Assessment of heavy metals in Hibiscus sabdariffa calyces and Moringa oleifera leaves collected from different areas in Tanzania

Medicinal plants are known in prevention and curing of diseases. Contamination of medicinal plants by heavy metals is one of the factors affecting quality of medicinal products from medicinal plants. Heavy metals may enter the edible and medicinal plants through contaminated environment such as water bodies, air or soil. Hibiscus sabdariffa calyces and Moringa oleifera leaves are used in Tanzania as nutritional and disease-remedial herbal drinks. This study assessed heavy metal contamination of Hibiscus sabdariffa calyces and Moringa oleifera leaves collected from Dodoma region (Vikonje, Msanga, Nzuguni A and Nzuguni B village), Pwani region (Kongowe and Chalinze village) and Shinyanga region (Mwime village).Lead, arsenic, chromium, cadmium and mercury were analyzed by Atomic Absorption Spectrometry. Microsoft excel 2010 software were used to analyze the means of heavy metal concentrations. Levels of chromium in the calyces and leaves of H. sabdariffa and M. oleifera ranged between 0.029-0.221 ppm and 0.019-0.088 ppm respectively while Arsenic was 0.096-0.204 ppm and 0.096-0.219 ppm, respectively. Mercury and Lead were found only in leaves of M. oleifera, mercury was between 0.017 ppm and 0.042 ppm and lead was 0.056 ppm. Cadmium was not detected in all plant materials. The concentrations of heavy metals in the selected samples were statistically significant at (P<0.05). M. oleifera leaves and H. sabdariffa calyces, collected from different regions, had the low levels of heavy metals than recommended limits provided by the Tanzania Medicine and Medical Devices Authority (TMDA) and WHO.


INTRODUCTION
The term "heavy metals" refers to natural metallic elements with a density greater than 5g/cm 3 [1]. Heavy metals are naturally occurring components of the earth's crust that are, neither created nor destroyed, but are simply redistributed [2]. Heavy metals have adverse effects on human health and there-fore in Tanzania and the world in general. M. oleifera possesses an impressive range of medicinal uses and high nutritional value (Rockwood et al, 2013) [9]. Hibiscus is a genus of more than 200 annual and perennial species native to the subtropical and tropical regions of the world. Hibiscus sabdariffa L. commonly known as Roselle is an herbaceous plant that belongs to the family Malvaceae [10]. In Tanzania, H. sabdariffa is cultivated mainly in the central and coastal regions such as Dodoma and Pwani Region [11]- [13]. H. sabdariffa and M. oleifera have great values in the community for being used as traditional medicine for remedy of cancer, constipation, teeth and gum diseases, impotence, fatigue, malaria, typhoid fever, hypertension and diabetes [14] and they contain high amount of vitamin C, vitamins A and calcium, thus considered as nutritional food. H. sabdariffa and M. oleifera are used in forms of raw materials as vegetable, herbal tea, juice, syrups, flavoring and coloring agent [14]- [16].
Due to health hazards caused by these toxic metals, WHO advocates that herbs and herbal products should not be used without qualitative and quantitative analysis of their heavy metal contents [17]. The present study was carried out to assess the heavy metals contaminants in H. sabdariffa calyces and M. oleifera leaves collected from different regions in Tanzania in order to create awareness to cultivators of H. sabdariffa and M. oleifera about possible medicinal plant contaminations, especial heavy metals and provide data of safety levels of those metals in the investigated regions, as the main growing areas in Tanzania.

Study Design
The study was experimental where by H. sabdariffa calyces and M. oleifera leaves were analyzed for heavy metals contamination using Atomic Absorption spectrometer (AAS).

Sample Preparation
The air dried samples were ground into fine particles and sieved using 212 µm mesh at the Government Chemist Laboratory Agency (GCLA), Dar es Salaam. Powdered samples were stored in air tight container for experimental study.

Determination of Heavy Metals
The heavy metals in powdered samples were determined at the Government Chemist Laboratory Agency (GCLA), Mwanza.

Determination of lead, arsenic, cadmium and chromium
Powdered plant materials (1g each) were transferred into 100 ml beaker each. 40 ml of Aquaregia (3:1 HCl, HNO 3 ) followed by few ant-bumping were added to each beaker and the mixture was heated on the hot plate with 105°C for 1hr and left to near dryness (5 ml), removed and cooled. The digest was filtered by Whatman No 541 into 100 ml volumetric flask and then diluted with 0.1% Nitric acid up to the mark. Each plant sample was digested and analyzed in duplicate to confirm precision of the result. The blank solution was prepared by taking a mixture of 30 ml HCl and 10ml HNO 3 and treating similarly as that of the sample.
Standard solutions with different concentrations of 1, 2, 3, 4, 6 ppm were prepared from 50ppm stock solutions for each metal. The Atomic Absorption spectrometer (AAS) was switched on and the required Acetylene gas turned on; for Pb, Cd and Cr while for As, Acetylene and nitrous gas was used. Metal specific lamp was inserted and AAS machine stabilized for 15 Minutes. Standards were analyzed to prepare calibration curves followed with blank and samples analysis in AAS.

Determination of mercury
Powdered plant materials (1g each) was mixed with 5 ml H 2 SO 4 , 2.5 ml conc. HNO 3 and 100 ml distilled water in a 250 ml conical flask and 20 ml 5% KMnO4 solution was added then solution was shaken until the purple color was persistent for at least 15 minutes. 8 ml of 5% K 2 S 2 O 8 solution was added, mixed thoroughly and, heated for two (2) hours in a water bath Standard solutions with different concentrations of 0.8, 1.6, 3.2, 6.4 ppb were prepared from 50ppm stock solution of mercury. Acid blank reagent (50%V/ or 6M HCl) and reductant reagent (stannous chloride) were used to increase sensitivity of AAS to mercury [18]. Mercury analysis in standards, blank and samples was performed by VP 100 Cold Vapor Atomic Absorption Spectrometry with detection limit of 0.0001µg/L (ppb).

Statistical Analysis
The means of heavy metal concentrations in each plant materials, were analyzed using Microsoft excel 2010 software.
Data were considered statistically differences at significance level p ≤ 0.05 and expressed in mean ± SD.

RESULT
The means of heavy metals concentrations in each plant materials, were analyzed using Microsoft excel 2010 software as used by Atinafu et al., 2015; Malum and Salihu, 2017 [19], [20]. The results were presented in mean concentration levels and standard deviations of each heavy metal (Pb, Hg, As, Cd and Cr) in the plant part material concerned as presented in the tables as follows: Table 3.1 shows the concentration of heavy metals in H. sabdariffa calyces. The mean range concentration levels were between 0.029 to 0.221 ppm. The highest concentration of chromium (0.221ppm) was detected in H. sabdariffa calyces from Nzuguni B and lowest (0.029ppm) from Msanga, arsenic (0.204ppm) from Vikonje and 0.096 ppm from Msanga, mercury (0.038ppm) from Kongowe. Lead and cadmium were not detected. Mean concentrations of heavy metals detected in H. sabdariffa calyces were statistically different (p<0.05) and were below the recommended limit set by WHO and TMDA in medicinal plants [17], [21]. Table 3.2 shows the concentration of heavy metals (Pb, Hg, As, Cd and Cr) in M. oleifera leaves. The mean range concentration levels were between 0.017 to 0.219 ppm. The highest concentration of chromium (0.088ppm) was detected in the leaves of M. oleifera from Chalinze and Nzuguni B and lowest (0.019ppm) from Msanga, arsenic (0.219ppm) from Nzuguni A and 0.096 ppm from Chalinze, mercury (0.042ppm) from Nzuguni B and 0.017ppm from Nzuguni A and Lead (0.056ppm) from Nzuguni B. Cadmium was not detected. Mean concentrations of heavy metals detected in M. oleifera leaves were statistically different (p<0.05) and were below the recommended limit set by WHO and TMDA in medicinal plants [17], [21].

DISCUSSION
The levels of heavy metals in medicinal plant samples collected from Kongowe, Nzuguni A, Vikonje and Msanga were found to contain relatively higher amount of arsenic than that of Chalinze, and Nzuguni B, while those collected from Mwime had no arsenic. The higher concentration of arsenic might be caused by contaminations from animal manure and pesticides applied to other crops such as millets and sun flower plants that were found cultivated together with medicinal plants in the same farm. The study by [5], [22], [23] found that, application of pesticides and manure in agriculture is the source of arsenic, lead and mercury in soil and crops. Arsenic causes cancer, damage to the skin, eyes and liver and affect kidneys and central nervous system [24], [25].
Chromium was detected in all medicinal plant samples except calyces sampled from Mwime and Kongowe. Cigarette smokes and sewage sources might be the main sources of chromium contamination [1], this is due to the reason that, most of medicinal plant farms are surrounded with residence areas. Chromium may results to cancer, drowsiness, fatigue, hair loss, insomnia, memory loss, recurrent infections, restlessness, vision disturbances [24].
Trace amount of mercury was found in M. oleifera and H. sabdariffa from Nzuguni A, Nzuguni B and Kongowe respectively and lead detected only in M. oleifera from Nzuguni B. The trace amounts of mercury and lead might be originated from pesticides applied to mixed crops and paints used for home decoration [5], [22], [23]. Exposure to lead is cumulative over time, high concentrations of lead in the body can cause cardiovascular disease and to children causes impaired development, reduced intelligence, loss of short-term memory, learning disabilities, coordination problems and finally death or permanent damage to the central nervous system, brain, and kidneys [22], [26], [27]. Short time exposure to mercury can cause lung damage, kidney damage and affect nervous system [25].
Cadmium was not detected in all medicinal plants; this could mean that there is low pollution with respect to cadmium in the study areas.
Generally, the detected heavy metals might be originated from human activities such as application of pesticides and animal manures in crops, painting, cigarette smokes and sewage disposal or natural sources such as rock weathering and wind-borne soil particles.

CONCLUSION
The plant materials of M. oleifera leaves and H. sabdariffa calyces, collected from different regions, had the low levels of heavy metals than recommended limits provided by TMDA and WHO. Even though the plant materials had low levels of heavy metals, it is further recommended to carry out other safety assessments for these plant materials from the studied sites to ensure safety of consumers.

CONFLICTS OF INTEREST
No competing interests exist.