Elemental Characterization of Medicinal Plants and Soils

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Journal of Oleo ScienceCopyright ©2019 by Japan Oil Chemists’ Societydoi : 10.5650/jos.ess19004J. Oleo Sci. 68, (5) 443-461 (2019)

Elemental Characterization of Medicinal Plants and Soils from Hazarganji Chiltan National Park and Nearby Unprotected Areas of Balochistan, PakistanShaista Anjum1, Zahoor Ahmed Bazai1, Sabeena Rizwan2, Cinzia Benincasa3, Khalid Mehmood4, Naila Siddique5, Ghazala Shaheen1, Zahid Mehmood4, Muhammad Azam4, and Ashif Sajjad4＊

1 Department of Botany, University of Balochistan, Sariab Road-87300, Quetta, PAKISTAN.2 Department of Chemistry, Sardar Bahadur Khan, Women’s University, Brewery Road, Quetta, PAKISTAN.3 CREA Research Centre for Olive, Citrus and Tree Fruit, C.da Li Rocchi, 87036 Rende (CS), ITALY.4 Institute of Biochemistry, University of Balochistan, Sariab Road-87300, Quetta, PAKISTAN. 5 Chemistry Division, Directorate of Science PINSTECH, P. O. Nilore, Islamabad 45650 PAKISTAN.

1 INTRODUCTIONAmong the protected areas, national parks and nature

reserves cover about 12％ of the earth’s surface, which acts as a backbone for biodiversity conservation. National parks provide a primary source of safe ecosystem services in addition to their recreational role, essential for healthy well-being. These national parks, also, serve as a diversified habitat for many valuable medicinal plants. Many research-ers focused on the study of important medicinal plants present in National parks of Pakistan and document their

＊Correspondence to: Ashif Sajjad, Institute of Biochemistry, University of Balochistan, Sariab Road-87300, Quetta, PAKISTAN. E-mail: [email protected], [email protected] February 25, 2019 (received for review January 7, 2019)Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 onlinehttp://www.jstage.jst.go.jp/browse/jos/　　http://mc.manusriptcentral.com/jjocs

various uses. The uses and the characteristics of about fifty medicinal plants from Margalla Hills National park at Islam-abad, Pakistan, were investigated1）. Further surveys were conducted to assay valuable medicinal plants present in Machyara National park at Azad Jammu and Kashmir, Paki-stan2, 3）. Such studies clearly indicate the importance of medicinal plants as a major source for the preparation of thousands of herbal products4）. The medicinal plants are considered non toxic and are consumed in raw form, either as a powder or a decoction. Some researchers, who have

Abstract: The aim of this work was to evaluate the variability in elemental composition of seven medicinal plants and their respective soils belonging to protected and nearby unprotected sites of the Hazarganji Chiltan National Park. The medical plants under study were Achillea wilhelmsii C. Koch, Peganum harmala Linn, Sophora mollis (Royle) Baker, Perovskia atriplicifolia Benth, Seriphidium quettense (Podlech.) Ling, Hertia intermedia (Bioss) O. Ktze, and Nepeta praetervisa Rech. F. Macro (C, H, N, S, K, Ca), micro (Cl, Cu, Fe, Mn, and Zn), beneficial (Al, Co, Na), others (As, Br, Cr, Cs, Hf, Pb, Rb, Sb, Sr, Sn, V and Th) and rare earth elements (Ce, Eu, La, Lu, Nd Sc, Sm, Tb and Yb) were characterized by means of standard organic elemental and instrumental neutron activation methodologies and by flame atomic absorption spectroscopy. Results showed that, among macro nutrients, carbon concentration was the highest element in both plant and soil samples followed by H and K. Elements such as Cl, Na and Fe were detected in considerably good amounts; all the other elements were found in trace quantities. Principal component analysis (PCA) was applied to identify spatial variation in elemental composition of medicinal plants, in which 80-90% of the total variance in whole set of data was found. In particular, the findings highlighted the presence of essential and beneficial elements such as C, H, N, K, Ca, Fe, Mn and Na, in samples from protected sites, while potentially dangerous elements such as Al, As, Br and Cr were detected in samples from unprotected sites. These results emphasized on the need for rational exploitation of valuable medicinal plants and supporting protected areas as an excellent source of biodiversity conservation.

Key words: protected area, medicinal plants, elemental composition, organic elemental analyzer (CHNS-O), flame atomic absorption spectroscopy (FAAS), instrumental neutron activation analysis (INAA).

S. Anjum, Z. A. Bazai, S. Rizwan et al.

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explored the bioactive components of plants, have reported that plants growing in a contaminated natural environment can lead to the accumulation of undesirable levels of some elements in the flora and fauna5, 6）. In fact, edaphic factors and climatic conditions, in which plants grow, serves as a source of elements and, this is essential to quantify the es-sential elements of medicinal plants, as well as to quantify the toxic inorganic hazardous elements if present in food preparations7, 8）. As the soil is the primary source of ele-ments for plants, quantification of its elements is a pre-req-uisite. Uptakes of elements present in medicinal plants may cause disturbance to the normal body functions if con-sumed for longer periods without any restrictions. Toxic elements such as heavy metals may cause serious detri-mental effects because of their lower excretion rates. Certain elements such as iron, zinc, copper, chromium and manganese are considered essential because of their vital role in physiological and biological pathways. These ele-ments could even be toxic if taken above their permissible limits9, 10）. Most researchers, working on medicinal plants, have raised their concern regarding the cultivation of me-dicinal plants in places that any kind of climatic or edaphic contamination may occur. Therefore, in order to ensure the beneficial properties of medicinal plants it is essential to quantify their elemental composition along with other phy-tochemical analysis11）.

The aim of the present study was to carry out elemental composition analyses of seven medicinal plants growing wild in protected and nearby unprotected sites of the Na-tional park located in the Mastung district of Balochistan, a south western province of Pakistan. The elemental compo-sitions of their respective soils were also evaluated. The results of the present study will lead to contribute in the selection of the right medicinal plants cultivation, as well as in their harvesting sites. The experimental obtained data will be compared with those presented in the literature. Moreover, the data could be further used for the synthesis of new herbal drugs, with known elemental compositions, to be used in the treatment of various diseases with minimal and/or non-redundant effects on human health.

2 EXPERIMENTAL2.1 Study area

Hazarganji Chiltan National Park（HCNP）is one of the most important National parks located in the Mastung district（30°7’N, 66°54’E）and it considered as a hot spot of Balochistan, a south western province of Pakistan12）. The area, covering about 27,421 ha, is part of the Sulaiman Mountains. Its climate is Mediterranean with rainfall and snow in the winter season and occasional precipitation in spring and summer（250 mm precipitation per annum）13）. The area was declared protected in 198014）. HCNP is con-

sidered one of the most valuable assets of the country for its rich biodiversity and huge variation of medicinal and ar-omatic plants. For this study, two sites were selected and the choice was based on the level of disturbance caused by various anthropogenic and topographic factors. The pro-tected sites, covered by good vegetation, were positioned at a distance of 4-5 km from the main high way. The unpro-tected areas, located nearby the National park and the Na-tional high way, were exposed to different biotic and abiotic stresses. In protected sites, unlike those unprotect-ed, many practices that are considered destructive for the natural habitat have been banned. Among those, the exces-sive exploitation of wild flora by grazing, uprooting of plants and firewood production. But not only, many recre-ational activities such as camping and hiking have also been banned to avoid deforestation and the removal of vegetation cover, as well as access to off-road vehicles, heavy trucks and road bikes.

2.2 Soil sampling and processingBoth protected and unprotected sites were further sub-

divided into different strata by following the stratified random sampling technique. With the help of the area to-po-sheets, five sampling strata were individuated in pro-tected area and four sampling strata in unprotected ones. From these strata, soil collection was performed at a depth of 0-10 cm by means of clean and proper scientific tools. Briefly, soil samples were carried out from five sampling cores of each stratum of both sites and then mixed to get a reasonably representative soil sample of each site. Com-posite soil samples were processed by removal of plant debris, stones and all other non-soil materials and were allowed to dry at room temperature. Afterwards, in order to prevent any contamination, soil samples were passed through 2 mm diameter plastic sieve and stored in cleaned polyethylene bottles.

2.3 Plant sampling and processingPlants were selected in accordance to their uses by the

local communities of the interested area. A total of seven plant species namely Achillea wilhelmsii C. Koch, Hertia intermedia（Bioss）O.Ktze, Nepeta praetervisa Rech. F., Peganum harmala Linn, Perovskia atriplicifolia Benth, Seriphidium quettense（Podlech.）Ling., and Sophora mollis（Royle）Baker were collected from protected and unprotected sites（Table S1）. Plant samples were carefully collected from each stratum of each site by random sam-pling technique. Approximately 25 samples of each select-ed plant species were collected from each stratum. Mature and healthy leaves were hand plucked, separated from each plant and, in order to remove any kind of dust and contamination, properly washed with tap water and dis-tilled water. Leaves were then mixed to make a representa-tive sample for each site and immediately oven dried at

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60℃ for 48 hours for moisture removal. In order to avoid any metal contamination, dried plant samples were manu-ally crushed in to fine powder in a ceramic pestle and mortar, and stored in clean polyethylene containers.

2.4 Organic elemental analysisC, H, N, and S were determined by CHNS-O analyzer

（Flash 2000 Thermo Scientific）. For this purpose, 10 mg of each sample was placed in tin capsules and completely oxi-dized, at 950℃, to their elemental gases（CO2, H2O, N2 and SO2）. The excess of oxygen was retained. The resultant combustion products were mechanically homogenized in a gas control zone and separated in a gas chromatographic column. Finally, eluted gases were conveyed to a thermal conductivity detector and amounts of N, H, C and S ob-tained. Helium and oxygen, at 99.99％ of purity, were used as carrier gas and to perform the oxidation, respectively.

2.5 Flame atomic absorption spectroscopyCu and Pb were determined by FAAS that involves the

digestion of sample prior analysis. In this regard, digestion was carried out following two analytical procedures. In particular, for soil samples, the digestion procedure con-sisted in treating 1 g of sample with 10 mL of 50％ HNO3 at 95℃; the boiling point was avoided. After sample was cooled, the process was repeated with an aliquot of 65％ HNO3 until the disappearance of brown fume. The solution was then reduced to 5 mL by evaporation. Afterwards, 10 mL of 30％ H2O2 was added very slowly to avoid sample loss. The sample was then treated with 10 mL of 37％ HCl at 95℃ for 15 min. Finally, the digested sample was filtered and diluted with deionized water. The digestion procedure of plant samples consisted in soaking overnight, in a 100 mL flask, 2 g of dry powdered plant sample with nitric, concentrated perchloric and concentrated sulfuric acids. The day after, the flask was heated for 40 min at 70℃. Further heat, at 160℃, allowed the solution to clear up with the formation of white fumes. Finally, the digested so-lution was diluted with distilled water, boiled for 15 min, and transferred to a 250 mL volumetric flask for subse-quent measurements. The final volume for both plant and soils digested was 50 mL. The flame atomic absorption spectrometer used was a Hitachi Model Z-2000 Polarized Zeeman. Quantitative determination of Cu and Pb was achieved by external calibration curve obtained by diluting a stock standard solution of Cu and Pb at 1000 mg/L.

2.6 Instrumental Neutron Activation AnalysisA total of 29 elements（Al, As, Br, Ca, Ce, Cl, Co, Cr, Cs,

Eu, Fe, Hf, K, La, Lu, Mn, Na, Nd, Rb, Sb, Sc, Sm, Sn, Sr, Tb, Th, V, Yb and Zn）were quantified using instrumental neutron activation analysis（INAA）. To prepare irradiation targets, 100 mg of each soil and plant samples were placed in pre-cleaned poly-ethylene vials for sequential, short, in-

termediate and long irradiation protocols（Table S2）. To evaluate the validity and quality of the analytical proce-dure, such as optimal irradiation, cooling and counting times using nuclear data for each studied isotope, certified reference materials（CRMs）were analyzed and the quality assurance（QA）parameters evaluated（Tables 1 and 2）. In general, 100 mg of plant and soil samples were weighed in pre-cleaned polyethylene vials. These samples were pre-pared as irradiation targets for four irradiation schemes se-quential, short, intermediate and long protocols. For quality assurance analyses of plant samples, two CRMs, from the National Institute of Standard and Technology（NIST）and one CRM, from the International Atomic Energy Agency（IAEA）, were used: NIST-SRM-1572（citrus leaves）, NIST-SRM-1573a（tomato leaves）and IAEA-SL1（Lake Sediments）. For quality assurance analyses of soil samples, three CRMs, from IAEA, were analysed: IAEA-SL1（Lake Sediments）, IAEA-SDM-2/TM（Marine Sediments）and IAEA-S7（Soil）. Samples and reference materials were irradiated by Atomic Research Reactor-2（PARR-2）in Paki-stan. The atomic reactor is a 27 kW tank in pool type min-iature reactor which serves as a neutron source with a thermal neutron flux density of 1×1012 cm－2 sec－1. The final optimized irradiation schemes for the determination of the elements in both plants and soil sample are given in Table S2. The experimental set-up mainly consisted of a high purity Germanium detector of Canberra（Model AL-30）linked through a spectroscopy amplifier（Ortec model 2010, Oak Ridge, USA）and attached to a PC based multi channel analyzer（Inter-technique model Pro-286e, Paris, France）. The resolution of the system for the 1332.5 keV 60 Co peak is 1.9 keV with peak-to-compton ratio of 40:1. Data were processed by the software Intergamma（version 5.03）. Final elemental concentration of each element was calculated from data files with important in-formation regarding peak energy, peak area etc through in house computer program “GammaCal”15）. Uncertainties in elemental concentrations were determined by applying error propagation rules using the uncertainties in weighing uncertainty in peak area, uncertainty in counting and of certified values of reference or control material.

2.7 Statistical AnalysisDescriptive statistics were used for the analysis of the

collected quantitative data. Mean and standard deviation from the triplicate values data obtained by CHNS-O and FAAS were obtained by SPSS statistics version 20.0 soft-ware. INAA data were used to measure elemental concen-trations at 95％ confidence level by using Gamma Cal com-puter program. To identify significant elements, the obtained overall data were subjected to principal compo-nent analysis（PCA）by using Multi-Variate Statistical Package（MVSP version 3.22）software.


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3 RESULTS AND DISCUSSIONEarth is a reservoir of 92 elements, and about 82 ele-

ments can be utilized by many plant species16）. In the present work, a total of 35 elements were quantified, in soil samples（31 elements）and in selected medicinal plants（33 elements）collected from protected and unprotected sites.

In the study, a wide range of variation in elemental concen-trations was observed not only in protected and unprotect-ed sites but among plant species as well. The collected plants were, also, analyzed for other parameters such as phytochemical constituents including phenolic content and protein concentration. The results, however, are not dis-

Table 1　QA Data for Biological Samples.

ElementIAEA-SL1 NIST-SRM-1572 NIST-SRM-1573a

Certified Values Measured Values Certified Values Measured Values Certified Values Measured ValuesAl (89000) 91170±27890 92.0±14.7 90.7±27.6 598±2 582±141As 27.50±1.45 31.22±8.86 3.10±0.30 0.112±0.004Ba 639±27 635±178 21.0±2.9 21.3±6.1 (63.0) 63.0±17.6Br 6.82±0.87 8.44±5.04 (8.20) 8.13±4.18 (1300) 1280±820Ca (2500) 31500±1008 29700±8910 50500±900 50250±15080Ce 117.0±8.5 118.9±55.3 (0.28) 0.29±0.15 (2.00) 1.85±0.65Cl (10.00) (414) 414±103 (6600) 5860±1460Co 19.80±0.75 18.21±7.58 (0.020) 0.020±0.012 0.57±0.02 0.57±0.33Cr 104.0±4.5 111.6±37.5 0.80±0.20 0.77±0.20 1.99±0.06 1.9±0.5Cs 7.01±0.44 7.81±1.52 (0.098) 0.126±0.101 (0.05) 0.05±0.02Eu (1.60) 1.62±0.88 (0.010) 0.012±0.007 0.04±0.03Fe 67400±850 66800±16000 90.0±9.9 88.9±16.9 368.0±7.0 364±90Hf 4.16±0.29 4.50±0.51 (0.14)Hg (0.13) 0.08±0.02 0.08±0.03 0.034±0.004 0.033±0.021K (15000) 16040±190 18200±600 17880±12880 2700±500 2810±970La 52.6±1.6 54.2±10.2 (0.19) 0.19±0.15 (2.30) 2.63±1.55Lu (0.54) 0.56±0.17Mg (29000) 5800±302 5720±1160 (12000) 13040±4310Mn 3460±80 3650±120 23.0±2.0 20.4±0.4 246.0±8.0 251.0±5.0Na 1720±60 1630±40 160±21 175±22 136.0±4.0 142±20Nd 43.8±1.4 42.6±5.7 1.1±0.7 1.9±1.1Rb 113.0±5.5 122.9±28.4 4.8±0.1 4.7±1.3 14.9±0.3 14.4±2.8Sb 1.31±0.06 1.19±0.53 (0.040) 0.044±0.028 0.06±0.01 0.06±0.03Sc 17.30±0.55 16.59±5.52 (0.010) 0.011±0.004 (0.10) 0.09±0.04Sm 9.25±0.26 8.77±2.00 (0.052) 0.056±0.019 (0.19) 0.17±0.06Sn (4.00) 4.20±2.43 (0.24) 0.20±0.12 1.18±1.42Sr (80.0) 88.0±24.5 100.0±2.0 98.9±37.4 (85.0) 86.0±79.3Ta (1.58) 1.65±0.33Tb (1.40) 1.25±0.69 0.08±0.05 0.13±0.07Th 14.00±0.50 15.06±5.71 0.02±0.01 (0.12) 0.10±0.06Ti 5170±185 5050±200V 170.0±7.5 189.2±32.5 0.84±0.01

Yb 3.42±0.32 3.52±0.72Zn 223.0±5.0 233.0±47.8 29.0±2.0 25.8±8.6 30.9±0.7 30.9±9.2

Data in parenthesis implies information values.


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cussed in this context. In general, there was a significant difference between the total phenol and protein contents that differentiate the plants collected from protected sites respect the plants collected from unprotected ones. The elemental profile of soil samples in present work, were cat-egorized as macro（C, H, N and K）, micro（Cu, Fe, Mn, and Zn）, beneficial（Al, Co and Na）, other trace（As, Br, Cr, Cs, Hf, Pb, Rb, Sb, Sn, V and Th）and rare earth elements（Ce, Eu, La, Lu, Nd, Sc, Sm, Tb and Yb）. The elemental profile of all selected medicinal plants revealed the presence of

various macro（C, H, N, S, K and Ca）, micro（Cl, Cu, Fe, Mn and Zn）, beneficial（Al, Co and Na）, trace（As, Br, Cr, Cs, Hf, Pb, Rb, Sb, Sn, Sr, V and Th）and rare earth elements（Ce, Eu, La, Lu, Sc, Sm and Yb）indicating the importance of wild flora. Overall results depicted a noticeable variation in the concentration of all studied elements in the selected plants and their associated soil samples（Tables 3-5）.

3.1 Macro-elementsThe importance of carbon（C）assimilation in plant

Table 2　QA Data for Soil Samples.

ElementIAEA-SL1 IAEA-SDM-2/TM IAEA-S7

Certified Values Measured Values Certified Values Measured Values Certified Values Measured ValuesAl (89000) 91170±27890 32000±19900 38760±19230 47000±3500 47520±18220As 27.50±1.45 31.22±8.86 18.30±0.95 18.95±1.76 13.40±0.85 14.68±4.62Ba 639±27 635±178 252.0±39.5 159.0±32.5Br 6.82±0.87 8.44±5.04 65.70±10.30 67.21±18.70 7.00±3.50Ce 117.0±8.5 118.9±55.3 54.30±4.30 55.19±9.92 61.00±6.50 69.06±10.34Co 19.80±0.75 18.21±7.58 13.60±0.55 12.88±1.56 8.90±0.85 8.94±1.17Cr 104.0±4.5 111.6±37.5 77.2±9.4 83.3±16.7 60.0±12.5 66.0±12.9Cs 7.01±0.44 7.81±1.52 8.05±1.29 8.64±1.44 5.40±0.75 5.51±0.97Eu (1.60) 1.62±0.88 0.85±0.20 0.78±0.29 1.00±0.20 1.12±0.41Fe 67400±850 66800±16000 27100±1750 29220±3180 25700±550 25390±3170Hf 4.16±0.29 4.50±0.51 2.83±0.48 3.14±0.58 5.10±0.35 5.51±1.06K (15000) 16040±190 17600±2100 17500±6720 12100±700 11080±3360La 52.6±1.6 54.2±10.2 26.20±2.20 29.62±4.31 28.00±1.00 28.21±5.28Lu (0.54) 0.56±0.17 0.24±0.07 0.24±0.17 0.30±0.15 0.34±0.19Mg (29000) 29000±5200 29740±3260 11300±400 11400±930Mn 3460±80 3650±120 1170.0±45.0 1147.7±29.4 631.0±23.0 645.1±61.2Na 1720±60 1630±40 13500±1250 12970±720 2400±100 2510±420Nd 43.8±1.4 42.6±5.7 24.6±10.3 24.1±20.4 30.0±6.0 25.8±8.7Rb 113.0±5.5 122.9±28.4 99.7±14.5 51.0±4.5Sb 1.31±0.06 1.19±0.53 0.99±0.17 0.92±0.45 1.70±0.20 1.57±0.73Sc 17.30±0.55 16.59±5.52 10.30±0.75 10.51±1.47 8.30±1.05 8.76±1.17Sm 9.25±0.26 8.77±2.00 4.27±0.81 4.38±0.66 5.10±0.35 5.36±0.80Sn (4.00) 4.20±2.43 8.00±7.90 8.59±4.96 12.60±10.47Sr (80.0) 88.0±24.5 540.0±29.0 108.0±5.5Ta (1.58) 1.65±0.33Tb (1.40) 1.25±0.69 0.52±0.05 0.56±0.24 0.60±0.20 0.66±0.29Th 14.00±0.50 15.06±5.71 8.15±0.95 8.41±1.17 8.20±1.10 8.18±1.16Ti 5170±185 5050±200V 170.0±7.5 189.2±32.5 91.2±12.7 105.9±65.4 66.0±7.0 63.2±43.4

Yb 3.42±0.32 3.52±0.72 1.62±0.26 1.43±0.49 2.40±0.35 2.27±0.85Zn 223.0±5.0 233.0±47.8 74.8±3.2 70.3±16.9 104.0±6.0 89.0±38.2

Data in parenthesis implies information values.


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growth is inevitable as being a major component of photo-synthesis17）. In the present study, the mean values of C, ranged from 46.06％（Sophora mollis）to 37.30％（Peganum harmala）for protected and 43.68％（Sophora mollis）to 34.93％（Peganum harmala）for unprotected sites（Table 4）. The results obtained were in-line with the previously reported ones18）. Higher concentrations of C, from 45 to 51％ and from 45.75 to 55.05％ were, also, re-corded in other herbs, shrubs and tree species19）. Similarly, in soil samples, carbon concentration was found relatively higher（3.71％）in samples from protected sites then in samples from unprotected（2.61％）ones（Table 3）. These results provide an insight to understand mitigating role of protected area against climate change. Soil carbon in

closely co-related to the climatic conditions: if soil can store more carbon then, there it will be less CO2 in the at-mosphere18）.

Hydrogen（H）, another important element or macronutri-ent, in Sophora mollis and Peganum harmala, was mea-sured in a range between 6.19 and 5.02％ in protected samples and between 5.97 and 4.78％ in unprotected samples（Table 4）. Relatively higher H percentages were measured in the other medicinal plants20）. H concentration in soil samples from protected sites was 0.30％, whereas in unprotected sites it was 0.23％（Table 3）. The data ob-tained from carbon and hydrogen analyses have indicated the importance that protected areas have in terms of biodi-versity conservation as a long term strategy; in fact, the

Table 3　 Elemental composition of soil samples of protected and unprotected sites from Hazarganji Chiltan National Park（HCNP）of Balochistan, Pakistan by CHNS-O analyzer, FAAS and INAA.

ElementsCHNS-O and FAAS

ElementsINAA

Protected site Unprotected site Protected site Unprotected siteC(%) 3.71±0.72 2.61±0.65 Al (mg/kg) 29930±5610 28470±2220H(%) 0.30±0.10 0.23±0.10 As (mg/kg) 4.67±1.08 4.26±0.58N(%) 0.05±0.02 0.02±0.01 Br (mg/kg) ND 6.41±1.06S(%) ND ND Ce (mg/kg) 44.00±2.1 41.00±3.6

Cu(ppm) 13.43±0.71 15.00±0.01 Co (mg/kg) 11.23±0.50 10.20±0.44Pb(ppm) 2.49±0.70 2.50±0.71 Cr (mg/kg) 89.40±4.1 91.10±4.4

Cs (mg/kg) 3.63±0.18 3.43±0.14Eu (mg/kg) 0.91±0.02 0.90±0.03Fe (mg/kg) 26370±570 23320±410Hf (mg/kg) 4.71±0.31 5.78±0.23K (mg/kg) 15530±840 16100±1090La (mg/kg) 21.04±1.94 21.52±0.97Lu (mg/kg) 0.21±0.04 0.22±0.06Mn (mg/kg) 510±100 420±30Na (mg/kg) 7990±90 8060±160Nd (mg/kg) 27.20±7.0 20.30±5.0Rb (mg/kg) 67.80±10.4 60.20±8.1Sb (mg/kg) 0.70±0.23 0.80±0.02Sc (mg/kg) 8.71±0.34 8.00±0.11Sm (mg/kg) 4.51±0.07 4.06±0.18Sn (mg/kg) 8.43±1.81 8.77±1.99Tb (mg/kg) 0.69±0.15 0.89±0.08Th (mg/kg) 7.64±0.15 7.13±0.15V (mg/kg) 42.40±17.9 42.00±10.6

Yb (mg/kg) 1.60±0.18 2.84±1.25Zn (mg/kg) 113.50±9.1 103.40±6.5

Mean±Standard Deviation, N=3Concentrations in mg/kg on dry basis at 95% confidence interval, ND: Not Detected


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concentration of these two macro elements were found higher in both plants and associated soils collected from protected sites when compared to unprotected ones.

Sulfur（S）, as macronutrient, is a major constituent of amino acids including methionine, cysteine and cystine and is essential for plant’s normal growth and development21）. The mean values of S varied from 0.04％（Sophora mollis）to 0.62％（Peganum harmala）in plants from protected sites while, it varied from 0.01（Peganum harmala）to 0.40％（Perovskia atriplicifolia）in plants from unprotect-ed sites（Table 4）. These results were similar to those pre-viously detected20）.

Among macronutrients, nitrogen（N）is the most impor-tant element as it is a principal constituent of almost all amino acids, proteins and co-enzymes22, 23）. N percentage detected in soils from protected sites was 0.05％; its per-centage in soils from unprotected sites was 0.02％（Table 3）. A similar trend of total N content was previously ob-served24）. Similarly, N concentrations were in a range of 1.70％（Peganum harmala）to 4.93％（Nepeta praetervi-sa）in plants from protected sites and 1.32％（Peganum harmala）to 4.60％（Nepeta praetervisa）in plants from unprotected ones（Table 4）. Comparatively, lower amount of N was measured in medicinal plants by Obratov-Petković et al. in 200625）and, higher percentages were documented in other medicinal plants20）. Nitrogen uptake by plants not only depends on its availability in the external medium but, also, affects the uptake of various other elements. Accord-ingly, optimum supply of nitrogen to plants, ensures optimum uptake of other nutrients like K, P, Mg, Fe, Mn and Zn, which evidences N as a major limiting nutrient for plant growth and development26）. Nonetheless, similar trend of interactions was observed by the results of this re-

search.Potassium（K）was quantified in concentration of 15,530

mg/kg in soils from protected sites and 16100 mg/kg in soils from unprotected ones（Table 3）. These values were found to be higher when compared to the ones found in the liter-ature27）. However, the concentrations of K obtained for plants were in agreement with those reported by Jabeen et al. in 20108）and Volpe et al. in 201528）. In particular, K values ranged from 52620 mg/kg（Peganum harmala）to 8900 mg/kg（Sophora mollis）for the plants from protected sites and from 36690 mg/kg（Peganum harmala）to 8780 mg/kg（Sophora mollis）for the plants from unprotected ones（Table 5）. The concentration ranges for potassium observed in the medicinal plants under investigation were in-line with the ones already reported28－31）. However, higher and lower K contents in medicinal plants were docu-mented by Hassan et al. in 201532）and others33, 34）.

Calcium（Ca）is required in large amounts in plant cells, it has significant role in membrane stability along with other Ca-dependent metabolic processes35, 36）. Ca concentrations were found higher in plant samples collected from protect-ed sites than in plant samples collected in unprotected ones. In protected sites, the highest concentration of Ca was measured in Nepeta praetervisa（35,270 mg/kg）, while the lowest was found in Perovskia atriplicifolia（16,590 mg/kg）. In unprotected sites, a concentration range of 25,020 mg/kg（Hertia intermedia）to 13,700 mg/kg（Perovskia atriplicifolia）was measured（Table 5）. Similar concentration ranges were reported in other me-dicinal plants28, 31, 37）. Low and extremely high Ca concentra-tion were, also, reported in Pakistani medicinal plants33, 34）.

Table 4　 Elemental composition of selected medicinal plants of protected and unprotected sites from Hazarganji Chiltan National Park（HCNP）of Balochistan, Pakistan by CHNS-O analyzer and FAAS.

Elements

Plant species

Seriphidium quettense

Perovskia atriplicifolia

Achillea wilhelmsii

Hertiaintermedia

Peganum harmala

Sophora Mollis

Nepetapraetervisa

(a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b)

C(%)42.55±1.17

41.42±0.64

45.45±0.18

42.55±0.80

42.38±0.08

42.34±0.09

39.12±0.93

38.13±0.02

37.30±0.68

34.93±1.15

46.06±1.00

43.68±0.30

40.02±0.08

38.59±1.39

H(%)5.50±0.36

5.41±0.34

5.85±0.35

5.48±10

5.74±0.26

5.60±0.26

5.28±0.13

5.10±0.20

5.02±0.01

4.78±0.24

6.19±0.08

5.97±1.15

5.19±0.80

5.01±0.99

N(%)2.86±0.01

2.12±0.01

3.21±0.81

2.83±0.03

3.20±1.10

2.47±0.02

3.05±0.01

3.02±0.01

4.93±0.01

4.60±0.01

3.20±0.17

2.98±0.11

1.70±0.02

1.32±0.17

S(%)0.19±0.06

0.14±0.02

0.08±0.04

0.01±0.008

0.22±0.08

0.18±0.07

0.32±0.02

0.31±0.01

0.62±0.03

0.40±0.17

0.04±0.02

0.02±0.01

0.11±0.10

0.05±0.006

Cu(ppm)14.44±0.07

11.17±0.83

6.89±0.14

6.45±0.43

10.65±0.18

9.19±0.85

5.88±0.18

4.35±0.07

9.96±0.13

3.49±0.14

6.89±0.43

5.29±0.14

11.36±0.62

10.00±0.28

Pb(ppm)0.40±0.31

1.00±0.85

0.40±0.28

0.60±0.01

0.20±0.11

0.70±0.42

ND0.40±0.02

0.20±0.11

0.40±0.01

ND0.20±0.01

0.69±0.41

0.69±0.41

(a) Protected site (b) Unprotected site. Mean±Standard Deviation, N=3 ND: Not Detected


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Table 5　�Elemental composition of selected medicinal plants of protected and unprotected sites from Hazarganji Chiltan National Park（HCNP）of Balochistan, Pakistan by INAA.

Elements

Plant species

Seriphidium quettense Perovskia atriplicifolia Achillea wilhelmsii Hertia

intermedia Peganum harmala Sophora mollis

Nepeta praetervisa

(a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b)

Al(mg/kg) 1680±210

2550±250

1120±110

1150±570

1390±60

1760±300

1150±120

1280±40

380±80

800±60

550±70

760±60

2730±320

6610±680

As (mg/kg) ND ND ND ND ND ND ND ND ND ND ND 0.83±0.13 ND 1.70

±0.28

Br (mg/kg) 29.30±1.4

38.50±2.4

9.70±1.3

37.30±3.5

34.10±3.4

50.80±3.3

122.60±7.5

265.70±18.3

64.80±5.7

75.20±13.4

4.50±1.0

4.70±0.8

5.30±1.1

6.60±0.5

Ca (mg/kg) 17250±1470

14670±3870

16590±5080

13700±2790 ND ND 32380

±1432025020±10840

27820±7520

24290±12970 ND ND 35270

±1390 ND

Ce(mg/kg) 1.72±0.15

2.08±0.14

1.07±0.19

1.48±0.20

1.66±0.41

1.72±0.19

1.32±0.27

1.75±0.09

0.38±0.09

0.56±0.20

0.48±0.08

0.91±0.31

3.09±1.07

7.12±1.31

Cl(mg/kg) 10640±380

11600±20 ND ND 10820

±3010980±950

14030±220

16640±630

19140±230

32630±2250 ND ND 2710

±19302770±1150

Co(mg/kg) 0.49±0.03

0.60±0.03

0.29±0.02

0.38±0.04

0.50±0.08

0.50±0.04

0.72±0.09

0.92±0.06

0.23±0.01

0.33±0.05

0.16±0.03

0.20±0.01

0.60±0.03

1.44±0.09

Cr(mg/kg) 6.99±0.53

13.28±1.74

2.22±0.13

4.89±0.36

9.45±2.69

10.44±2.91

5.96±1.97

7.47±2.78

1.35±0.48

2.51±0.54

3.35±0.68

5.13±1.81

17.64±1.61

34.17±3.36

Cs(mg/kg) 0.13±0.04

0.20±0.06

0.13±0.04

0.11±0.03

0.10±0.014

0.09±0.011

0.07±0.007

0.07±0.012

0.04±0.007

0.08±0.03

0.09±0.03

0.06±0.02

0.50±0.16

0.21±0.07

Eu(mg/kg) 0.03±0.006

0.05±0.009

0.03±0.006

0.02±0.003

0.03±0.011

0.04±0.014

0.02±0.008

0.03±0.008

0.01±0.002

0.01±0.006

0.02±0.006

0.01±0.005

0.10±0.044

0.04±0.020

Fe(mg/kg) 1220±120

930±80

810±90

640±60

1160±120

1010±1740

810±80

720±110

393±15

300±40

491±30

352±51

3460±230

1360±55

Hf(mg/kg) 0.13±0.014

0.36±0.06

0.15±0.02

0.10±0.011

0.12±0.03

0.26±0.05

0.20±0.05

0.31±0.04

0.09±0.02

0.05±0.003

0.11±0.02

0.04±0.01

0.73±0.13

0.38±0.03

K(mg/kg) 32250±1980

25640±1810

35010±2040

27100±1580

29150±1120

36570±1450

255470±960

26200±790

52620±2400

36690±1750

8900±810

8780±460

16920±890

20000±1110

La(mg/kg) ND ND ND ND ND ND ND ND ND ND ND ND 5.50±0.67

2.34±1.07

Lu(mg/kg) ND ND ND ND ND ND ND ND ND ND ND ND ND 0.01±0.003

Mn(mg/kg) 97.40±4.5

92.00±10.3

53.90±3.5

51.70±16.9

108.60±1.2

90.60±8.3

59.50±0.7

56.30±0.4

85.70±3.6

61.90±1.0

41.40±1.7

30.10±1.7

101.30±7.8

59.20±6.8

Na(mg/kg) 4480±30

3660±140

1100±70

930±30

2600±90

980±30

3580±200

2570±100

14110±50

7700±360

300±30

150±10

1920±80

1080±50

Rb(mg/kg) 5.41±1.10

6.40±1.47

6.34±1.15

5.55±1.15

11.25±1.32

7.39±0.74

8.01±0.32

8.11±2.11

14.60±1.82

7.80±0.80

2.78±0.45

2.05±0.10

12.27±0.35

7.25±0.88

Sb(mg/kg) 0.04±0.002

0.04±0.001

0.04±0.002 ND 0.16

±0.100.03±0.02

0.07±0.05 ND 0.04

±0.020.03±0.01

0.03±0.01

0.04±0.01

0.16±0.01

0.05±0.01

Sc(mg/kg) 0.32±0.03

0.43±0.03

0.28±0.03

0.22±0.02

0.31±0.05

0.36±0.02

0.25±0.01

0.21±0.03

0.09±0.01

0.13±0.01

0.15±0.01

0.11±0.02

1.23±0.06

0.47±0.01

Sn(mg/kg) 0.15± 0.02 ND ND 0.11

±0.01 ND ND ND ND ND ND ND ND ND ND

Sm(mg/kg) ND ND ND ND 0.06±0.02 ND ND ND ND ND ND ND ND ND

Sr(mg/kg) 41.80±9.7

47.20±12.8

140.50±31.9

41.20±9.4

121.50±11.9

72.70±8.6

105.40±10.2

148.00±18.2

117.60±17.3

91.70±10.0

65.70±8.2

47.50±5.7

103.40±12.6

78.80±13.7

Th(mg/kg) 0.29±0.05

0.37±0.05

0.21±0.05

0.17±0.04

0.25±0.06

0.38±0.01

0.21±0.02

0.20±0.05

0.07±0.01

0.08±0.01

0.14±0.01

0.09±0.01

1.09±0.05

0.44±0.04

V(mg/kg) 3.62±1.31

4.29±0.06 ND ND ND ND ND ND ND ND ND ND 10.92

±2.70 ND

Yb(mg/kg) ND ND ND ND ND ND ND ND ND ND ND ND 0.23±0.06

0.07±0.01

Zn(mg/kg) 24.30±0.7

23.20±0.7

53.00±1.7

61.30±39.6

157.10±30.8

174.50±12.8

121.90±5.9

102.90±15.8

35.80±7.6

45.10±28.6

42.80±34.3

33.80±28.7

329.10±312.3

140.00±107.2

(a) Protected site (b) Unprotected site. Concentrations in mg/kg on dry basis at 95% confidence interval, ND: Not Detected


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3.2 Micro-elementsChlorine（Cl）, an essential micronutrient for plants, is in-

volved in the splitting of water molecules during photosyn-thesis38）and in the enhancing of the evolution of oxygen and photophosphorylation39）. The concentration values of Cl found in the plant samples collected from protected sites were ranged from 19,140 mg/kg（Peganum harmala）to 2,710 mg/kg（Nepeta praetervisa）. The concentration values of Cl found in the plant samples collected from un-protected sites were from 32,630 mg/kg（Peganum harmala）to 2,770 mg/kg（Nepeta praetervisa）（Table 5）. Despite the fact that Cl concentration was relatively lower in plant samples collected from protected sites, the overall concentration range for Cl was much higher when com-pared with previous research on other medicinal plant species30, 40）.

Copper（Cu）is associated with many photosynthetic enzymes in plants41）but, the exact function is still unknown. In addition, Cu regulates alkaloid accumulation in medici-nal plants and restrains ethylene biosynthesis42）Cu content ranged between 14.44 ppm（Seriphidium quettense）to 5.88 ppm（Hertia intermedia）in plants collected from protected sites and 11.17 ppm（Seriphidium quettense）to 3.49 ppm（Hertia intermedia）in plants collected from un-protected ones（Table 4）. According to Mishra et al. in 201221）Cu content varies between 2-20 ppm, which it is in agreement with our results. China and Singapore have set, in a previous work, the permissible limits for Cu in medici-nal plants（20-150 ppm）43）. Comparing our results with those limits, the Cu content found in the medicinal plants under investigation resulted to be below the lower limit. Moreover, our results were in agreement with those found in the available literature32, 44）. Higher concentration ranges in medicinal plants were, also, reported45－49）. Cu level in soil samples collected from protected site was 13.43 ppm, whereas in soils collected in unprotected sites was 15.00 ppm（Table 3）. All the values were lower than the values found in the literature28, 44）.

Iron（Fe）is one of the most well known element associat-ed to the activity of enzyme related to the functioning of the heme50）. The concentrations of Fe found in the plants collected from protected sites were in a range of 3460 mg/kg（Nepeta praetervisa）and 393 mg/kg（Peganum harmala）, while plants collected from unprotected sites were in a range of 1360 mg/kg（Nepeta praetervisa）and 300 mg/kg（Peganum harmala）（Table 5）. Lower Fe con-centrations were reported in the literature34, 53, 51）. The per-missible limit for edible plants was set at 20 mg/kg52）; no limits are, however, set for medicinal plants. Similar or even higher ranges of Fe were detected in other medicinal plant8, 47, 49, 53）. Concerning the soil samples, relatively high amounts of Fe have been detected in samples collected in protected sites（26,370 mg/kg）compared to the samples collected in unprotected ones（23,320 mg/kg）（Table 3）.

These results strongly contradict the results previously re-ported by other authors44, 54）, but give a logical reason of the presence of this element in the plants associated with these soils.

Manganese（Mn）, along with other essential elements, is also required by higher and lower plants for oxygen evolu-tion as being a part of catalytic mangano-protein complex55）. The optimal level of Mn content required for normal growth of medicinal plants such as Mentha spicata was found at 2.5 mg/kg, beyond which a significant decline in growth and development was detected56）. From the data obtained, the maximum level of Mn content was found in Achillea wilhelmsii（108.60 mg/kg）, whereas the minimum level was found in Sophora mollis（41.40 mg/kg）both col-lected in protected sites. In unprotected areas, the value of Mn detected was in a range of 92.00 mg/kg（Seriphidium quettense）and 30.10 mg/kg（Sophora mollis）（Table 5）. Considering the permissible limit of Mn being 2 mg/kg for edible plants52）, these results indicate toxic levels of this element in the medicinal plants under observation. Al-though for medicinal plants the limit is not established yet, it is noteworthy to explore the levels of Mn in medicinal plants as these are consumed by most of the poor rural communities in developing countries. The results obtained in this work agreed with the results previously reported in the literature8, 47）in fact, relatively higher levels of Mn were observed by other researchers in medicinal plants31, 57）and comparatively low concentrations were documented in some medicinal plants28, 44, 58）. For soils, Mn concentration was 510 mg/kg in samples collected in protected sites and 420 mg/kg in samples collected in unprotected ones（Table 3）. These results were in agreement with the Mn concen-tration range previously reported54）. However, other ranges of values for this element has been reported as well27, 44）.

Zinc（Zn）is another essential micronutrient which role, in plant growth, is the promoting of carbon incorporation and consumption in terpene biosynthesis, saccharide accu-mulation, free radical confiscation and activation of antioxi-dant enzyme. The concentrations of Zn detected in plants collected from protected sites were between 329.10 mg/kg（Nepeta praetervisa）and 24.30 mg/kg（Seriphidium quettense）. Plants from unprotected sites showed values of Zn of 174.50 mg/kg for Achillea wilhelmsii and 23.20 mg/kg for Seriphidium quettense（Table 5）. Relatively higher concentrations of Zn were found in plant samples when compared to the results of other medicinal plants8, 27, 28, 31, 33, 34, 37, 46, 47, 51, 53, 58）. The permissible limit of Zn in edible plants was set at 27.40 mg/kg52）. With the excep-tion of Seriphidium quettense, all plants investigated were found to accumulate Zn, but no limits are established for medicinal plants yet43）. For soils, the concentration of Zn was 113.50 mg/kg for samples collected in protected sites and 103.40 mg/kg for samples collected in unprotect-ed ones（Table 3）. Both lower and higher levels of Zn were


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452

documented by other researcher27, 44, 54, 59）.

3.3 Bene�cial elementsAlthough some evidences have shown that aluminum

（Al）, in low concentrations, is favorable for the growth and development of plants, the exact mechanism is not well clear60）. Al concentrations, among the investigated medici-nal plant samples collected from protected sites, were found in high levels（2,730 mg/kg）in Nepeta praetervisa and low levels（380 mg/kg）in Peganum harmala. In me-dicinal plant samples collected from unprotected sites, Al were found in higher levels（6,610 mg/kg）in Nepeta praetervisa and low levels in（760 mg/kg）in Sophora mollis（Table 5）. Relatively lower concentrations of Al in medicinal plants were also reported61）. For soils, the samples collected from protected sites, showed somewhat lower amounts of Al（28,470 mg/kg）when compared to samples collected from unprotected sites（29,930 mg/kg）（Table 3）. The concentration ranges of the detected Al values were much lower than that found in the literature54）. Considering that the daily intake of Al by an adult must be in a range of 7-10 mg/day, the results obtained in this study were quite alarming as the results previously recorded by other authors31）. High levels of Al in plant cells may cause many detrimental effects as it may interfere with phos-phate metabolism and various other enzymatic processes62）. Al may, also, retard Fe uptake specially interfere with Mg＋ ion63）, while in human bodies, high doses of Al can cause very serious disorders like neurological dementia and Par-kinson disease64）.

Cobalt（Co）is considered as a vital element as being part of many enzymes and co-enzymes and may influence the plant’s metabolism to different degrees depending upon its prominence and levels in the associated soils65）. A typical range of Co in soil may vary from 0.3-20 mg/kg66）. The results obtained showed a Co concentration of 11.23 mg/kg in soil samples from protected sites and 10.20 mg/kg from unprotected sites（Table 3）. These values were lower than those previously reported54）. As the absorption and uptake of Co from the soil to the plants is controlled by different mechanism, every plant species may assimilate different concentration of Co65）. In the investigated medicinal plants, Co concentration varied from 0.72 mg/kg（Hertia interme-dia）to 0.16 mg/kg（Sophora mollis）for samples collected from protected sites, and from 1.44 mg/kg（Nepeta praetervisa）to 0.20 mg/kg（Sophora mollis）for samples collected from unprotected sites（Table 5）. These concen-trations were in agreement with those previously reported for other medicinal plants31）and were relatively higher respect to those reported for some other Pakistani medici-nal plants34, 46－48, 53）. Although, yet no standard limits have been set for Co in medicinal plants. Evidences showed that Co, if accumulated in relatively higher concentrations, has adverse effects on plant morphology67）. Elevated levels of

Co in plant interfere with Fe and Cd inducing Fe deficiency and suppressing Cd uptake by roots68）.

Sodium（Na）is an essential element which plays a vital role along with K and Cl for normal distribution of fluids in both extra and intra-cellular cells of the body while serving as an electrolyte. Decrease in biomass, photosynthetic rate and protein content was observed in some medicinal plants with increase of Na concentration69）. From the results ob-tained in this work, Na/K ratio was found low enough in all medicinal plants analyzed indicating adequate and bal-anced quantities of these essential elements. Na concentra-tion ranged between 14,110 mg/kg（Sophora mollis）to 300 mg/kg（Sophora mollis）for samples collected from pro-tected sites and, 7,700 mg/kg（Peganum harmala）to 150 mg/kg（Sophora mollis）for samples collected from unpro-tected sites（Table 5）. Lower concentrations were found by other authors28, 31, 33, 34, 53）. For soils, Na concentration was 7,990 mg/kg in samples collected from protected sites and 8,060 mg/kg for samples collected from unprotected ones（Table 3）. These results were relatively higher than those found in the literature54）.

3.4 Other trace elementsArsenic（As）is a toxic element that was detected in me-

dicinal plants samples collected from some unprotected sites. In Sophora mollis and Nepeta praetervisa its con-centration was found at a level of 0.83 mg/kg and 1.70 mg/kg, respectively（Table 5）. These values were higher than those found in the literature31, 53）. In the associated soil samples, the concentration of As was 4.26 mg/kg and 4.67 mg/kg, for samples collected from protected and unpro-tected sites, respectively（Table 3）. These concentrations strongly agreed with those available in the literature54）. Ac-cording to the Swedish Environmental Protection Agency, As in soil should be below 10 mg/kg70）. The presence of As in soils and edible plants is of great concern due to its extreme toxic nature.

The levels of Chromium（Cr）detected in this study were relatively high: 91.10 mg/kg of Cr were detected in soils collected in unprotected sites and 89.40 mg/kg of Cr were detected in soils collected in protected sites（Table 3）. All these results were in disagreement with the levels of Cr re-ported from other authors44, 59）. A typical range of Cr in soil is from 0.9 mg/kg to 1,500 mg/kg66）. Comparing our values with these values, the levels of Cr in the soils under inves-tigation are below the safe limits. Similarly, plants growing in protected sites exhibited values in a range of 17.64 mg/kg（Nepeta praetervisa）to 1.35 mg/kg（Peganum harmala）and 34.17 mg/kg（Nepeta praetervisa）to 2.51 mg/kg（Peganum harmala）if they were collected from un-protected sites（Table 5）. The results were in-line with the findings of other researchers8, 34）and disagreed with other published work on medicinal plants31, 37, 44, 57, 59, 71）. The con-centration of Cr in plants under normal conditions is


J. Oleo Sci. 68, (5) 443-461 (2019)

453

usually less than 1.0 mg/kg72）. Therefore, the plants under investigation have found to have toxic levels of this trace element but, no standard limits of Cr concentration have been established for medicinal plants. Cr in general is a toxic metal and can cause extreme damage to plants by suppressing antioxidant enzyme and thus inducing oxida-tive stress inside plant tissues and declining antioxidant properties of the plants73）. A medicinal plant under Cr stress may have a low antioxidant efficacy and may not justify well with its therapeutic properties when consumed for a particular ailment.

Cesium（Cs）is present in earth’s Crust as trace element in very low quantity（3 mg/kg）and is absorbed by plant roots. Although exact role of Cs is not known inside plant metabolism, it may inhibit plant development if assimilated in large concentrations74－76）. From the results obtained in this study, samples collected from protected sites showed a concentration of Cs between 0.13 mg/kg（Perovskia atri-plicifolia and Seriphidium quettense）and 0.04 mg/kg（Peganum harmala）. In samples collected from unpro-tected sites its range varied from 0.21 mg/kg（Nepeta praetervisa）to 0.06 mg/kg（Sophora mollis）（Table 5）. These results were in agreement with those previously re-ported for other medicinal plants31）, while higher levels were also documented53）. The concentration of Cs in the soils collected from protected and unprotected sites was 3.63 mg/kg and 3.43 mg/kg, respectively（Table 3）.

The levels of Hafnium（Hf）detected in the samples ana-lyzed were much lower than those reported in the litera-ture77）. Hf was found at 4.71 mg/kg in soils collected from protected sites and 5.78 mg/kg in soils collected in unpro-tected sites（Table 3）. Among the selected medicinal plants, Hf was found in a range between 0.73 mg/kg（Nepeta praetervisa）and 0.09 mg/kg（Peganum harmala）in samples collected from protected sites and between 0.38 mg/kg（Nepeta praetervisa）and 0.04 mg/kg（Sophora mollis）in samples collected from unprotected sites（Table 5）. The lower levels of Hf, were in agreement while higher levels disagreed with the results found by others31）.

Lead（Pb）is present abundantly in our environment but, is actually a non essential rather than a toxic element. The soils analyzed showed a content of Pb of 2.49 ppm and 2.50 ppm if collected from protected and unprotected sites, re-spectively（Table 3）, while higher concentrations were found in the literature44）. The plants analyzed showed a content of Pb between a range of 0.69 ppm and 0.20 ppm for samples collected from protected sites and between a range of 1.00 ppm and 0.20 ppm for samples collected from unprotected sites（Table 4）. The permissible limit of Pb for medicinal plant is set at 10 ppm43）therefore, all the medicinal plants under investigation exhibit a lower level of this element. These results agreed with those reported by other authors58, 78）

and disagreed while those detected by others8, 31, 34, 47）.

Rubidium（Rb）content ranged between 14.60 mg/kg, in Peganum harmala, and 2.78 mg/kg, in Sophora mollis, when the samples were collected from protected sites and between 8.11 mg/kg, in Hertia intermedia, and 2.05 mg/kg, in Sophora mollis, when the samples were collected from unprotected sites（Table 5）. The overall range is still much lower when compared to the results previously de-tected27, 31, 49, 59）. In the soil samples collected from protect-ed and unprotected sites, Rb concentrations were found to be 67.80 mg/kg and 60.20 mg/kg, respectively（Table 3）. These results were lower when compared with the concen-trations reported by Kogo et al. in 200954）but higher when compared with the concentrations reported by other authors27, 59）.

Antimony（Sb）is a trace element present in very low concentrations in natural environment. In earth’s crust its concentration range is 0.2-0.3 mg/kg and, in general is less than 1.0 mg/kg79, 80）. Sb contents in soil samples collected from protected and unprotected sites were 0.70 mg/kg and 0.80 mg/kg, respectively（Table 3）. Higher concentration of Sb may be attributed to the anthropogenic sources. In the medicinal plants under investigation, Sb was found in a range of 0.16 mg/kg in Achillea wilhelmsii and Nepeta praetervisa and 0.03 mg/kg in Sophora mollis when the plants were collected from protected sites, and between 0.05 mg/kg in Nepeta praetervisa and 0.03 mg/kg in Achil-lea wilhelmsii and Peganum harmala when the samples were collected from unprotected ones（Table 5）. The results obtained were in agreement with those present in the literature31, 49）. Although the levels of Sb that have been detected in the samples were lower enough, the biological function of this trace element inside living tissues is not well known and it could be toxic or even carcinogenic when absorbed at elevated levels81）.

The results obtained for bromine（Br）and strontium（Sr）are shown in Table 5 for medicinal plants. Among all the investigated medicinal plants collected from protected sites, Br and Sr concentrations were higher in Hertia in-termedia and Perovskia atriplicifolia（122.60 mg/kg of Br and 140.50 mg/kg of Sr, respectively）. The lowest levels were detected in Sophora mollis（4.50 mg/kg of Br and 65.70 mg/kg of Sr）. Similarly, in the samples collected from unprotected sites, higher levels were found in Hertia in-termedia（265.70 mg/kg of Br and 148.00 mg/kg of Sr）, while lowest levels were detected in Sophora mollis and Perovskia atriplicifolia（4.70 mg/kg of Br and 41.20 mg/kg of Sr）. Br concentrations were found moderate to ex-tremely high when compared with the results previously reported31, 49, 81）. Sr concentrations agreed with the results of other researchers27, 49, 71）. Regarding the soils, Sr was not detected in any of the samples and Br was found in samples collected only from unprotected sites at a level of 6.41 mg/kg（Table 3）.

Tin（Sn）was detected at a concentration of 8.43 mg/kg in


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the soil samples collected from protected sites, and 8.77 mg/kg in the soil samples collected from unprotected ones（Table 3）. These concentrations were lower than those found in the literature83）. In the plants, Sn was detected only in two samples collected from unprotected sites: in Seriphidium quettense at a concentration of 0.15 mg/kg, and in Perovskia atriplicifolia at a concentration of 0.11 mg/kg（Table 5）.

Vanadium（V）is considered among the twenty most abundantly found element in Earth’s mantle along with copper and lead, but its importance in human health is still unknown84）, even if an excess levels of this element may cause cancer85）. In the soils analyzed, V concentrations were 42.00 mg/kg for the samples collected from protected sites, and 42.40 mg/kg for the samples collected from un-protected ones（Table 3）. According to the European Union, the established safe limit for V in soil is between 90 mg/kg and 500 mg/kg86）therefore, the soils under investiga-tion were well below these values. In the plants analyzed, V concentrations was detected in Seriphidium quettense at a concentration of 3.62 mg/kg when collected from pro-tected sites, and 4.92 mg/kg when collected from unpro-tected ones, and in Nepeta praetervisa collected from un-protected sites at a concentration of 10.92 mg/kg（Table 5）. The results were in agreements with those previously re-ported71）. However, our results were relatively higher when compared with those in the literature31, 49, 87）.

Thorium（Th）is widely distributed in natural environ-ment and, as a radionuclide, is one of the major component of biosphere present in almost all soils and plants. Its con-centration in soil varies depending upon soil type and site of collection, parent rock material, climatic conditions and seasonal variations; however, a typical range of concentra-tion is from 2 mg/kg to 12 mg/kg77）. Soils collected from protected sites showed a concentration of Th of 7.64 mg/kg; the samples collected from unprotected sites showed a concentration of Th of 7.13 mg/kg（Table 3）. These values cascade almost to the average range of normal concentra-tion of Th. Likewise, in plants Th concentration also varies widely due to many factors including soil type, plant species, plant tissues, concentration in soil, chemical form in soil etc88）. In the medicinal plants collected from pro-tected sites its concentration varied in a range of 1.09 mg/kg, for Nepeta praetervisa, and 0.07 mg/kg, for Peganum harmala. For samples collected from unprotected sites, Th concentration varies from 0.44 mg/kg, for Nepeta praetervisa, and 0.08 mg/kg, for Peganum harmala（Table 5）. These results agreed with those found by other authors49）.

3.5 Rare earth elementsRare earth elements（REEs）are also trace metals and,

unlike their name, are widely spread in geosphere. In the soils under investigation, nine REEs（Ce, Eu, La, Lu, Nd,

Sc, Sm, Tb. Yb）were quantified. Ce, Eu, Nd, Sc and Sm were found in slightly higher concentrations in samples collected from protected sites samples than in samples col-lected from unprotected sites, whereas La, Lu, Tb, Yb were detected in slightly higher amount in samples collected from unprotected sites than in samples collected from pro-tected sites（Table 3）. Seven REEs（Ce, Eu, La, Lu, Sc, Sm Yb）were identified and quantified in medicinal plants growing on protected and unprotected soils. Furthermore, it was evident from the results that Ce, Eu and Sc were all found in the investigated medicinal plants showing a varia-tion in their levels. In Nepeta praetervisa and Peganum harmala collected from protected sites, the concentra-tions of Ce varies from 3.09 mg/kg to 0.38 mg/kg, the con-centrations of Eu varies from 0.10 mg/kg to 0.01 mg/kg and the concentrations of Sc varies from 1.23 mg/kg to 0.09 mg/kg, respectively. These concentrations, in Nepeta praeter-visa and Peganum harmala collected from unprotected sites were as follows: for Ce was from 7.12 mg/kg to 0.56 mg/kg, 0.05 mg/kg to 0.01 mg/kg for Eu and 0.47 mg/kg to 0.11 mg/kg for Sc（Table 5）. La and Yb were detected in Nepeta praetervisa in concentrations of 5.50 mg/kg and 0.23 mg/kg for samples collected from protected sites while, 2.34 mg/kg and 0.07 mg/kg for samples collected from unprotected sites. Lu was detected in Nepeta praetervisa collected from unprotected sites in a concen-tration of 0.01 mg/kg（Table 5）. REEs have a wide range of positive effects on overall growth of root system, seed ger-mination, biomass and secondary metabolite production and absorption of mineral nutrients and other metals in medicinal plants89）. Similarly, like other trace elements, REEs may cause phyto-toxicity when accumulated in large amounts90）. As reported in the literature, absorption of REEs by root system may also regulate or alter the absorp-tion of other mineral elements91）. REEs must be identified and quantified thoroughly in medicinal plants in order to get a clear picture before they can be recommended for therapeutic purposes. From the results obtained it was evident that a variation in the elemental profile between samples collected from protected and unprotected sites in-dicating a relationship between concentration of elements and their sampling locations. Moreover, dissimilarities in the elemental concentration of plant samples collected from the same site clearly decipher that the elemental profile of each plant is conditional and is attributed to the plant genotype, physical and chemical properties of soil and ability of plant to absorb selective elements92）.

3.6 Spatial variation analysis of elements in selected medicinal plants using a multivariate statistical tool - Principal component analysis

To examine multidimensional overview of a multivariate data, it is crucial to use an exploratory tool like principal component analysis（PCA）. PCA can divulge hidden struc-


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tures of a large data set, and thus make it possible to iden-tify the potential outcome more precisely93）. In the present work, all data were divided in to two groups for PCA. In particular, elements with high concentration were named as Major Elements, and elements with relatively low con-centrations were named as Trace and Rare Earth Elements.

3.7 Principal component analysis of Major ElementsThe elemental concentrations of Major Elements（C, H, S,

N, K, Ca, Al, Fe, Cl and Na）were used as variables in the PCA. By extracting the principal components（PCs）with significant Eigen values greater than 1, three principal components were produced. PC1 with highest Eigen value of 4.34 was explaining the maximum variability in the ob-tained set of data followed by PC2（2.87）and PC3（1.64）. These three principal components contributed for 80.38％ of the total variance. The first, second and third compo-nents showed a variance of 39.44％, 26.06％ and 14.88％, respectively. PCA Euclidean bi-plot, in Fig. 1, shows the spatial variability between the Major Elements, indepen-

dent variables, and the medicinal plants investigated de-pendent variables. PCA variable loadings showed a positive correlation on PC1 with C and H, and negative ones with Na, Cl, S and N. PC2 was positively correlated with Fe and Ca. PC3 showed a positive correlation with Al, N, H and C. From PCA case score, it is evident that Perovskia atri-plicifolia and Sophora mollis samples collected from pro-tected sites and Perovskia atriplicifolia and Sophora mollis collected from unprotected sites, have the highest concentration of C and H. All the other plant samples showed lower amount of these two major elements. From the scores of PC2, it is evident that Nepeta praetervisa collected from protected sites, followed by Nepeta praetervisa collected from unprotected sites, have the highest concentration of Fe and Ca. All the other plant samples showed lower amount of these two elements. PCA Euclidean bi-plot divided the medicinal plants in three main groups. The first group is composed by Perovskia at-riplicifolia, Sophora mollis, Seriphidium quettense and Achillea wilhelmsii, all being correlated with C and H.

Fig. 1　 2D Euclidean Biplot of PCA showing spatial compositional variability in Major Elements in selected medicinal plants of Hazarganji Chiltan National Park. 1. Seriphidium quettense（Protected）2. Seriphidium quettense（Unprotected）3. Perovskia atriplicifolia（Protected）4. Perovskia atriplicifolia（Unprotected）5. Achillea wilhelmsii（Protected）6. Achillea wilhelmsii（Unprotected）7. Hertia intermedia（Protected）8. Hertia intermedia（Un-protected）9. Peganum harmala（Protected）10. Peganum harmala（Unprotected）11. Sophora mollis（Protected）12. Sophora mollis（Unprotected）13. Nepeta praetervisa（Protected）14. Nepeta praetervisa（Unprotected）


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The second group is composed by Peganum harmala and Hertia intermedia, correlated with K, Na, N, S and Cl and, the third group is composed by only one sample, Nepeta praetervisa, correlated with Al, Fe and Ca.

3.8 Principal component analysis of Trace and Rare Earth Elements

PCA was applied to the whole set of Trace and REEs ele-ments in order to get a clear explicable overview of the main information. Principal components（PCs）with Eigen values greater than 1 were extracted which led to the for-mation of five PCs. PC1 had the highest Eigen value, 12.16; PC2, PC3, PC4 and PC5 were 3.28, 3.05, 1.85 and 1.17, re-spectively. All five principal components accounted for 89.62％ of the total variance. In particular, PC1 showed the maximum variation in the data set representing 50.66％, PC2 accounted for 13.67％, PC3 for 12.69％, PC4 for 7.72％ and PC5 for 4.87％. Spatial compositional variability in Trace and Rare Earth Elements in selected medicinal plants was represented in PCA Euclidean bi-plot（Fig. 2）. PCA variable loadings explained a positive correlation on PC1 with Ce, Co, Cr, Cs, Eu, Hf, La, Sc, Th, V, Yb and Zn, while a negative correlation was shown with Br and Sn.

Similarly, PC2 was positively correlated with Cu and Pb. From case scores of PC1, it is evident that the concentra-tion of all trace elements, with the exception of Br and Sn, was higher in Nepeta praetervisa collected from protected sites, followed by the same variety collected from unpro-tected sites. Similarly, case scores of PC2 showed that Cu and Pb were higher in Seriphidium quettense collected from protected sites, followed by Seriphidium quettense, Nepeta praetervisa, Perovskia atriplicifolia and Sophora mollis collected from unprotected sites. PCA Elucidean bi-plot divided the medicinal plants in three main groups. The first group contains Seriphidium quettense and Achillea wilhelmsii, correlated with Cu, Pb, Mn, Sm, Sn, Sb and Rb. The second group contains Perovskia atriplicifolia, Sophora mollis, Peganum harmala and Hertia intermedia, correlated with Sn and Br. The third, finally, includes only Nepeta praetervisa, correlated with As, Ce, Co, Cr, Cs, Eu, Hf, La, Lu, Sb, Sc, Th, V, Yb and Zn. The overall PCA results showed that rela-tively higher concentration of essential, trace and REEs like C, H, Fe, Ca, Ce, Co, Cr, Ce, Eu, Hf, La, Sc, Th, V, Yb and Zn distinguished the samples collected from protected sites. PCA, further, revealed that the elemental composi-

Fig. 2　 2D Euclidean Biplot of PCA showing spatial compositional variability in Trace and REEs Elements in selected medicinal plants of Hazarganji Chiltan National Park.1. Seriphidium quettense（Protected）2. Seriphidium quettense（Unprotected）3. Perovskia atriplicifolia（Protected）4. Perovskia atriplicifolia（Unprotected）5. Achillea wilhelmsii（Protected）6. Achillea wilhelmsii（Unprotected）7. Hertia intermedia（Protected）8. Hertia intermedia（Unprotected）9. Peganum harmala（Protected）10. Peganum harmala（Unprotected）11. Sophora mollis（Protected）12. Sophora mollis（Unprotected）13. Nepeta praetervisa（Protected）14. Nepeta praetervisa（Unprotected）


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tion of medicinal plants may be influenced by many factors including plant species, origin, seasonal variations, climatic and edaphic conditions. Moreover, from this work, it was evident that Nepeta praetervisa is a potential outlier due to its accumulation affinity for trace and REEs.

4 CONCLUSIONGood quality control of medicinal plants is essential as

these are consumed without any processing and restric-tions and, thus, may cause hazardous effects. The results obtained in this study have showed a wide variability of macro, micro and other trace elements in samples collected from both protected and unprotected sites. This study has revealed that all selected medicinal plant species were a good source of many essential elements such as K, Ca, Na, Fe, etc., however, significantly toxic levels of some trace elements were also documented, which may cause various other health hazards in human if consumed for long period of time. Overall findings agreed to the fact that elemental composition of medicinal plants was conditional and may influence by multiple factors including soil as a prime source of trace and REEs followed by air and water serving as the major source of some macro elements. Any contami-nation/pollution by anthropogenic contributions or natural processes altering elemental composition of soil, air or water ultimately influences elemental profile of plants which may attribute to the differences in elemental profile of same plant species of different sites. Differences in the elemental profile of different species of same site further indicated plant genotype and absorption affinity towards a particular element and group of elements as another im-portant factor that may influence elemental profile of me-dicinal plants. Thus, external factors such as soil composi-tion and environmental dispersion of elements and internal factors such as plant genotype and selective absorption ability may be considered as determining factors of ele-mental profile of medicinal plants. Furthermore, in plants from protected sites, relatively good quantities of essential elements were found compared to those of samples col-lected from unprotected sites. The latter, have shown a comparatively higher affinity in accumulating some REEs and potentially dangerous elements such as Pb and As. These levels, although in relatively low concentrations, can highlight the positive impact that the national park has on plants. In fact, they can be considered medicinal plants with potential therapeutic effects. Furthermore, having protected sites contributes to the conservation of plant biodiversity. To the best of authors’ knowledge, this is for the very first time that such a comprehensive elemental analysis of medicinal plants and their associated soils from Balochistan, a South West province of Pakistan, have been carried out. As ethno-medicinal plants are one of the most

valuable assets of the region, there is a dire need to investi-gate and study the best management practices to endure the impacts of anthropogenic and climatic catastrophes and to provide the local communities with greater benefits on sustained basis.

ACKNOWLEDGEMENTSWe are thankful to the Chemistry Division, Directorate of

Science PINSTECH, Islamabad for its help during the ana-lytical procedures and to Higher Education commission, Pakistan, for financial assistance through MS leading to PhD（Indigenous）scholarship program.

Supporting InformationThis material is available free of charge via the Internet

at http://dx.doi.org/jos.68.10.5650/jos.ess.19004

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Elemental Characterization of Medicinal Plants and Soils