Journal of Trace Elements in Medicine and Biology 31 (2015) 98–106

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ANALYTICAL METHODOLOGY

Content of trace elements and chromium speciation in Neem powder and tea infusions ˇ canˇcar a,b , Radmila Milaˇciˇc a,b,∗ Breda Novotnik a,b , Tea Zuliani a , Janez Sˇ a b

Department of Environmental Sciences, Joˇzef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Joˇzef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia

a r t i c l e

i n f o

Article history: Received 10 January 2015 Accepted 13 April 2015 Keywords: Neem powder Trace elements Chromium speciation Enriched stable isotopic solutions of chromium High performance liquid chromatography–inductively coupled plasma mass spectrometry

a b s t r a c t Total concentrations of selected trace elements in Neem powder and in Neem tea were determined by inductively coupled plasma mass spectrometry (ICP-MS). The data revealed that despite high total concentrations of the potentially toxic elements Al and Ni in Neem powder, their amounts dissolved in Neem tea were low. Total concentrations of the other toxic elements Pb, As and Cd were also very low and do not represent a health hazard. In contrast, total concentrations of the essential elements Fe, Cu, Zn, Se Mo and Cr in Neem powder were high and also considerable in Neem tea. Consuming one cup of Neem tea (2 g per 200 mL of water) covers the recommended daily intakes for Cr and Se and represents an important source of Mo and Cu. Speciation analysis of Cr by high performance liquid chromatography (HPLC) coupled to ICP-MS with the use of enriched Cr isotopic tracers to follow species interconversions during the analytical procedure demonstrated that toxic Cr(VI) was not present either in Neem powder or in Neem tea. Its concentrations were below the limits of detection of the HPLC–ICP-MS procedure applied. The speciation analysis data confirmed that even Cr(VI) was added, it was rapidly reduced by the presence of antioxidants in Neem leaves. By the use of enriched Cr isotopic spike solutions it was also demonstrated that for obtaining reliable analytical data it is essential to apply the extraction procedures which prevent Cr species interconversions, or to correct for species transformation. © 2015 Elsevier GmbH. All rights reserved.

Introduction Neem (Azadirachta indica A. Juss) is an evergreen tree in the mahogany family Meliaceae. The leaves, fruit and bark of thee Neem tree, which all possess beneficial medicinal properties, have been used traditionally in Ayurvedic medicine in India. More than 135 chemical compounds have been isolated, but only a few have been tested for their medicinal properties [1]. Extracts from different parts of the Neem plant were found to have antifungal [2], acaricidal [3], antibacterial, antisecretorial, antihaemorrhagic [4], antiviral [5], larvicidal [6], insecticidal and antifeedant [7] activities. Due to its beneficial properties, Neem is widely used in the personal hygiene and cosmetic industry, and in insecticide and pesticide production. Moreover, it is plant useful in combating deforestation and desertification [8].

∗ Corresponding author at: Department of Environmental Sciences, Joˇzef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. Tel.: +386 1 477 3560; fax: +386 1 2519 385. E-mail address: [email protected] (R. Milaˇciˇc). http://dx.doi.org/10.1016/j.jtemb.2015.04.003 0946-672X/© 2015 Elsevier GmbH. All rights reserved.

Since some trace elements are essential for normal functioning of the human organism and several have health promoting properties, Neem extracts have been subjected to numerous trace element analyses, most commonly by atomic absorption spectroscopy [9–11] and neutron activation analysis [12–14]. Among the essential and toxic elements Zn, Cu, Fe, Co, Ni, Al and Cr were frequently determined in different Neem extracts. Oxidation state is the most important factor determining the toxicity of Cr and its essentiality towards living organisms. In general, trivalent Cr (Cr(III)) is considered to be essential, while hexavalent Cr (Cr(VI)) is toxic [15,16]. In several studies, the content of Cr has been determined in digested Neem leaf samples [9,11] and in aqueous extracts of Neem leaves [11]. Determination of total Cr concentrations in Neem leaves provides data which is important for estimating its contribution to daily intake, but to evaluate the biological effects of Cr on humans, or its potential toxicity, speciation analysis is mandatory. Regarding Cr(VI), consumption of foodstuffs is considered to be safe, since Cr(VI) is rapidly reduced in these matrices by organic matter [15]. Despite this well-known fact, several articles have been published reporting the presence of Cr(VI) in plants [17] tea [18,19] and bread samples [20]. The authors used alkaline

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99

Table 1 ICP-MS operating parameters for determination of element concentrations. Parameter

Type/value

Sample introduction Nebuliser Spray chamber Skimmer and sampler

Miramist Scott Ni

Plasma conditions Forward power Plasma gas flow Carrier gas flow Dilution gas flow QP bias Oct bias Cell entrance Cell exit Deflect Plate bias Sample uptake rate Data acquisition parameters Isotopes monitored Isotopes of internal standards

He mode (4.5 mL He min−1 )

No gas mode

1.05 L min−1 0.10 L min−1 −15.0 V −18.0 V

0.75 L min−1 0.45 L min−1 −3.6 V −8.0 V

−60 V −2.2 V −60 V

−50 V 13.4 V −40 V

27

111

1550 W 15.0 L min−1

−40.0 V

0.3 mL min−1

45

Al, 51 V, 52 Cr, 56 Fe, 59 Co, 60 Ni, 63 Cu, 66 Zn, 75 As, 78 Se, 95 Mo Sc, 72 Ge, 103 Rh

extraction procedures similar to that reported by James and coworkers [21] for the extraction of Cr(VI) from solid samples, or hot water to prepare Neem tea infusions. The content of Cr in the extracts determined by electrothermal atomic absorption spectrometry (ETAAS) was ascribed to Cr(VI), without the use of any speciation analysis for the identification of Cr(VI) [17,18,20]. Chen et al. [19] performed speciation analysis of Cr in tea leaves infusions by separation of Cr(III) from Cr(VI) using a pre-concentration of positively charged Cr(III) species on the negatively charged surface of titanium dioxide nanotubes, while negatively charged Cr(VI) remained in solution. The fraction of Cr in the tea infusion which was not adsorbed on the nanotube sorbent was ascribed as Cr(VI). The authors did not consider that in tea infusion Cr(III) is complexed by available organic ligands, forming negatively charged and neutral complexes, which are not retained by the negatively charged nanotube surface. Assigning the Cr content that was not adsorbed as Cr(VI) may lead to erroneous interpretation of the data. To check whether data reporting the existence of Cr(VI) in tea infusions and bread was obtained as an artefact of inappropriate methodology, Novotnik and co-workers [22] repeated the experiments of Mandiwana et al. [18] and Soares et al. [20]. By applying speciation analysis using high performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC–ICP-MS) and enriched stable Cr isotopes to follow species interconversions during the extraction procedures, the authors proved that in the presence of antioxidants and organic matter, Cr(VI) cannot exist in tea infusions and bread samples. The importance of the use of adequate analytical tools in Cr speciation was ˇ canˇcar and Milaˇciˇc [23]. emphasised also in a review article by Sˇ Since Neem leaf powder is rich in essential trace elements and can also contain elevated Cr concentrations, the aim of the present work was to determine the total concentrations of selected trace elements in Neem leaf powder and tea infusions by ICP-MS. Further, in Neem tea infusions, speciation of Cr(VI) was performed by HPLC–ICP-MS using enriched stable isotopic tracer solutions of 50 Cr(VI) and 53 Cr(III) to follow species interconversions during the extraction procedures. Experimental Instrumentation Total elemental concentrations were determined by an inductively coupled plasma mass spectrometer (ICP-MS), model 7700x

45

Cd, 118 Sn, 121 Sb, 137 Ba, 201 Hg, 208 Pb Sc, 72 Ge, 103 Rh

Table 2 ICP-MS operating parameters for chromium speciation. Parameter

Type/value

Sample introduction Nebuliser Spray chamber Skimmer and sampler

Miramist Scott Ni

Plasma conditions Forward power Plasma gas flow Carrier gas flow Dilution gas flow Total carrier gas flow HECM QP bias Oct bias Cell entrance Cell exit Deflect Plate bias Sample uptake rate

1550 W 15.0 L min−1 0.64 L min−1 0.53 L min−1 1.17 L min−1 10 mL He min−1 −97 V −100 V −130 V −150 V −80 V −150 V 1.5 mL min−1

Data acquisition parameters Isotopes monitored in Cr speciation Isotopes of internal standards Total acquisition time

50

Cr, 52 Cr,53 Cr Sc, 72 Ge 599 s 45

from Agilent Technologies (Tokyo, Japan). ICP-MS operating parameters are presented in Table 1. For HPLC separations an Agilent (Tokyo, Japan) series 1200 quaternary pump equipped with a Rheodyne model 7725i (Cotati, CA, USA) sample injection valve fitted with a 0.05 mL injection loop was used. An anion-exchange FPLC column of Mono Q 5/5 GL (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden) (column dimensions 5 mm × 50 mm, matrix polystyrene/divenyl benzene, pH stability 2–12, particle size 10 ␮m) was applied for separation of Cr species. After the chromatographic separation, Cr was detected by ICP-MS. The outlet of the chromatographic column was directly connected to the nebuliser of the ICP-MS instrument. ICP-MS operating conditions for Cr speciation are presented in Table 2. Data were treated with Agilent MassHunter software. Data processing was based on peak area. Experimental working conditions for ICP-MS were optimised for plasma robustness and sensitivity using a High Matrix Introduction (HMI) system. To eliminate the polyatomic interferences of chlorine and carbon on m/z 52 and 53, the High Energy Collision Mode (HECM) was applied [24].

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A CEM Corporation (Matthews, NC, USA) MARS 5 Microwave Acceleration Reaction System was used for digestion of samples. A WTW (Weilheim, Germany) 330 pH meter was employed to determine the pH. A Mettler AE 163 (Zürich, Switzerland) analytical balance was used for all weighing. Reagents and materials Ultrapure 18.2 M cm water obtained from a Direct-Q 5 Ultrapure water system (Millipore Watertown, MA, USA) was used for preparation of samples and reagents. Merck (Darmstadt, Germany) suprapur nitric and hydrofluoric acids and ultrapure hydrochloric acid were applied in digestion of samples. The alkaline extraction solution was prepared from Merck suprapur sodium carbonate. Merck suprapur sodium chloride was used for the preparation of eluent in the chromatographic procedure and magnesium chloride (analytical reagent grade) to prevent Cr(III) oxidation during the alkaline extraction. Enriched 50 Cr and 53 Cr isotopes in the form of Cr2 O3 were purchased from Oak Ridge National Laboratory (Oak Ridge, TN, USA) and were used for the preparation of 50 Cr(VI) and 53 Cr(III) isotopic spike solutions [25]. The composition of the enriched 50 Cr isotope was determined to be 96.04 ± 0.05%, 3.40 ± 0.02%, 0.25 ± 0.01% and 0.03 ± 0.01% for the isotopes 50, 52, 53 and 54, respectively, while of that of the enriched 53 Cr isotope 0.040 ± 0.005%, 2.81 ± 0.02% 52, 95.58 ± 0.02% and 0.160 ± 0.005% for the isotopes 50, 52, 53 and 54, respectively. A natural isotopic abundance Cr(III) solution (Cr(NO3 )3 in 2–3% HNO3 ) containing 1000 ± 4 mg L−1 of Cr and a natural abundance Cr(VI) solution (K2 CrO4 in water) containing 1000 ± 2 mg L−1 of CrO4 2− were obtained from Merck. Ge (1000 ± 2 mg L−1 in water) and Sc (1000 ± 2 mg L−1 in 5% nitric acid), both purchased from Merck, were used as internal standards. Samples were filtered using 0.45 ␮m Minisart filters (Sartorius Stedim Biotech GmbH, Goettingen, Germany). The accuracy of the determination of total concentrations of different elements in Neem powder was checked by the analysis of the certified reference material SRM 1573a Tomato leaves purchased from NIST (National Institute of Standards & Technology, Gaithersbury, USA). The certified reference materials CRM 544, Cr(III), Cr(VI) species and total Cr in lyophilised solution and CRM 545, Cr(VI) and total leachable Cr in welding dust loaded on a filter, obtained from the Community Bureau of Reference (BCR, Geel, Belgium), were used to check the accuracy of the Cr(VI) determination. Neem powder was provided by Bio Era d. o. o. (Lepajci, Croatia). Chromatographic separation Chromatographic separation of Cr species was performed according to a previously developed and validated procedure [24,25]. Briefly, 0.05 mL of sample was injected onto the column. Linear gradient elution from 100% water to 100% 0.7 mol L−1 NaCl was applied for 10 min at a flow rate of 1.5 mL min−1 . The eluate from the column was connected on-line to the ICP-MS. After separation the column was regenerated at the same flow rate with 2 mol L−1 NaCl for 3 min and equilibrated with water in the next 7 min. Sample preparation Preparation of sample digests for determination of total element concentrations in Neem powder For the determination of total element concentrations in Neem powder approximately 0.25 g of sample was transferred to a Teflon vessel and 4 mL of concentrated HNO3 , 0.1 mL of HF and 1 mL H2 O2 were added. The Teflon vessel was subjected to closed vessel microwave assisted digestion [26]. After the digestion, the content

was transferred to 20 mL polyethylene tubes. Before determination of elements concentrations by ICP-MS, samples were diluted for at least 10 times. Preparation of aqueous extracts of Neem powder for the determination of total element and Cr(VI) concentrations in Neem tea infusions Two different Neem powder infusions were prepared. For daily consumption, the manufacturer recommends ¼ of a teaspoon of Neem powder per cup of water, while for short-term consumption in medicinal treatments, one tablespoon. So, 0.1 or 2 g of sample was weighed into a glass beaker and 200 mL of boiling water added. The infusions were immediately doubly spiked with 10 ng mL−1 of both 50 Cr(VI) and 53 Cr(III), left to brew for 5 min and filtered. As soon as the Neem powder infusions had cooled to room temperature, speciation analysis was performed by the HPLC–ICP-MS procedure. Chromatograms of Cr were recorded at m/z 50, 52 and 53. For the determination of pH and element contents in Neem powder infusions, an aqueous extract (2 g of sample per 200 mL of water) was prepared without addition of Cr isotopic spike solutions and the total concentration of elements determined by ICP-MS. Preparation of alkaline extracts for the determination of total Cr(VI) concentration in Neem powder For determination of the total Cr(VI) content in Neem powder (sum of sparingly soluble and soluble Cr(VI)) an alkaline extraction of the solid sample similar to that proposed by James et al. [21] was performed. 0.1 or 2.0 g of Neem powder was weighed into a glass beaker and 200 mL of solution containing 0.1 mol L−1 Na2 CO3 and 0.1 mol L−1 MgCl2 , which was previously heated to 70 ◦ C, was added. The addition of MgCl2 provoked precipitation of Cr(III) in alkaline solution and thus prevented its oxidation to Cr(VI) during the extraction procedure [27]. To follow species interconversions during the analytical procedure the sample was immediately spiked with 10 ␮g L−1 of both 50 Cr(VI) and 53 Cr(III), kept for the next 3 min at 70 ◦ C and filtered. After the alkaline extract had cooled to room temperature, the pH was measured and the Cr(VI) content determined by HPLC–ICP-MS at m/z 50, 52 and 53. In order to demonstrate that at highly alkaline pH and elevated temperature species interconversions may occur in the sample analysed, the same experiments were performed with the addition of boiling solutions of spiked alkaline extracts. It should be stressed that for minimising the procedural blanks in speciation analysis, effective cleaning of the column resin and the use of chemicals of suprapur quality is mandatory. All the analyses were performed in triplicate. Results and discussion Quality control The accuracy of the analytical procedure for the determination of total element concentrations and determination of Cr(VI) in aqueous solutions and alkaline extracts was checked by analyses of the reference materials SRM 1573a, Tomato leaves, CRM 544, Cr(VI) in lyophilised solution and CRM 545, Cr(VI) in welding dust. The results are presented in Table 3. As evident, good agreement was found between the determined and the reported certified values, confirming the accuracy of the analytical procedures applied. Total concentration of elements in Neem powder and its aqueous extracts Neem powder, made from Neem tree leaves, is known for its health-promoting properties and is an important source of

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Table 3 Determination of total element concentrations in SRM 1573a Tomato leaves by ICP-MS and Cr(VI) in certified reference material CRM 544, Cr(VI) in lyophilised solution and in CRM 545, Cr(VI) in welding dust, by HPLC–ICP-MS (number of replicates = 3). Element

Al Fe Co Cu Ni Cd V Zn As Se Cr Cr(VI)

SRM 1573a

CRM 544 Certified (mg kg−1 )

Determined (␮g L−1 )

Certified (␮g L−1 )

Determined (␮g L−1 )

Certified (␮g L−1 )

595 ± 363 ± 0.589 ± 4.73 ± 1.64 ± 1.53 ± 0.830 ± 31.2 ± 0.110 ± 0.056 ± 1.98 ± –

598 ± 368 ± 0.57 ± 4.70 ± 1.59 ± 1.52 ± 0.835 ± 30.9 ± 0.112 ± 0.054 ± 1.99 ± –

– – – – – – – – – – – 23.5 ± 0.7

– – – – – – – – – – – 22.8 ± 1.0

– – – – – – – – – – – 40.5 ± 0.6

– – – – – – – – – – – 40.2 ± 0.6

5 4 0.001 0.01 0.05 0.02 0.005 0.2 0.002 0.001 0.01

12 7 0.02 0.14 0.07 0.04 0.010 0.7 0.004 0.003 0.06

Table 4 Total element concentrations and concentration of elements in aqueous extracts of Neem powder determined by ICP-MS. Element

Al Fe Co Cu Ni Cd V Zn As Se Mo Pb Cr

CRM 545

Determined (mg kg−1 )

Total concentration (mg kg−1 ) 3100 1510 1.56 19.9 17.8 0.034 9.3 16.2 0.63 0.54 1.57 4.6 197

± ± ± ± ± ± ± ± ± ± ± ± ±

10 40 0.02 0.2 0.8 0.001 0.4 0.7 0.02 0.01 0.06 0.1 5

Concentration in aqueous extract (mg kg−1 ) 2.1 6.7 0.162 2.3 0.38 0.0059 0.028 6.1 0.126 0.170 0.086 0.083 0.19

± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.3 0.001 0.1 0.01 0.0001 0.001 0.4 0.002 0.004 0.004 0.003 0.01

Table 5 Total concentrations of elements in Neem leaves determined in the present study and the reported literature data.

Percentage (%) of element in aqueous extract

Element

0.1 0.44 10.4 11.6 2.1 17.3 0.29 37.6 20.0 31.5 5.4 1.8 0.1

Al

trace elements. In order to estimate the extent of accumulation of essential and potentially toxic trace elements in Neem leaves, the total concentrations of selected elements were determined after microwave-assisted digestion of Neem powder by ICP-MS. In addition, concentrations of the same elements were also determined in Neem tea infusions in order to evaluate their dietary intake. These results are presented in Table 4, while in Table 5, concentrations of trace elements found reported in the literature in different parts of the Neem plant are reviewed for comparison. As can be seen from Table 4, Al and Fe are present in elevated concentrations (3100 and 1500 mg kg−1 , respectively). The total concentration of Al found is about three times higher than those determined in Neem leaves by Sahito et al. [11] and Garg et al. [13] and of Fe six times higher than those determined by Garg et al. [13] (Table 5). Total Cr and Ni concentrations (Table 4) are also very high (200 and 18 mg kg−1 , respectively) and about sixty times higher for Cr and six times for Ni than the total concentrations reported for Neem tea leaves by Sahito et al. [11] (Table 5). The Zn concentration (16 mg kg−1 ) found is comparable to the literature data, while the Cu concentration (20 mg kg−1 ) is about three times higher than that found by Garg et al. [13]. The concentrations of the other trace elements determined, which range from 0.03 mg kg−1 to 9 mg kg−1 for Cd and V, respectively are in general comparable to those reported in the cited literature. From the data of Table 5 it can be seen that the contents of elements in Neem leaves varies significantly between studies, most likely because of different geographical origin of the Neem tree, the different age of the leaves and different protocols used in their processing. Similar to Neem leaves, concentrations of Al, Ni and Cr in leaves of the tea plant Camellia sinensis, which is also an accumulator of many metals, vary significantly and depend

Present study

Reported literature data −1

Concentration (mg kg−1 )

Reference

3100

72 553–889 960 116–309

[14] [11] [13] [12]

Fe

1500

10.5 43–69 72 256

[9] [11] [14] [13]

Co

1.56

0.12 0.51 4.3–5.7

[13] [14] [11]

Cu

19.9

ND 3.3–4.8 6.49

[9,14] [11] [13]

Ni

17.8

ND 2.9–4.3

[9] [11]

Cd

0.34

ND 0.6–1.4

[9,14] [11]

V

9.3

1.19 1.66–4.26

[13] [12]

Zn

16.2

2.04 10.1 34.8 45.6–63.6

[9] [14] [13] [11]

Pb

4.6

ND 1.3–2.5

[9] [11]

Cr

197

ND 1.47 2.0–2.8

[9] [13] [11]

Concentration (mg kg

)

ND: not detected.

strongly on the plant origin and leaf processing. Thus the reported total Al concentrations in tea leaves of C. sinensis ranged from 200 to 1200 mg kg−1 [28,29], total Ni concentrations between 1 and 15 mg kg−1 [30,31], and total Cr concentrations between 0.1 and 2 mg kg−1 [22]. In consumption, a tea infusion is made from Neem powder. Thus, it is important to know the concentrations of elements extracted by hot water. As evident from Table 4, less than 0.1% of potentially toxic Al is extractable during tea infusion preparation. The concentration of Al in the aqueous Neem extract is 2 mg kg−1 , comparable to Al concentrations in infusions of C. sinensis tea leaves reported by [28] and Kralj et al. [29]. Although around 20% of total Cd and As are

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extractable by hot water, the concentrations of these toxic elements in Neem tea infusion were very low (0.006 and 0.1 mg kg−1 , respectively), due to their low total contents in Neem powder. Pb, another toxic element, also does not represent a health hazard, since only 0.08 mg kg−1 is extractable with hot water (less than 2% of its total concentration). Currently, there is no tolerable upper intake level of Ni specified in any guideline or legislation concerning food safety, but the general opinion prevails that for individuals sensitised to Ni, oral intakes of Ni should be kept lower than 500 mg per day (recommendation of the European Food Safety Authority) [32]. Regarding the extractable Ni concentration, it can be seen (Table 4) that only 0.4 mg kg−1 of Ni (2% of its total content) is present in the Neem tea infusion. This concentration is about 10 times lower than the Ni content in infusions of C. sinensis tea leaves [30,31]. In drinking one cup of Neem tea prepared for therapeutic purposes (2 g of Neem powder per 200 mL of water), the amount of Ni consumed is 80 ␮g, which represents an almost negligible contribution to Ni daily dietary intake. Among the essential elements, the Neem tea infusion represents a dietary source of Cu (2.3 mg kg−1 , 10% of total content in Neem leaf powder), Se (0.17 mg kg−1 , 32% of total content), Mo (0.09 mg kg−1 , 10% of total content) and Fe (7 mg kg−1 , 0.5% of total content). Zn, which is extractable from Neem powder to the highest extent (38%), was found in the infusion at a concentration of 6 mg kg−1 . On the other hand, a very low extractable portion was found for Cr (0.1% of total content). Its content in Neem tea infusion (0.2 mg kg−1 ) is comparable to concentrations reported for Neem extracts by Sahito et al. [11], which ranged from 0.14 to 0.98 mg kg−1 . The concentration in the Neem tea infusion determined in the present study is also comparable or even higher than Cr concentrations determined in infusions of C. sinensis tea leaves [22], although in tea infusions the extractable amounts of Cr ranged between 10 and 40%. To conclude, despite the very high total concentrations of Al, Fe, Cr and Ni in Neem leaf powder, their very low hot water extractability (0.1–2%) indicates, that Neem leaf processing, in particular grinding in a stainless steel mill, most probably contributes to their elevated total concentrations in Neem powder. The National Research Council in the USA has issued Adequate Intakes (AI) and Recommended Dietary Allowances (RDA) for daily intakes of essential microelements; these are considered to be sufficient to meet the requirements of health. For Fe, which is an active component of haemoglobin, a biomolecule responsible for the transport of oxygen to body tissues, the RDA is 18 or 12 mg per day for men or women, respectively (National Research Council) [33]. When one cup of Neem tea is consumed for therapeutic purposes (2 g of Neem powder per 200 mL of water), the Fe intake is 1.3 mg, which represents about 10% of the RDA. Zn is an essential trace element required for the normal activity of more than 300 enzymes involved in the maintenance of the structural integrity of proteins and regulation of gene expression. It also has an essential role in the immune system. The RDA for Zn is 11 and 8 mg per day for men and women, respectively (National Research Council) [33]. By consuming one cup of Neem tea, the Zn intake is 1.2 mg, representing 10% of the RDA. Mo is a cofactor in some enzymes in humans. The RDA for Mo for adult men and women is 45 ␮g per day (National Research Council) [33]. One cup of the Neem tea investigated contains 17 ␮g of Mo, representing about 40% of the RDA. Cu is component of numerous metallooxidases that catalyse the reduction of molecular oxygen. The RDA for Cu for adult men and women is 900 ␮g per day (National Research Council) [33]. By drinking one cup of Neem tea, 430 ␮g of Cu is consumed, which is around 50% of the RDA. Se is incorporated into different proteins which are important antioxidant enzymes. The antioxidant properties of some selenoproteins prevent cellular damage from free radicals, while some other selenoproteins regulate thyroid function and play a role in maintaining the immune system. The RDA for

adult men and women is 40 ␮g per day (National Research Council) [34]. In one cup of Neem tea 35 ␮g of Se is present, representing about 90% of the RDA. The role and impact of Cr in living organisms depends primarily on its chemical forms. The high toxicity of Cr(VI) is well documented, while Cr(III) is an essential micronutrient for glucose and lipid metabolism. The AI for Cr for adult men and women is 35 ␮g per day (National Research Council) [33]. By drinking one cup of Neem tea 40 ␮g of Cr is consumed, which completely covers the daily needs for Cr. Based on the above findings it may be concluded that despite the high total Al and Ni contents, their concentrations in Neem tea infusion are low. Total Pb, As and Cd concentrations are also low and in particular their extractable portions in Neem tea infusion. Therefore, toxic elements extracted in Neem infusions do not represent any health hazard. On the other hand, the essential elements (Fe, Cu, Zn, Se Mo and Cr) are present in high concentrations in Neem powder and in substantial concentrations also in Neem tea infusions. A Neem tea infusion thus represents significant contribution to daily requirements for Mo and Cu, and rich source of Se and Cr. A Neem tea infusion is a particularly important source of Cr, since Cr in foodstuffs is commonly present in very low concentrations [26,35]. Speciation of Cr In matrices with a high content of organic matter toxic Cr(VI) is rapidly reduced [15,22]. Because of this well-known fact, it can be expected that Cr(VI) cannot exist either in Neem powder or in aqueous Neem extracts. In order to confirm this hypothesis, speciation analysis of Cr was performed in the present investigation by HPLC–ICP-MS in aqueous and alkaline Neem powder extracts. Samples were prepared as described in sections Preparation of aqueous extracts of Neem powder for the determination of total element and Cr(VI) concentrations in Neem tea infusions and Preparation of alkaline extracts for the determination of total Cr(VI) concentration in Neem powder. The pH of aqueous and alkaline Neem powder extracts was 6.0 ± 0.1 and 11.0 ± 0.1, respectively. On the Mono Q anion-exchange column, Cr(VI) is quantitatively eluted from 420 to 470 s within a wide pH range from acidic (pH 3) to highly alkaline pH values (pH 13), while the separation of Cr(III) on the column strongly depends on pH and its particular chemical forms. In aqueous solutions at pH below 4, Cr(III) exists as the [Cr(H2 O)6 ]3+ species, which is eluted with the solvent front. In the pH range from 6 to 12, Cr(III) is present mainly as neutral Cr(OH)3 , a precipitate which is strongly adsorbed on the column resin [24,25]. Neutral Cr(III) complexes with different organic ligands are also retained by the column resin, while negatively charged Cr(III) complexes with organic acids are eluted at around 200 s and are well separated from Cr(VI) [22]. At a pH higher than 12, a small portion of Cr(OH)3 is re-dissolved, forming a soluble Cr(OH)4 − complex, which is eluted from 200 to 250 s, whereas the remaining Cr(III), present as Cr(OH)3 , is adsorbed on the column resin. The adsorbed Cr(III) species do not influence the subsequent chromatographic separations, due to their efficient elution from the column resin with 2 mol L−1 NaCl in the regeneration step. Cr has four naturally occurring isotopes 50 Cr, 52 Cr, 53 Cr and 54 Cr, with abundances of 4.351%, 83.789%, 9.501% and 2.365%, respectively [36]. So, by applying ICP-MS as a detector for the Cr species separated, it is possible to use enriched Cr stable isotopic solutions as tracers to monitor species transformation during the analytical procedures and for accurate quantification of Cr(VI) by isotope dilution (ID)-ICP-MS [23,27]. In the present investigation, species interconversions in aqueous and alkaline Neem extracts were followed by the use of enriched 50 Cr(VI) and 53 Cr(III) isotopic spike solutions. Typical chromatograms of Cr species in aqueous solutions at pH 6 and 11, doubly spiked with 10 ␮g L−1 of 50 Cr(VI) and 10 ␮g L−1 of

B. Novotnik et al. / Journal of Trace Elements in Medicine and Biology 31 (2015) 98–106 12000

12000

200

300

400

500

6000

3000

3000

0 m/z 52 m/z 50 m/z 53

0 m/z 52 m/z 50 m/z 53

600

0

100

200

Time (sec)

300

400

500

600

Time (sec) 12000

C

100

200

300

D

400

500

9000

CPS

9000 6000

0

12000

6000

3000

3000

0 m/z 52 m/z 50 m/z 53

0 m/z 52 m/z 50 m/z 53

600

Time (sec)

0

100

200

300

400

500

CPS

100

9000

CPS

6000

0

B

9000

CPS

A

103

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Fig. 1. Typical chromatograms of Cr species in aqueous solutions obtained by the HPLC–ICP-MS procedure, recorded at m/z 50, 52 and 53. (A) Blank sample at pH 6, (B) aqueous solution at pH 6, doubly spiked with 10 ␮g L−1 of 50 Cr(VI) and 10 ␮g L−1 of 53 Cr(III), (C) blank sample at pH 11, and (D) aqueous solution at pH 11, doubly spiked with 10 ␮g L−1 of 50 Cr(VI) and 10 ␮g L−1 of 53 Cr(III).

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and the corresponding blank chromatograms obtained by the HPLC–ICP-MS procedure are presented in Fig. 1. As can be seen from chromatograms recorded at m/z 53, the elution profile for 53 Cr(III) at pH 6 is the same for the blank (Fig. 1A) and 53 Cr(III) isotopic solution (Fig. 1B), since at this pH 53 Cr(III) is strongly adsorbed by the column resin. The small broadened peak eluted with the solvent front at m/z 53 (Fig. 1A and B) arises from metal parts of the chromatographic system (the contribution of Cr of natural abundance (9.501%) to m/z 53). From the elution profiles recorded at m/z 50, it is evident that at pH 6 50 Cr(VI) is quantitatively eluted from 420 to 470 s (Fig. 1B), while in the blank sample (Fig. 1A) no detectable Cr species are eluted at m/z 50. Chromatograms recorded at m/z 52 (Fig. 1A and B) further indicate that Cr is eluted in broadened peaks with the solvent front. These Cr peaks arise from metallic parts of the chromatographic system (the contribution of Cr of natural abundance (83.789%) to m/z 52). The small peak eluted from 420 to 470 s observed at m/z 52 arises from the contribution of the 50 Cr(VI) isotopic spiking solution (3.40%) to m/z 52. At pH 11, Cr(III) is adsorbed by the column resin, so in chromatograms recorded at m/z 53, no detectable Cr peaks are observed for the blank sample (Fig. 1C) or the 53 Cr(III) isotopic solution (Fig. 1D). From the elution profiles recorded at m/z 52 it may be seen that at pH 11 50 Cr(VI) is quantitatively eluted from 420 to 470 s (Fig. 1D), while in the blank sample (Fig. 1C) no detectable Cr species are eluted at m/z 50. The chromatograms recorded at m/z 52 demonstrate that at pH 11 the Cr peaks eluted with the solvent front (Fig. 1C and D) are lower than those at pH 6 (Fig. 1A and B), since the blank arising from metallic parts of the HPLC system is lower (the degree of leaching of Cr from stainless steel is lower at alkaline pH). A small peak eluted from 420 to 470 s observed at m/z 52 (Fig. 1D) arises from the reagent blank (0.1 M Na2 CO3 ) and the contribution of the 50 Cr(VI) isotopic solution to m/z 52. The speciation procedure used in this work is highly selective and sensitive and has successfully been applied for the determination of Cr(VI) in a variety of environmental and biological samples

[22,27,37,38]. The limits of detection (LOD) and of quantification (LOQ) for the determination of Cr(VI) at m/z 52 were calculated as the concentration that provides a signal (peak area) equal to 3s and 10s of the blank sample in the chromatogram, respectively. At pH 6, the LOD and LOQ were found to be 0.03 and 0.10 ␮g Cr(VI) L−1 , respectively, while at pH 11, they were 0.06 and 0.20 Cr(VI) L−1 , respectively. The repeatability of measurement for Cr(VI) tested on six consecutive speciation analyses of aqueous solutions of natural abundance Cr(VI) (10 ␮g L−1 , pH 6.0) applying the HPLC–ICP-MS procedure was found to be ±1.5%. Linearity of measurement for the determination of Cr(VI) by HPLC–ICP-MS was obtained over a concentration range from the LOQ to 500 ␮g L−1 . Speciation of Cr in aqueous extracts of Neem powder To support the hypothesis that Cr(VI) cannot exist in aqueous infusions of Neem powder, speciation analysis of samples doubly spiked with enriched isotopic solutions of 10 ng L−1 53 Cr(III) and 10 ng L−1 50 Cr(VI) was performed by HPLC–ICP-MS. Aqueous infusions were prepared as described in section Preparation of aqueous extracts of Neem powder for the determination of total element and Cr(VI) concentrations in Neem tea infusions from 0.1 g or 2 g of Neem powder per 200 mL of boiling water. The corresponding concentrations of Cr in these Neem infusions were 0.095 or 1.9 ␮g Cr L−1 , respectively. The chromatograms recorded at m/z 50, 52 and 53 are presented in Fig. 2. From the chromatogram of Fig. 2A (0.1 g of Neem powder per 200 mL of boiling water), recorded at m/z 53, it is evident that the majority of added 53 Cr(III) was eluted with the solvent front, since at pH 6 the added 53 Cr(III) was, due to the high content of organic acids in Neem tea infusions, solubilised. Part of the 53 Cr(III) was also complexed by the available organic ligands, forming negatively charged or neutral complexes. These negatively charged 53 Cr(III) species were eluted as small peaks from around 120 to 415 s, while the neutral 53 Cr(III) complexes were adsorbed by the column resin. From the chromatogram of Fig. 2B (2 g of Neem powder per 200 mL of boiling water) recorded at m/z 53 it can be seen

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Fig. 2. Chromatograms of Cr species in aqueous extract (pH 6) of Neem powder doubly spiked with 10 ␮g L−1 of 50 Cr(VI) and 10 ␮g L−1 of 53 Cr(III) obtained by the HPLC–ICPMS procedure, recorded at m/z 50, 52 and 53. (A) 0.1 g of Neem powder extracted with 200 mL of boiling water for 3 min and (B) 2 g of Neem powder extracted with 200 mL of boiling water for 5 min.

that due to 20 times the higher amount of Neem powder used in preparation of the aqueous infusion, and consequently higher content of organic substances, only a small amount of added 53 Cr(III) is eluted with the solvent front, since the majority is complexed by the available organic ligands. The negatively charged 53 Cr(III) complexes are eluted in several small peaks from around 120 to 415 s, while the neutral 53 Cr(III) complexes were adsorbed by the column resin. From the data of Fig. 2A recorded at m/z 50 it is further evident that about 70% of the added 50 Cr(VI) is reduced in the Neem tea infusion prepared from 0.1 g of Neem powder per 200 mL of water due to the presence of organic molecules, which are strong reducing agents. The newly formed 50 Cr(III) is partially eluted with the solvent as positively charged 50 Cr(III) species and partially adsorbed by the column resin. In the Neem tea infusion made from 2 g of Neem powder per 200 mL of water (Fig. 2B), the added 50 Cr(VI) is completely reduced during the extraction procedure by the presence of higher amounts of antioxidants, confirming our hypothesis that Cr(VI) cannot exist in Neem tea infusions. After being reduced, the newly formed 50 Cr(III) species were mainly adsorbed by the column resin as neutral complexes with organic molecules and to a lesser extent eluted as negatively charged 50 Cr(III) complexes. From the chromatograms of the Neem tea infusions recorded at m/z 52 (Fig. 2A and B), it can be seen that the charged Cr(III) species present in Neem tea infusions were eluted with the solvent front, while neutral Cr(III) complexes present in the infusion were adsorbed by the column resin. It is clearly evident that in the Neem tea infusions analysed no detectable Cr(VI) was observed at m/z 52. Its concentrations were below the LOD (0.03 ␮g Cr(VI) L−1 ). The experimental results of the present study are in accordance with our previous investigations performed on speciation of Cr in 13 tea infusions made from C. sinensis and other herbal plants [22] where we also evidently demonstrated that Cr(VI) cannot exist in tea infusions which are rich in organic matter. Speciation of Cr in alkaline extracts of Neem powder In addition to water soluble Cr(VI), it is important to know the total (sparingly soluble) Cr(VI) content in Neem powder, which may potentially be formed during processing of Neem leaves. For determination of total Cr(VI) concentrations in solid matrices, alkaline based extractions are commonly applied [21,39]. In the present work, alkaline (0.1 mol L−1 Na2 CO3 containing 0.1 mol L−1 MgCl2 ) extracts, doubly spiked with 10 ng L−1 53 Cr(III) and 10 ng L−1 50 Cr(VI), were prepared from 0.1 g or 2 g of Neem powder per 200 mL of extracting solution at 70 ◦ C and speciation analysis performed by the HPLC–ICP-MS procedure. MgCl2 was added to precipitate Cr(III) in alkaline solution in order to prevent its oxidation during the extraction procedure. The results of these experiments are presented in Fig. 3.

As can be seen from the chromatograms of Fig. 3A and B recorded at m/z 53, the added 53 Cr(III) was precipitated and adsorbed on the column resin (only a small 53 Cr(III) peak is eluted with the solvent front in Fig. 3B). It can also be seen that no oxidation occurred during the extraction procedure (no detectable 53 Cr(VI) was formed from 53 Cr(III)). The chromatogram recorded at m/z 50 (Fig. 3A) further indicates that due to the high pH, added 50 Cr(VI) was not reduced during the extraction procedure, despite the presence of organic substances. With a higher content of organic matter (Fig. 3B) about 35% of added 50 Cr(VI) was reduced, regardless the high pH, by antioxidants present in the Neem powder. From the chromatograms of the alkaline Neem extracts recorded at m/z 52 (Fig. 3A and B) it may be seen that charged Cr(III) complexes, which are present in the Neem tea infusion, were eluted with the solvent front. The small peaks eluted from 420 to 470 s correspond to traces of Cr(VI) arising from the reagent blank (0.1 M Na2 CO3 and 0.1 M MgCl2 ) and from the contribution of the 50 Cr(VI) isotopic spiking solution to m/z 52. Therefore, Cr(VI) was not detected in the alkaline Neem tea infusion analysed. Its concentrations were below the LOD (0.06 ␮g Cr(VI) L−1 ), confirming that Cr(VI) is not present in Neem powder. These observations agree with the data of our previous study [22], where we demonstrated that Cr(VI) cannot exist in tea leaves and bread samples, matrices rich in organic matter. Artefacts in Cr speciation Mandiwana et al. [18] used a boiling solution of 0.1 mol L−1 Na2 CO3 for the extraction of total Cr(VI) from tea leaves. In order to demonstrate that artefacts in Cr speciation may occur if unsuitable extraction procedures are used, the same experiments were performed as described in section Speciation of Cr in alkaline extracts of Neem powder, with the exception that a boiling alkaline extracting solution was added to Neem powder (in section Speciation of Cr in alkaline extracts of Neem powder the extraction temperature was 70 ◦ C). This study was performed to demonstrate that extractions made with boiling alkaline solutions, without any control of possible species transformation, could lead to wrong interpretation of the analytical data. The results are presented in Fig. 4. From the chromatogram recorded at m/z 53 (Fig. 4A) it is clearly evident that despite the addition of MgCl2 , the higher temperature (100 ◦ C) and highly alkaline conditions provoked oxidation of 53 Cr(III) during the extraction procedure by the dissolved oxygen in the sample extract prepared from 0.1 g of Neem powder. About 30% of added 53 Cr(III) was oxidised to 53 Cr(VI). In the presence of higher content of organic matter in the Neem alkaline infusion prepared from 2 g of Neem powder (Fig. 4B), the oxidation of added 53 Cr(III) was prevented by the higher concentration of antioxidants, so no detectable 53 Cr(VI) was formed.

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Fig. 4. Chromatograms of Cr species in alkaline extract (0.1 mol L−1 Na2 CO3 containing 0.1 mol L−1 MgCl2 , pH 11) of Neem powder doubly spiked with 10 ␮g L−1 of 50 Cr(VI) and 10 ␮g L−1 of 53 Cr(III) obtained by the HPLC–ICP-MS procedure, recorded at m/z 50, 52 and 53. (A) 0.1 g of Neem powder extracted with 200 mL of boiling alkaline extract for 3 min. (B) 2 g of Neem powder extracted with 200 mL of boiling alkaline extract for 3 min.

On the other hand, at elevated temperatures reduction processes are also more intensive. As evident from the chromatogram recorded at m/z 50 (Fig. 4A), about 10% of added 50 Cr(VI) was reduced, while in the Neem tea infusion with a higher amount of organic matter (Fig. 4B), 95% of added Cr(VI) was reduced, despite the highly alkaline pH. The elution profiles of Cr at m/z 52 (Fig. 4A) further indicate that, due to the elevated extraction temperature, Cr(III) present in the Neem alkaline extract was partially oxidised to Cr(VI). The pronounced Cr(VI) peak eluted from 420 to 470 s does not reflect its presence in the Neem extract. It arises from the reagent blank, the contribution of the 50 Cr(VI) spike to m/z 52 and to a great extent is an artefact of oxidation of Cr(III) caused by the high extraction temperature in highly alkaline media. In the presence of higher amounts of organic matter in the Neem tea infusion (Fig. 4B), reducing conditions prevail, preventing oxidation of Cr(III). The above data demonstrate that an extraction procedure such as that used by Mandiwana et al. [18] causes Cr species transformation. In their work, these authors also did not apply any speciation analysis and simply assumed the extractable Cr to be Cr(VI). Therefore, it must be stressed that the use of inappropriate analytical methodology [20] or speciation procedures, without considering the specific behaviour of Cr in complex sample matrices [19], can lead to erroneous and misleading interpretation of the analytical data. As demonstrated in the present study, and highlighted by several investigators [23,39], it is of paramount importance that potential species interconversions during the speciation procedure are evaluated for each sample matrix. Using isotopically enriched Cr spike solutions it is possible to optimise the extraction procedure to prevent Cr species interconversions, or to correct for any species transformation occurring during the analytical procedures and to quantify Cr species by speciated ID-ICP-MS [22,27,37,40,41]. Isotopically enriched Cr spike solutions represent a powerful tool in speciation analysis of Cr in environmental and biological samples.

Conclusions Neem tree leaves, fruits and bark have been traditionally used in Ayuverdic medicine in India. Today, the Neem plant is used in several therapeutical and industrial preparations, stimulating the interest of many scientists to investigate this medicinal plant. Neem is recommended to be consumed daily if it is prepared from 0.1 g of powder per 200 mL of boiling water, or in limited applications for therapeutic purposes when prepared from 2 g of powder per 200 mL of boiling water. Among other beneficial properties, consumption of Neem tea infusions may represent a source of essential trace elements. So, in the present study the total concentrations of selected elements in the leaves and their concentrations in the tea infusions were determined by ICP-MS. The results demonstrated that total Al, Fe, Ni and Cr concentrations in Neem powder were higher than those found reported in the previous literature for Neem leaves. The origin of the high total concentrations of these elements is most likely the processing of Neem leaves and their grinding in stainless steel mills. Nevertheless, the extractable concentrations in hot water were low for Al and Ni (2 mg kg−1 and 0.4 mg kg−1 , respectively). Other toxic elements are also extractable only in negligible amounts and in the Neem tea infusion were 0.006, 0.1, 0.08 and 0.4 mg kg−1 for Cd, As, Pb and Ni, respectively. As such, they do not represent any health hazard. On the contrary, the essential elements are present in a Neem tea infusion in amounts that significantly contribute to their recommended dietary daily intake. Consumption of one cup of Neem tea infusion prepared for therapeutic purposes thus covers 10% of the daily requirements for Fe and Zn, 40% for Mo, 50% for Cu, 90% for Se, and 100% for Cr. To examine the hypothesis that toxic Cr(VI) cannot exist in a Neem tea infusion and in Neem leaf powder, speciation analyses were performed in this work by HPLC–ICP-MS using enriched 53 Cr(III) and 50 Cr(VI) isotopic solutions to species interconversions to be followed during the analytical procedure. For this purpose, aqueous and alkaline Neem powder extracts were analysed. The

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data proved that Cr(VI) does not exist either in the Neem tea infusion, or in Neem leaves, since it was readily reduced by the presence of antioxidants. Cr(VI) concentrations were below the LOD of the speciation procedure applied (0.03 and 0.06 ␮g L−1 , for aqueous and alkaline extracts, respectively). It was further demonstrated that in complex sample matrices it is essential to consider the specific behaviour of Cr in the sample analysed in order to correctly interpret the speciation analysis data. It is also necessary to apply extraction procedures that prevent Cr species interconversions, or to correct for species transformation occurring during the analytical procedure. Only by applying reliable Cr speciation procedures is correct evaluation of the analytical data possible. Conflict of interest statement None of the authors have any conflict of interest. Acknowledgements This work was supported by the Ministry of Higher Education, Science and Technology of the Republic of Slovenia (Programme group P1-0143). We thank Dr. Anthony R. Byrne for linguistic corrections. Special thanks go to Mrs. Sandra Tomljenovic´ from Bio Era d.o.o., Lepajci, Croatia, for providing the Neem powder for this research. References [1] Biswas K, Chattopadhyay I, Banerjee RK. Biological activities and medicinal properties of neem (Azadirachta indica). Curr Sci 2002;82:1336–45. [2] Islam MM, Shams MI, Ilias GNM, Hannan MO. Protective antifungal effect of neem (Azadirachta indica) extracts on mango (Mangifera indica) and rain tree (Albizia saman) wood. Int Biodeterior Biodegrad 2009;63:241–3. [3] Du Y-H, Jia R-Y, Yin Z-Q, Pu Z-H, Chen J, Yang F, et al. Acaricidal activity of extracts of neem (Azadirachta indica) oil against the larvae of the rabbit mite Sarcoptes scabiei var. cuniculi in vitro. Vet Parasitol 2008;157:144–8. [4] Thakurta P, Bhowmik P, Mukherjee S, Hajra TK, Patra A, Bag PK. Antibacterial, antisecretory and antihemorrhagic activity of Azadirachta indica used to treat cholera and diarrhea in India. J Ethnopharmacol 2007;111:607–12. [5] Parida MM, Upadhyay C, Pandya G, Jana AM. Inhibitory potential of neem (Azadirachta indica Juss) leaves on dengue virus type-2 replication. J Ethnopharmacol 2002;79:273–8. [6] Dua VK, Pandey AC, Raghavendra K, Gupta A, Sharma T, Dash AP. Larvicidal activity of neem oil (Azadirachta indica) formulation against mosquitoes. Malar J 2009;8:124 http://www.malariajournal.com/content/8/1/124 [7] Hasan F, Ansari MS. Toxic effects of neem-based insecticides on Pieris brassicae (Linn.). Crop Prot 2011;30:502–7. [8] Ogbuewu LP, Odoemenam HO, Obikaonu MN, Opara OO, Emenalom MC, Uchegbu MC, et al. The growing importance of Neem (Azadirachta indica A. Juss) in agriculture, industry, medicine and environment: a review. Res J Med Plant 2011;5:230–324. [9] Ata S, Farooq F, Javed S. Elemental profile of 24 common medicinal plants of Pakistan and its direct link with traditional uses. J Med Plants Res 2011;5:6164–8. [10] Benzo Z, Zoltan T, Murillo M, Quintal M, Salas J, Marcano E, et al. Determination of trace manganese and Ni in neem oil by ETA–AAS with emulsion sample introduction. J Am Oil Chem Soc 2006;83:401–5. [11] Sahito SR, Memon MA, Kazi TG, Kazi GH, Jakhrani MA, Haque QU, et al. Evaluation of mineral contents in medicinal plant Azadirachta indica (Neem). J Chem Soc Pak 2003;25:139–43. [12] Balaji T, Acharya RN, Nair AGC, Reddy AVR, Rao KS, Naidu GRK, et al. Determination of essential elements in ayurvedic medicinal leaves by k0 standardized instrumental neutron activation analysis. J Radioanal Nucl Chem 2000;243:783–8. [13] Garg AN, Kumar A, Nair AGC, Reddy AVR. Analysis of some Indian medicinal herbs by INAA. J Radioanal Nucl Chem 2007;271:611–9. [14] Samudralwar DL, Garg AN. Minor and trace elemental determination in the Indian herbal and other medicinal preparations. Biol Trace Elem Res 1996;54:113–21.

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