|Iranian Journal of Pharmaceutical Research (2005)
Received: October 2004
Accepted: April 2005
Copyright ? 2005 by School of Pharmacy
Mitra Mehrabania*, Mohammadreza Shams-Ardakanib, Alireza Ghannadic, ?Nasrolah Ghassemi Dehkordic and Seyyed Ebrahim Sajjadi Jazic
aDepartment of Pharmacognosy, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran. bDepartment of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. Department of Pharmacognosy, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.
* Corresponding author: email@example.com
Echium amoenum Fisch. and C.A. Mey. (Boraginaceae) is a very popular medicinal plant which is used as a tonic, tranquillizer, diaphoretic, cough remedy, sore throat and pneumonia in Iran?s traditional medicine. Callus culture of medicinal plants is one of the ways for production of secondary metabolites. In this study, callus culture of E. amoenum and its major secondary metabolite were investigated. The callus culture of E. amoenum was initiated and established from seeds in MS media with three different ratios of plant growth regulatories: kinetin, 2,4-D and NAA. Methanolic extracts of freeze-dried calluses were compared by TLC and HPLC. The major secondary metabolite was separated by preparative HPLC and the structure of this pure compound was elucidated by UV, IR, one and two dimensional 1H and 13C-NMR and Mass spectroscopy. Rosmarinic acid was identified by various spectroscopic methods from callus culture of E. amoenum. Rosmarinic acid is widespread within the plant cell tissue culture of the Lamiaceae and Boraginaceae families, although in insignificant quantities. Rosmarinic acid has an antimicrobial, antiviral, and anti-inflammatory effect, which makes it a valuable product for the pharmaceutical and cosmetic industries.
Echium genus (Boraginaceae) has 4 species in Iran (1) and only dried violet?blue petals of Echium amoenum Fish. & C.A. Mey. have medicinal uses in Iran (2, 3). E. amoenum is a biennial or perennial herb indigenous to the narrow zone of northern part of Iran and Caucasus, where it grows at an altitude ranging from 60 to 2200 m (1). This medicinal plant has long been used as a tonic, tranquillizer, diaphoretic, a remedy for cough, sore throat and pneumonia in traditional medicine of Iran (2, 4).
The production of secondary metabolites through a cell culture technology of renowned medicinal plants has been a challenging subject for many researchers. It has been established that cell cultures, obtained from plants of Boraginaceae family such as Lithospermum erythrorhizon and Anchusa officinalis, produce considerable amounts of rosmarinic acid (5, 6) and L. erythrorhizon also produces shikonin and a new brown benzoquinone (7, 8). Other secondary metabolites (e.g. quinones and naphthoquinones) were isolated from cell cultures of Echium lycopsis (9, 10) and even their antimicrobial activities investigated (11).
In this study callus culture of E. amoenum and its major secondary metabolite were investigated.
Seeds of E. amoenum for production of callus were collected from a farm in Rabor at 200 km South of Kerman in June 2000. Voucher specimens of plant (No. 1001) were authenticated and then deposited in Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran.
Initiation of calli and cell cultures:
The callus culture was produced from seedling. The seeds of E. amoenum were germinated as follows. First, the surfaces were sterilized by shaking in 30% (w/v) aqueous hydrogen peroxide solution containing 1% (v/v) Tween 20 for 2.5 min and after washing by sterilized water, placed within 96˚ ethanol for 20 second and then washed again and incubated in 10?90 mm sterilized glass petri dishes lined with two sheets of filter paper which contained 20 ml of distillated water. The petri dishes were kept in the dark at a temperature of 25? 2˚C until the seeds germinated. After obtaining the strong seedlings (up to 2 cm long), for producing callus, the sterile hypocotyls apical buds and adjacent leaf pairs were cut with an sterile scalpel and then aseptically transferred to screw-cap wide-mouth bottles containing 50 ml of Murashige and Skoog (MS) with Gamborge vitamins solid media, containing 1 mg/l of 1-naphtalen acetic acid (NAA) , 0.5 mg/l of 2,4- dichlorophenoxy acetic acid (2,4-D) and 0.5 mg/l of kinetin (kin). Cultures were maintained at 25?2˚C under a 16/8 h light / dark photo period, with a light radiation of ca. 10000 lux provided by cool white fluorescent tubes (GE, 20 W). After three subcultures, a homologues callus mass was divided to three parts and then transferred to MS with Gamborg vitamins solid media with three different combinations of growth regulators, as follows: NAA 2 mg/l+kin 0.7 mg/l (S1); NAA 1 mg/l+2,4-D 0.5 mg/l+kin 0.7 mg/l with 15% coconut milk (S2), and NAA 1 mg/l+2,4-D 0.5 mg/l+kin 1.2 mg/l (S3).
Preparation of medium:
Murashige and Skoog (MS) with Gamborg vitamins medium (Sigma, 4.4 g), sucrose (BDH, 30 g), agar ?agar (Merck, 12 g), de-ionized water and growth regulators (as needed, Sigma) were employed for preparation of one liter of solid medium and then pH adjusted to 5.7. Finally 50 ml of the medium poured into containers, then autoclaved at 121˚C / 1.8 bar for 15 min (12).
Preparation of callus tissues and extraction:
Every 21 days the young and healthy callus culture were subcultured. In 44 sequential subcultures, sufficient amounts of three different calluses (according to difference of growth regulators): S1, S2 and S3, were dried by a freeze drier (Snijders-Ly-5-FM). Their dried weight percentage were then calculated. They were then extracted with methanol in a Soxhlet apparatus under reduced pressure at a temperature of 30˚C, separately. The resulting methanolic extracts were filtrated and concentrated in vacuum and percentage of total dried extracts determined.
TLC analysis- The S1, S2 and S3 callus extracts were analyzed by TLC (40 μl of 100 mg extract/10 ml methanol) on Merck TLC GF254 plates (10?10 cm) with 1, 2-dichloroethane- methanol- acetic acid-water (54:28:11:7) as the eluent. TLC plates were sprayed by Natural Products (NP) reagent (ROTH) (0.6% in methanol) and the spots visualized under UV366 light.
High performance liquid chromatography (HPLC) analysis- A Waters
HPLC system, equipped with preparative LC-4000, and UV-Vis dual λ 2487
spectrophotometric detector, was used for analytical and preparative HPLC
analysis of isolated fractions by column chromatography. An analytical
μBondapack C18 (10 μm) stainless steel column (4.6?250 mm) was used for this purpose. Flow
rate was 1 ml/min and the injected volume was 20 μl of 100 mg extract/10
ml methanol. The column used for preparative HPLC was a PrepLC? μBondapak
C18 (10 μm) (25?200
mm). The injected
volume was 1000 μl and flow rate was 5 ml/min. Mobile phase was methanol (A) and 5% formic acid (B), and a linear gradient from 100% B to 45% A in 175 min was applied. The detector was preset at 280 nm and 350 nm. The major compound was collected by Waters II fraction collector and after removal of the solvent, the residue was subjected to spectroscopic methods.
Ultraviolet (UV) spectroscopy- The UV absorption spectra (220-400 nm) of purified compound (in methanol) was recorded using a Secomam S-1000 UV/Vis spectrophotometer.
Infrared (IR) spectroscopy- The IR spectrum of the purified compound (in KBr) was recorded using a Perkin-Elmer 650 IR spectrophotometer.
Electron impact-mass spectroscopy (EI-MS)? Mass spectra of the purified compound (in DMSO) were recorded using an electron impact (EI) mode at 45 and 70 eV in Finnigan-mat TQS 70EI and Shimadzu Qp 1100EX EI quadruple mass, respectively. The source, probe and scanning temperatures used in this study, were 200, 100-300 and 25-30˚C, respectively.
Nuclear magnetic resonance (NMR) spectroscopy- The NMR spectra were recorded using a Bruker DRX 500 Avence spectrometer. 1H-NMR (at 500 MHz) and 1H-1H (COSY) and 1H-13C (HETEROCOSY) correlation, DEPT 135˚ and 13C-NMR(125 MHz) spectroscopic data were collected at room temperature in d6- DMSO. Chemical shifts (δ, ppm) were reported relative to tetramethylsilane (TMS) as the internal standard.
Results and Discussion
The percentage of high growth rate, dried weights and total dried extracts of E. amoenum calluses were obtained on the S1 (table 1). It seems that on increase in all three cases depends on a high amount of NAA. Coconut milk as a source of cytokinins, compared to the use of higher amounts of kinetin, (S2 vs. S3) results in an increase in all three cases. The calluses of S1, S2 and S3 extracts were fragile and yellowish white; solid and greenish yellow; proportionally fragile and creamy, respectively.
In TLC analysis of S1, S2 and S3 calluses extracts, at an Rf of 0.7, a major spot with quenching in UV254 and bright yellow fluorescence in UV366 (after spraying the NP reagent), was appeared. S1 had the largest spot. By HPLC, this major compound (Rt = 110 min) was separated and its? structure elucidated by UV, IR, NMR and Mass spectra. All the data confirmed that this compound is rosmarinic acid (figure 1). Absorption maxima of UV spectra were 330 nm and a shoulder at 290 nm, as reported for this compound in the literature (13). The IR spectrum showed that the compound has OH groups (3180 and four peaks at 3350-3550 cm ?1, acidic C=O (1740 cm ?1), esteric C=O (1720 cm?1) and aromatic rings (1615,1540,1465 cm?1). The mass fragment profile at 45 eV showed a very small molecular radical ion (M+.) at m/z of 360.2 (intensity = 0.1%) wich was not present as it decomposed at 70 eV. m/z=123 was the base peak at 45 eV and had 75% intensity at 70 eV, belonging to C7H7O2+ (13). NMR data of the compound confirming the structure of rosmarinic acid (13, 14) were as follow:
1H-NMR (500 MHz in d6- DMSO ): δ12.90 (1H, s, OH-18), 9.68 (1H, s, OH-4), 9.20 (1H, s, OH-15), 8.81 (1H, s, OH-3), 8.75 (1H, s, OH-14), 7.47 (1H, d, J=16(7,8)Hz, H-7), 7.06 (1H, s, H-2), 7.01 (1H, d, J=8(6,5)Hz, H-6), 6.77 (1H, d, J=8(5,6)Hz, H-5), 6.69 (1H, s, H-13), 6.64 (1H, d, J=7.6(14,15)Hz, H-16), 6.53 (1H, d, J=7.6(15,14)Hz, H-17), 6.24 (1H, d, J=16(8,7)Hz, H-8), 5.03 (1H, dd, J=8.6(17,16)Hz, J=4(17,16)Hz, H-10), 2.99 (1H, dd, J=14(16,16)Hz, J=4(16,17)Hz, H-11), 2.91(1H, dd, J=14(16,16)Hz, J=8.6(16,17)Hz, H-11).
13C-NMR and DEPT 135˚ (125 MHz in d6- DMSO): δ171.84 (s, C-18), 166.81 (s, C-9), 149.49 (s, C-15), 146.73 (d, C-7), 146.47 (s, C-14), 145.79 (s, C4), 144.85 (s, C-3), 128.28 (s, C12), 126.20 (s, C-1), 122.45 (d, C-6), 120.90 (d, C-17), 117.55 (d, C-13), 116.64 (d, C-5), 116.25 (d, C-16), 115.74 (d, C-2), 114.18 (d, C-8), 73.84 (d, C-10), 37.01(t, C-11).
Rosmarinic acid is accumulated in undifferentiated plant cell cultures such as Zataria multiflora (15), Coleus blumei (16), Salvia fruticosa (17), Thymus vulgaris (18) and Lavandula officinalis (19) from the Lamiaceae family and Lithospermum erythrorhizon and Anchusa officinalis from the Boraginaceae family (5, 6). This study is the first report on the accumulation of rosmarinic acid in one of the Echium species. In previous reports, quinone derivatives have been obtained from cell cultures of these plants (9, 10).
For the first time rosmarinic acid has been reported from other species of Echium, Echium vulgare (20) and according to the chemtaxonomic rules, isolation rosmarinic acid from callus culture of E. amoenum could be expected. An increase in the level of rosmarinic acid accumulation was shown to be dependent on the sucrose content (5, 15, 16) and nitrate ions (19). In this investigation, increasing the NAA, as a growth regulators, or the addition of coconut milk, as a nutrient containing cytokinins, could increase the accumulation of rosmarinic acid in callus culture cells of E. amoenum.
The authors would like to acknowledge the financial support by the Research Council of Isfahan University of Medical Sciences for this work.
Mozaffarian V. A Dictionary of Iranian Plant Names. Farhang Moaser, Tehran (1996) 198
Hooper D. Useful Plants and Drugs of Iran and Iraq. Field Museum of Natural History, Chicago (1937) 115
Delorme P, Jay M and Ferry S. Inventaire Phytochimique des Boraginaceae Indigenes. Planta Medica (1977) 11: 5-11
Amin Gh. Popular Medicinal Plants of Iran. Iranian Research Institute of Medicinal Plants, Tehran (1991) 80
Misawa M. Plant tissue culture: an alternative for production of useful metabolites. Daya Publishing House, Dehli (1997) 60-62
Yamamoto H, Inoue K and Yazaki K. Caffeic acid oligomers in Lithospermum erythrorhizon cell suspension cultures. Phytochemistry (2000) 53: 651-657
Bulgakov VP, Kozyrenko MM, Fedoreyev SA and Zhuravlev YN. Shikonin production by para-fluorophenylalanine resistant cells of Lithospermum erythrorhizon. Fitotrapia (2001) 72: 394-401
Fukui H, Feroj HAFM, Tomohiro U and Masaharu K. Formation and sceration of a new brown benzoquinon by hairy root culture of Lithospermum erythrorhizon. Phytochemistry (1998) 47:1037-1039
Inouye H, Matsumura H, Kawasaki M, Inoue K, Tsukada M and Tabata M. Two quinone from callus culture of Echium lycopsis. Phytochemistry (1981) 20: 1701-1706
Fukui H, Tsukada M, Hajime M and Tabata M. Formation of stereoisomeric mixture of naphthoquinone derivatives in Echium lycopsis callus culture. Phytochemistry (1983) 22: 453-456
Tabata M, Tsukada M and Fukui H. Antimicrobial activity of quinone derivatives from callus culture of Echium lycopsis. Planta Med. (1982) 44: 234-236
Asghari Gh, Lackwood B and Houshfar Gh. Production of volatile sulphides in Allium porrum cell culture. DARU (2002) 10: 137-140
Wettasinghe M, Shahidi F, Amarowicz R and Abou-Zaid MM. Phenolic acids in defatted seeds of borage (Borago officinalis). Food Chemistry (2001) 75: 49-56
Lu Y and Yeap Foo L. Rosmarinic acid derivatives from Salvia officinalis. Phytochemistry (1999) 51: 91-94
Mohagheghzadeh A, Shams-Ardakani MR and Ghannadi AR. Rosmarinic acid from Zataria multiflora tops and in vitro cultures. Fitoterapia (2004) 75: 315-321
Petersen M and Simmonds MSJ. Molecules of Interest Rosmarinic acid. Phytochemistry (2003) 62 : 121?125
Karam NS, Jawad FM, Arikat NA and Shibili RA. Growth and rosmarinic acid accumulation in callus cell suspension and root cultures of wild Salvia fruticosa. Plant Cell Tissue and Organ Culture (2003) 73: 117-121
Hussain AA, Mansour BMM, Toaima N, Craker L and Shetty K. Tissue culture selection for phenolic and rosmarinic acid in Thyme. J. Herbs Species Medicinal Plants (2001) 8: 31-42
Ilieva M and Pavlov A. Rosmarinic acid production by Lavandula vera MM. cell suspension culture nitrogen effect. J. Microbiol. Biotech. (1999) 15: 711-714
Kuruuzum-Uz A, Guvenalp Z, Stroch K, Demirezer LO and Zeeck A. Phytochemical and antimicrobial investigation of Echium vulgare growing in Turkey. Biochemical Systematics and Ecology (2004) 32: 833?836