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Journal of the American College of Nutrition, Vol. 23, No. 6, 763S-767S (2004)
Published by the American College of Nutrition

Effect of Magnesium on Essential Oil Formation of Genetically Transformed and Non-Transformed Chamomile Cultures

Éva Szöke, DSc, Emöke Máday, PhD, Sandor A. Kiss, CSc, Lara Sonnewend, MD and Éva Lemberkovics, CSc

Semmelweis University, Department of Pharmacognosy, Budapest (E.S., E.M., L.S., E.L.), HUNGARY
University of Szeged, Department of Inorganic and Analytical Chemistry (S.A.K.), Szeged, HUNGARY

Address reprint requests to: Éva Szöke, D.Sc., Semmelweis University, Department of Pharmacognosy, H-1085 Budapest Üllöi str. 26., HUNGARY. E-mail: szokee{at}drog.sote.hu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: The importance of chamomile (Chamomilla recutita) is widely known in classical and folk medicine, with the largest group of its effective substances forming the essential oil (chamazulene, {alpha}-bisabolol, trans-ß-farnesene, spathulenol, cis/trans-en-in-dicycloethers). The increasing need for plant-derived high quality drugs cannot be provided by their collection in the wilderness.

Method A: To preserve the genome of Szabadkigyó. wild type having high (–)-{alpha}-bisabolol content, we used biotechnological methods.

Results: The roots of organized culture contained ß-eudesmol, which we have identified in the intact roots. Our gas-chromatographic and mass-spectroscopic studies showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristics of the intact plant. We identified berkheyaradulene, geranyl-isovalerate and cedrol, as new components in these cultures. Magnesium (Mg) (370 and 740 mg/l MgSO4) has a positive effect on the growth of organized cultures and also on the quality and quantity of essential oil production.

Method B: Another possible source of variants is available by the genetic transformation of organized cultures by infection with Agrobacterium rhisogenes. With this method, we cultivated chamomile infected by A4-Y clone and investigated the essential oil production by hairy root cultures cultivated on solid and liquid MS B-5 media. The main component of the essential oil of hairy root cultures was trans-ß-farnesene.

Results: We identified {alpha}-selinene, as a new component in these hairy roots. We studied the growth rate of A4-Y clone on the cited media, containing MgSO4 concentrations: 0; 185; 370 and 740 mg/l. The cultures grew most in medium containing 740 mg/l of MgSO4. Essential oil content was compared from hairy root cultures of different Mg containing media and measured by GC and GC-MS methods. Mg has a similar effect on hairy roots as on organized cultures.

Key words: chamomile, Chamomilla recutita, essential oil, magnesium, organized culture, Agrobacterium rhizogenes, hairy root


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
The pharmacologic effect of medicinal drugs or extracts cannot be attributed only to their bioactive components but to mineral elements as well, since the preparation of extracts results in the presence of both organic and inorganic compounds.

The importance of chamomile, Chamomila recutita (Asteraceae) is widely known in both classical and folk medicine. The plant owes its therapeutic activity to groups of effective substances which give the plant a complex effect [1]. Chamomile is important mostly for its antiphlogistic activity but macro and micro elements can increase its therapeutic activity. The largest group of its medically important compounds are the essential oils, primarily the chamazulene, (–)-{alpha}-bisabolol, bisabololoxides, bisabolonoxide A, farnesene, spathulenol and the cis/trans-en-in-dicycloethers. Flavonoids, coumarins, mucilages, mono- and oligosacharids also have pharmacologic effects. Some compounds are restricted to special organs; others can be detected in almost all parts of the plant [2].

Need for high quality drugs derived from plants cannot be provided only by their collection in the wilderness. For several years the systematic and controlled cultivation of chamomile types has been promoted worldwide [3]. Organized chamomile cultures grow and develop well on Murashige-Skoog (MS) medium [4] which contains Mg. Previously, the effect of Mg in different concentrations was investigated on organized chamomile cultures, and it was established that Mg enhances both the biomass formation and essential oil production of organized cultures. Growth of organized cultures obtained from floral callus tissue of chamomile is minimal on Mg-free medium. The biomass formation and essential oil production is most significant on the medium with twice the MgSO4 concentration: it is about six times higher than that of the control culture, since Mg causes a significant increase in both the fresh mass and the essential oil content [5].

Genetic transformation of organized cultures by infection with virulent strains of Agrobacterium rhisogenes provides another possible source of variants. This new area led us to investigate the possibility of using "hairy roots" for the production of root-specific metabolites for production of essential oil. Mg is indispensable for plant development. In connection with the utilization of natural Mg sources in medicinal plants, understanding the effect of increasing the Mg supply provides an opportunity to increase efficiency of medicinal plant cultivation. In the course of our experiment we followed the changes in the essential oil output and composition parallel with the biomass production, with special regard to the quantity of components considered therapeutically important [6,7].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
Plant Material
Chamomilla recutita (L.) Rausch., Asteraceae.
Wild chamomile populations were obtained from soily areas of Vésztö, Szabadkígyós and the National Park in Hortobágy. They have common morphological features and are rich in (–)-{alpha}-bisabolol. The improved Degumil and polyploid BK-2 type was cultivated in Kerepes.

Sterile Chamomile Cultures.
Sterile chamomile plants were obtained by sterilization of seeds of intact plants [8]. Seedlings were then cultivated at 2500 Lux (16 hours light, 8 hours dark photoperiod) at 26° C, on solid 1/2 Murashige-Skoog (MS) hormone-free media [4]. To determine the effect of MgSO4 we added it to the culture medium in half (185 mg/l) and twice (740 mg/l), the concentration of the 370 mg/l concentration of control prescribed by Murashige-Skoog [9].

Hairy Root Cultures.
The transformed root cultures of chamomile were obtained by infection of sterile two-months-old organized cultures with Agrobacterium rhizogenes strains A 4, 15834 and R 1601. The antibiotic carbenicillin (800 mg/l) and the combination of cefotaxime (250 mg/l) with ampicillin (1000 mg/l) were added to several subcultures to eliminate the bacteria. The bacteria-free hairy roots were cultivated on solid and then in liquid hormone-free B5 and MS medium [4,10].

Extraction and Investigation of the Essential Oil
The essential oil from roots, herbs, and flowers was extracted by steam distillation with apparatus according to the Ph.Hg.VII. The essential oil content was measured gravimetrically [11]. The qualitative and quantitative composition of the essential oil was examined by gas chromatograph (GC) and mass spectrometer (GC-MS).

Gas Chromatographic (GC) Parameters
FISONS GC 8000; Column: 30 m x 0.32 mm, I.D; 0.25 µm; Stationary phases: DB-1701 and DEXm; Column temp.: 60–230°C, 8°C/min, 230°C, isotherm 3 min.; Detector: flame ionisation, 240°C, Carrier gas: Nitrogen, pN2 = 50 kPa, V = 6.8 m3/min; Injector: 200°C; Injection splitless: 10 sec. The percentage (%) evaluation of oil components was carried out on basis of peak-area by Chrom Card computer programme.

GC-MS Parameters (GC-MS: Finnigan GCQ)
Gas Chromatographic Parameters.
Finnigan GC; column: 30 m, I.D.: 0.22 mm; Film thickness: 0.25 µm; Stationary phase: BPX5; Column temp.: 60–230°C, 8°C/min, 230°C isotherm 3 min.; Carrier gas: He, pHe = 40.0 psi; Carrier speed: 40 cm/s; Injector: 200°C, Injection splitless: 6 s, Injected solution volume 4 µl.

MS-Parameters.
Detector: Finnigan MS; Start: 3 min after injection; Mode: EI: 70eV; Mass rate: 40–650 z/e; Scanning: 1 analyse/s; Evaluation: Finnigan GCQ 2.0 computer program.

Investigation of Element Content
For the determination of macro- and microelement content of pulverized, dried drugs, atomic-emission spectrometry (AES) was used [12,13]. Determination of metal ion composition and quantity was by an inductive coupled plasma (ICP)- AES, 22 elements were examined. For standardization of the apparatus, Merck ICP standards of similar composition and matrix as sample solutions were used. Three exact calibrations with 3-second-integration time, background correction and blank substraction were applied. Spectrometer: Thermo Jarrell Ash, type Atom Scan 25 ICP with generator (2 kW; 27.12 MHz) exciting argon plasma to 8000–10000 °K. The optical system is composed of a Czerny-Turner vacuum monochromator and two fotoelectron multipliers.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
Occurrence of macro and micro elements in chamomile species contribute to their therapeutic activity and increase their value. Potassium (K) and sodium (Na) as well as calcium (Ca) and Mg have antagonistic effects and are interdependent [14–20], which is why the ratios of these components were also investigated. Most of the mineral elements were most plentiful in the inflorescence of wild chamomile species; ratios of Ca/Mg in the cultivated type (Degumil) are many-fold higher than in wild chamomile populations. It is remarkable that the ratio of Ca/Mg in the herbs and roots of the cultivated Degumil chamomile examined exceeds that of the inflorescence, but these values are lower in the organized cultures (Fig. 1).



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  		Fig. 1. Ca/Mg ratio in the inflorescence of the cultivated and wild chamomiles and in the sterile culture of the cultivated Degumil chamomiles.

 
The organized cultures of chamomile were produced from callus tissues of inflorescence origin on Murashige-Skoog medium of mineral- and vitamin composition. After being transferred several times, the cultures were raised on hormone-free MS medium. In order to determine the effect of MgSO4 we again added it to the culture medium at half and twice the concentrations of that prescribed by Murashige-Skoog [4]. The control cultures were kept on Mg-free culture medium. We characterized the growth activity and essential oil formation of the tissues. The fresh weight values characteristic of the different MgSO4 concentrations were obtained by weighing the fresh herbs and roots (Fig. 2).



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  		Fig. 2. Effect of MgSO4 on fresh weights (A) and essential oil content (B) and composition (C) of sterile organized cultures from cultivated (BK-2) chamomile herb.

 
Comparing the composition of the essential oil of the intact and organized roots and herbs, we could establish that the composition is similar but intact plants are richer in the percentile distribution of the components. In the essential oil of the root, we first identified (by MS) the ß-eudesmol component known for its liver-protective activity. Our GC and GC-MS studies showed that sterile cultures generated the most important terpenoid and polyin compounds, characteristic of the intact plant. We identified berkheyaradulene, geranyl-isovalerate and cedrol as new components in these cultures. Mg (370 and 740 mg/l MgSO4) enhances the growth of organized cultures and also improves the quality and quantity of essential oil production (Fig. 2).

As MgSO4 has an exceptionally positive effect on rooting of Chamomila recutita organized cultures, it is interesting to demonstrate its effect on the growth of roots of organized cultures and genetically transformed hairy root cultures. Comparing the production of total essential oil of hairy roots growing on four different media mentioned above, we could determine a higher percentage essential oil content on MS media than that on B5 medium. The highest value belonged to cultures on liquid MS medium, which means a five times higher value compared to the content of the intact root (0.04%). In addition, the total essential oil content on both MS media greatly exceeds that of the control (MgSO4 370 mg/l) which is a root of organized cultures and was also cultivated on solid MS medium.

Some of the essential oil components of hairy roots were also determined by mass-spectrometry, namely two farnesene isomeres (trans-ß-farnesene and {alpha}-farnesene) and two other terpene components (cedrol and geranyl-isovalerate). Qualitatively, according to gas-chromatography, the composition of the essential oil of hairy root cultures on different media were similar to each other, but their proportions differed. Cultures on MS medium are rich in tr-ß-farnesene, while the main components of cultures on B5 medium are the terpene molecules. As previously discussed, the highest essential oil content belongs to cultures grown on liquid MS medium, and it is again the liquid MS medium cultures which show the greatest similarity with the control, not only in content of the main component, tr-ß-farnesene, but also in distribution of essential oil components. The control organized culture was again derived from the original root.

Because of the advantageous properties of cultures on liquid and solid MS media, cultures growing on these media were chosen for our further Mg investigations. We studied the effect of different Mg concentrations (Mg-free culture, 185 mg/l, 370 mg/l and 740 mg/l of MgSO4 in the medium) on the linear growth of the hairy-root clones during 40 days on solid MS medium. The growth on Mg-free medium was minimal; growth was similar at half and control Mg concentration; the greatest biomass production was observed on medium with double (740 mg/l) the Mg concentration. Hairy root cultures growing on liquid MS media also had the biomass formation increase with the Mg concentration, which correlates with the results of linear growth of cultures on solid MS medium (Table 1).


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  		Table 1. Magnesium Effect on Biomass Formation of Hairy Root Cultures (D/400 Clone) on MS-2 and B-5 Media

 
The total essential oil content of hairy roots increased with the Mg concentration, but in contrast, the relative percentile distribution of the main essential oil components (e.g. tr-ß-farnesene, {alpha}-farnesene) decreased, while that of other terpenes increased. These results correspond to those found previously in the organized chamomile cultures. The composition of the essential oil of the hairy root cultures growing on liquid MS media of different Mg concentration is similar but they differ in proportion: cultures at 185 mg/l Mg-concentration are rich in tr-ß-farnesene while at 740 mg/l Mg-concentration the main component is berkheyaradulene (sesquiterpene component of 204 molecule weight), which occurs in the intact plant as a minor component (Table 2).


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  		Table 2. Effect of Mgso4 on Essential Oil Components of Hairy Root Cultures (Chamomile Degumil) Cultivated on MS Medium

 

   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
ACKNOWLEDGMENTS
 REFERENCES
 
Addition of MgSO4 to the solid and liquid MS media respectively, favorably affects the biomass and essential oil production of chamomile organized cultures and hairy root cultures. The total essential oil content of hairy roots increases with higher Mg concentration, but in contrast, the relative percentile distribution of the main essential oil components (e.g. tr-ß-farnesene, {alpha}-farnesene) decreases while that of other terpenes increases. These results correspond to those in the organized chamomile cultures.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors express their appreciation to Dr. Zsolt Muskáth and István Antal (Semmelweis University Department of Pharmaceutics, Budapest) for helping with the GC/MS examinations. The authors also thank the help of Dr. Klára Szentmihályi (Central Research Institute for Chemistry of the Hungarian Academy of Sciences) for determination of macro- and micro element content by atomic-emission spectrometry.

Received August 5, 2004.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 

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This Article
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