Article pubs.acs.org/JAFC
Biofortification of Soy (Glycine max (L.) Merr.) with Strontium Ions Ireneusz Sowa,*,† Magdalena Wójciak-Kosior,† Maciej Strzemski,† Sławomir Dresler,‡ Wojciech Szwerc,† Tomasz Blicharski,§ Grazẏ na Szymczak,∥ and Ryszard Kocjan† †
Department of Analytical Chemistry, Medical University of Lublin, Chodźki 4a, 20-093 Lublin, Poland Department of Plant Physiology, Institute of Biology and Biochemistry, Maria Curie-Skłodowska University, 20-033 Lublin, Poland § Orthopaedics and Rehabilitation Clinic, Medical University Lublin, Lublin, Poland ∥ Botanical Garden of Maria Curie-Skłodowska University in Lublin, Sławinkowska 3, 20-810 Lublin, Poland ‡
ABSTRACT: Soy (Glycine max (L.) Merr.) is an annual plant cultivated worldwide mostly for food. Moreover, due to its pharmacological properties it is widely used in pharmacy for alleviating the symptoms of osteoporosis. The aim of the present study was to investigate the biofortification of soy treated with various concentrations of strontium. Soy was found to have a strong capacity to absorb Sr2+ (bioconcentration factor higher than 1). A positive linear correlation (R2 > 0.98) between the amount of strontium in the growth medium and its content in the plant was also observed. Moreover, at a concentration of 1.5 mM, strontium appeared to be nontoxic and even stimulated plant growth by approximately 19.4% and 22.6% of fresh weight for shoots and roots, respectively. Our research may be useful to obtain vegetable products or herbal preparations containing both phytoestrogens and strontium to prevent postmenopausal osteoporosis. KEYWORDS: soy, Glycine max, strontium, osteoporosis, biofortification
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INTRODUCTION Glycine max (L.) Merr. (Fabaceae) is an annual plant cultivated worldwide, mainly in the United States (35%), Brazil (27%), Argentina (19%), China (6%), and India (4%). Production thereof is estimated to be approximately 280 million tons per year. Soybeans are a rich source of proteins, saponins, phospholipids, fiber, phytic acid, anthocyanins, amino acids, organic acids, and flavonoids,1−4 and they are used as highquality food. Moreover, soy holds an important position in production of animals feed and oils.5,6 Several reports describe positive effects of soybean consumption for the prevention of various diseases, e.g., traditional diet based on soy may lower the risk of breast cancer cases in Asian women.7,8 In recent years, soy and soy products have become the subject of extensive phytochemical and pharmacological investigations for their potential application in the therapy of cardiovascular diseases and certain forms of cancer.9,10 Their antioxidant activity has been also reported.11−13 Additionally, data suggest that isoflavones, the major phenolic compounds present in soy, protect against postmenopausal osteoporosis associated with endogenous estrogen deficiency.14 Isoflavones are often used as an alternative to synthetic modulators of estrogen receptors, which are currently applied in hormone replacement therapy.15−17 Pharmaceutical and dietary preparations containing soy extract are commonly used to alleviate the symptoms of menopause and reduce the risk of diseases, whose etiology is associated with a low level of estrogen. The reduction of bone mass and deterioration of bone structure resulting in an increased risk of fractures are the main symptoms of osteoporosis.18 Usually, high doses of calcium carbonate with vitamin D3 are used in the therapy.19 On the other hand, strontium salts, e.g., ranelate, have shown unique pharmacological effects on bone resorption and formation and © XXXX American Chemical Society
the ability to induce differentiation of osteoblasts with simultaneous inhibition of osteoclast differentiation; hence, they may be useful for treatment of osteoporosis.20−22 Strontium occurs in nature at a concentration of 0.033%, mostly as celestite and strontianite. However, its amount may reach even several thousands of ppm, e.g., near roads with heavy traffic. Moreover, the high concentration of toxic, radioactive forms of strontium may be found in areas after nuclear weapon tests or nuclear accidents. Soil-to-plant transfer of strontium varies significantly depending on pH of soil and the type of biota.23 Some plant species possess high ability to absorb strontium ions, and they may be used for phytoremediation.24 On the other hand, the accumulation of stable strontium can be exploited for biofortification of plants. The present study investigated biofortification of soy with Sr2+. Soy is an important component of the human diet, and accumulation of this ion may be useful to obtain vegetable products or herbal preparations containing both phytoestrogens and strontium to prevent postmenopausal osteoporosis.
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MATERIALS AND METHODS
Chemicals and Reagents. All components of Hoagland’s medium and Sr(NO3)2 were from POCH (Gliwice, Poland). Suprapure nitric acid was obtained from Merck (Darmstadt, Germany). Water was deionized and purified by ULTRAPURE Milipore Direct-Q 3UV-R (Merck). The efficiency of this process was checked conductometrically; the resistivity of water was 18 MΩ-cm. Strontium atomic spectroscopy standard solution was purchased from Sigma-Aldrich (St. Louis, MO, USA). Received: March 18, 2014 Revised: May 16, 2014 Accepted: May 19, 2014
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dx.doi.org/10.1021/jf501257r | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Table 1. Content of Strontium in Different Parts of Glycine max Treated with Various Sr Concentrations (±SD)a
Plant Material. Soybeans seeds (Glycine max (L.) Merr.) were supplied from the Botanical Garden in Lublin. The plants were germinated from seeds on wet filter paper in a thermostat-controlled chamber. After 7 days, the seedlings were transferred into Hoagland’s nutrient solution for 3 days, and then the growth medium was removed. The composition of Hoagland’s solution was modified by addition of Sr2+ ions to obtain appropriate concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mM). Additionally, iron citrate was removed and Mg(NO3)2·6H2O instead of MgSO4·7H2O was used in order to avoid precipitation of insoluble strontium salt. Twenty plants for each concentration of Sr were grown in modified Hoagland’s medium for another 14 days at a day/night cycle of 16/8 h and 24/17 °C, respectively, relative humidity 60−70%, and photosynthetic photon flux density of 150 μmol/m2 s. The nutrient solution with Sr2+ ions was changed every 3 days. After harvest, the plants were separated into roots and shoots and accurately weighed. Some material was frozen in liquid nitrogen and stored at −80 °C prior to analysis of the content of chlorophylls and carotenoids. Atomic Absorption Spectrometry (AAS) Procedure. Fresh roots were washed with deionized water three times to remove Sr2+adsorbed on the surface. Five roots and two shoots were taken per replication. The plant material was dried at 80 °C to constant weight and pulverized. The shoots and roots were accurately weighed (0.5 and 0.3 g respectively) and mineralized in a microwave-assisted highpressure digestion closed system (UniClever BM-1, 350 W, Plazmotronika, Poznań, Poland) with the use of a mixture containing 4 mL of 65% nitric acid and 10 mL of deionized water. The mineralization parameters were as follows: the power of the microwave generator at 60%, 80%, and 100% during 10, 15, and 20 min, respectively. The mineralizates were weighed. Sr2+ contents were determined by flame atomic absorption spectrometry (HighResolution Continuum Source atomic absorption spectrometer ContrAA 700, Analytik Jena AG, Germany). A xenon lamp working in an optimized Hot-Spot-Mode and a CCD array detector (185−900 nm) with high quantum efficiency and increased UV-sensitivity were employed. The measurements were performed at λ = 460.733 nm, and the concentration range of strontium for the calibration curve was from 0 to 20 mg/L (n = 5). The validation parameters obtained were as follows: correlation coefficient R = 0.9999, the value of characteristic concentration for 1% of absorbance was 0.04513 mg/L, calibration equation y = (−0.0001968 + 0.0580274)/(1 + 0.0201765x), and precision expressed as relative standard deviation (% RSD) 0.7−2.9%. Chlorophyll and Carotenoid Analysis. Chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Car) were extracted with 80% acetone, and their contents were spectrophotometrically determined (Shimadzu UV 160A) at λ = 663, λ = 646, and λ = 470 nm, respectively. The concentrations (mg/g) were calculated according to Wellburn.25 Statistical Analysis. Statistical analysis was conducted using the STATISTICA ver.10 (StatSoft, Inc., USA) program. Data was analyzed by ANOVA, and the significances of differences were examined using Fisher’s LSD test. The statistical confidence was set at p = 0.05.
content of Sr (mg/kg) DW concn of Sr in medium (mM)
shoot (n = 10)
0.5 1 1.5 2 2.5 3 a
175.06 351.06 673.85 885.80 1014.85 1246.13
± ± ± ± ± ±
10.21 17.12 31.19 40.05 50.43 65.63
root (n = 4) 18.40 37.39 85.20 111.26 147.20 180.22
± ± ± ± ± ±
1.97 2.61 4.20 5.11 7.07 9.94
The concentration of Sr in control was below the limit of detection.
Figure 1. Linear correlation between of the strontium content in the growth medium and its content in dry weight of Glycine max.
Table 2. Strontium Distribution and Uptake by Glycine max Treated with Various Sr Concentrations (±SD)a BCF Sr concn in medium (mM) 0.5 1 1.5 2 2.5 3
shoot (S/M) 3.97 4.01 5.13 5.06 4.63 4.74
±0.10 ±0.11 ±0.16 ±0.17 ±0.11 ±0.18
root (R/M) a a b b c bc
0.42 0.43 0.65 0.64 0.67 0.69
±0.010 ±0.010 ±0.017 ±0.020 ±0.014 ±0.012
TF (S/R) a a b b bc c
9.45 9.39 7.91 7.96 6.89 6.91
±0.32 ±0.33 ±0.29 ±0.24 ±0.31 ±0.23
a a b b c c
a
Values followed by the same letters were not significantly different (p < 0.05).
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such components of soil as other ions could influence the uptake of strontium. Accumulation and Translocation of Strontium in soy. The accumulation of strontium in plants strongly depends on its concentration in the growth medium and the type of biota. Its content may vary from a few to even more than 2000 ppm.23 Moreover, differences in the amount of strontium between above-ground and under-ground plant parts have been observed,28−30 with higher concentrations generally present in shoot.23,31 As our experiments showed, soy has a pronounced tendency toward accumulation of Sr2+ in shoots. Its content ranged from 87.4% to 90.4% of the total amount of the ions in the plants (Table 1). A positive linear correlation (R2 > 0.98) between the amount of strontium in the growth medium and its content in the plant was also observed (Figure 1).
RESULTS AND DISCUSSION Phytoextraction of metals is a common effect which may be used for phytoremediation of soil contaminated by toxic elements or radionuclides26 or for biofortification with microelements, for instance essential for plant physiology.27 Here we present the results of the influence of strontium fertilization on accumulation of the metal in Glycine max. Our research may be useful to obtain phytoestrogen-containing vegetable products or herbal formulations enriched with strontium to alleviate the risk of osteoporosis effects for postmenopausal women. In our investigations, hydroponic cultivation was employed to ensure controlled conditions of the plant culture; otherwise, B
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Table 3. Effect of Treatment with Various Concentrations of Strontium on Glycine max Biomass (n = 20)a FW
DW
shoot
a
concn (mM)
g
0 0.5 1 1.5 2 2.5 3
1.924 1.990 2.160 2.298 2.121 1.756 1.735
root SD
±0.096 ±0.109 ±0.109 ±0.110 ±0.109 ±0.093 ±0.089
shoot
g ab ac cd d ad be e
SD ±0.091 ±0.099 ±0.096 ±0.112 ±0.081 ±0.076 ±0.076
1.518 1.570 1.595 1.862 1.356 1.271 1.265
g ab a a d bc c c
root SD
±0.019 ±0.020 ±0.022 ±0.023 ±0.021 ±0.017 ±0.016
0.312 0.318 0.324 0.358 0.303 0.287 0.274
g a ab ac bc ad ae de
SD ±0.006 ±0.006 ±0.007 ±0.007 ±0.006 ±0.004 ±0.004
0.092 0.092 0.097 0.113 0.090 0.074 0.072
a a a c a b b
The values followed by the same letters were not significantly different (p < 0.05).
The ability to accumulate a metal from the medium can be estimated by the bioconcentration factor (BCF), expressed as the ratio of the ion content in plant (S, shoot, or R, root) to its concentration in the environment (M, medium). The tendency toward translocation of a metal in the plant can be described by the transfer factor (TF), which is defined as the ratio of the ion content in shoots to roots.32 The calculated BCF and TF values are presented in Table 2. The TF for strontium content in the shoots was 6.89−9.39 times higher than in the roots, which proves that Sr2+ are effectively transferred to the above parts of the plants and accumulated therein. The active transport from the roots to shoots decreased at higher concentrations and resulted in lower values of TF. As it can be seen in Table 2, the BCF depends on the content of the metal in the medium. The BCF for the roots was increased for all the investigated concentrations of strontium; however, it reached the highest value at 1.5−2 mM of Sr2+ for the shoots (BCF = 5.13−5.06). A value of the BCF higher than 1 indicates that soy has a strong capacity to absorb strontium.29 Effect of Strontium on Soy Growth. The average fresh (FW) and dry (DW) weight of biomass, of both the root and the shoot, obtained after 24 days of cultivation (including 14 days of treatment with various concentrations of strontium) and the weight of the control cultivated without strontium addition are shown in Table 3. At concentrations up to 1.5 mM, strontium stimulated plant growth by approximately 19.42% FW (14.70% DW) and 22.62% FW (22.66% DW) for the shoots and roots, respectively (Table 3). However, toxic effects occurred at concentrations higher than 2 mM. The total amount of fresh and dry weight at concentration 3 mM was lower by approximately 12.84−14.37%, in comparison to the control. A similar tendency was observed by Moyen and Roblin29 for hydroponic cultivation of maize. This study on maize showed that the moisture content remained at a constant level; the FW/water ratio was from 1.13 to 1.14.
Figure 2. Tolerance index for the shoots and roots of Glycine max to different concentrations of strontium.
Figure 3. Effect of different strontium concentrations on the content of chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Car) and on the chlorophyll a/b ratio in Glycine max.
Table 4. Coefficient of Correlation between the Indexes of Glycine max and the Strontium Treatment (n = 10) correlation coeff
Sr content in plant
Sr content in medium Sr content in plant
0.969
Sr content in medium Sr content in plant
0.9930
FW 0.867 0.961 −0.861 −0.834
DW
chlorophyll content
Concentration Range 0−1.5 mM 0.801 −0.544 0.918 −0.558 Concentration Range 1.5−3.0 mM −0.840 −0.294 −0.822 −0.255 C
chlorophyll a/b ratio
carotene content
−0.124 −0.133
−0.382 −0.395
0.986 0.994
−0.010 −0.004
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(5) Yoshida, H.; Hirakawa, Y.; Murakami, Ch.; Mizushina, Y.; Yamade, T. Variation in the content of tocopherols and distribution of fatty acids within soya bean seeds (Glycine max L.). J. Food Compos. Anal. 2003, 16, 429−440. (6) Şensöz, S.; Kaynar, I. Bio-oil production from soybean (Glycine max L.); fuel properties of Bio-oil. Ind. Crop Prod. 2006, 23, 99−105. (7) Messina, M. Legumes and soybeans: overview of their nutritional profiles and health effects. Am. J. Clin. Nutr. 1999, 70, 439−450. (8) Lee, Y. B.; Lee, H. J.; Kim, C. H.; Lee, S. B.; Sohn, H. S. Soy Isoflavones and Soyasaponins: Characteristics and Physiological Functions. Agric. Chem. Biotechnol. 2005, 48, 49−57. (9) Wu, A. H.; Yu, M. C.; Tseng, C. C.; Pike, M. C. Epidemiology of soy exposures and breast cancer risk. Br. J. Cancer 2008, 98, 9−14. (10) Ahn-Jarvis, J. H.; Riedl, K. M.; Schwartz, S. J.; Vodovotz, Y. Design and Selection of Soy Breads Used for Evaluating Isoflavone Bioavailability in Clinical Trials. J. Agric. Food Chem. 2013, 61, 3111− 3120. (11) Prakash, D.; Upadhyay, G.; Singh, B. N.; Singh, H. B. Antioxidant and free radical-scavenging activities of seeds and agriwastes of some varieties of soybean (Glycine max). Food Chem. 2007, 104, 783−790. (12) Silva, L. R.; Pereira, M. J.; Azevedo, J.; Gonçalves, R. F.; Valentão, P.; Guedes de Pinho, P.; Andrade, P. B. Glycine max (L.) Merr., Vigna radiata L. and Medicago sativa L. sprouts: A natural source of bioactive compounds. Food Res. Int. 2013, 50, 167−175. (13) Zieliń ski, H. Contribution of Low Molecular Weight Antioxidants to the Antioxidant Screen of Germinated Soybean Seeds. Plant Food Hum. Nutr. 2003, 58, 1−20. (14) Wildman, R. E. C. Handbook of nutraceuticals and functional foods. In: Hendrich, S.; Murphy, P. A. Isoflavones; CRC Press: Boca Raton, FL, 2006; pp 23−54. (15) Cornwell, T.; Cohick, W.; Raskin, I. Dietary phytoestrogens and health. Phytochemistry 2004, 65, 995−1016. (16) Booth, N. L.; Overk, C. R.; Yao, P.; Totura, S.; Deng, Y.; Hedayat, A. S.; Bolton, J. L.; Pauli, G. F.; Farnsworth, N. R. Seasonal variation of red clover (Trifolium pratense L., Fabaceae) isoflavones and estrogenic activity. J. Agric. Food Chem. 2006, 54, 1277−1282. (17) Taku, K.; Melby, M. K.; Nishi, N.; Omori, T.; Kurzer, M. S. Soy isoflavones for osteoporosis: An evidence-based approach. Maturitas 2011, 70, 333−338. (18) Marquis, P.; Roux, C.; De la Loge, C.; Diaz-Curiel, M.; Cormier, C.; Isaia, G.; Badurski, J.; Wark, J.; Meunier, P. J. Strontium ranelate prevents quality of life impairment in post-menopausal women with established vertebral osteoporosis. Osteoporosis Int. 2008, 19, 503−510. (19) Rizzoli, R.; Boonen, S.; Brandi, M. L.; Burlet, N.; Delmas, P.; Reginster, J. Y. The role of calcium and vitamin D in the management of osteoporosis. Bone 2008, 42, 246−249. (20) Marie, P. J. Strontium ranelate: a novel mode of action optimizing bone formation and resorption. Osteoporosis Int. 2005, 16, 7−10. (21) Marie, P. J.; Felsenberg, D.; Brandi, M. L. How strontium ranelate, via opposite effects on bone resorption and formation, prevents osteoporosis. Osteoporosis Int. 2011, 22, 1659−1667. (22) Stepan, J. J. Strontium ranelate: in search for the mechanism of action. J. Bone Miner. Metab. 2013, 31, 606−612. (23) Kabata-Pendias, A. Trace Elements in Soils and Plants, 4th ed.; CRC Press: Boca Raton, FL, 2010; pp 135−146. (24) Eapen, S.; Singh, S.; Thorat, V.; Kaushik, C. P.; Raj, K.; D’Souza, S. F. Phytoremediation of radiostrontium (90Sr) and radiocesium (137Cs) using giant milky weed (Calotropis gigantea R.Br.) plants. Chemosphere 2006, 65, 2071−2073. (25) Wellburn, A. R. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 1994, 144, 307−313. (26) Singh, S.; Eapen, S.; Thorat, V.; Kaushik, C. P.; Raj, K.; D’Souza, S. F. Phytoremediation of 137cesium and 90strontium from solutions and low-level nuclear waste by Vetiveria zizanoides. Ecotoxicol. Environ. Saf. 2008, 69, 306−311.
The tolerance index (TI) is commonly used to quantify tolerance of plant organs to metals. It is calculated from the formula: TI = (FWS or R/FWcontrol) 100%. As can be seen in Figure 2, the concentration of strontium in the range 0.5−1.5 mM turned out to be nontoxic. This finding may be explained by the physical and chemical similarity of strontium to calcium, which is an essential component for plant growth and can be partially replaced by Sr2+.33 The TI for the roots was significantly lower than that for the shoots at the higher concentration of strontium. Inhibition of root growth is one of the fastest physiological reactions to stress.34,35 Effect of Strontium on the Content of Plant Pigments. It has been reported that strontium significantly affected the content of plant pigments depending on its amount and time of exposure.29,33 Figure 3 shows changes in the chlorophyll a, b, and carotene content after 24 days of cultivation (including 14 days of treatment with the increasing concentrations of strontium). The total amount of plant pigments decreased at the 0.5 mM concentration of strontium, in comparison to plants without Sr. However, the changes in the plant pigments were not correlated with the metal concentration. The differences were not significant (p > 0.05). In comparison to the control, a decrease in the chlorophyll a/b ratio was observed. This finding may be explained by greater sensitivity of Chla than Chlb to strontium. The present data showed a positive correlation between the Sr concentration in the media and Sr content in plants. Sr concentrations up to 1.5 mM stimulated plant growth; however, at a higher concentration, a negative correlation between concentration and fresh and dry weight was observed (Table 4). Soy has a relatively high ability (BCF >1) to accumulate strontium, which may be useful to obtain phytoestrogencontaining vegetable products or herbal formulations for prevention postmenopausal osteoporosis. In vivo and in vitro studies showed high efficiency of strontium salts in osteoporosis therapy and lack of their toxicity in animals and human.36 Our further investigations will focus on examining the activity and toxicity of extracts from soy enriched with strontium.
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AUTHOR INFORMATION
Corresponding Author
*Tel/fax: +48 81 5357350. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Alezandro, M. R.; Granato, D.; Lajolo, F. M.; Genovese, M. I. Nutritional aspects of second generation soy foods. J. Agric. Food Chem. 2011, 59, 5490−5497. (2) Jiao, Z.; Si, X.; Zhang, Z.; Li, G.; Cai, Z. Compositional study of different soybean (Glycine max L.) varieties by 1H NMR spectroscopy, chromatographic and spectrometric techniques. Food Chem. 2012, 135, 285−291. (3) Song, J.; Liu, Ch.; Li, D.; Gu, Z. Evaluation of sugar, free amino acid, and organic acid compositions of different varieties of vegetable soybean (Glycine max (L.) Merr). Ind. Crop Prod. 2013, 50, 743−749. (4) Mebrahtu, T.; Mohamed, A.; Wang, C. Y.; Andebrhan, T. Analysis of Isoflavone Contents in Vegetable Soybeans. Plant Food Hum. Nutr. 2004, 59, 55−61. D
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(27) Zhang, H.; Yang, H.; Wang, Y.; Gao, Y.; Zhang, L. The response of ginseng grown on farmland to foliar-applied iron, zinc, manganese and copper. Ind. Crop Prod. 2013, 45, 388−394. (28) Sasmaz, A.; Sasmaz, M. The phytoremediation potential for strontium of indigenous plants growing in a mining area. Environ. Exp. Bot. 2009, 67, 139−144. (29) Moyen, C.; Roblin, G. Uptake and translocation of strontium in hydroponically grown maize plants, and subsequent effects on tissue ion content, growth and chlorophyll a/b ratio: comparison with Ca effects. Environ. Exp. Bot. 2010, 68, 247−257. (30) Wang, D.; Wen, F.; Xu, C.; Tang, Y.; Luo, X. The uptake of Cs and Sr from soil to radish (Raphanus sativus L.) - potential for phytoextraction and remediation of contaminated soils. J. Environ. Radioact. 2012, 110, 78−83. (31) Bonanno, G. Trace element accumulation and distribution in the organs of Phragmites australis (common reed) and biomonitoring applications. Ecotoxicol. Environ. Saf. 2011, 74, 1057−1064. (32) Tsukada, H.; Takeda, A.; Takahashi, T.; Hasegawa, H.; Hisamatsu, S.; Inaba, J. Uptake and distribution of 90Sr and stable Sr in rice plants. J. Environ. Radioact. 2005, 81, 221−231. (33) Chen, M.; Tang, Y. L.; Ao, J.; Wang, D. Effects of strontium on photosynthetic characteristics of oilseed rape seedlings. Russ. J. Plant Physiol. 2012, 59, 772−780. (34) Schützendübel, A.; Schwanz, P.; Teichmann, T.; Gross, K.; Langenfeld-Heyser, R.; Godbold, D.; Polle, A. Cadmium induced changes in antioxidative systems, hydrogen peroxide content, and differentiation in scots pine roots. Plant Physiol. 2001, 127, 887−898. (35) Drążkiewicz, M.; Baszyński, T. Growth parameters and photosynthetic pigments in leaf segments of Zea mays exposed to cadmium, as related to protection mechanisms. J. Plant Physiol. 2005, 162, 1013−1021. (36) Cianferotti, L.; D’Asta, F.; Brandi, M. L. A review on strontium ranelate long-term antifracture efficacy in the treatment of postmenopausal osteoporosis. Ther. Adv. Musculoskeletal Dis. 2013, 5 (3), 127−139.
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dx.doi.org/10.1021/jf501257r | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Biofortification of Soy (Glycine max (L.) Merr.) with Strontium Ions Ireneusz Sowa,*,† Magdalena Wójciak-Kosior,† Maciej Strzemski,† Sławomir Dresler,‡ Wojciech Szwerc,† Tomasz Blicharski,§ Grazẏ na Szymczak,∥ and Ryszard Kocjan† †
Department of Analytical Chemistry, Medical University of Lublin, Chodźki 4a, 20-093 Lublin, Poland Department of Plant Physiology, Institute of Biology and Biochemistry, Maria Curie-Skłodowska University, 20-033 Lublin, Poland § Orthopaedics and Rehabilitation Clinic, Medical University Lublin, Lublin, Poland ∥ Botanical Garden of Maria Curie-Skłodowska University in Lublin, Sławinkowska 3, 20-810 Lublin, Poland ‡
ABSTRACT: Soy (Glycine max (L.) Merr.) is an annual plant cultivated worldwide mostly for food. Moreover, due to its pharmacological properties it is widely used in pharmacy for alleviating the symptoms of osteoporosis. The aim of the present study was to investigate the biofortification of soy treated with various concentrations of strontium. Soy was found to have a strong capacity to absorb Sr2+ (bioconcentration factor higher than 1). A positive linear correlation (R2 > 0.98) between the amount of strontium in the growth medium and its content in the plant was also observed. Moreover, at a concentration of 1.5 mM, strontium appeared to be nontoxic and even stimulated plant growth by approximately 19.4% and 22.6% of fresh weight for shoots and roots, respectively. Our research may be useful to obtain vegetable products or herbal preparations containing both phytoestrogens and strontium to prevent postmenopausal osteoporosis. KEYWORDS: soy, Glycine max, strontium, osteoporosis, biofortification
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INTRODUCTION Glycine max (L.) Merr. (Fabaceae) is an annual plant cultivated worldwide, mainly in the United States (35%), Brazil (27%), Argentina (19%), China (6%), and India (4%). Production thereof is estimated to be approximately 280 million tons per year. Soybeans are a rich source of proteins, saponins, phospholipids, fiber, phytic acid, anthocyanins, amino acids, organic acids, and flavonoids,1−4 and they are used as highquality food. Moreover, soy holds an important position in production of animals feed and oils.5,6 Several reports describe positive effects of soybean consumption for the prevention of various diseases, e.g., traditional diet based on soy may lower the risk of breast cancer cases in Asian women.7,8 In recent years, soy and soy products have become the subject of extensive phytochemical and pharmacological investigations for their potential application in the therapy of cardiovascular diseases and certain forms of cancer.9,10 Their antioxidant activity has been also reported.11−13 Additionally, data suggest that isoflavones, the major phenolic compounds present in soy, protect against postmenopausal osteoporosis associated with endogenous estrogen deficiency.14 Isoflavones are often used as an alternative to synthetic modulators of estrogen receptors, which are currently applied in hormone replacement therapy.15−17 Pharmaceutical and dietary preparations containing soy extract are commonly used to alleviate the symptoms of menopause and reduce the risk of diseases, whose etiology is associated with a low level of estrogen. The reduction of bone mass and deterioration of bone structure resulting in an increased risk of fractures are the main symptoms of osteoporosis.18 Usually, high doses of calcium carbonate with vitamin D3 are used in the therapy.19 On the other hand, strontium salts, e.g., ranelate, have shown unique pharmacological effects on bone resorption and formation and © XXXX American Chemical Society
the ability to induce differentiation of osteoblasts with simultaneous inhibition of osteoclast differentiation; hence, they may be useful for treatment of osteoporosis.20−22 Strontium occurs in nature at a concentration of 0.033%, mostly as celestite and strontianite. However, its amount may reach even several thousands of ppm, e.g., near roads with heavy traffic. Moreover, the high concentration of toxic, radioactive forms of strontium may be found in areas after nuclear weapon tests or nuclear accidents. Soil-to-plant transfer of strontium varies significantly depending on pH of soil and the type of biota.23 Some plant species possess high ability to absorb strontium ions, and they may be used for phytoremediation.24 On the other hand, the accumulation of stable strontium can be exploited for biofortification of plants. The present study investigated biofortification of soy with Sr2+. Soy is an important component of the human diet, and accumulation of this ion may be useful to obtain vegetable products or herbal preparations containing both phytoestrogens and strontium to prevent postmenopausal osteoporosis.
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MATERIALS AND METHODS
Chemicals and Reagents. All components of Hoagland’s medium and Sr(NO3)2 were from POCH (Gliwice, Poland). Suprapure nitric acid was obtained from Merck (Darmstadt, Germany). Water was deionized and purified by ULTRAPURE Milipore Direct-Q 3UV-R (Merck). The efficiency of this process was checked conductometrically; the resistivity of water was 18 MΩ-cm. Strontium atomic spectroscopy standard solution was purchased from Sigma-Aldrich (St. Louis, MO, USA). Received: March 18, 2014 Revised: May 16, 2014 Accepted: May 19, 2014
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Table 1. Content of Strontium in Different Parts of Glycine max Treated with Various Sr Concentrations (±SD)a
Plant Material. Soybeans seeds (Glycine max (L.) Merr.) were supplied from the Botanical Garden in Lublin. The plants were germinated from seeds on wet filter paper in a thermostat-controlled chamber. After 7 days, the seedlings were transferred into Hoagland’s nutrient solution for 3 days, and then the growth medium was removed. The composition of Hoagland’s solution was modified by addition of Sr2+ ions to obtain appropriate concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mM). Additionally, iron citrate was removed and Mg(NO3)2·6H2O instead of MgSO4·7H2O was used in order to avoid precipitation of insoluble strontium salt. Twenty plants for each concentration of Sr were grown in modified Hoagland’s medium for another 14 days at a day/night cycle of 16/8 h and 24/17 °C, respectively, relative humidity 60−70%, and photosynthetic photon flux density of 150 μmol/m2 s. The nutrient solution with Sr2+ ions was changed every 3 days. After harvest, the plants were separated into roots and shoots and accurately weighed. Some material was frozen in liquid nitrogen and stored at −80 °C prior to analysis of the content of chlorophylls and carotenoids. Atomic Absorption Spectrometry (AAS) Procedure. Fresh roots were washed with deionized water three times to remove Sr2+adsorbed on the surface. Five roots and two shoots were taken per replication. The plant material was dried at 80 °C to constant weight and pulverized. The shoots and roots were accurately weighed (0.5 and 0.3 g respectively) and mineralized in a microwave-assisted highpressure digestion closed system (UniClever BM-1, 350 W, Plazmotronika, Poznań, Poland) with the use of a mixture containing 4 mL of 65% nitric acid and 10 mL of deionized water. The mineralization parameters were as follows: the power of the microwave generator at 60%, 80%, and 100% during 10, 15, and 20 min, respectively. The mineralizates were weighed. Sr2+ contents were determined by flame atomic absorption spectrometry (HighResolution Continuum Source atomic absorption spectrometer ContrAA 700, Analytik Jena AG, Germany). A xenon lamp working in an optimized Hot-Spot-Mode and a CCD array detector (185−900 nm) with high quantum efficiency and increased UV-sensitivity were employed. The measurements were performed at λ = 460.733 nm, and the concentration range of strontium for the calibration curve was from 0 to 20 mg/L (n = 5). The validation parameters obtained were as follows: correlation coefficient R = 0.9999, the value of characteristic concentration for 1% of absorbance was 0.04513 mg/L, calibration equation y = (−0.0001968 + 0.0580274)/(1 + 0.0201765x), and precision expressed as relative standard deviation (% RSD) 0.7−2.9%. Chlorophyll and Carotenoid Analysis. Chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Car) were extracted with 80% acetone, and their contents were spectrophotometrically determined (Shimadzu UV 160A) at λ = 663, λ = 646, and λ = 470 nm, respectively. The concentrations (mg/g) were calculated according to Wellburn.25 Statistical Analysis. Statistical analysis was conducted using the STATISTICA ver.10 (StatSoft, Inc., USA) program. Data was analyzed by ANOVA, and the significances of differences were examined using Fisher’s LSD test. The statistical confidence was set at p = 0.05.
content of Sr (mg/kg) DW concn of Sr in medium (mM)
shoot (n = 10)
0.5 1 1.5 2 2.5 3 a
175.06 351.06 673.85 885.80 1014.85 1246.13
± ± ± ± ± ±
10.21 17.12 31.19 40.05 50.43 65.63
root (n = 4) 18.40 37.39 85.20 111.26 147.20 180.22
± ± ± ± ± ±
1.97 2.61 4.20 5.11 7.07 9.94
The concentration of Sr in control was below the limit of detection.
Figure 1. Linear correlation between of the strontium content in the growth medium and its content in dry weight of Glycine max.
Table 2. Strontium Distribution and Uptake by Glycine max Treated with Various Sr Concentrations (±SD)a BCF Sr concn in medium (mM) 0.5 1 1.5 2 2.5 3
shoot (S/M) 3.97 4.01 5.13 5.06 4.63 4.74
±0.10 ±0.11 ±0.16 ±0.17 ±0.11 ±0.18
root (R/M) a a b b c bc
0.42 0.43 0.65 0.64 0.67 0.69
±0.010 ±0.010 ±0.017 ±0.020 ±0.014 ±0.012
TF (S/R) a a b b bc c
9.45 9.39 7.91 7.96 6.89 6.91
±0.32 ±0.33 ±0.29 ±0.24 ±0.31 ±0.23
a a b b c c
a
Values followed by the same letters were not significantly different (p < 0.05).
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such components of soil as other ions could influence the uptake of strontium. Accumulation and Translocation of Strontium in soy. The accumulation of strontium in plants strongly depends on its concentration in the growth medium and the type of biota. Its content may vary from a few to even more than 2000 ppm.23 Moreover, differences in the amount of strontium between above-ground and under-ground plant parts have been observed,28−30 with higher concentrations generally present in shoot.23,31 As our experiments showed, soy has a pronounced tendency toward accumulation of Sr2+ in shoots. Its content ranged from 87.4% to 90.4% of the total amount of the ions in the plants (Table 1). A positive linear correlation (R2 > 0.98) between the amount of strontium in the growth medium and its content in the plant was also observed (Figure 1).
RESULTS AND DISCUSSION Phytoextraction of metals is a common effect which may be used for phytoremediation of soil contaminated by toxic elements or radionuclides26 or for biofortification with microelements, for instance essential for plant physiology.27 Here we present the results of the influence of strontium fertilization on accumulation of the metal in Glycine max. Our research may be useful to obtain phytoestrogen-containing vegetable products or herbal formulations enriched with strontium to alleviate the risk of osteoporosis effects for postmenopausal women. In our investigations, hydroponic cultivation was employed to ensure controlled conditions of the plant culture; otherwise, B
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Table 3. Effect of Treatment with Various Concentrations of Strontium on Glycine max Biomass (n = 20)a FW
DW
shoot
a
concn (mM)
g
0 0.5 1 1.5 2 2.5 3
1.924 1.990 2.160 2.298 2.121 1.756 1.735
root SD
±0.096 ±0.109 ±0.109 ±0.110 ±0.109 ±0.093 ±0.089
shoot
g ab ac cd d ad be e
SD ±0.091 ±0.099 ±0.096 ±0.112 ±0.081 ±0.076 ±0.076
1.518 1.570 1.595 1.862 1.356 1.271 1.265
g ab a a d bc c c
root SD
±0.019 ±0.020 ±0.022 ±0.023 ±0.021 ±0.017 ±0.016
0.312 0.318 0.324 0.358 0.303 0.287 0.274
g a ab ac bc ad ae de
SD ±0.006 ±0.006 ±0.007 ±0.007 ±0.006 ±0.004 ±0.004
0.092 0.092 0.097 0.113 0.090 0.074 0.072
a a a c a b b
The values followed by the same letters were not significantly different (p < 0.05).
The ability to accumulate a metal from the medium can be estimated by the bioconcentration factor (BCF), expressed as the ratio of the ion content in plant (S, shoot, or R, root) to its concentration in the environment (M, medium). The tendency toward translocation of a metal in the plant can be described by the transfer factor (TF), which is defined as the ratio of the ion content in shoots to roots.32 The calculated BCF and TF values are presented in Table 2. The TF for strontium content in the shoots was 6.89−9.39 times higher than in the roots, which proves that Sr2+ are effectively transferred to the above parts of the plants and accumulated therein. The active transport from the roots to shoots decreased at higher concentrations and resulted in lower values of TF. As it can be seen in Table 2, the BCF depends on the content of the metal in the medium. The BCF for the roots was increased for all the investigated concentrations of strontium; however, it reached the highest value at 1.5−2 mM of Sr2+ for the shoots (BCF = 5.13−5.06). A value of the BCF higher than 1 indicates that soy has a strong capacity to absorb strontium.29 Effect of Strontium on Soy Growth. The average fresh (FW) and dry (DW) weight of biomass, of both the root and the shoot, obtained after 24 days of cultivation (including 14 days of treatment with various concentrations of strontium) and the weight of the control cultivated without strontium addition are shown in Table 3. At concentrations up to 1.5 mM, strontium stimulated plant growth by approximately 19.42% FW (14.70% DW) and 22.62% FW (22.66% DW) for the shoots and roots, respectively (Table 3). However, toxic effects occurred at concentrations higher than 2 mM. The total amount of fresh and dry weight at concentration 3 mM was lower by approximately 12.84−14.37%, in comparison to the control. A similar tendency was observed by Moyen and Roblin29 for hydroponic cultivation of maize. This study on maize showed that the moisture content remained at a constant level; the FW/water ratio was from 1.13 to 1.14.
Figure 2. Tolerance index for the shoots and roots of Glycine max to different concentrations of strontium.
Figure 3. Effect of different strontium concentrations on the content of chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Car) and on the chlorophyll a/b ratio in Glycine max.
Table 4. Coefficient of Correlation between the Indexes of Glycine max and the Strontium Treatment (n = 10) correlation coeff
Sr content in plant
Sr content in medium Sr content in plant
0.969
Sr content in medium Sr content in plant
0.9930
FW 0.867 0.961 −0.861 −0.834
DW
chlorophyll content
Concentration Range 0−1.5 mM 0.801 −0.544 0.918 −0.558 Concentration Range 1.5−3.0 mM −0.840 −0.294 −0.822 −0.255 C
chlorophyll a/b ratio
carotene content
−0.124 −0.133
−0.382 −0.395
0.986 0.994
−0.010 −0.004
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(5) Yoshida, H.; Hirakawa, Y.; Murakami, Ch.; Mizushina, Y.; Yamade, T. Variation in the content of tocopherols and distribution of fatty acids within soya bean seeds (Glycine max L.). J. Food Compos. Anal. 2003, 16, 429−440. (6) Şensöz, S.; Kaynar, I. Bio-oil production from soybean (Glycine max L.); fuel properties of Bio-oil. Ind. Crop Prod. 2006, 23, 99−105. (7) Messina, M. Legumes and soybeans: overview of their nutritional profiles and health effects. Am. J. Clin. Nutr. 1999, 70, 439−450. (8) Lee, Y. B.; Lee, H. J.; Kim, C. H.; Lee, S. B.; Sohn, H. S. Soy Isoflavones and Soyasaponins: Characteristics and Physiological Functions. Agric. Chem. Biotechnol. 2005, 48, 49−57. (9) Wu, A. H.; Yu, M. C.; Tseng, C. C.; Pike, M. C. Epidemiology of soy exposures and breast cancer risk. Br. J. Cancer 2008, 98, 9−14. (10) Ahn-Jarvis, J. H.; Riedl, K. M.; Schwartz, S. J.; Vodovotz, Y. Design and Selection of Soy Breads Used for Evaluating Isoflavone Bioavailability in Clinical Trials. J. Agric. Food Chem. 2013, 61, 3111− 3120. (11) Prakash, D.; Upadhyay, G.; Singh, B. N.; Singh, H. B. Antioxidant and free radical-scavenging activities of seeds and agriwastes of some varieties of soybean (Glycine max). Food Chem. 2007, 104, 783−790. (12) Silva, L. R.; Pereira, M. J.; Azevedo, J.; Gonçalves, R. F.; Valentão, P.; Guedes de Pinho, P.; Andrade, P. B. Glycine max (L.) Merr., Vigna radiata L. and Medicago sativa L. sprouts: A natural source of bioactive compounds. Food Res. Int. 2013, 50, 167−175. (13) Zieliń ski, H. Contribution of Low Molecular Weight Antioxidants to the Antioxidant Screen of Germinated Soybean Seeds. Plant Food Hum. Nutr. 2003, 58, 1−20. (14) Wildman, R. E. C. Handbook of nutraceuticals and functional foods. In: Hendrich, S.; Murphy, P. A. Isoflavones; CRC Press: Boca Raton, FL, 2006; pp 23−54. (15) Cornwell, T.; Cohick, W.; Raskin, I. Dietary phytoestrogens and health. Phytochemistry 2004, 65, 995−1016. (16) Booth, N. L.; Overk, C. R.; Yao, P.; Totura, S.; Deng, Y.; Hedayat, A. S.; Bolton, J. L.; Pauli, G. F.; Farnsworth, N. R. Seasonal variation of red clover (Trifolium pratense L., Fabaceae) isoflavones and estrogenic activity. J. Agric. Food Chem. 2006, 54, 1277−1282. (17) Taku, K.; Melby, M. K.; Nishi, N.; Omori, T.; Kurzer, M. S. Soy isoflavones for osteoporosis: An evidence-based approach. Maturitas 2011, 70, 333−338. (18) Marquis, P.; Roux, C.; De la Loge, C.; Diaz-Curiel, M.; Cormier, C.; Isaia, G.; Badurski, J.; Wark, J.; Meunier, P. J. Strontium ranelate prevents quality of life impairment in post-menopausal women with established vertebral osteoporosis. Osteoporosis Int. 2008, 19, 503−510. (19) Rizzoli, R.; Boonen, S.; Brandi, M. L.; Burlet, N.; Delmas, P.; Reginster, J. Y. The role of calcium and vitamin D in the management of osteoporosis. Bone 2008, 42, 246−249. (20) Marie, P. J. Strontium ranelate: a novel mode of action optimizing bone formation and resorption. Osteoporosis Int. 2005, 16, 7−10. (21) Marie, P. J.; Felsenberg, D.; Brandi, M. L. How strontium ranelate, via opposite effects on bone resorption and formation, prevents osteoporosis. Osteoporosis Int. 2011, 22, 1659−1667. (22) Stepan, J. J. Strontium ranelate: in search for the mechanism of action. J. Bone Miner. Metab. 2013, 31, 606−612. (23) Kabata-Pendias, A. Trace Elements in Soils and Plants, 4th ed.; CRC Press: Boca Raton, FL, 2010; pp 135−146. (24) Eapen, S.; Singh, S.; Thorat, V.; Kaushik, C. P.; Raj, K.; D’Souza, S. F. Phytoremediation of radiostrontium (90Sr) and radiocesium (137Cs) using giant milky weed (Calotropis gigantea R.Br.) plants. Chemosphere 2006, 65, 2071−2073. (25) Wellburn, A. R. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 1994, 144, 307−313. (26) Singh, S.; Eapen, S.; Thorat, V.; Kaushik, C. P.; Raj, K.; D’Souza, S. F. Phytoremediation of 137cesium and 90strontium from solutions and low-level nuclear waste by Vetiveria zizanoides. Ecotoxicol. Environ. Saf. 2008, 69, 306−311.
The tolerance index (TI) is commonly used to quantify tolerance of plant organs to metals. It is calculated from the formula: TI = (FWS or R/FWcontrol) 100%. As can be seen in Figure 2, the concentration of strontium in the range 0.5−1.5 mM turned out to be nontoxic. This finding may be explained by the physical and chemical similarity of strontium to calcium, which is an essential component for plant growth and can be partially replaced by Sr2+.33 The TI for the roots was significantly lower than that for the shoots at the higher concentration of strontium. Inhibition of root growth is one of the fastest physiological reactions to stress.34,35 Effect of Strontium on the Content of Plant Pigments. It has been reported that strontium significantly affected the content of plant pigments depending on its amount and time of exposure.29,33 Figure 3 shows changes in the chlorophyll a, b, and carotene content after 24 days of cultivation (including 14 days of treatment with the increasing concentrations of strontium). The total amount of plant pigments decreased at the 0.5 mM concentration of strontium, in comparison to plants without Sr. However, the changes in the plant pigments were not correlated with the metal concentration. The differences were not significant (p > 0.05). In comparison to the control, a decrease in the chlorophyll a/b ratio was observed. This finding may be explained by greater sensitivity of Chla than Chlb to strontium. The present data showed a positive correlation between the Sr concentration in the media and Sr content in plants. Sr concentrations up to 1.5 mM stimulated plant growth; however, at a higher concentration, a negative correlation between concentration and fresh and dry weight was observed (Table 4). Soy has a relatively high ability (BCF >1) to accumulate strontium, which may be useful to obtain phytoestrogencontaining vegetable products or herbal formulations for prevention postmenopausal osteoporosis. In vivo and in vitro studies showed high efficiency of strontium salts in osteoporosis therapy and lack of their toxicity in animals and human.36 Our further investigations will focus on examining the activity and toxicity of extracts from soy enriched with strontium.
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AUTHOR INFORMATION
Corresponding Author
*Tel/fax: +48 81 5357350. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
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