Profiling of various elements in Haloxylon griffithiiand Convolvulus leiocalycinus using atomic absorption spectroscopy and flame photometry

The concentration of nine different elements including heavy metals were analyzed in selected medicinal plants i.e. Haloxylon griffithii and Convolvulus leiocalycinus. The mineralization of plant samples was done using wet digestion method in which three strong acids were used i.e. nitric acid (HNO3), sulphuric acid (H2SO4) and perchloric acid (HClO4) followed by filtration and analyzed through flame atomic absorption. For sodium and potassium, flame photometer was used and samples were diluted to 100 folds for obtaining results within the range of the instrument. The decreasing order of concentration of elements in H. griffithii was K>Na>Fe>Ni>Mn>Cu>Co>Pb>Cd whereas, in C. leiocalycinus was K>Na>Fe>Cu>Mn>Ni>Co>Pb>Cd. The study revealed that heavy metals accumulate in the plants with different concentrations and their concentrations were within the permissible ranges established by international organizations. This will provide knowledge in the invention of many new drugs and the research helps the legislators to provide protection to the public regarding adverse effects of heavy metals.

In the past few years, the consumption of herbal medicine proliferates because of its ease of use and less side effects among the third world countries. The use of herbal medicines give cure against various ailments and fulfil the need of essential and non-essential minerals in the body [5]. Experimentally

Materials and methods Reagents and solutions
Different standards (1000ppm) of analytical grade were purchased from Merck, Marker for the analysis of heavy metals. Concentrated acids were used like perchloric acid (HClO4), nitric acid (HNO3) and sulphuric acid(H2SO4)which are the strong oxidizing agents used in the digestion of plants and were of analytical grade.

Instrument and glasswares
Atomic absorption spectrophotometer of Thermoelectron S4 AA system was used for the determination of heavy metals with hollow cathode lamps of different metals and air acetylene gas as a flame was used in the analysis of heavy metals. Whereas, Na and K was detected through flame emission spectroscopy of Jenway PFP7 for the above mentioned plants.
Various glass wares including round bottom flask, conical flask and beakers of pyrex glass were used and rinsed with deionized water before analysis.

Sample preparation Crushing and grinding
The collected plants were first dried in shady area then dried plants were crushed into powder with the help of electrical blender. These powdered forms of plants were further used for the analysis through atomic absorption spectrophotometer.

Digestion method
As various methods are used for the digestion of metals from plants but here wet digestion method was used followed the protocol of Zafar et al. [25].Each plant sample in powdered form 0.25g were taken in a 50ml round bottom flask then the mixture of acids with a total volume of 6.5ml i.e., 1ml conc. sulphuric acid, 5ml conc.nitric acid and 0.5ml conc.perchloric acid was added in a 50ml round bottom flask containing plant materials. Individually,each plant sample containing mixture of acids was heated on a hot plate (JENWAY 1000)at 80-85 degree Celsius until white fumes comes out from the flask. These white fumes indicate that the digestion was complete. Subsequently, added few drops of distilled water in it, removed it from the hot plate and allowed it to cool then transferred the solution to 50ml volumetric flask and made the volume up to the mark i.e. 50ml by pouring distilled water in it. Filtered the extract using filter paper Whatmann No.1 in labelled plastic bottles. These prepared solutions after the wet digestion were analyzed for the detection of elements using atomic absorption spectrophotometer of Thermoelectron S4 AA system with selective hollow cathode lamps. Dilutions of different concentrations were prepared from analytical grade stock standards of 1000ppm for the purpose of calibration. These dilutions were prepared immediately before running the samples. Deionized water was used throughout the investigation. FES ofJenwayPFP7, the technique was applied for the evaluation of sodium and potassium. For obtaining results within the range of flame photometer, the samples were diluted 100 folds with deionized water.

Results and discussion
The results are given below in table 1 and their comparison is given in figure 3. FAAS was used for the evaluation of different heavy metals because of its accuracy and FES is a simple method for the evaluation of sodium and potassium. For sodium and potassium, the dilution of samples was done using deionized water considering the range of calibration curves of different elements. As flame atomic absorption was attached with the computer device and it gives calculated concentration of various elements upon detection whereas flame photometer was not able to give the calculated concentrations so, the concentration of sodium and potassium were obtained through the regression equation. Satisfactory results were obtained through the wet digestion method for the mineralization of plant materials. This method eradicates the surrounding of organic compounds around the minerals, only the minerals are left behind in an aqueous solution by using the strong oxidizing agents. This method is simple and faster than other methods of digestion like dry ashing and microwave digestion. Nickel is essential for human body as well as for plants in a very less amount as it takes part in the production of insulin and its deficiency leads to liver malfunction. In high concentration, its toxicity leads to many diseases including loss of body weight, cardiac and liver problems. According to WHO, the permissible limit of Ni is 1.5mg/kg in the plants and for dietary intake it is 1mg/day [29].The mean concentration of Cobalt in H. griffithii and C. leiocalycinus were 0.7544µg/ml and 0.1871µg/ml respectively. Iron is a vital element for the production of red blood cells in the body. Low concentration of iron in the body leads to anemia [32]. The high concentration of iron cause damage to the tissues in humans [33].In plants, the acceptable limit of iron according to WHO is 20mg/kg while for human consumption it is 10 to 28mg/day [29]. The mean concentration of iron in H. griffithii and C. leiocalycinus were 4.8342µg/ml and 5.6601µg/ml respectively. Manganeseis a very vital element for the growth of both plants and animals. In mammals the reproductive and skeletal problems are due to the deficiency of this trace metal in the body. Excessive intake of manganese leads to many lung problems and affects the human brain [34]. The permissible limit of manganese according to WHO in plants is 200mg/kg and for human intake it is 11mg [29]. The mean concentration of iron in H. griffithii and C. leiocalycinus were 0.5174µg/ml and 0.2294µg/ml respectively. Cadmium is noxious for human health even in the low concentration, as it is a nonessential heavy metal. Learning debilities and hyperactivity in progenies all due to cadmium [35]. According to WHO, the permissible limit of cadmium in therapeutic plants is 0.3mg/kg [29]. The average concentration of cadmium in H. griffithii and C. leiocalycinus were 0.0126µg/ml and 0.0139µg/ml respectively. Lead is a non-essential heavy metal. It is accumulated in the bones and teeth which causes the bones to stiff and weakens the wrists and limbs. It affects the reproductive, renal, immune and nervous system of the body through the deposition of lead in the soft tissues. The safety limit of lead for human consumption is 1.5 ppm [32, 36] and 10mg/kg in medicinal plants [29]. The average concentration of lead in H. griffithii and C. leiocalycinus were 0.0568µg/ml and 0.0309µg/ml respectively. Sodium is a transporter of amino acids, glucose and other metabolites in the cells of the body which in results produces energy. The deficiency of sodium in the body leads to mood swings, causing cramps in the muscles, dehydration in the body and hair loss. The recommended value of sodium for human intake is 13.8mg/day [37]. The average concentration of lead in H. griffithii and C. leiocalycinus was 819.83µg/ml and 423.83µg/ml respectively. Potassium maintains the balance of hormones, secretion of insulin and response of immune system.Potassium works to reduce the blood pressure, maintains the cell volume and also thecell signaling of the body. It helps in the treatment of liver and kidney problems.
For adult women, the permissible limit of potassium is 2300mg/day and for adult men it is 3100mg/day [38]. The average concentration of lead in H. griffithii and C. leiocalycinus were836.67µg/ml and 504.67µg/ml respectively.

Conclusion
This study revealed that the concentration of heavy metals in plants i.e. H. griffithii and C. leiocalycinuswere below their safety limits given by different international organizations like WHO and FAO. Nine elements including essential and non-essential elements were evaluated in these plants among which the concentration of sodium and potassium were found to be higher in both plants and concentration of heavy metals were within the permissible ranges. These plants are safe to use as traditional medicines and therefore are not at risk. The accurate and precise evaluation of metals are very necessary. The results obtained in this investigation will be supportive for the manufacturers in the synthesis of new drugs with different combination of medicinal plants for curing various ailments.