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Effect of saline irrigation water on agronomical and phytochemical characters of chamomile (Matricaria recutita L.)
Scientia Horticulturae 116 (2008) 437–441
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Effect of saline irrigation water on agronomical and phytochemical characters of chamomile (Matricaria recutita L.) Kambiz Baghalian a,b,*, Ali Haghiry b, Mohammad Reza Naghavi c, Abodollah Mohammadi d a
Department of Horticulture, Faculty of Agriculture, Islamic Azad University, Karaj Branch, P.O. Box 31876-44511, Karaj, Iran Iranian Academic Center for Education, Culture & Research (ACECR), Institute of Medicinal Plants Research, Iran c Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Tehran, Karaj, Iran d Department of Agronomy and Plant Breeding, Faculty of Agriculture, Islamic Azad University, Karaj Branch, Iran b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 August 2007 Received in revised form 5 January 2008 Accepted 25 February 2008
In arid and semi-arid regions, where water availability is a major limitation in crop production, using alternative water resources, such as saline water is one way to utilize lands. Chamomile (Matricaria recutita L.) as an annual medicinal herb may be considered as an economic substitute for field crops irrigated with fresh water since it has adaptability to wide range of climate and soil. A field examination was conducted during 2004–2005 using complete randomized block design with four replications in order to evaluate the effects of saline irrigation water on morphological characters, mineral content, oil quantity (content, yield), oil composition and apigenin content of chamomile. In each plot, 0.6 g/m2 of seeds were grown in 4 rows. The irrigation water had five different salinity levels (0, 4, 8, 12 and 16 dS m1). The investigated characters through cultivation were fresh weight of flower (g), dry weight of flower (g), dry weight of aerial stems (g), dry weight of root (g), oil yield (kg/h), oil content (%), oil quality and apigenin content (%). After harvesting, the content of minerals (Na+, Cl, K+, Ca2+, Mg2+) were evaluated in aerial parts and roots of each plot. Mean comparisons for fresh flower weight in different treatments showed that fresh flower yield decreased with increasing salinity and it was higher in control compared to others. Analysis of variance showed that saline irrigation water had no significant effect on oil quantity (yield and content), oil quality (chemical composition) or apigenin content. Our results showed that chamomile is able to maintain all its medical properties, under saline condition and could be cultivated economically in such conditions. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Medicinal value Secondary metabolites Apigenin Oil constituents Minerals
1. Introduction Saline soil can be defined as soil having an electrical conductivity of the saturated paste extract (ECe) of 4 dS m1 (4 dS m1 40 mM NaCl) or more. Salinity is a major factor reducing plant growth and productivity worldwide. It affects about 7% of the world’s total land area (Flowers et al., 1997; Zhu, 2002). Iran’s climate is mostly arid and semi-arid, where water availability is a major problem in crop production. Fifteen percent of total agricultural lands of Iran have salt in water or soil. In such conditions cultivation of resistant plants is one way to utilize these lands and therefore the selection of suitable crops, which could cope with these conditions, is a necessity (Mirmohamadimeibodi and Gharaiazi, 2002). * Corresponding author at: Department of Horticulture, Faculty of Agriculture, Islamic Azad University, Karaj Branch, P.O. Box 31876-44511, Karaj, Iran. Tel.: +98 2122580227; fax: +98 2122580227. E-mail address: [email protected] (K. Baghalian). 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.02.014
Chamomile (Matricaria recutita L.) may be considered as an economic substitute for field crops irrigated with fresh water since it has adaptability to wide range of climate and soil (Svab, 1992; Ram and Misra, 2004). Chamomile is an annual herb with short but widespread roots. It varies in size (from small to 60 cm) depending on the locality and the soil. The flowers are collected from May to July (Applequist, 2002). Chamomile flower is an official drug (recognized by government authority) in the pharmacopoeia of 26 countries. At present this plant is known as one of the main medicinal herbs and is listed in major pharmacopoeia such as United States Pharmacopoeia (USP, 2004) and British Pharmacopoeia (2002). Furthermore, chamomile is widely used as a medicinal herb in Europe. The European Scientific Cooperative for Phytotherapy (ESCOP) is producing comprehensive scientific reviews and suggested regulatory texts for herb use. One of the first herbs for which they produced such a document was chamomile (Blumenthal, 2004).
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One of the main reasons for pharmaceutical characters of chamomile is related to different classes of active constituents including essential oils and flavonoids (Wichtl, 1994; Szoke et al., 2004). The plant contains 0.24–1.9% essential oils, from which over 120 components have been identified. The major oil components include a-()-bisabolol and chamazulene which directly reduce inflammation and are both mild antibacterials. The essential oil also contains bisabolol oxides A and B, farnesene and abisabolonoxide A, which have anti-inflammatory and antispasmodic actions (Achterrath-Tuckermann et al., 1980; Jakolev et al., 1983). Apigenin-7-glucoside, a flavonoid glycoside in chamomile, inhibits transcriptional activation of inducible COX-2 and nitric oxide synthase, via inhibition of NFkappaB (Repcak and Martonfi, 1995; Avallone et al., 2000). Due to the foregoing, chamomile’s world market is growing. However, worldwide production figures are difficult to find, due to the small scale of chamomile farming and the fact that statistics generally do not quote chamomile separately from other herbs. At present this plant is mainly produced in countries with low labor costs and exported to industrial countries. In 2002, number of acres cultivated for chamomile worldwide is estimated at 50,000 and the cost ranges between 3 and 12 dollars per pound depending on quality (Brester et al., 2002). In Iran, chamomile holds a very special position. Chamomile naturally grows in west, northwest and southern part of Iran (Podlech, 1986). Its consumption as a folklore medicine has a long history. Medicinal importances of this species are also on the rise and at present seven pharmaceutical products are produced from chamomile in Iran under license of Ministry of Health. Its cultivation has been increased steadily in recent years (Baghalian, 2000). Biosynthesis of secondary metabolites is not only controlled genetically but it also is affected strongly by environmental influences (Naghdi Badi et al., 2004). In line with the foregoing, environmental variables affect essential oil and its composition in chamomile (Mann and Staba, 1986). There are extensive data on the effect of environment on content and composition of its secondary metabolites (Galambosi and Repecak, 1999; Franz, 2000; Salamon, 2000; Galambosi, 2001). Letchamo and Vomel (2001) have shown that temperature could increase content of a()-bisabolol and bisabolol oxides A and B. We found that performance of chamomile on saline water has not been well studied or documented and there are only a few reports on the effect of water salinity on morphological characters and oil composition of chamomile (Cellarova et al., 1986; Afzali et al., 2006). The aim of this work was to study the effect of saline irrigation water on agronomical and phytochemical characters, in order to support breeding program and use of chamomile as an economic substitute for field crops irrigated with fresh water. 2. Materials and methods 2.1. Field site description A field examination was conducted using complete randomized block design with four replications at the Experimental Station of Institute of Medicinal Plant, Karaj, Iran during 2004–2005. The geographical location of the station was 358470 N and 508560 E with 126 m altitude. The soil properties of the location are presented in Table 1. Seeds of the Iranian natural population of chamomile, provided by Institute of Medicinal Plant, Karaj, Iran, were sown in November 2004 by hand. These seeds were originally collected from natural sources and have since been cultivated every year for
Table 1 Chemical and physical characteristics for soil of experimental field Analysis
Soil depth 0–30 cm
ECe (dS/m) pH TNV (%) OC (%) Total N (%) Available P (ppm) Available K (ppm) Clay (%) Silt (%) Sand (%) Texture Zn (ppm) Fe (ppm) Mn (ppm) Cu (ppm)
0.93 7.9 8.5 0.82 0.08 36.2 49.8 16 22 62 Silt-loam 0.6 5.74 11.2 0.7
purpose of seed production. For each experimental plot, 0.6 g/m2 of seeds were grown in 4 rows with 50 cm apart and 3 m length. 2.2. Irrigation water The experimental treatments consisted of four salinity levels of the irrigation water (4, 8, 12 and 16 dS m1) in addition to control (0 dS m1). The salinity levels were obtained by addition of appropriate amount of NaCl to water and were adjusted by a portable Ec meter instrument. The different irrigation treatments started in the beginning of spring after the seedlings started their growth and development. Each plot received 40 l of water nine times from the middle of April to the end of June 2005. In each plot, 25 g nitrogenous fertilizer as ammonium nitrate was applied. 2.3. Field investigated characters Chamomile has a continuous flowering habit. Plants flowering started from middle of May and lasted to end of June 2005. Therefore, for all plants in each plot, several harvests were done, 7– 10 days apart, depending on the weather conditions, and total fresh weight of harvested flowers was measured. Dry flower weight of each plot was calculated after drying the flowers at room temperature (20–25 8C). After the flowering period, plants (root and stem) were collected and dry weights of stem and root were recorded for all plants in each plot. 2.4. Content of minerals After harvesting, content of minerals (Na+, K+, Ca2+, Mg2+, Cl) were evaluated in stems and roots of each plot. Two g of dried material was used for test solution preparing, according to method described by Baker and Suhr (1982). Sodium and potassium concentration was determined by Flame photometer (Jenway PFP7) and automatic titration method was used for chloride determination (Adriano and Doner, 1982). Calcium and Magnesium contents were measured using complexometry method which is a practical technique used to determine the concentration of metal ions. In a titration, the volume of EDTA as a standard solution of a complex-forming reagent needed to react exactly with the metal ions in a defined volume of the unknown solution is measured (Waling et al., 1989). 2.5. Analysis of essential oils Oils were extracted by hydro distillation of flowers using Clevenger-type apparatus for 3 h (British Pharmacopoeia, 2002).
K. Baghalian et al. / Scientia Horticulturae 116 (2008) 437–441
439
The oils were dried over anhydrous sodium sulphate and kept at 4 8C until they were analyzed. Qualitative and quantitative analyses of oils were performed by Agilent gas chromatography model 6890 N, equipped with MSD model 5973 N and fused silica capillary column (HP-5MS, 30 m 0.25 mm). The GC oven temperature was held at 50 8C for 2 min, then programmed from 50 8C to 200 8C at a rate of 3 8C min1 and from 200 8C to 280 8C at a rate of 7 8C min1, held for 2 min at 280 8C, using He as the carrier gas (1.0 ml min1). The temperature of injector was 250 8C. The percentage composition of the essential oils was computed from GC peak areas without correction factors. Qualitative analysis was based on a comparison of retention times and indexes on both columns and mass spectra with corresponding data available in the literature (Adams, 1995) and computer mass spectra libraries. 2.6. Apigenin-7-glucoside determination An HPLC method used for quantification of apigenin-7-glucoside determination according to the method described in United States Pharmacopoeia (2004). HPLC instrument consisted KNUWER pump k-1001 and KNUWER C18 column (150 mm 4.6 mm). The isocratic mobile phase was variable mixtures of solution A (0.005 M monophosphate potassium adjusted with phosphoric acid (5%) to pH = 2.55 0.05) and solution B (acetonitril–methanol (65:35)) which programmed at a flow rate of 1 ml min1. Peaks were detected at 335 nm, and recorded by a KNUWER UV-visible detector model K-2501. Standard solution was produced by dissolving accurately weighted quantity of apigenin-7-Glucoside (Fluka, purity 97%) in methanol and diluting to obtain a solution having concentration of 0.25 mg/ml of standard.1.0 g chamomile from each of 20 treatments were accurately weighted and used for preparing test solution according to the method described in United State Pharmacopoeia (2004). After preparing test and standard solutions, equal volumes (20 ml) of them were separately injected to the HPLC. The percentage of apigenin-7-glucoside was calculated based on the fallowing expression: Apigenin-7-glucoside ð%Þ ¼ 20ðC=WÞðr u =r s Þ in which C is the concentration, in mg per ml, of apigenin-7glucoside in standard solution; W is weight in g, of chamomile taken for test solution; ru and rs are the apigenin-7-glucoside responses obtained from the test solution and the standard solution, respectively. 2.7. Data analysis After agronomical and phytochemical evaluation, quantitative analyses were carried out using the computer software SPSS
Fig. 1. Duncan result for fresh flower weight in different treatments (different letters on the top of columns indicate statistically significant differences).
version 10.0. Data were analyzed by ANOVA and the means of results were compared by Duncan’s multiple range tests. 3. Results 3.1. Agronomical evaluation The results indicated that fresh flower weight was the only morphological trait, which was significantly different in salinity levels. This result is not inconsistent with results reported before (Prasad et al., 1997). Duncan results showed non-significant effect of salinity levels on dry flower weight, while fresh flower yield decreased with increasing salinity and it was higher in control compared to others (Fig. 1). 3.2. Mineral content Mineral nutrition is mainly affected by salinity and leads to ionic imbalance and nutrition problems. In other words, salinity alters the ion transport and contents of ions such as K+, Ca2+ and with an antagonist like effect (Cramer and Schmidt, 1995). In this study mineral content evaluation showed that chloride and sodium were affected significantly in both root and stem. Furthermore Ca2+ and Mg2+ accumulation had been increased significantly in stem. This is in agreement with results obtained by Prasad et al. (1997). Saline irrigation water had no significant effect on K+ accumulation in both root and shoot. Comparison of Na+ and Cl accumulation in root and stem showed that increasing salinity leads to increasing in accumulation (Fig. 2). It is worth noting that concentration of both minerals in stem was higher than root.
Fig. 2. Comparison of Na+ (left) and ClS (right) content (%) in stem and root (different letters on the top of columns indicate statistically significant differences).
K. Baghalian et al. / Scientia Horticulturae 116 (2008) 437–441
440 Table 2 Duncan multiple test for phytochemical characters Salinity levels (dS m1)
0 4 8 12 16
Oil quantity
Oil composition (%)
Apigenin (%)
Yield (kg/h)
Content (%)
BoB
BnA
Ch
BoA
Bo
Fa
0.04 0.03 0.03 0.05 0.03
0.037 0.037 0.032 0.041 0.027
8.881(a) 10.2(a) 14.8(a) 11.25(a) 10.87(a)
7.17(a) 7.54(a) 8.3(a) 6.64(a) 7.66(a)
18.76(a) 19.53(a) 20(a) 19.71(a) 19.55(a)
30.82(a) 32.15(a) 29.7(a) 33.7(a) 33.36(a)
1.03(a) 1.07(a) 0.91(a) 2.01(a) 1.27(a)
0.17(a) 0.1(a) 0(a) 0(a) 0.3(a)
(a) (a) (a) (a) (a)
(a) (a) (a) (a) (a)
0.28(a) 0.26(a) 0.28(a) 0.25(a) 0.27(a)
BoB (a-bisabololoxide B), BnA (a-bisabolonoxide A), Ch (chamazulene), BoA (a-bisabolol oxide A), Bo (a-bisabolol), Fa (trans-b-farnesene), letter (a) indicates statistically non-significant differences between treatments.
Fig. 3. HPLC chromatogram of apigenin in test solution (right) and standard solution (left).
3.3. Qualitative and quantitative analysis of essential oils Analysis of variance showed that saline irrigation water had no significant effect on oil quantity (yield and content) and medically important constituents such as a-bisabololoxide B, a-bisabolonoxide A, chamazulene, a-bisabolol oxide A, a-bisabolol and transb-farnesene (Table 2). 3.4. Apigenin evaluation When the percentage of total apigenin was analyzed by HPLC (Fig. 3), surprisingly we found that salinity had no significant effect on apigenin content and therefore, on its spasmolytic activity (Table 2). 4. Discussion In this study, the effect of saline water on morphological and phytochemical characters of chamomile, as a medicinal plant, were investigated. The results in agronomical evaluation suggest that cultivation of chamomile on a large scale could successfully represent an acceptable agronomical yield. It is worth noting that dry flower weight was not significant. This could be contributed to different water accumulation between different treatments. As chamomile flowers are marketed in dry form, this means that salinity has no significant effect on economical flower yield. The difference in a plant’s response to a given level of salinity is dependent upon the concentration and composition of the ions in solution as well as the genotype that is exposed to the salinity (Greenway and Munns, 1980). Higher accumulations of Na+ and Cl in the stem than those in the root are probably due to the translocation of minerals to stem which causes the plant to overcome salinity. Similar results have been reported in studies conducted on other crop plants such as rice (Aslam et al., 1996), pepper (Cornillon and Palliox, 1997) and wheat (Ha and Schmidhalter, 1997). Qualitative and quantitative analysis of essential oils showed that under saline condition, quantitative and qualitative value of
oil, as a medically important factor in chamomile, could present an acceptable performance. These results are not consistent with the findings of Ram et al. (1999) and Prasad et al. (2006) which reported that increasing sodium ions significantly affects oil content and composition. Apigenin variations, as a flavonoid, are considered as a phytochemical adaptation to the abiotic and biotic environment (Dixon and Paiva, 1995). There are extensive data that suggest that apigenin synthesis is influenced by different factors such as UV light radiation, drought, ozone, phytopathogens and insectdeterrent (Nikolova and Ivancheva, 2005), however we did not find any reports treating the subject of apigenin change in response to salinity. Furthermore, we did not find any significant effect on apigenin content among different salinity levels. This means that production and accumulation of apigenin, as an economically important aspect of chamomile, could be done in a usual manner when this plant is treated with salinity. In summary, through our study, the results showed that chamomile could present acceptable agronomical yield with sufficient medicinal properties. Therefore, cultivation of chamomile on a large scale could be successfully accomplished on saline water, where cultivation of field crops irrigated with fresh water is not possible. Identification of plant mechanisms for salt tolerance and breeding of new cultivars, represents essential strategies for reducing salinity effects in agriculture (Poustini and Siosemardeh, 2004). Furthermore, it seems that we need to undertake further studies, in different locations and with durations longer than one year, in order to obtain more reliable results and also to clarify physiological mechanism of salinity tolerance in chamomile. Acknowledgments We gratefully acknowledge the financial support of Islamic Azad University, Karaj Branch, through this research program. Further we express our special thanks to Iranian Academic Center
K. Baghalian et al. / Scientia Horticulturae 116 (2008) 437–441
for Education, Culture & Research (ACECR), Institute of Medicinal Plants Research for their valuable support and cooperation.
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Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Effect of saline irrigation water on agronomical and phytochemical characters of chamomile (Matricaria recutita L.) Kambiz Baghalian a,b,*, Ali Haghiry b, Mohammad Reza Naghavi c, Abodollah Mohammadi d a
Department of Horticulture, Faculty of Agriculture, Islamic Azad University, Karaj Branch, P.O. Box 31876-44511, Karaj, Iran Iranian Academic Center for Education, Culture & Research (ACECR), Institute of Medicinal Plants Research, Iran c Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Tehran, Karaj, Iran d Department of Agronomy and Plant Breeding, Faculty of Agriculture, Islamic Azad University, Karaj Branch, Iran b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 August 2007 Received in revised form 5 January 2008 Accepted 25 February 2008
In arid and semi-arid regions, where water availability is a major limitation in crop production, using alternative water resources, such as saline water is one way to utilize lands. Chamomile (Matricaria recutita L.) as an annual medicinal herb may be considered as an economic substitute for field crops irrigated with fresh water since it has adaptability to wide range of climate and soil. A field examination was conducted during 2004–2005 using complete randomized block design with four replications in order to evaluate the effects of saline irrigation water on morphological characters, mineral content, oil quantity (content, yield), oil composition and apigenin content of chamomile. In each plot, 0.6 g/m2 of seeds were grown in 4 rows. The irrigation water had five different salinity levels (0, 4, 8, 12 and 16 dS m1). The investigated characters through cultivation were fresh weight of flower (g), dry weight of flower (g), dry weight of aerial stems (g), dry weight of root (g), oil yield (kg/h), oil content (%), oil quality and apigenin content (%). After harvesting, the content of minerals (Na+, Cl, K+, Ca2+, Mg2+) were evaluated in aerial parts and roots of each plot. Mean comparisons for fresh flower weight in different treatments showed that fresh flower yield decreased with increasing salinity and it was higher in control compared to others. Analysis of variance showed that saline irrigation water had no significant effect on oil quantity (yield and content), oil quality (chemical composition) or apigenin content. Our results showed that chamomile is able to maintain all its medical properties, under saline condition and could be cultivated economically in such conditions. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Medicinal value Secondary metabolites Apigenin Oil constituents Minerals
1. Introduction Saline soil can be defined as soil having an electrical conductivity of the saturated paste extract (ECe) of 4 dS m1 (4 dS m1 40 mM NaCl) or more. Salinity is a major factor reducing plant growth and productivity worldwide. It affects about 7% of the world’s total land area (Flowers et al., 1997; Zhu, 2002). Iran’s climate is mostly arid and semi-arid, where water availability is a major problem in crop production. Fifteen percent of total agricultural lands of Iran have salt in water or soil. In such conditions cultivation of resistant plants is one way to utilize these lands and therefore the selection of suitable crops, which could cope with these conditions, is a necessity (Mirmohamadimeibodi and Gharaiazi, 2002). * Corresponding author at: Department of Horticulture, Faculty of Agriculture, Islamic Azad University, Karaj Branch, P.O. Box 31876-44511, Karaj, Iran. Tel.: +98 2122580227; fax: +98 2122580227. E-mail address: [email protected] (K. Baghalian). 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.02.014
Chamomile (Matricaria recutita L.) may be considered as an economic substitute for field crops irrigated with fresh water since it has adaptability to wide range of climate and soil (Svab, 1992; Ram and Misra, 2004). Chamomile is an annual herb with short but widespread roots. It varies in size (from small to 60 cm) depending on the locality and the soil. The flowers are collected from May to July (Applequist, 2002). Chamomile flower is an official drug (recognized by government authority) in the pharmacopoeia of 26 countries. At present this plant is known as one of the main medicinal herbs and is listed in major pharmacopoeia such as United States Pharmacopoeia (USP, 2004) and British Pharmacopoeia (2002). Furthermore, chamomile is widely used as a medicinal herb in Europe. The European Scientific Cooperative for Phytotherapy (ESCOP) is producing comprehensive scientific reviews and suggested regulatory texts for herb use. One of the first herbs for which they produced such a document was chamomile (Blumenthal, 2004).
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One of the main reasons for pharmaceutical characters of chamomile is related to different classes of active constituents including essential oils and flavonoids (Wichtl, 1994; Szoke et al., 2004). The plant contains 0.24–1.9% essential oils, from which over 120 components have been identified. The major oil components include a-()-bisabolol and chamazulene which directly reduce inflammation and are both mild antibacterials. The essential oil also contains bisabolol oxides A and B, farnesene and abisabolonoxide A, which have anti-inflammatory and antispasmodic actions (Achterrath-Tuckermann et al., 1980; Jakolev et al., 1983). Apigenin-7-glucoside, a flavonoid glycoside in chamomile, inhibits transcriptional activation of inducible COX-2 and nitric oxide synthase, via inhibition of NFkappaB (Repcak and Martonfi, 1995; Avallone et al., 2000). Due to the foregoing, chamomile’s world market is growing. However, worldwide production figures are difficult to find, due to the small scale of chamomile farming and the fact that statistics generally do not quote chamomile separately from other herbs. At present this plant is mainly produced in countries with low labor costs and exported to industrial countries. In 2002, number of acres cultivated for chamomile worldwide is estimated at 50,000 and the cost ranges between 3 and 12 dollars per pound depending on quality (Brester et al., 2002). In Iran, chamomile holds a very special position. Chamomile naturally grows in west, northwest and southern part of Iran (Podlech, 1986). Its consumption as a folklore medicine has a long history. Medicinal importances of this species are also on the rise and at present seven pharmaceutical products are produced from chamomile in Iran under license of Ministry of Health. Its cultivation has been increased steadily in recent years (Baghalian, 2000). Biosynthesis of secondary metabolites is not only controlled genetically but it also is affected strongly by environmental influences (Naghdi Badi et al., 2004). In line with the foregoing, environmental variables affect essential oil and its composition in chamomile (Mann and Staba, 1986). There are extensive data on the effect of environment on content and composition of its secondary metabolites (Galambosi and Repecak, 1999; Franz, 2000; Salamon, 2000; Galambosi, 2001). Letchamo and Vomel (2001) have shown that temperature could increase content of a()-bisabolol and bisabolol oxides A and B. We found that performance of chamomile on saline water has not been well studied or documented and there are only a few reports on the effect of water salinity on morphological characters and oil composition of chamomile (Cellarova et al., 1986; Afzali et al., 2006). The aim of this work was to study the effect of saline irrigation water on agronomical and phytochemical characters, in order to support breeding program and use of chamomile as an economic substitute for field crops irrigated with fresh water. 2. Materials and methods 2.1. Field site description A field examination was conducted using complete randomized block design with four replications at the Experimental Station of Institute of Medicinal Plant, Karaj, Iran during 2004–2005. The geographical location of the station was 358470 N and 508560 E with 126 m altitude. The soil properties of the location are presented in Table 1. Seeds of the Iranian natural population of chamomile, provided by Institute of Medicinal Plant, Karaj, Iran, were sown in November 2004 by hand. These seeds were originally collected from natural sources and have since been cultivated every year for
Table 1 Chemical and physical characteristics for soil of experimental field Analysis
Soil depth 0–30 cm
ECe (dS/m) pH TNV (%) OC (%) Total N (%) Available P (ppm) Available K (ppm) Clay (%) Silt (%) Sand (%) Texture Zn (ppm) Fe (ppm) Mn (ppm) Cu (ppm)
0.93 7.9 8.5 0.82 0.08 36.2 49.8 16 22 62 Silt-loam 0.6 5.74 11.2 0.7
purpose of seed production. For each experimental plot, 0.6 g/m2 of seeds were grown in 4 rows with 50 cm apart and 3 m length. 2.2. Irrigation water The experimental treatments consisted of four salinity levels of the irrigation water (4, 8, 12 and 16 dS m1) in addition to control (0 dS m1). The salinity levels were obtained by addition of appropriate amount of NaCl to water and were adjusted by a portable Ec meter instrument. The different irrigation treatments started in the beginning of spring after the seedlings started their growth and development. Each plot received 40 l of water nine times from the middle of April to the end of June 2005. In each plot, 25 g nitrogenous fertilizer as ammonium nitrate was applied. 2.3. Field investigated characters Chamomile has a continuous flowering habit. Plants flowering started from middle of May and lasted to end of June 2005. Therefore, for all plants in each plot, several harvests were done, 7– 10 days apart, depending on the weather conditions, and total fresh weight of harvested flowers was measured. Dry flower weight of each plot was calculated after drying the flowers at room temperature (20–25 8C). After the flowering period, plants (root and stem) were collected and dry weights of stem and root were recorded for all plants in each plot. 2.4. Content of minerals After harvesting, content of minerals (Na+, K+, Ca2+, Mg2+, Cl) were evaluated in stems and roots of each plot. Two g of dried material was used for test solution preparing, according to method described by Baker and Suhr (1982). Sodium and potassium concentration was determined by Flame photometer (Jenway PFP7) and automatic titration method was used for chloride determination (Adriano and Doner, 1982). Calcium and Magnesium contents were measured using complexometry method which is a practical technique used to determine the concentration of metal ions. In a titration, the volume of EDTA as a standard solution of a complex-forming reagent needed to react exactly with the metal ions in a defined volume of the unknown solution is measured (Waling et al., 1989). 2.5. Analysis of essential oils Oils were extracted by hydro distillation of flowers using Clevenger-type apparatus for 3 h (British Pharmacopoeia, 2002).
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The oils were dried over anhydrous sodium sulphate and kept at 4 8C until they were analyzed. Qualitative and quantitative analyses of oils were performed by Agilent gas chromatography model 6890 N, equipped with MSD model 5973 N and fused silica capillary column (HP-5MS, 30 m 0.25 mm). The GC oven temperature was held at 50 8C for 2 min, then programmed from 50 8C to 200 8C at a rate of 3 8C min1 and from 200 8C to 280 8C at a rate of 7 8C min1, held for 2 min at 280 8C, using He as the carrier gas (1.0 ml min1). The temperature of injector was 250 8C. The percentage composition of the essential oils was computed from GC peak areas without correction factors. Qualitative analysis was based on a comparison of retention times and indexes on both columns and mass spectra with corresponding data available in the literature (Adams, 1995) and computer mass spectra libraries. 2.6. Apigenin-7-glucoside determination An HPLC method used for quantification of apigenin-7-glucoside determination according to the method described in United States Pharmacopoeia (2004). HPLC instrument consisted KNUWER pump k-1001 and KNUWER C18 column (150 mm 4.6 mm). The isocratic mobile phase was variable mixtures of solution A (0.005 M monophosphate potassium adjusted with phosphoric acid (5%) to pH = 2.55 0.05) and solution B (acetonitril–methanol (65:35)) which programmed at a flow rate of 1 ml min1. Peaks were detected at 335 nm, and recorded by a KNUWER UV-visible detector model K-2501. Standard solution was produced by dissolving accurately weighted quantity of apigenin-7-Glucoside (Fluka, purity 97%) in methanol and diluting to obtain a solution having concentration of 0.25 mg/ml of standard.1.0 g chamomile from each of 20 treatments were accurately weighted and used for preparing test solution according to the method described in United State Pharmacopoeia (2004). After preparing test and standard solutions, equal volumes (20 ml) of them were separately injected to the HPLC. The percentage of apigenin-7-glucoside was calculated based on the fallowing expression: Apigenin-7-glucoside ð%Þ ¼ 20ðC=WÞðr u =r s Þ in which C is the concentration, in mg per ml, of apigenin-7glucoside in standard solution; W is weight in g, of chamomile taken for test solution; ru and rs are the apigenin-7-glucoside responses obtained from the test solution and the standard solution, respectively. 2.7. Data analysis After agronomical and phytochemical evaluation, quantitative analyses were carried out using the computer software SPSS
Fig. 1. Duncan result for fresh flower weight in different treatments (different letters on the top of columns indicate statistically significant differences).
version 10.0. Data were analyzed by ANOVA and the means of results were compared by Duncan’s multiple range tests. 3. Results 3.1. Agronomical evaluation The results indicated that fresh flower weight was the only morphological trait, which was significantly different in salinity levels. This result is not inconsistent with results reported before (Prasad et al., 1997). Duncan results showed non-significant effect of salinity levels on dry flower weight, while fresh flower yield decreased with increasing salinity and it was higher in control compared to others (Fig. 1). 3.2. Mineral content Mineral nutrition is mainly affected by salinity and leads to ionic imbalance and nutrition problems. In other words, salinity alters the ion transport and contents of ions such as K+, Ca2+ and with an antagonist like effect (Cramer and Schmidt, 1995). In this study mineral content evaluation showed that chloride and sodium were affected significantly in both root and stem. Furthermore Ca2+ and Mg2+ accumulation had been increased significantly in stem. This is in agreement with results obtained by Prasad et al. (1997). Saline irrigation water had no significant effect on K+ accumulation in both root and shoot. Comparison of Na+ and Cl accumulation in root and stem showed that increasing salinity leads to increasing in accumulation (Fig. 2). It is worth noting that concentration of both minerals in stem was higher than root.
Fig. 2. Comparison of Na+ (left) and ClS (right) content (%) in stem and root (different letters on the top of columns indicate statistically significant differences).
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440 Table 2 Duncan multiple test for phytochemical characters Salinity levels (dS m1)
0 4 8 12 16
Oil quantity
Oil composition (%)
Apigenin (%)
Yield (kg/h)
Content (%)
BoB
BnA
Ch
BoA
Bo
Fa
0.04 0.03 0.03 0.05 0.03
0.037 0.037 0.032 0.041 0.027
8.881(a) 10.2(a) 14.8(a) 11.25(a) 10.87(a)
7.17(a) 7.54(a) 8.3(a) 6.64(a) 7.66(a)
18.76(a) 19.53(a) 20(a) 19.71(a) 19.55(a)
30.82(a) 32.15(a) 29.7(a) 33.7(a) 33.36(a)
1.03(a) 1.07(a) 0.91(a) 2.01(a) 1.27(a)
0.17(a) 0.1(a) 0(a) 0(a) 0.3(a)
(a) (a) (a) (a) (a)
(a) (a) (a) (a) (a)
0.28(a) 0.26(a) 0.28(a) 0.25(a) 0.27(a)
BoB (a-bisabololoxide B), BnA (a-bisabolonoxide A), Ch (chamazulene), BoA (a-bisabolol oxide A), Bo (a-bisabolol), Fa (trans-b-farnesene), letter (a) indicates statistically non-significant differences between treatments.
Fig. 3. HPLC chromatogram of apigenin in test solution (right) and standard solution (left).
3.3. Qualitative and quantitative analysis of essential oils Analysis of variance showed that saline irrigation water had no significant effect on oil quantity (yield and content) and medically important constituents such as a-bisabololoxide B, a-bisabolonoxide A, chamazulene, a-bisabolol oxide A, a-bisabolol and transb-farnesene (Table 2). 3.4. Apigenin evaluation When the percentage of total apigenin was analyzed by HPLC (Fig. 3), surprisingly we found that salinity had no significant effect on apigenin content and therefore, on its spasmolytic activity (Table 2). 4. Discussion In this study, the effect of saline water on morphological and phytochemical characters of chamomile, as a medicinal plant, were investigated. The results in agronomical evaluation suggest that cultivation of chamomile on a large scale could successfully represent an acceptable agronomical yield. It is worth noting that dry flower weight was not significant. This could be contributed to different water accumulation between different treatments. As chamomile flowers are marketed in dry form, this means that salinity has no significant effect on economical flower yield. The difference in a plant’s response to a given level of salinity is dependent upon the concentration and composition of the ions in solution as well as the genotype that is exposed to the salinity (Greenway and Munns, 1980). Higher accumulations of Na+ and Cl in the stem than those in the root are probably due to the translocation of minerals to stem which causes the plant to overcome salinity. Similar results have been reported in studies conducted on other crop plants such as rice (Aslam et al., 1996), pepper (Cornillon and Palliox, 1997) and wheat (Ha and Schmidhalter, 1997). Qualitative and quantitative analysis of essential oils showed that under saline condition, quantitative and qualitative value of
oil, as a medically important factor in chamomile, could present an acceptable performance. These results are not consistent with the findings of Ram et al. (1999) and Prasad et al. (2006) which reported that increasing sodium ions significantly affects oil content and composition. Apigenin variations, as a flavonoid, are considered as a phytochemical adaptation to the abiotic and biotic environment (Dixon and Paiva, 1995). There are extensive data that suggest that apigenin synthesis is influenced by different factors such as UV light radiation, drought, ozone, phytopathogens and insectdeterrent (Nikolova and Ivancheva, 2005), however we did not find any reports treating the subject of apigenin change in response to salinity. Furthermore, we did not find any significant effect on apigenin content among different salinity levels. This means that production and accumulation of apigenin, as an economically important aspect of chamomile, could be done in a usual manner when this plant is treated with salinity. In summary, through our study, the results showed that chamomile could present acceptable agronomical yield with sufficient medicinal properties. Therefore, cultivation of chamomile on a large scale could be successfully accomplished on saline water, where cultivation of field crops irrigated with fresh water is not possible. Identification of plant mechanisms for salt tolerance and breeding of new cultivars, represents essential strategies for reducing salinity effects in agriculture (Poustini and Siosemardeh, 2004). Furthermore, it seems that we need to undertake further studies, in different locations and with durations longer than one year, in order to obtain more reliable results and also to clarify physiological mechanism of salinity tolerance in chamomile. Acknowledgments We gratefully acknowledge the financial support of Islamic Azad University, Karaj Branch, through this research program. Further we express our special thanks to Iranian Academic Center
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for Education, Culture & Research (ACECR), Institute of Medicinal Plants Research for their valuable support and cooperation.
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