Chemical Composition of Seeds of Two Endemic Lathyrus L. Species from Turkey

Genus Lathyrus L. belonging to Fabaceae (Leguminosae) family consisted of 77 taxa at the species, subspecies, and variety level at Turkey and 25 of that were known as endemic. Lathyrus egirdiricus H.Genc & A.Sahin and Lathyrus tefennicus H. Genç & A. Şahin were endemic species and described at the date of 2008 and 2011, respectively. The seeds of Lathyrus tefennicus and Lathyrus egirdiricus were collected in Tefenni of Burdur province and Eğirdir of Isparta province in May of 2016 at Turkey. The concentration of Cd, Cr, Ni, Cu, Mn, Zn, Fe, Ca, Mg, Na and K in the Lathyrus tefennicus and Lathyrus egirdiricus seeds were determined by Atomic Absorption Spectrometry. A microwave oven was used for the sample digestion. The accuracy of proposed method was confirmed by the analysis of the certified reference material (EnviroMAT Contaminated Soil). All results were given at 95% of confidence level. The presence of different concentrations of elements in the seeds of both species strengthens the belief that this data can be used in plant taxonomy.

The heavy metals were transferred from the soil to plant and following accumulated in the seeds, cereals, fruits, roots, stalks and leaves. To investigate the physiological activity of the plant, the concentrations of Cr, Mn, Mo, Zn, Fe, Co, Cu, Al, Ni should be determined (Filipiak-Szok et al., 2015;Heredia et al., 1016). When the plant included excessive or low amount of these metals, it caused to a negative effect for the plant health . Because some metals such as Pb, Ni, and Co hinder to ecesis as well as make prohibition effects on the plants (Mohamed et al., 2017), determination of metals in the plants were important.
There were some analytical methods such as flame atomic absorption spectrometry (FAAS), graphite furnace atomic absorption spectrometry (GFAAS), inductively coupled plasma optical emission spectrometry (ICPOES), and inductively coupled plasma mass spectrometry (ICPMS) were used for the determination of metals in any samples (Santos Júnior et al., 2017). Among them, FAAS was frequently used because of its some advantages such as more speed, lower cost and higher accessibility (Elik et al., 2017).
In this study, determinations of 11 different metals such as Cd, Cr, Ni, Cu, Mn, Zn, Fe, Ca, Mg, Na and K in the both Lathyrus tefennicus and Lathyrus egirdiricus seeds were performed by FAAS method. The microwave method was used to digest seeds because of its rapid and repeatable properties (Tüzen, 2003). Lathyrus tefennicus an endemic species was found in Tefenni of Burdur province at Turkey and growed in Pinus L. forests. The flowering and fruiting time was between May and July. The colour and size of seeds were varied from dark brown to dark green with the rough-surfaced and 2.9-3.8 × 4.8-6 mm with a 1.5-3.2 mm long hilum, recpectively (Genç & Şahin, 2011). Furthermore, Lathyrus egirdiricus an endemic species was found about 3 km far from Eğirdir of Isparta province at Turkey. The colour of its flower was light purple and bloom in between May and July. However, the colour of seed was varied from light brown to dark brown with rough-surfaced. The size of seed is 6-8 per legume, 2 ×4 mm, hilum 0.5-1 mm (Genç & Şahin, 2008). The accuracy of the method was performed by using certified reference material (EnviroMAT Contaminated Soil) at 95% confidence level with good agreement. It was thought that the data obtained in this study could be used for plant taxonomy based on chemotaxonomics.

Material
The seeds of Lathyrus tefennicus and Lathyrus egirdiricus were collected from natural habitat in Tefenni (Burdur) and Eğirdir (Isparta) in May 2016.

Reagents and apparatus
All reagents were of analytical reagent grade. The stock solutions (1000 mg/L) of Cd, Cr, Ni, Cu, Mn, Zn, Fe, Ca, Mg, Na and K were prepared from Cd(NO 3 n), NaCI (Sigma-Aldrich) and KNO 3 (Merck), respectively. 18.2 MΩ.cm deionized water (PURIS, Expe-up) was used for all Journal of Biology and Life Science ISSN 2157-6076 2018 experimental studies. The concentrated HNO 3, HCI, and HF solutions used for the digestion step were supplied from Merck, Riedel de Haë n and Merck, respectively. A flame atomic absorption spectrometry (ATI Unicam 939 model) was used for determination of all metals. The operating conditions in determination step were shown in Table 1. The microwave digestion system (CEM, Mars 6 240/50 model) was used to digest both the seeds and certified reference material (EnviroMAT Contaminated Soil). The digestion conditions for the seeds and certified reference material were given in Table 2.

Digestion procedure
The seeds were first washed with distilled water and then dried at the room temperature. Afterwards, they were homogenized using a porcelain agate and stored in polyethylene bottles until experiment. During the digestion, 1.0 g of seed samples and 0.2 g of the certified reference material (EnviroMAT Contaminated Soil) were introduced into a Teflon vessel. Then, 3 mL of concentrated HNO 3 , 3mL of concentrated H 2 O 2 , 1 mL of concentrated HCIO 4 and 3 mL of deionized water were added on all samples, respectively. After digestion procedure given in Table 2, the samples were transferred into polyethylene bottles and kept in the refrigerator until analysis.

Concentrations of metals in the seeds
The concentrations of some metals such as Cd, Cr, Ni, Cu, Mn, Zn, Fe, Ca, Mg, Na and K were determined using by FAAS after digestion procedure. The standard solutions were prepared between 0.2 and 10 mg/L depending on metals and the absorbance values of the metals were measured in both standard and sample solutions using the calibration method. The linear equations of calibration graphs for all metals were given in Table 3. The concentrations of metals in the Lathyrus tefennicus and Lathyrus egirdiricus were calculated by using these linear equations depending on metals.
Cd in plants decreased the plant cultivation and microorganism variety (Sobariu et al., 2017). The Cr(III) was an essential metal and played important roles in the biological systems. However, the Cr(VI) was toxic for plants, animals and human beings. In general, Cr was transferred from the soil to plant (Sobariu et al., 2017). Ni was a serious pollutant due to burn of coal and petroleum and using of excessive fertilizer. The Ni concentration in the plant was increased with increasing Ni concentration in the soil. Its concentration in the plants was affected by the climate and soil pH. The excessive amount of Ni slowed down the plant growth (Kabala & Singh, 2001). Cu was an essential metal for all creatures. Cu was attended in some important biological mechanisms such as carbohydrate and lipid metabolism. However, the excessive amount of Cu could cause to harmful effects on the blood and kidney (Xiang et al., 2010). Mn was another essential metal and played an important role in the growth of plants. Because of this, the Mn salt was added to the soil (Lemos et al., 2009). Zn was toxic for plant growth when it was taken at higher than essential amount. In addition to this, the adsorption of Cd by the plant was diminished in the soil because of Zn (Pehlivan et al., 2008). Fe was essential for all creatures and it had significant functions for some processes such as DNA synthesis, oxygen and electron transport. The high amount of Fe could cause the tissue damage (Maiga et al., 2005). Ca was an essential metal for plant growth and development. Additionally, Ca played the crucial role in regulation of membrane permeability, stabilization of tissues and quality of plants (Tuna & Özer, 2005). Mg was accepted as the iron of the plant world. Similar to the Fe-hemoglobin relationship in humans, Mg entered the chlorophyll structure in plants (Solak Görmüş & Ergene, 2004). Na played an important role to adjust the blood volume, pressure, the osmotic balance and the pH value in the body. However, higher amounts of Na may cause to hypertension, stroke, stomach cancer, osteoporosis and kidney diseases (Hwang et al., 2017). K played some important roles such as regulate of plant water potential, form and transport of proteins, carbohydrates, fats and convert of amino acids into proteins. K was the most important element affecting the quality parameters of the plant such as color, odor, aroma, durability, hardness, resistance against diseases and storage period (Kacar & Katkat, 1998).
The concentration of metals in both Lathyrus tefennicus and Lathyrus egirdiricus and other plants in the literature were compared and the results were given in Table 4.

Method validation
The accuracy of the proposed method developed was checked by applying certified reference material (EnviroMAT Contaminated Soil) for Cu determination. The Cu amount was found as 174 mg/kg in the certified reference material and there was a good agreement between found and certified values at 95% confidence level with -8.9% relative error.

Conclusion
To the best of our knowledge, this was the first study to determine metal composition of new Lathyrus tefennicus and Lathyrus egirdiricus seeds. The amounts of Cd, Cr, Ni, Cu, Mn, Zn, Fe, Ca, Mg, Na and K in the seeds were determined using by FAAS after digestion step with microwave oven. Significant toxic metals such as Cd, Cr and Ni were not found in seeds. Cu were determined as 4.71±0.09 mg/kg and 3.74±0.09 mg/kg in Lathyrus tefennicus and Lathyrus egirdiricus, respectively. Mn values were found as 9.65±0.40 mg/kg in Lathyrus tefennicus and 11.0±0.28 mg/kg in Lathyrus egirdiricus. Zn were found as 25.0±0.32 mg/kg and 42.0±1.3 mg/kg in Lathyrus tefennicus and Lathyrus egirdiricus, respectively. The amounts of Fe were found as 66.0±3.0 mg/kg and 51.0±3.0 mg/kg in Lathyrus tefennicus and Lathyrus egirdiricus seeds, respectively. The amounts of Ca were identified as 760±62.0 mg/kg and 1343±98.0 mg/kg in Lathyrus tefennicus and Lathyrus egirdiricus seeds, respectively. The Mg amounts were determined as 1374±38.0 mg/kg and 1026±82.0 mg/kg in Lathyrus tefennicus and Lathyrus egirdiricus seeds, respectively. The concentrations of Na were found as 6650±59.0 mg/kg and 4967±251 mg/kg in Lathyrus tefennicus and Lathyrus egirdiricus seeds, respectively. The amounts of K in Lathyrus tefennicus and Lathyrus egirdiricus seeds were found as 5306±202 mg/kg and 5919±210 mg/kg, respectively. Furthermore, the accuracy of method was confirmed by the analysis of certified reference material (EnviroMAT Contaminated Soil). According to results, it was decided that 2 different endemic Lathyrus taxa were also different for the chemical compositions based on t-test calculations at 95% confidence level. Additionally, the presence of elements at different   0.17±0.01mg/kg (in smaller coix seeds)