4%) and ␣-phellandrene (8.7%). Owing to the multiple uses of the essential oil, the market demand has been gradually increasing, leading to annual production of 50 tons per year (Lubbe and Verpoorte, 2011). However, the erosion of its natural environments and the over harvest fragmented the natural distribution of A. archangelica to such extent that it is now considered an endangered species (Vashistha et al., 2006). This species is under cultivation trial at 2200 m asl (lower altitude than natural habitat). The aim of present work was to analyze the essential oil composition of the rhizomes of A. archangelica, growing at three different altitudes (two wild/natural and one cultivated sample), in a quest to promote cultivation as a more sustainable approach to meet market demand. We found that dillapiole and nothoapiole were the major constituents in the essential oil in this study which are never reported so far from A. archangelica. 2. Material and methods 2.1. Plant material Wild samples of A. archangelica were collected from Dayara, Uttarakashi (3500 m asl, 30◦ 50 N Lat. and 78◦ 33 E Long.) and Tungnath, Rudraprayag, (3300 m asl, 30◦ 14 N Lat. and 79◦ 22 E Long.) in Uttarakhand, India. A third set of plants was obtained from cultivation trials (seeds were collected from Tungnath to raise seedlings) carried out at the High Altitude Plant Physiology Research Centre at Pothiwasa (2200 m asl, 30◦ 28 N Lat. and 79◦ 16 E Long.), in the Rudraprayag district of Uttarakhand, India. Rhizomes were uprooted from senescent (winter dormant) plants during the second week of October from these three locations. Voucher specimens were authenticated by Dr. P. Prasad of the Botanical Survey of India, Dehradun, and deposited in the same herbarium (BSD 030-032). Rhizomes were taken to the laboratory and washed with tap water to remove soil particles, and then they were chopped (into 4–5 cm size pieces) and used fresh for the extraction of the essential oil by hydro-distillation in a Clevenger-type apparatus for 3 h. After decanting, oil samples were dried with anhydrous Na2 SO4 and stored at 4 ◦ C prior to analysis. 2.2. Purification of the major constituents, dillapiole and nothoapiole A portion of the essential oil (50 mg) was placed on a 20 × 20 preparative silica gel plate (Merck, Kieselgel 60F254 ) and eluted with pentane-Et2 O 95:5. Spots were visualized by an UV lamp (254 nm). Dillapiole and nothoapiole were desorbed from silica gel using Et2 O. After solvent evaporation, the two fractions obtained were used for the characterization of the two compounds. Their identification was obtained by GC/MS analysis and by NMR experiments as described below, as well as by comparing results with literature data (Rahman et al., 1999; Benevides et al., 1999; Nitta et al., 2006). 2.3. GC/FID and GC/MS analysis GC/FID analysis was carried out by using a Perkin Elmer Clarus 500 GC equipped with a 30 m × 0.32 mm Elite-5MS capillary column (0.32 m film thickness). Samples were injected (0.5 l) in the
“split” mode (1:30) with a column temperature program of 40 ◦ C for 5 min, then increased to 280 ◦ C at 4 ◦ C/min and finally held at this temperature for 10 min. Injector and detector were set at 250 ◦ C and 300 ◦ C, respectively, and the carrier gas was He with a head pressure of 12.0 psi. GC/MS analysis was carried out using a Perkin Elmer Clarus 500 GC equipped with a Clarus 500 mass spectrometer using the same capillary column and chromatographic conditions as for the GC/FID analysis. Mass spectra were acquired over 40–500 amu range at 1 scan/s with ionizing electron energy of 70 eV, and ion source at 200 ◦ C. The transfer line was set at 300 ◦ C, while the carrier gas was He at 1.0 ml/min. The identification of the oil components was performed by the determination of their retention indices (RI), by comparison with authentic reference compounds as well as with published mass spectra (Adams, 2007) and by peak-matching library search (NIST, 2000). Retention indices (RI) were calculated using a n-alkane series (C6 –C32 ) under the same GC conditions as for the samples. The relative amount (%) of individual components of the oil was expressed as percent peak area relative to total peak area from the GC/FID analysis of the whole extracts.
2.4. NMR analysis 1H
and 13 C NMR were measured on a Bruker AV-300 spectrometer at the operating frequencies of 300.13 and 75.13 MHz, respectively. The samples were examined as solutions in CDCl3 in 5 mm tubes at 25 ◦ C; tetramethylsilane was used as internal reference and chemical shifts were expressed in parts per million (ppm). Multiplicities and assignment of 13 C chemical shifts were made with the aid of DEPT. The complete assignments of all resonances were determined by 2D-HMBC experiments.
3. Results and discussion Essential oil content varied between 0.28–0.35% being minimum 0.28% in Pothivasa, 0.33% in Tungnath and maximum 0.35% in Dayara, which is within the range of previous studies (Hornok, 1992; Holm et al., 2012). Present study revealed that dillapiole and nothoapiole were the major constituents in the essential oil among the samples of A. archangelica collected from different altitudes, an occurrence never reported so far. These results are listed in Table 1, in which >98% of the total essential oil content was characterized by means of GC-FID and GC–MS analysis. Table 1 also presents retention index (RI) of each constituents. GC trace of volatile fraction is also presented in Fig. 1. A maximum of eleven compounds identified in the essential oil sample from Pothivasa nursery (cultivated source, 2200 m asl), whereas the number of compounds were lower in the samples collected from natural populations from Dayara (3500 m asl) and Tungnath (3300 m asl) (Table 1). Dillapiole appeared as the major constituent of the essential oil in the samples collected from Pothivasa and Tungnath, whereas nothoapiole was the major constituent in the samples collected from Dayara. These aroma compounds were not reported so far from this species, therefore, chemical structures of dillapiole and nothoapiole were elucidated by GC/MS and NMR, and shown in Fig. 2. However, several reports are available on the chemical composition of the essential oil from the rhizomes of A. archangelica growing in different parts of the world, showing ␣-pinene, limonene, p-cymene, ␦-3-carene, ␣-phellandrene, -phellandrene and bornyl acetate as the major constituents (Chalchat and Garry, 1997; Pasqua et al., 2001; Nivinskienë et al., 2005; Sigurdsson et al., 2005; Wedge et al., 2009; Holm et al., 2012; Fraternale et al., 2014). As a part of our phytochemical investigation on the plants growing at high altitudes of Northwestern Himalaya, given the variability in
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403
Table 1 Essential oil composition of rhizomes of A. archangelica L. growing at three different altitudes. No.
RI taba
RIb
Compound
Percentage (%) ADayara(3500 m asl)
BTungnath(3300 m asl)
CPothiwasa(2200 m asl)
1 2 3 4 5 6 7 8 9 10 11 12 13
932 946 974 1020 1024 1054 1086 1174 1287 1500 1517 1620 1758c
930 950 978 1022 1026 1053 1084 1171 1285 1495 1515 1618 1759
␣-Pinene Camphene -Pinene p-Cymene Limonene -Terpinene Terpinolene Terpinen-4-ol Bornyl acetate Bicyclogermacrene Myristicin Dillapiole Nothoapiole Total
– – – – tr tr – 0.24 – – 0.91 35.93 62.81 99.89
– – – – – 0.31 0.53 – 0.25 – 8.10 66.14 21.94 99.27
0.33 0.09 0.22 0.51 1.14 1.07 0.94 – – 1.30 0.75 91.55 0.14 98.01
tr- traces (<0.01%). a RI tab, Retention index from Adams, 2007. b RI calculated by GC using n-alkane series under the same conditions as for samples. c From Kapoor et al., 2010.
12
13
12
13
A 11
Detector response
8
11
B
6
7
9
12
C
1
2
3
4 10
5
6
10
7
20
30
11
Time ( i )
13 40
Fig. 1. GC trace of volatile fraction of rhizome of A. archangelica from three different locations: (A) Dayara; (B) Tungnath and (C) Pothiwasa (For peak identification see Table 1).
the composition of the plant’s essential oil, the present study was designed as a chemotypic screening of this species (Table 2). It is worth to note that dillapiole was previously reported to be a major component in the essential oil of another plant species ´ 2010) related to Foeniculum vulgare (Radulovic´ and Blagojevic, A. archangelica as it belongs to the same family Apiaceae. Other important constituents reported from all the three A. archangelica samples were ␥-terpinene and myristicin, being the latter a possible biosynthetic precursor of the apioles. Few other monoterpenic constituents were detected at low percentages only, either in one or two samples only. The presence of other common monoterpene constituents detected in our samples, such as ␣-pinene, limonene and p-cymene resulted, as mentioned before, in other studies on
this species (Chalchat and Garry, 1997; Nivinskienë et al., 2005; Wedge et al., 2009), although in those cases they were detected at much higher percentages. The same happened with other less abundant monoterpenes such as camphene, ␥-terpinene, terpinolene, terpinene-4-ol and bornyl acetate (Pasqua et al., 2001). The predominance of monoterpenes resulted at the utmost in the reports of Nivinskienë et al. (2005), in the seeds (fruits) of this species collected from three Lithuanian habitats, as -phellandrene was the main constituent in all essential oil samples obtained, followed by ␣-pinene and germacrene D (Pasqua et al., 2001), who studied the relationship between root differentiation and the accumulation of essential oils in A. archangelica and found ␣-pinene as the main constituent, followed by ␦-3-carene.
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Table 2 1 H (␦, mult, J Hz) and 13 C (␦) NMR data (CDCl3 ) of Dillapiole and Nothoapiole. Dillapiole 1
1 2 3 4 5 6 7 1 2 3 OCH3 − 4 OCH3 − 5 OCH3 − 7
H
– 5.95, 2H, s – – – – 6.37, 1H, s 3.34, 2H, dd, (1.3, 6.5) 5.91, 1H, m 5.04, 2H, m 3.78, 3H, s 4.03, 3H, s –
Fig. 2. Chemical structure of dillapiole and nothoapiole identified in A. archangelica rhizome essential oil.
In summary, presence of dillapiole and nothoapiole as major constituents may be treated as chemotype in A. archangelica. These major constituents identified from the essential oil in present study are important aroma chemicals of industrial use. Variation in the chemical composition of the essential oil among different altitudes may be attributed to adaptation to particular habitats. In addition, age of the plants and genetic variability may show some effect on the composition of the essential oil. Finally, it is our hope that the results of this analysis will contribute to encourage further investigation on the chemotypic nature and chemical composition of plants adapted to hard environments, both for research and conservation purposes. Acknowledgements The authors are thankful to Prof. A.R. Nautiyal, Director, HAPPRC, Srinagar for providing facilities for this work. We are also thankful to Mr. Arvind Badoni (JRF), HAPPRC, Srinagar for help during this work. Financial support from National Medicinal Plant Board, Govt. of India, New Delhi is acknowledged. Present financial support to first author by All India Coordinated Research Project on MAP&B, DMAPR, Anand, Gujarat is also acknowledged. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed. Allured Publishing Corporation, Carol Stream, IL. Alonso, J.R., 1998. Tratado de Fitomedicina. ISIS Ediciones S.R.L., Buenos Aires, Argentina, pp. 264–267. Anon, 1985. The Wealth of India: A Dictionary of Indian Raw Material and Industrial Products, vol. 1:A. Publication and information Directorate CSIR, New Delhi, pp. 275–276. Benevides, P.J.C., Sartorelli, P., Kato, M.J., 1999. Phenylpropanoids and neolignans from Piper regnellii. Phytochemistry 52, 339–343. Chalchat, J.C., Garry, R.P., 1997. Essential oil of Angelica roots (Angelica archangelica L.). Optimization of distillation, location in plant and chemical composition. J. Essent. Oil Res. 9, 311–319.
Nothoapiole 13
1
C
144.5 101.0 135.8 137.5 144.2 125.9 102.6 33.8 137.3 115.4 61.0 59.8 –
H
– 5.91, 2H, s – – – – – 3.34, 2H, dd, (1.4, 6.5) 5.95, 1H, m 5.02, 2H, m 3.78, 3H, s 3.96, 3H, s 3.90, 3H, s
13
C
144.9 101.1 134.3 133.7 136.4 118.6 144.9 28.2 137.6 114.2 61.3 59.9 60.2
Duke, J., 1987. Handbook of Medicinal Herbs. CRC Press Inc., Boca Raton, USA. Duke, J.A., 2002. CRC Handbook of Medicinal Herbs, 2nd ed. CRC Press, Boca Raton, FL. Fraternale, D., Flamini, G., Ricci, D., 2014. Essential oil composition and antimicrobial activity of Angelica archangelica L. (Apiaceae) roots. J. Med. Food 17 (9), 1043–1047. Fraternale, D., Flamini, G., Ricci, D., 2016. Essential oil composition of Angelica archangelica L. (Apiaceae) roots and its antifungal activity against plant pathogenic fungi. Plant Biosyst. 150 (3), 558–563. Gaur, R.D., 1999. Flora of the District Garhwal North-West Himalaya (With Ethenobotanical Notes). Transmedia, Srinagar (Garhwal) India. Holm, Y., Galambosi, B., Heikkinen, J., Galambosi, Z., Hiltunen, R., 2012. Essential oil composition of Angelica archangelica root from Northern Finland. Rev. Clin. Pharmacol. Med. Ther. 10 (2), 58. Hornok, L., 1992. Angelica. In: Cultivation and Processing of Medicinal Plants. John Wiley & Sons, Chichester, UK, pp. 147–150. Hutchens, A.R., 1992. A Handbook of Native American Herbs. Shambhala Publications, Boston, MA. Kapoor, I.P., Singh, B., Singh, G., De Heluani, C.S., De Lampasona, M.P., Catalan, C.A.N., 2010. Chemistry and antioxidant activity of essential oil and oleoresins of black caraway (Carum bulbocastanum) fruits: part 69. J. Sci. Food Agric. 90, 385–390. Louis, M.O., 2002. In: DerMarderosian, A., Beutler, J.A. (Eds.), The Review of Natural Products. , 2nd ed. Facts and Comparisons Publishing Group. Lubbe, A., Verpoorte, R., 2011. Cultivation of medicinal and aromatic plants for specialty industrial materials. Ind. Crops Prod. J. 34, 785–801. Murray, M.T., 1995. The Healing Power of Herbs, 2nd ed. Prima Publishing, Rocklin, CA, pp. 43–49. NIST/EPA/NIH, 2000. NIST/EPA/NIH Mass Spectral Database, Version 2.1, PerkinElmer Instrument LLC, Copyright 2000. Nitta, M., Kobayashi, H., Ohnishi-Kameyama, M., Nagamine, T., Yoshida, M., 2006. Essential oil variation of cultivated and wild Perilla analyzed by GC/MS. Biochem. Syst. Ecol. 34, 25–37. Nivinskienë, O., Butkienë, R., Mockutë, D., 2005. Chemical composition of seed (fruit) essential oils of Angelica archangelica L. growing wild in Lithuania. J. Essent. Oil Res. 17, 373–377. Pasqua, G., Barbara, M., Andrea, S., Renato, M., 2001. In vitro root differentiation and essential oil accumulation in Angelica archangelica. In Vitro Cell. Dev. Biol. Plant 37, 763–766. Pavela, R., 2010. Antifeedant activity of plant extracts on Leptinotarsa decemlineata Say. and Spodoptera littoralis Bois. larvae. Ind. Crops Prod. J. 32, 213–219. ´ N.S., Blagojevic, ´ P.D., 2010. A note on the volatile secondary metabolites Radulovic, of Foeniculum vulgare Mill (Apiaceae). Facta Univ. 8, 25–37. Rahman, A., Choudhary, M.I., Hayat, S., Khan, M., Ahmad, A., Malik, S., 1999. Spatozoate and varninasterol from the brown alga Spatoglossum variabile. Phytochemistry 52, 495–499. Sarker, S.D., Nahar, L., 2004. Natural medicine: the genus Angelica. Curr. Med. Chem. 11, 1479–1500. Sigurdsson, S., Ögmundsdottir, H.M., Gudbjarnason, S., 2005. The cytotoxic effect of two chemotypes of essential oils from the fruits of Angelica archangelica L. Anticancer Res. 25, 1877–1880. Stanchev, S., Milenkov, B., Hesse, M., 1993. A short synthesis of 15-pentadecanolide. Tetrahedron Lett. 34, 6107–6108. Vashistha, R., Nautiyal, B.P., Nautiyal, M.C., 2006. Conservation status and morphological variations between populations of Angelica glauca Edgew and A. archangelica Linn. in Garhwal Himalaya. Curr. Sci. 91, 1537–1542. Wedge, D.E., Klun, J.A., Tabanca, N., Demirci, B., Ozek, T., Baser, K.H., Liu, Z., Zhang, S., Cantrell, C.L., Zhang, J., 2009. Bioactivity-guided fractionation and GC/MS fingerprinting of Angelica sinensis and Angelica archangelica root components for antifungal and mosquito deterrent activity. J. Agric. Food Chem. 28, 464–470.
Contents lists available at ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Variation in the essential oil composition of Angelica archangelica from three different altitudes in Western Himalaya, India Rajendra S. Chauhan a,∗ , Mohan C. Nautiyal a , Roberto Cecotti b , Mariella Mella c , Aldo Tava b a
High Altitude Plant Physiology Research Center, H.N.B. Garhwal University, Srinagar, Uttarakhand, India Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie, CREA-FLC, Viale Piacenza, Lodi, Italy c Dipartimento di Chimica Organica Università di Pavia, v.le Taramelli 10, 27100 Pavia, Italy b
a r t i c l e
i n f o
Article history: Received 6 May 2016 Received in revised form 24 August 2016 Accepted 25 August 2016 Keywords: Adaptation Angelica archangelica Chemotypes Dillapiole Essential oil MAPs Nothoapiole
a b s t r a c t Angelica archangelica L. (Apiaceae) is one of the important perennial medicinal and aromatic plant, distributed in mid hill region of Europe and Asia. The essential oil composition of the rhizomes of Angelica archangelica from three different altitudes of Western Himalaya was investigated. Essential oils were predominantly composed of dillapiole and nothoapiole quantified by GC and identified based on their GC/MS spectra and NMR data. Composition of the essential oil varied greatly with altitude of collection as well as previous studies. Dillapiole was quantified between 35.–91.5% of the total essential oil, while nothoapiole was quantified between 0.1–62.8% of the total essential oil, which is not reported so far. Such great variations in the composition of the essential oil may be attributed to existence of chemotype and adaptation of the species to particular habitat. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Angelica archangelica L. (Apiaceae) is a perennial medicinal and aromatic plant species (MAPs), 60–230 cm tall, commonly known as European angelica. It is native to Europe and northern Asia, from where it was later introduced to other parts of the globe as important MAPs, as well as for ornamental purposes (Anon, 1985; Gaur, 1999). More southerly, it is common in the Western Himalayan region of India encompassing Kashmir, Himachal Pradesh, Uttarakhand and Sikkim Himalaya, between the altitudes 2600–3900 m asl. This species grows on poor soils, on steep slopes or near the river banks, preferably in moist environments (Alonso, 1998). A. archangelica has long been associated to magic of protection and healing when tried as a remedy against the Black Plague epidemics (Alonso, 1998). Tea made from roots of A. archangelica has been used as a folk remedy for stomach cancer (Duke, 1987). This plant used as a carminative, a gastric stimulant, rheumatic and skin dis-
∗ Corresponding author. Present address: College of Horticulture, VCSG Uttarakhand University of Horticulture & Forestry, Bharsar, Pauri Garhwal, Uttarakhand, 246 123, India. E-mail addresses: [email protected], [email protected] (R.S. Chauhan). http://dx.doi.org/10.1016/j.indcrop.2016.08.044 0926-6690/© 2016 Elsevier B.V. All rights reserved.
orders (Louis, 2002), treat respiratory problems as well as a tonic to improve disease recovery (Hutchens, 1992). Roots used internally in the treatment of gastrointestinal disorders, including gastric ulcers, as well as anorexia, migraine, bronchitis, chronic fatigue and menstrual complaints. It had been stimulated gastric and pancreatic secretions (Alonso, 1998), antiseptic and antidepressant properties (Anon, 1985; Gaur, 1999; Alonso, 1998; Louis, 2002; Hutchens, 1992; Sarker and Nahar, 2004). Recent sources have further substantiated it as an antiseptic, expectorant, emmenagogue and diuretic (Murray, 1995; Duke, 2002). A. archangelica exert an antifeedant activity against the larvae of two major plant pathogens such as Leptinotarsa decemlineata Say. and Spodoptera littoralis Bois (Pavela, 2010). A. archangelica oil showed good antimicrobial activity against Clostridium difficile, Clostridium perfringens, Enterococcus faecalis, Eubacterium limosum, Peptostreptococcus anaerobius and Candida albicans (Fraternale et al., 2014) and in vitro antifungal activity against Fusarium sp., Botrytis cinerea and Alternaria solani (Fraternale et al., 2016). Besides the described therapeutic uses, all parts of A. archangelica have been extensively employed as food flavorings, spices and condiments. The essential oil from the root is also an ingredient in liquors and in high-grade perfumery, notably to impart a musky note as well as a fixative (Stanchev et al., 1993). In general, essen-
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tial oils, flavonoids, tannin, resins, silica, carbohydrates, coumarins, organic acids and terpenes are reported from A. archangelica. This species is mainly cultivated for the production of root essential oil; content of which varies between 0.25–1.16% (Hornok, 1992; Holm et al., 2012). Root essential oils are mainly composed of ␣-phellandrene, -phellandrene, ␣-pinene, ␦-3-carene, limonene, etc. Fraternale et al. (2014) reported that root oil mainly contains ␣-pinene (21.3%), ␦-3-carene (16.5%), limonene (16.4%) and ␣-phellandrene (8.7%). Owing to the multiple uses of the essential oil, the market demand has been gradually increasing, leading to annual production of 50 tons per year (Lubbe and Verpoorte, 2011). However, the erosion of its natural environments and the over harvest fragmented the natural distribution of A. archangelica to such extent that it is now considered an endangered species (Vashistha et al., 2006). This species is under cultivation trial at 2200 m asl (lower altitude than natural habitat). The aim of present work was to analyze the essential oil composition of the rhizomes of A. archangelica, growing at three different altitudes (two wild/natural and one cultivated sample), in a quest to promote cultivation as a more sustainable approach to meet market demand. We found that dillapiole and nothoapiole were the major constituents in the essential oil in this study which are never reported so far from A. archangelica. 2. Material and methods 2.1. Plant material Wild samples of A. archangelica were collected from Dayara, Uttarakashi (3500 m asl, 30◦ 50 N Lat. and 78◦ 33 E Long.) and Tungnath, Rudraprayag, (3300 m asl, 30◦ 14 N Lat. and 79◦ 22 E Long.) in Uttarakhand, India. A third set of plants was obtained from cultivation trials (seeds were collected from Tungnath to raise seedlings) carried out at the High Altitude Plant Physiology Research Centre at Pothiwasa (2200 m asl, 30◦ 28 N Lat. and 79◦ 16 E Long.), in the Rudraprayag district of Uttarakhand, India. Rhizomes were uprooted from senescent (winter dormant) plants during the second week of October from these three locations. Voucher specimens were authenticated by Dr. P. Prasad of the Botanical Survey of India, Dehradun, and deposited in the same herbarium (BSD 030-032). Rhizomes were taken to the laboratory and washed with tap water to remove soil particles, and then they were chopped (into 4–5 cm size pieces) and used fresh for the extraction of the essential oil by hydro-distillation in a Clevenger-type apparatus for 3 h. After decanting, oil samples were dried with anhydrous Na2 SO4 and stored at 4 ◦ C prior to analysis. 2.2. Purification of the major constituents, dillapiole and nothoapiole A portion of the essential oil (50 mg) was placed on a 20 × 20 preparative silica gel plate (Merck, Kieselgel 60F254 ) and eluted with pentane-Et2 O 95:5. Spots were visualized by an UV lamp (254 nm). Dillapiole and nothoapiole were desorbed from silica gel using Et2 O. After solvent evaporation, the two fractions obtained were used for the characterization of the two compounds. Their identification was obtained by GC/MS analysis and by NMR experiments as described below, as well as by comparing results with literature data (Rahman et al., 1999; Benevides et al., 1999; Nitta et al., 2006). 2.3. GC/FID and GC/MS analysis GC/FID analysis was carried out by using a Perkin Elmer Clarus 500 GC equipped with a 30 m × 0.32 mm Elite-5MS capillary column (0.32 m film thickness). Samples were injected (0.5 l) in the
“split” mode (1:30) with a column temperature program of 40 ◦ C for 5 min, then increased to 280 ◦ C at 4 ◦ C/min and finally held at this temperature for 10 min. Injector and detector were set at 250 ◦ C and 300 ◦ C, respectively, and the carrier gas was He with a head pressure of 12.0 psi. GC/MS analysis was carried out using a Perkin Elmer Clarus 500 GC equipped with a Clarus 500 mass spectrometer using the same capillary column and chromatographic conditions as for the GC/FID analysis. Mass spectra were acquired over 40–500 amu range at 1 scan/s with ionizing electron energy of 70 eV, and ion source at 200 ◦ C. The transfer line was set at 300 ◦ C, while the carrier gas was He at 1.0 ml/min. The identification of the oil components was performed by the determination of their retention indices (RI), by comparison with authentic reference compounds as well as with published mass spectra (Adams, 2007) and by peak-matching library search (NIST, 2000). Retention indices (RI) were calculated using a n-alkane series (C6 –C32 ) under the same GC conditions as for the samples. The relative amount (%) of individual components of the oil was expressed as percent peak area relative to total peak area from the GC/FID analysis of the whole extracts.
2.4. NMR analysis 1H
and 13 C NMR were measured on a Bruker AV-300 spectrometer at the operating frequencies of 300.13 and 75.13 MHz, respectively. The samples were examined as solutions in CDCl3 in 5 mm tubes at 25 ◦ C; tetramethylsilane was used as internal reference and chemical shifts were expressed in parts per million (ppm). Multiplicities and assignment of 13 C chemical shifts were made with the aid of DEPT. The complete assignments of all resonances were determined by 2D-HMBC experiments.
3. Results and discussion Essential oil content varied between 0.28–0.35% being minimum 0.28% in Pothivasa, 0.33% in Tungnath and maximum 0.35% in Dayara, which is within the range of previous studies (Hornok, 1992; Holm et al., 2012). Present study revealed that dillapiole and nothoapiole were the major constituents in the essential oil among the samples of A. archangelica collected from different altitudes, an occurrence never reported so far. These results are listed in Table 1, in which >98% of the total essential oil content was characterized by means of GC-FID and GC–MS analysis. Table 1 also presents retention index (RI) of each constituents. GC trace of volatile fraction is also presented in Fig. 1. A maximum of eleven compounds identified in the essential oil sample from Pothivasa nursery (cultivated source, 2200 m asl), whereas the number of compounds were lower in the samples collected from natural populations from Dayara (3500 m asl) and Tungnath (3300 m asl) (Table 1). Dillapiole appeared as the major constituent of the essential oil in the samples collected from Pothivasa and Tungnath, whereas nothoapiole was the major constituent in the samples collected from Dayara. These aroma compounds were not reported so far from this species, therefore, chemical structures of dillapiole and nothoapiole were elucidated by GC/MS and NMR, and shown in Fig. 2. However, several reports are available on the chemical composition of the essential oil from the rhizomes of A. archangelica growing in different parts of the world, showing ␣-pinene, limonene, p-cymene, ␦-3-carene, ␣-phellandrene, -phellandrene and bornyl acetate as the major constituents (Chalchat and Garry, 1997; Pasqua et al., 2001; Nivinskienë et al., 2005; Sigurdsson et al., 2005; Wedge et al., 2009; Holm et al., 2012; Fraternale et al., 2014). As a part of our phytochemical investigation on the plants growing at high altitudes of Northwestern Himalaya, given the variability in
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403
Table 1 Essential oil composition of rhizomes of A. archangelica L. growing at three different altitudes. No.
RI taba
RIb
Compound
Percentage (%) ADayara(3500 m asl)
BTungnath(3300 m asl)
CPothiwasa(2200 m asl)
1 2 3 4 5 6 7 8 9 10 11 12 13
932 946 974 1020 1024 1054 1086 1174 1287 1500 1517 1620 1758c
930 950 978 1022 1026 1053 1084 1171 1285 1495 1515 1618 1759
␣-Pinene Camphene -Pinene p-Cymene Limonene -Terpinene Terpinolene Terpinen-4-ol Bornyl acetate Bicyclogermacrene Myristicin Dillapiole Nothoapiole Total
– – – – tr tr – 0.24 – – 0.91 35.93 62.81 99.89
– – – – – 0.31 0.53 – 0.25 – 8.10 66.14 21.94 99.27
0.33 0.09 0.22 0.51 1.14 1.07 0.94 – – 1.30 0.75 91.55 0.14 98.01
tr- traces (<0.01%). a RI tab, Retention index from Adams, 2007. b RI calculated by GC using n-alkane series under the same conditions as for samples. c From Kapoor et al., 2010.
12
13
12
13
A 11
Detector response
8
11
B
6
7
9
12
C
1
2
3
4 10
5
6
10
7
20
30
11
Time ( i )
13 40
Fig. 1. GC trace of volatile fraction of rhizome of A. archangelica from three different locations: (A) Dayara; (B) Tungnath and (C) Pothiwasa (For peak identification see Table 1).
the composition of the plant’s essential oil, the present study was designed as a chemotypic screening of this species (Table 2). It is worth to note that dillapiole was previously reported to be a major component in the essential oil of another plant species ´ 2010) related to Foeniculum vulgare (Radulovic´ and Blagojevic, A. archangelica as it belongs to the same family Apiaceae. Other important constituents reported from all the three A. archangelica samples were ␥-terpinene and myristicin, being the latter a possible biosynthetic precursor of the apioles. Few other monoterpenic constituents were detected at low percentages only, either in one or two samples only. The presence of other common monoterpene constituents detected in our samples, such as ␣-pinene, limonene and p-cymene resulted, as mentioned before, in other studies on
this species (Chalchat and Garry, 1997; Nivinskienë et al., 2005; Wedge et al., 2009), although in those cases they were detected at much higher percentages. The same happened with other less abundant monoterpenes such as camphene, ␥-terpinene, terpinolene, terpinene-4-ol and bornyl acetate (Pasqua et al., 2001). The predominance of monoterpenes resulted at the utmost in the reports of Nivinskienë et al. (2005), in the seeds (fruits) of this species collected from three Lithuanian habitats, as -phellandrene was the main constituent in all essential oil samples obtained, followed by ␣-pinene and germacrene D (Pasqua et al., 2001), who studied the relationship between root differentiation and the accumulation of essential oils in A. archangelica and found ␣-pinene as the main constituent, followed by ␦-3-carene.
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Table 2 1 H (␦, mult, J Hz) and 13 C (␦) NMR data (CDCl3 ) of Dillapiole and Nothoapiole. Dillapiole 1
1 2 3 4 5 6 7 1 2 3 OCH3 − 4 OCH3 − 5 OCH3 − 7
H
– 5.95, 2H, s – – – – 6.37, 1H, s 3.34, 2H, dd, (1.3, 6.5) 5.91, 1H, m 5.04, 2H, m 3.78, 3H, s 4.03, 3H, s –
Fig. 2. Chemical structure of dillapiole and nothoapiole identified in A. archangelica rhizome essential oil.
In summary, presence of dillapiole and nothoapiole as major constituents may be treated as chemotype in A. archangelica. These major constituents identified from the essential oil in present study are important aroma chemicals of industrial use. Variation in the chemical composition of the essential oil among different altitudes may be attributed to adaptation to particular habitats. In addition, age of the plants and genetic variability may show some effect on the composition of the essential oil. Finally, it is our hope that the results of this analysis will contribute to encourage further investigation on the chemotypic nature and chemical composition of plants adapted to hard environments, both for research and conservation purposes. Acknowledgements The authors are thankful to Prof. A.R. Nautiyal, Director, HAPPRC, Srinagar for providing facilities for this work. We are also thankful to Mr. Arvind Badoni (JRF), HAPPRC, Srinagar for help during this work. Financial support from National Medicinal Plant Board, Govt. of India, New Delhi is acknowledged. Present financial support to first author by All India Coordinated Research Project on MAP&B, DMAPR, Anand, Gujarat is also acknowledged. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed. Allured Publishing Corporation, Carol Stream, IL. Alonso, J.R., 1998. Tratado de Fitomedicina. ISIS Ediciones S.R.L., Buenos Aires, Argentina, pp. 264–267. Anon, 1985. The Wealth of India: A Dictionary of Indian Raw Material and Industrial Products, vol. 1:A. Publication and information Directorate CSIR, New Delhi, pp. 275–276. Benevides, P.J.C., Sartorelli, P., Kato, M.J., 1999. Phenylpropanoids and neolignans from Piper regnellii. Phytochemistry 52, 339–343. Chalchat, J.C., Garry, R.P., 1997. Essential oil of Angelica roots (Angelica archangelica L.). Optimization of distillation, location in plant and chemical composition. J. Essent. Oil Res. 9, 311–319.
Nothoapiole 13
1
C
144.5 101.0 135.8 137.5 144.2 125.9 102.6 33.8 137.3 115.4 61.0 59.8 –
H
– 5.91, 2H, s – – – – – 3.34, 2H, dd, (1.4, 6.5) 5.95, 1H, m 5.02, 2H, m 3.78, 3H, s 3.96, 3H, s 3.90, 3H, s
13
C
144.9 101.1 134.3 133.7 136.4 118.6 144.9 28.2 137.6 114.2 61.3 59.9 60.2
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