Estimation of Heavy Metals in Selected Medicinal Herbs

Ida Christi V E*1, Krishnaprabha C2, Blesson Sha Fogarty N I3, Sangilimuthu A4, Kumutha D1 1Faculty of Pharmacy, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India 2St.James college of Pharmaceutica Sciences, Thrissure, Kerala, India 3KMCH Institute of Health Sciences and Research , Coimbatore, Tamil Nadu, India 4Department of Biotechnology, Karpagam Academy of Higher Education, Coimbatore , Tamil Nadu, India


INTRODUCTION
The selected plants Moringa oleifera Lam.(MO),Achyranthes aspera Linn.(AA), Scoparia dulcis Linn.(SD) (Latha and Pari, 2005) ,Saropus androgynus Linn. (SA), (Gokhale et al., 2002) and Castus pictus Linn.(CP) are more useful in the traditional system of medicine for many diseases (Ezeamuzie et al., 1996). Herbal medicines when used for internal administration they must be standardize before formulation for the quantitative estimation of Physical parameters like heavy metals, Phytochemicals estimations. Heavy metals are the substances can be toxic when consumed by humans especially like Lead, Cadmium, Mercury, Chromium and Arsenic (Gasser et al., 2009). They are naturally present in the earth in different places in different concentration and it may absorbed and occur in plants (Karayil et al., 2014). Some of the metals are necessary and do some bene icial effect on human like iron and some do toxic effect selenium, arsenic (Graeme and Pollack, 1998a). Heavy metals can, in certain level, causes illness (Karadi et al., 2008), as well as carcinogenic, have adverse reproductive effects, (unfavorably impact nutritionon, and displace more geographically valuable metals such as magnesium and zinc (Duffus, 2003). This document is focused on the estimation of heavy metals like lead, mercury, arsenic, cadmium, selenium, chromium , cobalt and nickel. The guidelines developed to determine with accompanying explanations by the American Herbal Products Association (AHPA) and given the quantitative limits for it. As well as it debates relevant regulations about the prevalence of these chemicals in products sold in the United States. The heavy metals are wide spreader environmental contaminant and potential toxin that may adversely affect human health (Johri et al., 2010). When exposure to cadmium towards the respiratory or gastrointestinal tract (Rao et al., 2006) , important non-industrial source of exposure are cigarette smoke and food (from contaminated soil and water). The kidney is the main organ affected by chronic cadmium exposure and toxicity (Rai et al., 2001). Environmental cadmium exposure is also the signi icant contributory factor may cause the development of chronic kidney diseases, (Wang et al., 2007) . And in the presence of other metals also especially for diabetes, hypertension, therefore the souse and environmental impact of and efforts ti limit cd exposure, justify more attention (Graeme and Pollack, 1998a). Metals may produce toxic and sometimes produce effect as same as that of the essential elements in body and interact with metabolic process and produce problems in body (Pasquale et al., 1993).
Environmental Cd exposure may be a signi icant contributory factor to the development of chronic kidney disease, especially in the presence of other co-morbidities such as diabetes or hypertension; therefore, the sources and environmental impact of Cd, and efforts to limit Cd exposure, justify more attention (Graeme and Pollack, 1998b). Metals may produce toxic and sometime produce effect as same as that of the essential elements in body and interact with metabolic process and produce problems in body. This present study focused towards the importance of the quantitative evaluation of heavy metals in herbal products.

Collection and Authentication of Plant
The plants Moringa oleifera Lam. And Achyranthes aspera Linn., Saropus androgynus Linn., Castus pictus Linn, available locally and they were collected from in and around Coimbatore. The plant Scoparia dulcis Linn. was collected from Palakad district in Kerala. The botanical identity has been authenticated by the Director, Botanical Survey of India, Coimbatore (No:BSI/SRC/5/23/2011-12/Tech/376;BSI/SRC/5/23/2012-13/Tech/496). The voucher specimens had been submitted and preserved in herbarium for future reference.

Preparation of Drug Sample
For the estimation of heavy metals the ash from the plant leaves were prepared and the ash dissolved in dilute hydrochloric acid, iltered. The clear liquid was taken for the quantitative estimation of inorganic metals by lame photometer and Atomic Absorption Spectroscopic method (Becketta and Stenlakej, 1987).

Estimation of Inorganic Minerals by Flame photemeter Method
The alkalies and alkaline earth metals such as Na, K, and Ca can be determined by lame photometry. Flame emission photometry is also successfully used for estimation of certain transition elements such as copper, iron and manganese as the energy obtained by the lame is quite suf icient for the excitation of these elements. It is used by the lame to provide the energy of excitation to atoms introduced into the lame. The quantitative results can be obtained by plotting a calibration curve between the standard and sample concentration of the element and lame induction.
Liquid sample containing element, is aspirated into a lame to the formation of the liquid droplets. Evaporation of liquid droplets, resulting formation of salt residue. Then decomposition of residue to formation of free neutral atoms or radicals. These neutral atoms are excited by the thermal energy of the lame. The excited atoms which are unstable, quickly emit photons(radiation) and return to the lower energy state. The measurement of emitted radiation in terms of wavelength or intensity is basic of lame photometry. If E 2 and E 1 represent the energy of the higher and lower energy state. The radiation emitted during the changing energy level may be de ined Where h = planks constant, v= the frequency of emitted light.
Which is de ined as, We get From the above equation calculate the wave length of the emitted radiation which is characteristic of the particular element.
Weigh accurately 100 mg of sodium chloride and potassium chloride separately dissolve in 100 ml of distilled water. Prepare a series of standard solutions of sodium chloride, potassium chloride 10,20,30,40 and 50 µgm/ml concentrations. Switch the lame photometer and select the sodium ilter. Set the gas in lame and air pressure at 0.4 to 0.5 kg/cm 2 . Automize the lame intensity to 0% using distilled water with the knob. Automize the lame intensity to 100% using 50µgm/ml standard solution (highest concentrations). Measure the lame intensity of all the standard solutions with different concentrations like 10, 20, 30,40,50µg/ml, and unknown sample solution. Plot the graph between concentration and percent lame intensity. From the graph, make the percentage lame intensity of the known sample and by extrapolating, determine corresponded concentration (Becketta and Stenlakej, 1987).

Atomic Absorption method
This technique is closely related to lame emission spectrometry. They use a lame as the atomizer. The sample solution is aspirated into the lame same as lame emission spectrometry, here the element is converted to atomic vapor, it contains the atoms of the elements. Some are remain in the ground state. These ground state atoms can absorb radiation of a particular wavelength that is produced by a special source made from that element. The wavelengths of radiation given by the atoms in the lame is as identical in principle to absorption spectrometry (Abou-Arab et al., 1999) . The absorbance is directly proportional to the path length in the lame and to the concentration of atomic vapour in the lame. Both of the variables are dif icult to determine, but the path length in the lame can be held constant and the concentration of atomic vapour is directly proportional to the concentration of the analyte in the solution being aspirated. The calibration curve is prepared with concentration in the solution versus absorbance. The inorganic elements like magnesium, Zinc, Iron, Manganese, and copper like elements are estimated by Atomic absorption method. The heavy metals like mercury, Lead, Arsenic, Cadmium, Selenium, Chromiun, Nickel, Cobalt were also estimated by same Atomic Absorption method (Honary et al., 2007).The estimations were carried out in triplicate and calculated statistically.

Estimation of Inorganic Elements
The quantity of inorganic elements like Potassium, Manganese, Sodium, Calcium, Magnesium, Zinc, Iron and Copper were estimated by lame photometry and atomic absorption methods and the quantity present in the selected plant leaves were expressed in the Table 1.

Estimation of Heavy Metals
The selected plants leaves powder were estimated for the presence of Heavy metals by atomic absorption spectra method. The quantities of all heavy metals present in these plants were tabulated below.
The herbal drugs usage has been increased in recent years. The inorganic minerals which are necessary for human health were quanti ied and tabulated and also the quantity of heavy metals present in the selected plants leaves were estimated and tabulated. According to the WHO (2005) the standard quantity or limits of heavy metals in the medicinal plants were listed as Arsenic 3ppm, Lead 10ppm, Cadmium 0.3ppm, Chromium 1ppm and Mercury 0.03ppm. The results shows the quantity of these heavy metals present in the selected plants were within the limit and the results were tabulated in the Table 2, (Commission Regulation , 2006). The results of this study reveals that the concentration some of the inorganic minerals like Ca, Na, K, Cu, Mn, Mg and Zn present in the samples are in good quantity expressed in igure 1, because all these minerals are necessary for human life, where as the heavy metalsare within or remarkably much lower when comparing with the limits recommended by WHO. Different region of the world and countries set on maximum values for toxic metals in various quantities and now the world has recently been published by the WHO (WHO, 2007). In this context, the WHO propose some limit for Lead 10mg/kg and for Cadmium 0.3mg/kg in dried herbs (WHO, 2007).
These heavy metals determination was performed by using atomic absorption spectrometry (AAS). Here the concentration of lead was found to be much lesser quantity when compared with the standard limit 10mg/g of Lead (WHO, 1999). Because the more quantity of lead in herbal medicines will produce hazardous effect like deleterious effect on central nervous system (Khan et al., 2001), side effects in kidney and bones etc (Abou-Arab et al., 1999). Minerals like Copper and Zinc are important but there should be a limits, (de Souza et al., 2008) they were also determined and documented in the table no.1, that also found within the limit as per WHO guideline for copper 40mg/kg and Zinc 60mg/kg (FDA, 1993). Zinc is essential for plant growth and also for human body normal growth, brain development and bone formation. Copper is also playing important role in human body like oxidative defence system where as the concentration increases it will leads to toxic (Jones et al., 1977). Copper and lead are required for metabolic functions. The development of new blood vessels, for wound healing property is promoted by copper. Zinc enhances the human immune system, work as cofactor for some enzymes involved in metabolic pathways (Baker and Brooks, 1989). If the level increases it also leads to toxicity and interfere with copper metabolism (Maqsood and Khan, 2005). The arsenic and Cadmium content were determined and found that are within the permissible limit (WHO, 2003). Exposure in high dose they may produce nausea, vomiting, abnormal heart beat, damages blood vessels (Zhao et al., 2009). Arsenic poisoning leads to convulsion, confusion, vomiting, diarrhea, and in severe condition sit may leads to coma and death.

CONCLUSIONS
On basis of this experimental study, result shows the quantity of all the heavy metals estimated are below the permissible limit of WHO guideline. The result shows that all these selected plants are having the inorganic minerals in the considerable quantity and may helpful for health requirements. The result shows Moringa oleiferaLam. Achyranthes aspera Linn. Saropus androgynus Linn., Castus pictus Linn and Scoparia dulcis Linn. are safe for internal administration for their therapeutic values and will not produce any harmful effect by heavy metal toxicity. These plants are advisable for the development of new herbal formulations.