International Journal of Science and Healthcare Research Vol.5; Issue: 4; Oct.-Dec. 2020 Website: ijshr.com Original Research Article ISSN: 2455-7587

Levels of Some Heavy Metals in the Leaves of (Lawsonia inermis) within Lamu County, Kenya

1 2 Tsanuo Kassim Mohammed , Jaleny Paul Ochieng

1Department of Chemistry, Kenyatta University, Kenya 2Faculty of Biological and Physical Sciences, Tom Mboya University College, Kenya

Corresponding Author: Jaleny Paul Ochieng

ABSTRACT Among all the sampling sites, a significant difference in the level of nickel (p=0.021) was Henna plant scientifically known as Lawsonia recorded only within Mpeketoni site. From the inermis is an ethno botanical plant used majorly results of the study, it showed that henna leaves for preparation of called henna. The plant from Lamu County formed a good raw material contains a pigment called Laws one which for henna products due to their low level of lead, makes it suitable for preparation of henna used cadmium and nickel in comparison to the in painting or beautification of body parts. standard limits by WHO. Effective quality Henna painting of body parts is an art of control measures, proper selection of raw beautification which is commonly practised by material as well as good manufacturing practises the people of Lamu County and other parts of should be enhanced so as to minimise the levels the world. However, high levels of toxic heavy of these metals in henna products. metals such as lead (Pb), Cadmium (Cd) and Nikel (Ni) commonly found in henna products Key words: WHO, Heavy metal, Henna pose health threat to users. One of the sources of painting, Henna Products, Henna plant heavy metals in cosmetic henna products is the henna leaves. The aim of the study was to INTRODUCTION determine the level of heavy metals; Lead (Pb), Henna plant scientifically known as Cadmium (Cd) and Nickel (Ni) in henna leaves Lawsonia inermis belongs to the within Lamu County in order to assess their family of . It grows in the suitability as raw material for henna products tropical climates of Africa, northern with respect to the international standard limits. [1] A total of 60 henna leaf samples were randomly Australia, and southwest Asia , and well known for its cosmetic and therapeutic selected from Mpeketoni, Amu, Pate and [2] Kiunga sampling sites within Lamu County and virtues . It contains a pigment called Laws analysed for heavy metals using Flame Atomic one (2-hydroxy-1,4-naphthoquinone) and Absorption Spectroscopy (FAAS) after acid other compounds making leaves natural digestion of the samples. Analysis of variance source of colour for painting/decorating (ANOVA) using SPSS version 17.0 was used to body parts [3]. analyse the data. The least and highest mean Henna painting is an art which has levels (ppm) of heavy metals in the henna leaf been in practise for over 5000 years ago [4]. samples were as follows: Pb (0.04±0.01 to The ancient Egypt and its neighbouring 0.93±0.04), Cd (0.01±0.00 to 0.09±0.01) and Ni countries practised henna painting which (0.01±0.00 to 0.34±0.02). The pattern of overall later spread to Arabian countries and today, mean level of heavy metal accumulation in the [5] henna leaves followed the order: Pb > Ni > Cd. henna is used in all parts of the World . No significant difference in the overall mean “Henna” is English name which originated level of lead (p=0.155), cadmium (p=0.577) and from Arabic name hinna. For the indigenous nickel (p=0.355) in the henna leaves was noted. communities along the coastal strip of East

International Journal of Science and Healthcare Research (www.ijshr.com) 181 Vol.5; Issue: 4; October-December 2020 Tsanuo Kassim Mohammed et.al. Levels of some heavy metals in the leaves of henna plant (Lawsonia inermis) within Lamu County, Kenya

Africa, it is known as hina which means life henna fruit is a dry capsule which contains of beauty and happiness [6]. many seeds [5]. Lawsone (2-hydroxy-1, 4 naphthoquinone) is the tannin product which is responsible for the henna dye. From the henna leaves, lawsone is extracted at a concentration range of 1.0% to 1.5% [7]. The structure of lawsone is shown in Figure 1.1 Lawsone is a compound which imparts the characteristic red-orange pigmentation which makes the skin, nails and hair beautiful and attractive [8].The whole henna plant consists of xanthones, Figure 1.1: Structure of lawsone coumarins (fraxetin and scopletin), Henna plant grows to a height of flavanoides (luteolins, apigenin and their between 12 to 15 feet. The plant requires a glycosides) and steroids (β-sitosterol) [8]. temperature range of 10°C to 50°C. Henna The structures of these organic compounds plant grows well in dry soil than in damp are shown in Figure 1.2. soil. Henna leaves occur as decussate pairs which are oppositely arranged. A mature

Xanthone Fraxetin

Figure 1.2: Structures of organic compounds in henna plant

In addition to the organic leaves sampled in Pakistan [11,12]. Hence, compounds, henna plants absorb heavy plants growing in different areas accumulate metal ions from the soil and later different levels of heavy metals depending accumulate them in organs such as leaves on factors such as soil pollution level [13]. [9]. Plants growing in soils which are highly Cosmetic products are a possible polluted with heavy metals tend to source of heavy metals which may be found accumulate higher levels of the metals in the raw materials such as leaves or may compared to those growing in less polluted be added inadvertently during the soils [10].In Morocco, Ghanjaouri (2014) manufacturing process [14]. Henna products recorded the highest level of lead in henna have recorded different levels of heavy leaves at a concentration of 3.0 ppm while metals depending on their composition [15] Alwakeel (2008) recorded lead level at a Based on its chemical structure, range of 0.011 ppm to 1.528 ppm in henna henna dye molecule belongs to a class of

International Journal of Science and Healthcare Research (www.ijshr.com) 182 Vol.5; Issue: 4; October-December 2020 Tsanuo Kassim Mohammed et.al. Levels of some heavy metals in the leaves of henna plant (Lawsonia inermis) within Lamu County, Kenya called α-hydroxy-naphthoquinones. leaves also differ. Various henna products There are very rare reactions to lawsone are extensively used along the coastal strip compound in henna leaves and these are of Kenya for beautification and are believed mainly based on naphthoquinone sensitivity to be among the sources of heavy metals [8].The formulations of some premixed contamination which are toxic to both henna consist of heavy metals and synthetic humans and the environment [21].The dyes such as para-Phenylenediamine (PPD). possible sources of these heavy metals Such formulants cause toxic effects varying includehenna leaves and poor from mild effects such as irritation to more manufacturing processes [9]. Since the level severe effects such as allergy which is of heavy metals in henna leaves from Lamu manifested through blisters, lesions and is not known, it is difficult to control and sores on the skin [7]. Plate 2.0 shows the regulate the level of heavy metals in the adverse effects of some formulants used in henna products used by the residents of henna products. Lamu. Therefore, there is need to determine the levels of heavy metals: Lead, Cadmium and Nickel inhenna leaves in order to acertain the source of heavy metalscontamination of henna cosmetic products.

MATERIALS AND METHODS Research design The research adopted an experimental design which involved random sampling of henna leaves. Digestion of henna leaves followed a given protocol. Analysis of henna leaf samples for the level of heavy metals was done using a validated

Plate 1: Adverse effects of premixed henna. Henna page, AAS instrument. Measurements in the AAS Catherine (2006) were replicated to ensure high precision. Variations in the levels of heavy metals The contamination caused by heavy were inferred using statistical measures. metals in cosmetic products is an important Certified values of reference material and environmental and health issue since the [16] the set limits of heavy metals by standard metals are toxic . Some of the toxic bodies formed the controls upon which the effects of lead include haematological, experimental values were compared to. cardio-vascular and neurobehavioural complications such as encephalopathy, [17,18] Study area depression and malaise . Cadmium is Lamu is located to the north of known to trigger oestomalacia and Mombasa on latitude 2ºS and longitude respiratory tract effects such as pulmonary 40ºE and occupies a total area of 6,474.4 fibrosis, pneumonitis, emphysema and lung 2 [19] km that includes the mainland and over 65 cancer . Nickel is a strong allergen and it islands that form the archipelago. Most of is known to be the main cause of skin [20] the henna plants growing in Lamu are in sensitivity wild state and are scattered all over the Studies on heavy metal composition County. in henna leaves have been carried out in different countries. Due to the different Sampling sites level of soil pollution by heavy metals, the Henna leaves were sampled from composition of heavy metals in henna Mpeketoni, Amu, Pate and Kiunga sites.

International Journal of Science and Healthcare Research (www.ijshr.com) 183 Vol.5; Issue: 4; October-December 2020 Tsanuo Kassim Mohammed et.al. Levels of some heavy metals in the leaves of henna plant (Lawsonia inermis) within Lamu County, Kenya

The sites were selected based on their the North and Republic of Somali to the different anthropogenic and non- North East. Agriculture is practised in small anthropogenic sources of pollution that scale due to the prevailing semi-arid could possibly lead to heavy metal conditions. The major economic activities pollution. are fishing and honey harvesting [22]. Mpeketoni is in the western part of Lamu County. In this site there is heavy Sample size traffic and intensive farming of cash crops A total of sixty (60) henna leaves such as cotton, maize and cashewnuts. As a were randomly selected and analysed for result, agrochemicals such as fertilisers, lead, cadmium and nickel. The sample size pesticides and fungicides are highly used. was determined by an arbitrary sampling Amu was nominated by UNESCO as method shown in Equation 3.1 [23]. one of the world heritage centers. The town n = √N/2………………………………...3.1 lacks proper waste management system. Where: Due to poor sewerage and drainage system, n = Sample size waste water management is inefficient. N = Total population Some henna plants grow in the pavements along the open drainage system. Sampling and collection of henna leaves Pate is located to the eastern part of Based on their availability, a total of Lamu County. The site receives insufficient five (5) henna plants growing in different and unreliable rainfall annually hence farms in each of the four (4) sites were farming is only practised in small scale. The randomly selected. Three (3) henna leaves site is endowed with natural resources such were sampled from each henna plant. The as coal, oil and gas [22]. sampling scheme for the henna leaves is Kiunga boarders Garissa County to shown in Figure 3.1

Figure 3.1: Sampling scheme for henna leaves

The sampling sites were at least 5 Henna leaves were picked from the kilometer radius apart while the radius henna plants growing either in their wild or between the selected henna plants in each cultivated state in each of the sampling sites. site was at least 0.5 kilometer. This was The selected leaves from henna plants were done to minimize reproducibility of results of average size and age. Henna [23].The henna leaves were coded. For manufacturers do not prefer old henna example, leaf code LM1 represents henna leaves and young henna leaves due to their leaves (L) sampled from the first farm or low lawsone and high moisture content, henna plant (1) within Mpeketoni site (M) respectively [24]. The picked leaves were put (Appendix iii). in labeled perforated polythene bags. A

International Journal of Science and Healthcare Research (www.ijshr.com) 184 Vol.5; Issue: 4; October-December 2020 Tsanuo Kassim Mohammed et.al. Levels of some heavy metals in the leaves of henna plant (Lawsonia inermis) within Lamu County, Kenya taxonomist from KEMRI assisted in the method was adopted to treat henna leaf identification of the henna plants and sample because of its better recovery of ensured that the selected leaves were of lead, cadmium and nickel from henna leaves average age. [26]. A mass of 1.000 g of powdered henna leaf set was weighed using a BPS-4500-11 Reagents and solvents electronic balance from Adams equipment. All the reagents and chemical To avoid cross contamination a clean plastic substances used were of analytical grade spatula was used each time a different set of (AR) from Alpha chemicals. The reagents powdered henna sample was transferred and chemical substances used were nitric from the Teflon containers. The weighed acid, chloric acid, cadmium oxide, lead sample was put into a 50 ml beaker nitrate and nickel metal. Double distilled containing 6 ml of concentrated HNO3 and deionised water was used in dissolving the the mixture was gradually heated to 80°C in solids, dilution of solutions and rinsing of a fume chamber until the mixture remained apparatus. almost clear and was later cooled. Into the cooled mixture, 2 ml of 69% HClO4 was Cleaning of apparatus added and the mixture was heated over The glassware, Teflon and plastic water bath (direct heating of HClO4 mixture apparatus were soaked overnight in a can be explosive) and later was cooled. The soapless detergent in a plastic basin. The process was repeated until white fumes of apparatus were then rinsed with double HClO4 were produced. The sample was distilled deionised water and later soaked in boiled until the final volume of the mixture 10% (v/v) concentrated HNO3 for 3 days at was less than 5 ml. Thereafter, 10 ml of room temperature. Thereafter, the apparatus double distilled deionised water was added were rinsed thoroughly with double distilled into the digestate and the mixture was later deionised water and then allowed to dry in a filtered using Whatmans No. 1 filter paper shaded open air space which was covered to into a 50 ml volumetric flask which was prevent dust particles. The dried apparatus then topped up to the mark. A blank were later wrapped and stored in sealed digestate was prepared in the same way but polythene bags. without the sample.

Pre-treatment of henna leaf sample Preparation of stock and standard Three (3) leaves were picked from solutions each henna plant to form a set. The leaves Stock solutions of 1000 ppm of lead, were separately washed with double cadmium and nickel were prepared from distilled deionised water to remove soil their analytical grade metal salt, metal oxide particles. The clean leaves were chopped and pure metal, respectively [27]. into small pieces and then dried in an oven at a temperature range of 60°C to 65°C for Lead stock solution and its standards 72 hours and later cooled [25].The dried Lead stock solution (1000 ppm) was pieces of leaves were ground using a Wiley prepared by dissolving 1.598 g of Pb(NO3)2 mill to form a homogenous set of powdered into 1litre of double distilled deionised henna leaves. To avoid contamination, a water. Lead standard solutions of clean Wiley mill was used to grind a concentration range of 1.0, 2.0, 4.0, 8.0 and particular set of henna leaves [9]. Each set 10.0 ppm were prepared by serial dilution of was later packed in an air tight labeled the stock solution using the formula shown Teflon container awaiting digestion. in Equation 3.2

Henna leaf sample treatment V1C1=V2C2...... Eq 3.2 Nitric acid-chloric acid wet digestion Where:

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V1– Initial volume solutions of concentration range of 0.2, 0.6, C1 – Initial concentration 1.0, 1.5 and 5.0 ppm were prepared by serial V2– Final volume dilution of its stock solution using the same C2 – Final concentration procedure as for the standard solutions of For instance, to prepare 50 ml of 4 lead. ppm from the stock solution of lead, a working solution of 100 ml of 100 ppm lead Preparation of standard reference solution was prepared using the formula material (SRM) given in Equation 3.2 A certified standard reference V1 C1 = V2 C2 material No. 1572 citrus leaves (CL) from [28] V1 x 1000 ppm = 100 ml x100 ppm; NIST (1992) was used . Its solution was prepared the same way as the henna sample. Hence V1 = 100 ml x 100 ppm = 10 ml Methods validation 1000 ppm Calibration curve and regression analysis The 10 ml of the lead stock solution The prepared standards of each (1000 ppm) obtained was diluted to 100 ml metal and their corresponding absorbencies so as to prepare a working solution of 100 (Appendix i) were used to plot calibration ppm lead solution. Using the working curves. The product-moment correlation solution, the 50 ml of 4 ppm standard lead coefficient factor and the equation for the solution was prepared in the same way line of regression were derived from using Equation 3.2 calibration curve of each metal. Since, V1 C1 = V2 C2 V1 x 100 ppm = 50 ml x 4 ppm; Standard addition The AAS was validated by spiking Hence V1 = 50 ml x 4 ppm = 2 ml the samples with known standards and 100 ppm obtaining the percentage recovery as shown The 2 ml of 100 ppm working in Equation 3.3 solution of lead was diluted to 50 ml to prepare the 50 ml of 4 ppm standard solution of lead. Similar procedure was used to prepare the rest of the standard solutions Where: of lead. SAS ‒ Sample concentration after spiking SBS ‒ Sample concentration before spiking Cadmium stock solution and its SS ‒ Standard used for spiking standards Equal volumes of different Cadmium stock solution (1000 ppm) concentrations of the sample solution were prepared and separately spiked with was prepared by dissolving 1.142 g of [29] cadmium oxide in 20 ml of concentrated different amounts of the standard .The nitric acid and then topped up to 1litre. absorbencies of all the sample solutions Cadmium standard solutions of were measured in triplicate before and after concentration range of 0.1, 0.4, 0.6, 0.8, and spiking in order to obtain the percentage 1 ppm were prepared by serial dilution of its recovery. stock solution using the same procedure as the lead standards. Standard reference material The accuracy of FAAS was tested using a certified standard reference material Nickel stock solution and its standards [28] Nickel stock solution (1000 ppm) No. 1572 citrus leaves (CL) .The was prepared by dissolving 1.000 g of absorbencies of the reference material were nickel in 20 ml hot nitric acid and then run in triplicate. The t-test critical values topped up to 1litre. Nickel standard were compared with the calculated values so

International Journal of Science and Healthcare Research (www.ijshr.com) 186 Vol.5; Issue: 4; October-December 2020 Tsanuo Kassim Mohammed et.al. Levels of some heavy metals in the leaves of henna plant (Lawsonia inermis) within Lamu County, Kenya as to test the validity of the FAAS The actual concentration of heavy instrument. metals in henna leaf samples obtained from AAS was derived using Equation 3.5 Analysis of the sample by FAAS For dilutions, the actual weight was Before the analysis, the FAAS was obtained by multiplying the readout results set at its optimum conditions. The blanks from AAS with the dilution factor. were aspirated into the flame and their Data analysis absorbencies recorded. The limit of From the FAAS, concentrations of detection (LOD) was calculated by the heavy metals were recorded as triplicate obtaining a mean of ten blanks plus three measurement of their mean values with their times the blank’s standard deviation for corresponding standard deviations (SD). each metal as shown in Equation 3.4 The variation of levels of lead, cadmium and nickel in the various henna leaf samples was determined by a one-way ANOVA at Where: 95% confidence level. In SPSS (Statistical y ‒ Limit of detection package for social sciences) version 17.0, yB ‒ Blank signal SNK was used to compute the variation of sB ‒ Standard deviation of the blank. the levels of heavy metals in leaf henna The standard solutions and sample samples. A significant statistical test was set solutions were separately nebulised into the at p = 0.05 (α = 0.05). flame and their absorbencies were recorded in triplicate. The absorbencies obtained with RESULTS AND DISCUSSION the samples were used to calculate the Introduction unknown concentrations of the metals by The analytical results on the levels interpolation of their absorbencies on the of heavy metals in henna leaves were calibration curve for each metal. The blank, subjected to statistical tests of significance standard and sample solutions were run in and summarised in tables, linear and bar the AAS in an alternating manner so as to graphs. monitor the performance of the instrument. AAS validation results FAAS instrumentation Calibration curves and regression The model of FAAS used in the analysis analysis was Buck Scientific Model Figure 4.1 shows the standard 210VGP. The optimised operational calibration curve for lead. conditions of the machine are given in Table 3.2.

Working parameters for FAAS Operational conditions Element Lead Cadmium Nickel Lamp current (mA) 5 to 10 7 to 15 5 to 10 Slit width (nm) 1.0 0.7 0.2 Wavelength (nm) 283.3 324.8 232.0 Flow rate (litres/mm) 1.5 1.5 1.5 Flame temperature (°C) 2300 2300 2300 Detection limit (μg/g) 0.01 0.001 0.005

Calculation of concentration of heavy metals in henna samples

Figure 4.1: Calibration curve for lead

Figure 4.2 shows the standard calibration curve for cadmium.

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Followed by the Figures 4.1, 4.2 and 4.3, a positive correlation of absorbance versus concentration was observed. The correlation coefficient (R2) values were more than 0.977 indicating linearity on the calibration curves hence good instrumental stability which is necessary for analyte determination [30]. The intercept of the curves was close to zero indicating minimum matrix interference and their slopes were above zero meaning that the

Figure 4.2: Calibration curve for cadmium. instrument was sensitive. Followed by the Figures 4.1, 4.2 and 4.3, the curves were Figure 4.3 shows the standard used to quantify the heavy metals in the [29] calibration curve for nickel. henna leaves . Results from the blanks recorded detection limits for lead, cadmium and nickel at 0.02 ppm, 0.005 ppm and 0.009 ppm, respectively. These limits were below 0.1 ppm hence appropriate for the analysis of heavy metals [31].

Standard addition Table 4.1 shows the percentage recovery results for lead, cadmium and nickel after spiking a sample concentration with a standard solution.

Figure 4.3: Calibration curve for nickel

Results for standard addition method Element Sample concentration Standard used for Sample concentration after Percentage before spiking. Mean±SD spiking (μg/ml) spiking Mean±SD recovery (%) (μg/ml) (n=3) (μg/ml) (n=3) Lead 2.12±0.02 50 51.90±0.02 99 Cadmium 0.3±0.01 30 29.89±0.01 98 Nickel 1.0±0.03 20 20.80±0.03 99

For lead, there was 99% recovery their measured values using FAAS are given after spiking 2.12±0.02 μg/ml with 50 μg/ml in Table 4.2. of lead. For cadmium, there was 98% From Table 4.2 the certified level for recovery after spiking 0.3±0.01 μg/ml with lead was 1.62±0.12 µg/g while the measured 30 μg/ml of cadmium. For nickel, there was value was 1.59±0.27 µg/g. The calculated |t| 99% recovery after spiking 1.0±0.03 μg/ml value for 2 degrees of freedom was 0.005, with 20 μg/ml of nickel. Since the which was less than the critical value of |t| percentage recovery for all the metals were which was 4.303 at p=0.05. For cadmium within the acceptable range of 96% to and nickel, there was also no significant 105%, this indicated that the analytical difference between the certified values and method was accurate and reproducible [32]. the values obtained using this method since the calculated |t| values were lower than the Standard reference material critical values at 95% confidence level. The lead, cadmium and nickel Since the t-test critical values were more certified values of the reference material and than the calculated values it showed that the

International Journal of Science and Healthcare Research (www.ijshr.com) 188 Vol.5; Issue: 4; October-December 2020 Tsanuo Kassim Mohammed et.al. Levels of some heavy metals in the leaves of henna plant (Lawsonia inermis) within Lamu County, Kenya measured values were not significantly good accuracy of the instrument at 95% different from the certified values hence confidence level [29].

Results for standard reference citrus leaf (CL) material Reference material Element (n=3) Lead Mean±SD (μg/g) Cadmium Mean±SD (μg/g) Nickel Mean±SD (μg/g) Certified value (μg/g) 1.62±0.12 0.23±0.12 3.04±0.64 Measured value (μg/g) 1.59±0.27 0.21±0.35 2.98±0.42 Calculated t-test (|t|) 0.005 0.004 0.015 Critical value (υ=2) 4.303 4.303 4.303

Heavy metals in henna leaves from Lamu Table 4.3 shows the mean level of Level of lead, cadmium and nickel in lead, cadmium and nickel in leaves of henna henna leaves plants sampled from Mpeketoni, Amu, Pate and Kiunga sites within Lamu County.

Table 4:3: Mean level of lead, cadmium and nickel in henna leaves from Lamu Sampling Henna leaf Concentration Mean±SD (μg/g) (n=3) site code Pb Cd Ni a a LM1 0.08±0.01 0.01±0.01 0.02±0.01 a a LM2 0.05±0.01 BDL 0.06±0.01 b ab LM3 0.93±0.04 0.01±0.00 0.34±0.02

Mpeketoni LM4 BDL 0.09±0.01 BDL b a LM5 0.53±0.09 0.04±0.02 0.03±0.01 p-value p=0.051 p=0.650 p=0.021 Overall mean 0.40±0.11 0.04±0.01 0.11±0.04 a LA1 0.08±0.02 0.02±0.02 0.04±0.01 b LA2 0.17±0.02 0.05±0.01 0.01±0.00 LA3 BDL BDL BDL

Amu LA4 BDL 0.06±0.02 0.03±0.01 b LA5 0.35±0.08 0.03±0.01 0.02±0.01 p-value p=0.057 p=0.578 p=0.578 Overall mean 0.20±0.04 0.04±0.01 0.02±0.00 a LP1 0.06±0.01 0.04±0.01 0.01±0.01 LP2 BDL BDL BDL

LP3 BDL BDL 0.04±0.01 b Pate LP4 0.53±0.08 0.06±0.02 BDL a LP5 0.07±0.01 BDL 0.02±0.01 p-value p=0.153 p=0.578 p=0.512 Overall mean 0.22±0.04 0.05±0.01 0.02±0.00

LK1 BDL BDL 0.03±0.01

LK2 BDL 0.03±0.02 0.06±0.01 LK3 0.04±0.01 BDL BDL

Kiunga LK4 BDL BDL 0.02±0.01 LK5 0.05±0.02 0.04±0.02 BDL p-value p=0.736 p=0.747 p=0.612 Overall mean 0.05±0.00 0.03±0.01 0.03±0.01

Mean values followed by the same of 0.35±0.08 ppm while leaf LA1 recorded small letter(s) within the same column in the least level at a concentration of each sampling site do not differ significantly 0.08±0.02 ppm within Amu site. Within from one another (one-way ANOVA, SNK- Pate, leaf LP4 recorded the highest level of test, α = 0.05). Codes LM1, LA1, LP1 and LK1 lead at a concentration of 0.53±0.08 ppm represent henna leaves sampled from the which is similar to the level of lead recorded first henna plant in Mpeketoni, Amu, Pate in leaf LM5 from Mpeketoni. Within Kiunga and Kiunga sites, respectively. BDL‒Below only two leaves, LK3 and LK5 recorded lead detection limit. at a concentration of 0.04±0.01 ppm and From Table 4.3, the level of lead 0.05±0.02 ppm, respectively. There was no within Mpeketoni site ranged from significant difference in the level of lead in 0.05±0.01 ppm in leaf LM2 to 0.93±0.04 henna leaves sampled from all the sites: ppm in leaf LM3. The highest level of lead Mpeketoni (p=0.051), Amu (p=0.057), Pate was recorded in leaf LA5 at a concentration (p=0.153) and Kiunga (p=0.736).

International Journal of Science and Healthcare Research (www.ijshr.com) 189 Vol.5; Issue: 4; October-December 2020 Tsanuo Kassim Mohammed et.al. Levels of some heavy metals in the leaves of henna plant (Lawsonia inermis) within Lamu County, Kenya

Lead has not been shown to perform leaves sampled in Amu site (p=0.578), Pate any role in plant s metabolism although it site (p=0.512) and Kiunga site (p=0.612). occurs naturally in plants. Compared to However, a significant difference was noted other heavy metals, lead forms the highest in the level of nickel in henna leaves from percentage in the soil [13].The level of lead Mpeketoni site (p=0.021). in the soil as well as the form in which it Ahmad et al. (1994) recorded the occurs will determine its uptake by the highest nickel level in henna leaves from plants [33].For a plant that grows in less Faisalabad district, Pakistan at a polluted area, the level of lead is in the concentration of 0.18 ppm [24] which is range of 0.1 ppm to 10 ppm [10].Since the below the level of 0.34±0.02 ppm recorded level of lead obtained from this study falls from this study. In another study, Alwakeel within this range, the sites may be regarded (2008) analyzed nickel level in henna leaves as less polluted. sampled from henna plants growing in From Table 4.3, the level of Saudi Arabia and reported a range of 0.035 cadmium within Mpeketoni ranged from ppm to 0.073 ppm nickel [12] which falls 0.01±0.00 ppm in leaf LM1 and LM3 to within the range of 0.01±0.00 ppm to 0.09±0.01 ppm in leaf LM4. Within Amu, 0.34±0.02 ppm obtained from this study. leaf LA1 recorded the least level of cadmium Unlike lead and cadmium, nickel at a concentration of 0.02±0.02 ppm while was detected in most of the sampled henna leaf LA4 recorded the highest level of leaves. This may be due to the fact that cadmium at a concentration of 0.06±0.02 nickel is an essential element which is ppm. In Pate and Kiunga, the highest level important in plant’s metabolism. Nickel is a of cadmium was recorded in leaf LP4 and constituent of metallo‒enzymes such as LK5 at a concentration of 0.06±0.02 ppm and urease which metabolizes urea into useable 0.04±0.02 ppm, respectively. There was no ammonia within the plant. Nickel is significant difference in the level of phytotoxic at higher concentration [34]. cadmium in henna leaves sampled from all The different levels of heavy metals the sites: Mpeketoni (p=0.650), Amu in the henna leaves may be attributed to (p=0.578), Pate (p=0.578) and Kiunga different pollution levels of the soils under (p=0.747). which the henna plant is growing [35]. Both Alwakeel (2008) recorded levels of the anthropogenic and non-anthropogenic cadmium in henna leaves sampled from sources of pollution contribute to the level henna plants growing in Saudi Arabia at a of heavy metals in plants [34] (Ruchi et al., concentration range of 0.016 ppm to 0.019 2014). From Table 4.3, Mpeketoni site ppm [12] which is within the range of recorded the highest overall mean level of 0.01±0.01 ppm to 0.09±0.01 ppm obtained lead and nickel which can be attributed to from this study. Although considered a non- the use of agrochemicals such as fungicides essential element, cadmium is effectively and pesticides [36,37]. In addition, the heavy absorbed in roots and leaves of plants [33]. traffic in Mpeketoni as well as the effluents From Table 4.3, the level of nickel from car garages could contribute to the within Mpeketoni site ranged from high level of lead [38]. Non-point source 0.02±0.01 ppm in leaf LM1 to a significant contamination by the waste waters from the level of 0.34±0.02 ppm in leaf LM3. In both open sewerage and drainage system could Amu and Pate, leaf LA1 and LP3, lead to a higher overall mean level of respectively recorded the highest level of cadmium in Amu site [10]. In Pate, the nickel at a concentration of 0.04±0.01 ppm. highest overall mean level of cadmium Within Kiunga, the highest level of nickel might be attributed to the cadmium was noted in leaf LK2 at a concentration of composition of the underlying sedimentary 0.06±0.01 ppm. No significant difference rock, marine phosphites and phosphates was noted in the level of nickel in the henna which form the basement for coal and crude

International Journal of Science and Healthcare Research (www.ijshr.com) 190 Vol.5; Issue: 4; October-December 2020 Tsanuo Kassim Mohammed et.al. Levels of some heavy metals in the leaves of henna plant (Lawsonia inermis) within Lamu County, Kenya oil deposits [39]. Kiunga is a semi-arid area highest overall mean level at a concentration in which small scale farming is practised. of 0.24 ppm while cadmium recorded the The major economic activities include least overall mean level at a concentration honey harvesting and fishing, which do not of 0.04 ppm. No significant difference in the contribute much to pollution, hence the least overall mean level of lead (p=0.155), overall mean level of heavy metals cadmium (p=0.577) and nickel (p=0.355) in recorded. the henna leaves was noted. The order of accumulation of heavy metals in the henna Levels of accumulation of heavy metals in leaves was as follows: henna leaves Lead > Nickel > Cadmium This order is in agreement with that recorded by Ahmad et al. (1994) in Faisalabad district, Pakistan[24].The highest level of lead in the henna leaves may be attributed to the high pollution level of lead in soils compared to cadmium and nickel[13].The lowest level of cadmium could be due to the basic calcareous soils and limestone within Lamu[40]which could render cadmium unavailable to the roots and leaves of plants [33].

Comparison of level of heavy metals in Figure 4.4: Overall mean level of lead, cadmium and nickel in henna leaves from Lamu henna leaves with WHO limits Table 4.4 shows the overall mean The overall mean levels of heavy level of lead; cadmium and nickel in henna metals in Table 4.3 were subjected to tests leaves sampled from Lamu and the WHO of significance to show the pattern of recommended limits of the metals in herbal variation of heavy metals in the henna plants. leaves as shown in Figure 4.4 From Figure 4.4, lead recorded the

Table 4.4: Overall mean level of heavy metals in henna leaves from Lamu and WHO limits Element Concentration (Mean±SD) Recommended level ppm in the henna leaf samples (ppm) by WHO (2010) Lead 0.24±0.28 (n=36) 2.0 Cadmium 0.04±0.03 (n=36) 0.2 Nickel 0.05±0.02 (n=42) 1.68

From Table 4.4, the overall mean except for a case where lead was reported to levels of lead, cadmium and nickel in the a level as high as 3 ppm [11].The study has sampled henna leaves were lower at a revealed that the level of accumulation of concentration of 0.24±0.28 ppm, 0.04±0.03 heavy metals in henna leaves vary ppm and 0.05±0.02 ppm, respectively depending on the nature of the metal and the compared to the recommended level of 2.0 henna leaf sampled. Henna plants can be ppm, 0.2 ppm and 1.68 ppm, respectively by grown in controlled fields where the level of World Health Organization [30].Due to the heavy metals in the soils is kept at a lower level of heavy metals, the henna minimum level. In order to protect the leaves from Lamu form a good raw material henna users from the ill effects of heavy for henna products. metals, the henna manufacturers should Generally, the levels of heavy metals ensure that only the henna leaves with in henna leaves from Lamu are similar to heavy metal level below the standard limit those reported in other areas (section 4.3) are sampled as raw material for henna

International Journal of Science and Healthcare Research (www.ijshr.com) 191 Vol.5; Issue: 4; October-December 2020 Tsanuo Kassim Mohammed et.al. Levels of some heavy metals in the leaves of henna plant (Lawsonia inermis) within Lamu County, Kenya products. ppm). As raw materials, henna leaves recorded lower significant levels of Pb, Cd DISCUSSION and Ni than the henna products. This From the results of this study, it showed that henna leaves were not the only showed that henna leaves from Lamu source of heavy metals in the henna County formed a good raw material for products but more of these metals were henna products due to their low level of added during the manufacturing process lead, cadmium and nickel in comparison to The overall mean level of Ni in the standard limits by WHO. This study powdered henna products was 0.75 ppm concurs with a study by Mamoona and while paste henna products recorded a Maryam (2015) which reported lower level significant overall mean level of Ni at 1.28 of lead in henna leaves sampled from ppm. Hence the results showed that paste Pakistan at a concentration of 0.019 ppm, henna products contain higher level of Ni [41] but in contrast with a study in Morocco compared to powder henna products. by Ghanjouri (2014) which recorded the The levels of heavy metals in all highest level of lead in henna leaves at a henna products were determined and concentration of 3.00 ppm [11] which is compared with standard limit. In above the level of 0.93±0.04 ppm obtained comparison to Kenya Bureau of Standards from this study. Allergy to natural henna is (Kebs) set limit of 2 ppm, generally all not usual; however, use of poor quality raw sampled henna products recorded lower materials and the addition of para- levels Cd and Pb phenylenediamine (PPD) to the natural Recommendations henna during processing increase the risk of The henna leaves sampled in Lamu allergic contact dermatitis [42]. Effective County form a suitable raw material for the quality control measures, proper selection of manufacturer of henna products because raw material as well as good manufacturing they contain lower levels of heavy metals. practises should be enhanced so as to Since the levels of heavy metals in minimise the levels of these metals in henna henna leaves were lower compared to those products. in henna products, it is mostly likely that the heavy metals contamination witnessed CONCLUSIONS AND among henna products user could be due to RECOMMENDATIONS poor processing procedures, therefore Conclusions manufacturers should improve on their In henna leaves, the overall mean methodology to safeguard the end users. level of heavy metals were determined and The study should be extended to recorded as follows: Pb (0.24±0.08 ppm), determine the level of heavy metals in Cd (0.04±0.03 ppm) and Ni (0.05±0.02 henna cosmetics para-phenylenediamine, ppm). The set limit of heavy metals in common oxidative dye in premixed henna herbal plants by WHO was Pb (2.0 ppm), and a major cause of dermatitis. Cd (0.2 ppm) and Ni (1.68 ppm). Therefore, henna leaves recorded lower levels of Pb, ACKNOWLEDGEMENTS Cd and Ni compared to the limit by WHO. The authors sincerely thank the In henna leaves, the overall mean Almighty GOD for enabling them under level of heavy metals were determined and take this research successfully. Our special recorded as follows: Pb (0.24±0.08 ppm), thanks also go to Mr. Lucas Wasike of Cd (0.04±0.03 ppm) and Ni (0.05±0.02 KEMRI, Centre for Geographical Medicine ppm). In henna products, the overall mean Research, Kilifi for his assistance in the levels of heavy metals were determined and identification of henna plants as well as in recorded as follows: Pb (0.98±0.09 ppm), the selection of henna leaf samples for Cd (0.48±0.09 ppm) and Ni (1.02±0.18 analysis.

International Journal of Science and Healthcare Research (www.ijshr.com) 192 Vol.5; Issue: 4; October-December 2020 Tsanuo Kassim Mohammed et.al. Levels of some heavy metals in the leaves of henna plant (Lawsonia inermis) within Lamu County, Kenya

REFERENCES Cosmetics By Inductively Coupled 1. Gagandeep C, Sandeep G, Priyanka P. Plasma Atomic Emission Spectroscopy. Lawsonia inermis Linnaeus: a International journal of scientific & phytopharmacological review. Int J technology research. 2014; 3(10):104-2. Pharm Sci Drug Res. 2010; 2(2):91-8. 12. Alwakeel SS. Microbial and heavy 2. Gevrenova R. Determination of natural metals contamination of herbal colorants in plant extracts by high medicines. Research Journal of performance liquid chromatography. microbiology. 2008 Dec 1; 3(12):683- Journal of the Serbian Chemical Society. 91. 2010; 75 (7):903-15. 13. Khan SA, Khan L, Hussain I, Marwat 3. El Massoudi S, Benidir M, Chabir R, KB, Akhtar N. Profile of heavy metals Benjelloun M, El Ghadraoui L, in selected medicinal plants. Pakistan Errachidi F. Morphological, Journal of Weed Science Research. Biochemical, and Climatological 2008; 14(1-2):101-10. Analysis of Three Moroccan Henna 14. Alsaffar NM, Hussein HJ. Verities. The Scientific World Journal. Determination of heavy metals in some 2019 May 27; 2019. cosmetics available in locally markets. 4. Raboobee N. Sensitisation from PPD in IOSR J Environ Sci Toxicol Food temporary henna tattoos and subsequent Technol. 2014; 8(1):9-12. severe allergic contact dermatitis from 15. Nnorom IC, Igwe JC, Oji-Nnorom CG. hair dye. Curr Allergy Clin Immunol. Trace metal contents of facial (make-up) 2004; 17:194-5. cosmetics commonly used in Nigeria. 5. Rahman AU. Studies in Natural African Journal of Biotechnology. 2005; Products Chemistry: Bioactive Natural 4 (10). Products (Part XII). Elsevier; 2016 Sep 16. Swift JA. Fundamentals of human hair 6. science. Weymouth, U.K: Micelle 6. Packer, M. Henna source book: Origin Press; 1997. of henna. Winson publishing company. 17. Eman, M., Alissa, F. and Gordon, A. http://www crystalinks.com/henna.html. (2011). Heavy metal poisoning and Accessed 8.04.2014. cardiovascular diseases. Journal of 7. Deleo, Y. A. (2006). P- Toxicology.2011; 27: 76-98. Phenylenediamine. Dermatol publishers, 18. CDC. Centre for Disease Control and 4th street Manipur, India. Prevention. Preventing lead poisoning in 8. Kang IJ, Lee MH. Quantification of young children. Department of health para‐ phenylenediamine and heavy and human services, Atlanta, USA.2009. metals in henna dye. Contact dermatitis. 19. Kollmeier H, Seemann J, Wittig P, 2006 Jul; 55(1):26-9. Rothe G, Müller KM. Cadmium in 9. Mazoor, I. K. and Mahmoud, I. K. human lung tissue. International (2011). Study of heavy trace metals in archives of occupational and some medicinal-herbal plants of environmental health. 1990 Sep 1; 62 Pakistan. Pakistan Journal of (5):373-7. Botany.2011; 43: 2003-2009. 20. CDTSC (2004). California Department 10. Chibuike GU, Obiora SC. Heavy metal of Toxic Substances Control. Review of polluted soils: effect on plants and nickel and nickel compounds. bioremediation methods. Applied and 21. Al-Dayel O, Hefne J, Al-Ajyan T. environmental soil science. 2014 Oct; Human exposure to heavy metals from 2014. cosmetics. Oriental Journal of 11. Ghanjaoui ME, Cervera ML, El Rhazi Chemistry. 2011 Jan; 27(1):1-1. M, de la Guardia M. Assessment Of 22. LCIDP. Lamu County Integrated Trace Elements In Traditional Moroccan Development Plan. Economic Strategies

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within Lamu County, Kenya. 2013. and pharmacological aspects. Journal of 23. UNODC. United Nations Office on Ethnopharmacology, 2014 Aug 8; Drugs and Crime. Guidelines on 155(1):80-103. representative drug sampling. United 35. Al-Saleh I, Al-Enazi S, Shinwari N. nations publications. ISBN 978-92-1- Assessment of lead in cosmetic 148 241-6. New York.2009. products. Regulatory Toxicology and 24. Ahmed, I., Siddiqui, M. and Khan, Z. Pharmacology. 2009 Jul 1; 54(2):105- The major, minor and trace elements in 13. henna leaves. Journal of Chemistry 36. Kaara, J. N. Determination of heavy Society in Pakistan.1994; 16: 29-35. metals in sewage sludge, sewage 25. Ogunwande IA, Olawore NO. Volatile effluent, garden soils and food crops fractions from the leaf and flowers of grown in ordinary sewage-sludge “African Marigold”, Tagetes erecta Linn amended soils, Masters dissertation, from Nig. J. Essent. Oil Res. 2006; Kenyatta University, Nairobi, Kenya 18:366-8. (published).1992 26. Umar MA, Caleb H. Analysis of metals 37. Mitei, Y. C. Determination of heavy in some cosmetic products in FCT- metals in Kenyan cigarettes, tobacco Abuja, Nigeria. International Journal of leaves and intercropped plants by atomic Research in Cosmetic Science. 2013; absorption spectroscopy, M.Sc Thesis, 3(2):14-8. Kenyatta University, Nairobi, Kenya 27. Skoog, D. A. and Leary, J. J. Principles (published). 1996. of analytical chemistry. (4th edition). 38. Mutua, S. M. Lead, cadmium and zinc Harcourt Brace College publishers, Fort speciation in garage soils, their levels in Worth. Philadelphia.1992. Kales and water along Katothyani 28. NIST. National Institute of Standard and stream, Machakos town, Kenya, M.Sc Technology, Standard Materials Thesis, Kenyatta University, Nairobi, Programme, Gaithersburg M.D, Kenya (published). 2013. U.S.A.1992. 39. Muchena, F. N. and Ekirapa, A. E. Soils 29. Miller, J. C. and Miller, J. N. Statistics of the Magarini land settlement scheme for analytical chemistry. (2nd edition) (Kilifi District). Kenya soil survey. Site Ellis horwood limited. Chichester, west evaluation report No. P63. National sussex, po19 IEB, England.1998. Agricultural Research Laboratories.1992 30. Mendham, R. C., Renny, J. O. and 40. WHO (2010). World Health Barnes, K. Quantitative chemical Organisation. Exposure to lead: A major analysis. (6th edition). Prentice Hall, public health concern, Geneva, U.K. 1999. Switzerland. 31. Sawyer, W. and Beebe, A. Chemistry 41. Mamoona, N. and Maryam, N. experiment for instrumental methods. Determination of metal content in Wiley, New York.2007. Lawsonia inermis Linn by flame atomic 32. Kings, F. and Burgess, A. (1998). spectroscopy. International Journal of Nutrition for developing countries: A Healthcare Sciences. 2015; 3: 54-58. critical role on health. (2nd edition). New 42. Al-Suwaidi A, Ahmed H. Determination York: Oxford University Press Inc. of para-phenylenediamine (PPD) in 1998. henna in the United Arab Emirates. 33. Kabata, A. and Pendias, H. (1985). International Journal of Environmental Trace elements in soils and plants. CRC Research and Public Health. 2010 Apr; press, Inc. Florida 7(4):1681-93. 34. Ruchi, B. S., Deepak, K.S. and Catherine, C. J. Lawsonia inermis L. (Henna). Ethnobotanical, phytochemical

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APPENDICES Appendix II: Standards against absorbency for lead,cadmium

and nickel Appendix I: Sampled farms and henna leaf codes Metal Standards Absorbances Mean Sampling Sampled Henna plant Henna leaf absorbance site farm number code 1.0 0.002 0.002 0.003 0.0023 Mkunumbi Henna plant 1 L M1 2.0 0.004 0.004 0.004 0.0040 Mapenya Henna plant 2 L M2 Lead 4.0 0.006 0.006 0.005 0.0056 Mpeketoni Hongwe Henna plant 3 L M3 8.0 0.016 0.015 0.015 0.0153 Baharini Henna plant 4 L M4 10.0 0.020 0.020 0.020 0.0200 Kibaoni Henna plant 5 L M5 0.1 0.002 0.002 0.002 0.0020 Pamba roho Henna plant 1 L A1 0.4 0.009 0.010 0.010 0.0096 Uyoni Henna plant 2 L A2 0.6 0.011 0.012 0.012 0.0116 Amu Idabo Henna plant 3 L A3 Cadmium 0.8 0.012 0.012 0.012 0.0120 Matondoni Henna plant 4 L A4 1.0 0.017 0.017 0.017 0.0170 Langoni Henna plant 5 L A5 0.2 0.002 0.002 0.002 0.0020 Siyu Henna plant 1 L P1 0.6 0.007 0.006 0.007 0.0066 Nyabwogi Henna plant 2 L P2 1.0 0.015 0.015 0.015 0.0150 Pate Kandaani Henna plant 3 L P3 Nickel 1.5 0.020 0.021 0.020 0.0203 Mbwajumwali Henna plant 4 L P4 5.0 0.050 0.049 0.050 0.0496 Faza Henna plant 5 LP5 Dederi Henna plant 1 LK1 Kangwe Henna plant 2 LK2 How to cite this article: Mohammed TK, Kiunga Kiduruni Henna plant 3 LK3 Ochieng JP. Levels of some heavy metals in the Kombokombo Henna plant 4 LK4 Kwaiyuu Henna plant 5 LK5 leaves of henna plant (Lawsonia inermis) within Lamu County, Kenya. International Journal of

Science & Healthcare Research. 2020; 5(4): 181-195.

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International Journal of Science and Healthcare Research (www.ijshr.com) 195 Vol.5; Issue: 4; October-December 2020