Elemental Analysis of Galium incanum SM subsp Centrale Ehrend by X-ray Fluorescence Spectroscopy

Purpose: To evaluate the content of trace elements in Galium incanum SM. subsp. centrale Ehrend. Methods: The air-dried aerial parts of the plant material were used and its water extract (2 % w/v infusion) was analysed for trace elements using x-ray fluorescence (XRF ) spectrometry. Results: The aqueous extract depicted significant concentrations of macro- and micro-nutrients with heavy metal and metal oxide content of 4.07 - 6.02 and 3.19 - 4.01 % for powdered plant material and water extract, respectively. The contents of Ca (22840 ppm) and K (8204 ppm) were the highest among all the elements while Zn (45.9 ppm) and Fe (328 ppm) were also detected in significant amounts. Zn, Mn and Cu showed the highest content while those of Mg, Al, K, Ca, Fe and P lowest in the water extract. Conclusion: The presence of significant levels of Ca, K, Na, Fe, Zn, Mg, Mn and Cu in G. incanum subsp. centrale showed that this plant has notable nutrient elements. The traditional use of Gallium species as a diuretic may be attributed to its rich content of potassium.


INTRODUCTION
Rubiaceae, comprising 640 genera and over 10000 species in about 10 tribes distributed in the world, chiefly in tropical regions, form the fourth largest angiosperm family after Asteraceae, Orchidaceae and Leguminosae [1]Galium with 400 species distributed in both temperate and tropical regions is one of the largest genera of Rubiaceae [2].Galium is represented in Turkey by 101 species [3].These species are used to coagulate milk because of an enzyme in their chemical composition [4].Galium aparine L., G. cruciata ( L.) Scop.and G. verum L. are used as diuretic, chloretics, against diarrhea and in the treatment of some stomach complaints, gout and epilepsy in folk medicine [5].
It is well known that many trace elements play a vital role in human health.Most of the studies on medicinal plants pertain to their secondary metabolites viz.glycosides, alkaloids, phenolic componds, essential oils and other active components and their biological effects.Considering the importance of trace elements and for adequate safety evaluation, the present study was aimed to determine the presence and quantity of metallic elements in G. incanum SM. subsp.centrale Ehrend., an endemic species known as "yoghurt herb" in Turkey.

EXPERIMENTAL
The aerial parts of Galium incanum subsp.centrale were collected in July 2012 from Ödemiş, Bozdağ, İzmir, Turkey.Plant material was identified by Dr. Cenk Durmuşkahya, Celal Bayar University, Faculty of Education, School of Applied Science, Demirci, Manisa.A voucher specimen (no.1432) was deposited in the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Ege University, Izmir, Turkey.
The air-dried plant materials were cut into small pieces and ground into fine powder.The water extract of plant material was prepared as a 2 %w/v infusion and then filtered using Whatman filter paper no. 1 paper.The filtrate was concentrated using vacuum evaporator and kept at -20 °C until use.The powdered plant material and water extract were analysed for content of trace elements by energy dispersive x-ray fluorescence (XRF) spectroscopy.The elemental composition was determined by using SPECTRO IQ II (Ametek, Germany) with silicon drift detector SDD with resolution of 145 eV at 10 000 pulses.The primary beam was polarized by Bragg crystal and Highly Ordered Pyrolytic Graphite -HOPG target.The samples (0.1 g) were measured during 300 s at voltage of 25 kV and 50 kV, at current of 0.5 and 1.0 mA under helium atmosphere.

RESULTS
The elemental range for XRF spectroscopy is from sodium to uranium.Generally, it is difficult to quantify elements lighter than sodium.In this study, the concentrations of trace elements from sodium to uranium in the periodic table were determined in the water extract (WE) and powdered plant material (PM) of Galium incanum subsp.centrale by using XRF spectroscopy.The results showed various concentration of macroelements and microelements in the samples.An examination of the data from Table 1 shows that PM and WE contain Na, Mg, Al, Si, S, K, Ca, Fe, Cl, Mn and Zn and their oxides in various proportions.The results indicate that heavy metal and metal oxide content was 4.07 -6.02 and 3.19 -4.01 % for powdered plant material and water extract, respectively.

DISCUSSION
XRF technique is well suited for multielemental determinations in plant samples.The samples do not need any chemical treatment and any possible contamination is therefore avoided.XRF is one of the sensitive, rapid and simple analytical technique to study the essential element content of medicinal plants [10,11].Many trace elements play significant roles in various physiological and biochemical events.Excessive levels of these elements in medicinal plants could lead to toxicity.Food and Nutrition Board recommends calcium intake as 1000 mg/day wheras the recommended daily intake of sodium and potassium are 1500 mg/day and 2300 -3200 mg/day, respectively [12].Plants are important link in the transfer of trace elements from soil to humans [13].Fruits and vegetables are safe and valuable sources of minerals [14].In previous studies, medicinal plants are shown to posses some important elements which have both therapeutic and prophylactic properties [13][14][15][16][17].In addition to expressing their nutrient contents as the uncombined elements, metals are also expressed as their oxides.Therefore, in this study, the oxide forms of elements in G. incanum subsp.centrale were also determined.The concentration of Ca was determined as 22840 and 2917 ppm for PM and WE, respectively.CaO was measured as 31950 and 4081 ppm in PM and WE, respectively.Solubility depends on the form of metal (i.e., whether the metal exists in a free or complexed form).Metal transfer from herbs to final extract is both sample-dependent and solvent-dependent.This study was also aimed to investigate the metal content of the extract prepared from G. incanum subsp.centrale.The resulting metal concentrations in WE increased or decreased when compared with PM.For example, concentrations of Zn, Mn, and Cu increased whereas that of Mg, Al, K, Ca and P decreased in WE.
The role of inorganic elements in human metabolism has long been established.Macroand microelements influence many biochemical processes to maintain good health.Study of elements with respect to medicinal plants reveals that major and trace elements play significant roles in combatting diseases [17,18].Some metals in health products are as a result of adulteration.Many herbs are metal accumulators and take up both essential and non-essential metals from soil and environment during the growth.Thus, knowledge of metal content is also essential to evaluating the quality of herbal products.

CONCLUSION
The levels of Ca, K, Na, Fe, Zn, Mg, Mn and Cu in G. incanum subsp.centrale indicate that the plant is a notable source of nutrient elements with potential therapeutic benefits.Chemical constituents of Galium species have been published previously.To the best of our knowledge, there is no scientific report on the mineral content of this genus.The water extract prepared from the plant material accumulated various elements in higher levels than the plant material itself.It is, therefore, important for safety reasons that evaluation of both the plant material itself and its water extract are carried out.The data obtained from this study lends some support for the use of the plant in traditional medicine in Turkey.The results also suggest that the plant material may be useful in the formulation nutritional and/or therapeutic products.