VOLATILE TERPENES OF SANTOLINA CHAMAECYPARISSUS 403 FLAVOUR AND FRAGRANCE JOURNAL Flavour Fragr. J. 2005; 20: 403–406 Published online 4 May 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1440
Production of volatile terpenes by proliferating shoots and micropropagated plants of Santolina chamaecyparissus L. (cotton lavender) Ashok Ahuja,* S. K. Bakshi, S. K. Sharma, R. K. Thappa, S. G. Agarwal, S. K. Kichlu, R. Paul and M. K. Kaul Regional Research Laboratory (CSIR), Canal Road, Jammu-Tawi 180 001, India Received 15 August 2003; Revised 10 January 2004; Accepted 10 February 2004
ABSTRACT: The biosynthetic capacity of in vitro proliferating shoots and regenerated callus clones has been evaluated for essential oil production. On evaluation it was found that the essential oil isolated from foliage of proliferating shoots and regenerated plantlets was a complex mixture with 49 components, 25 of which were identified, corresponding to 80% of the total oil content. The analysis of the identified constituents included monoterpene hydrocarbon (43%), oxygenated monoterpene (31%), sesquiterpene hydrocarbons (7.4%) and oxygenated sesquiterpenes (4.0%). The major constituents were myrcene, limonene, (E)-linalool, (Z)-β -ocimene and β -caryophyllene oxide. Copyright © 2005 John Wiley & Sons, Ltd. KEY WORDS:
Santolina chamaecyparissus; essential oils; terpenoids; shoot cultures; plantlets
Introduction
Experimental
Santolina L. is a small genus of the large subfamily Asteraceae of the family Compositae and is native to the Mediterranean region. Among the various species, Santolina chamaecyparissus L. is the most common, most studied, and the only species cultivated on a small scale as a medicinal herb throughout Europe and in adjacent parts of Asia and Africa. The infusion, essence, powder and oil from the foliage, and to a lesser extent from the flower heads, are all widely used in folk and herbal medicine on account of their marked antihelmintic, antispasmodic and emmenagogic properties. Beside, it is claimed to be efficacious for the treatment of eye infections, in stimulating the proliferation of scar tissue and in relief of insect bites.1 The chemical composition of the essential oils of S. chamaecyparissus has been studied and variations have been reported in the yield and composition.2–13 Studies on tissue culture of S. chamaecyparissus have been scarce. Callus cultures have been established to study production of essential oils.14 To our knowledge, reports on the establishment of shoot cultures and plantlets for the production of essential oils by such systems are lacking. The present investigations aim to study the biosynthetic capacity of proliferating shoots and in vitro regenerants for the production of essential oils, and comparison with naturally grown plants.
Establishment of Multiple Shoot Cultures and Plantlets Regeneration
* Correspondence to: A. Ahuja, Regional Research Laboratory (CSIR) Canal Road, Jammu-Tawi-180 001, India. E-mail:
[email protected]
Copyright © 2005 John Wiley & Sons, Ltd.
The shoot apices were collected from uniformly grown plants available in the Germplasm Repository at the Regional Research Laboratory, Jammu, India. The material was kept under running tap water for 2 h with a few drops of Teepol. The material was surface-sterilized using 70% EtOH for 1 min, followed by 0.1% (w/v) HgCl2 and rinsed with sterile distilled water three times. The shoot apices were dissected to the size of 3–4 mm each and cultured on Murashige and Skoog15 (MS) agarsolidified medium supplemented with 3% sucrose and 6-benzyaminopurine (BAP), kinetin (Kn), naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), indolebutyric acid (IBA), either singly or in various combinations, using various concentrations (0.1–5.0 mg/l). The cultures were incubated under fluorescent light, using a 16 h/day light photoperiod at 25 ± 2 °C. Periodic transfers to fresh medium maintained multiple shoot cultures differentiated in MS medium containing BAP 1.0 mg/l after every 4 weeks. The in vitro regenerated shoots of different developmental stages were harvested for extraction of essential oil. The in vitro developed shoots from such cultures were subjected to rooting on MS medium supplemented with IAA or IBA (0.5–1.0 mg/1). The rooted shoots were removed carefully and hardened under greenhouse conditions (25 °C; RH 70–80%) and successfully transferred to the field and grown to the maturity. The foliage of in vitro-raised plants and naturally grown plants of the same age were extracted for essential oil.
Flavour Fragr. J. 2005; 20: 403–406
404
A. AHUJA ET AL.
Isolation of the Essential Oils The essential oil was obtained from aerial parts of naturally grown plants and tissue culture samples and field grown acclimatized plantlets by the hydro-distillation using a Clevenger-type apparatus. The oil was collected in benzene, as the percentage was very low. Triplicate distillations were performed in succession for each sample. The oil samples were stored at 4 °C until used for chemical analysis.
Analysis of the Essential Oils Essential oil samples were studied on Silica gel TLC plates using benzene and Pet Ether-Ethyl acetate (90:10) as the solvent system and 2% vanillin–sulfuric acid as visualization agent. Running reference samples under similar conditions identified compounds on TLC plates. Gas Chromatography (GC) analysis of the oil was carried out on NUCON Gas Chromatograph apparatus fitted with a fused silica capillary column coated with FFAP and helium as carrier gas. The compounds were identified by comparison of their relative retention times with those of known compounds run under similar conditions and by enrichment technique. GC–MS was recorded on QP-2000 Shimadzu Model fitted with fused Silica capillary column 30 meter long coated with SE-30. The GC oven temperature was programmed as follows: initial temperature 90°/2 min–220 °C with rise at the rate of 7 °C/min to 220 °C. The carrier gas was Helium with FID detector. The samples were injected using split sampling method, ratio 1:50. The composition in the relative percentage was computed by the normalization method from the GC peak areas.
Results and Discussion Green shining callus was initiated from shoot tips using MS medium supplemented with IAA (0.5 mg/l). Stock callus 1 month old, conditioned under white fluorescent light, showing signs of green colour and shoot primordia, was selected and transferred to bud induction medium, containing 0.2 mg/l IAA, for 4 weeks. The shoot buds thus differentiated were promoted into shoots on transfer to MS medium containing BAP (1 mg/l) under light incubation. After 3 weeks, elongated shoots (about 4– 5 cm) were isolated and placed on root initiation medium, MS medium with IAA or IBA (1 mg/l). After a total of 4 weeks, sufficient roots had formed for the plantlet to be ready for transplantation. Before transplantation to a sterile mixture of sand:soil (1:1), agar medium was carefully removed from the roots. The plantlets were acclimatized under very high humidity in a mist house, then ‘hardenedoff’ in greenhouse conditions. Well-acclimatized plants
Copyright © 2005 John Wiley & Sons, Ltd.
Table 1. Volatile terpenoid contents in essential oils isolated from naturally grown plants and tissue culture regenerated plants of Santolina chamaecyparissus Constituents Essential oil content* Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes
A
B
C
D
1.1 30.0 56.0 10.0 0.8
0.1 32.0 20.0 4.0 9.0
0.1 19.0 23.0 1.0 18.0
0.32 43.0 31.1 7.4 4.0
* ml/100 g. A, naturally cultivated plants (southern hills), after Garg et al. 2001; B, plants cultivated at Jammu site (subtropical); C, plants grown at srinagar site (temperate zone); D, tissue culture regenerated plants cultivated at Jammu site (subtropical).
Table 2. Contents of the essential oil in Santolina chamaecyparissus (cotton lavender) isolated from naturally cultivated mother plants in Jammu-subtropical (A), Srinagar-temperate zone (D), in vitro-proliferated shoots (B) and foliage of tissue culture-raised plants (C) Compounds (% in oil) Santolinatriene α-Pinene β -Pinene Myrcene Car-3-ene p-Cymene Limonene 1,8-Cineole (Z)-β -Ocimene (E)-β -Ocimene α-Terpinene Artemisia alcohol Linalool α-Thujone Cryptone Borneol Terpine-4-ol α-Terpineol β -Elemene β -Caryophyllene β -Gurjunene α-Humulene β -Caryophyllene oxide Globulol β -Oplopenone
RI
A
B
C
D
870 930 968 983 1009 1018 1020 1022 1030 1041 1047 1068 1084 1100 1140 1148 1160 1170 1391 1412 1420 1441 1565 1583 1590
0.50 0.2 0.1 7.0 0.3 0.3 11.7 0.5 13.5 5.4 8.8 0.2 1.9 0.70 1.7 1.6 2.6 2.1 0.3 2.0 0.9 0.6 2.0 1.6 1.0
— — — — — — 7.0 13.5 — 11.7 — — 5.4 — — 1.6 1.7 0.6 1.0 — — — — — —
0.30 0.20 tr 1.8 0.5 0.7 9.2 1.0 1.9 0.7 0.8 1.5 26.6 0.80 0.7 3.2 0.3 0.3 1.2 0.7 0.5 2.5 2.5 1.0 1.5
0.60 0.1 tr 7.9 0.5 0.9 9.6 0.8 10.2 1.0 1.4 1.7 11.5 2.8 0.7 2.1 0.5 0.2 0.15 1.0 0.5 2.3 2.7 1.2 1.5
Identification based on RI using SE-30 column and GC–MS. tr, traces (
