zingiberaceae , one of the largest families of the plant kingdom , is an important natural resource that provides many useful food products , spices , and traditional medicines to treat a variety of diseases [ 1 , 2 ] .
the consumption of herbal products for therapeutic purposes and to promote wellness is widely popular since people are greatly concerned about side effects of synthetic drugs .
herbs are being increasingly used in the pharmaceutical industry as raw materials for the preparation of herbal medicines .
arsenic is widely distributed in the earth 's crust and present at an average concentration of 2  mg / kg . around one - third of atmospheric flux of arsenic
arsenic is one of the first chemicals designated as a group 1 carcinogen   and well known to be poisonous to organisms .
the inorganic arsenic species ( as(iii ) and as(v ) ) are the most toxic forms of arsenic present in food .
previous studies have indicated that ingested inorganic arsenic is strongly associated with a wide spectrum of adverse health outcomes , primary cancers , and other chronic diseases .
the daily intake of total arsenic from food and beverages is generally in the range of 20300  g / day .
furthermore , joint food and agriculture organization / world health organization expert committee on food additives note that benchmark dose lower confidence limit for a 0.5% ( bmdl 0.5 ) of inorganic arsenic in human was 3.0  g / kg body weight per day .  
root or rhizome is a part of the plant that has high opportunity to contaminate with arsenic .
current research demonstrated that plants absorbed heavy metals from soil [ 12 , 13 ] .
the contaminants and residues of toxic metal arsenic may cause harm to the consumers of herbal medicines .
plants that grow in an arsenic - affected area may have a high level of arsenic .
the transfer of arsenic from soils to plants might be a key step in the route of arsenic entry into human body [ 14 , 15 ] .
experimental data had shown that a variety of vegetable crops accumulate arsenic by root uptake from soil deposited on the leaves , and gulz et al .   reported several edible plants grown in contaminated soils that accumulated high levels of arsenic .
the who   determined that arsenic concentration in plants grown in soils without arsenic - containing pesticides varied from 0.02 to 5  mg / kg ( dry weight ) , and in the arsenic soil contamination indicated root can contain higher levels of arsenic than other parts of plant .  
a previous study about rhizome of zingiber officinale ( ginger ) shows that arsenic level varied from not detected to 0.13  g / l .
other arsenic contamination reports of karadas and kara   show that arsenic levels in cumin and turmeric were 174  14 and 39  5  ng / g , dry wt , respectively .
vegetable crops in the contaminated region were found high in arsenic level ( dry wt ) in arum ranged from 74.3 to 89.2  mg / kg , in cabbage from 27.12 to 39.39  mg / kg and in pumpkin from 17.28 to 22.05  mg / kg . baroni et al .   analyzed sixty - four plant species , and the highest arsenic contents
investigated high arsenic levels in cumin and turmeric ( 47.86209.75 and 297.33280.9  ng / g , res . )
moreover , dry ginger exhibited mean arsenic content of 77.9  8.7  ng / g . in general ,
the highest concentration of arsenic was found in plant roots , the intermediate level in vegetative tissues ( leaves and stems ) , and the lowest level in reproductive tissue ( fruits and seeds ) [ 23 , 24 ] .
plants absorb arsenic fairly easily , so that high - ranking concentration may be present . among many of public
researches , most studies had focused on foods and not much on the information that was available on plants especially in the part of rhizomes although it was a high opportunity for arsenic accumulation .
for this reason , well - known rhizomes of zingiberaceae family that are used as food , dietary supplements and alternative medicines in thailand , that is , a. galanga , b. rotunda , c. longa , c. zedoaria , z. cassumunar , and z. officinale , were interesting to investigate their total and inorganic arsenic concentrations .
standard reference material ( srm ) 1568a ( rice flour ) was purchased from the national institute of standards and technology ( gaithersburg , md , usa ) . nitric acid ( hno3 ) and hydrochloric acid ( hcl ) were purchased from merck chemicals ( darmstadt , germany ) ; dimethylarsinic acid ( dma ) , hydrazine sulfate , hydrobromic acid , and other chemicals were obtained from sigma - aldrich ( st .
all standard solutions , reagents , and samples were prepared using deionized water ( 18  m  cm ) throughout the study . to remove possible arsenic residue contamination ,
all glasswares were washed thoroughly with tap water , air - dried , soaked in 10% ( v / v ) hno3 for 2024  h , and washed three times with deionized water .
six kinds of plants in the family zingiberaceae of which rhizomes are widely used for consumption and medication purposes were selected .
( kra - chaai ) , curcuma longa ( l. ) ( khamin - chan ) , curcuma zedoaria ( berg . ) roscoe ( khamin - oi ) , zingiber cassumunar roxb .
sixteen samples of each rhizome species were collected during december 2011 and january 2012 from eight provinces ( chiang rai , lampang , khon kaen , ubon ratchathani , ratchaburi , samut prakan , krabi , and songkhla ) in north , northeast , central and south thailand . totally , 96 samples were collected for investigation .
w. gritsanapan , and the voucher specimens ( ag111201 - 16 , br120101 - 16 , cl111201 - 16 , cz111201 - 16 , zc120101 - 16 , and zo120101 - 16 ) were deposited at department of pharmacognosy , faculty of pharmacy , mahidol university , thailand .
freeze drying is used to dry sliced rhizomes to keep them in a stable condition .
the dried samples were grinded into powder with a porcelain mortar and pestle and passed through a fine mesh sieve .
powder of lyophilized samples was kept in air tight containers at 4c and protected from light until analysis .
the lyophilized sample preparation for determination of total arsenic was performed by acid digestion procedure described by muoz et al . .
an accurate weight ( 0.5  0.01  g ) of each lyophilized sample was mixed with 1  ml of an ashing suspension ( 20% ( w / v ) mg(no3)26h2o and 2% ( w / v ) mgo in water ) and 5  ml of 50% ( v / v ) hno3 .
the mixture was evaporated on a hot plate to dryness and mineralized at 450c in a furnace .
the resulting white ash was dissolved in 5  ml of 6  n hcl and 5  ml of a freshly prepared reducing solution ( 5% ( w / v ) ki and 5% ( w / v ) ascorbic acid ) .
the solution was left for 30  min , and then 10  ml of 50% ( v / v ) hcl was added to the solution .
the solution was filtered through a whatman no . 1 filter paper into a 25  ml volumetric flask and adjusted to volume with 50% ( v / v ) hcl .
an accurate weight ( 1.0  0.01  g ) of each lyophilized sample was placed in a 50  ml screw - capped centrifuge tube ; 4.1  ml of water was added to the sample and mixed until completely moistened . in order to hydrolyze as(iii ) from the thiol groups of proteins ,
18.4  ml of concentrated hcl was added to the moistened sample , shaken for 1  h , and left overnight ( 12 to 15  h ) . a reducing agent ( 1  ml of 1.5% ( w / v ) freshly prepared hydrazine sulfate and 2  ml of hydrobromic acid )
was added to the sample tube and vortexed for 2  min . for extraction of inorganic arsenic
, 10  ml of chloroform was added to the tube , vortexed for 3  min , and inverted for 1  min . to break the emulsion formed during the extraction , the tube was centrifuged at 1,000  g ( measured in gravity  force or g - force ) for 10  min using an eppendorf bench top centrifuge 5810 ( hamburg , germany ) .
, the combined chloroform phase was filtered through a syringe filter with a 25  mm ptef membrane , pore size 0.45  m ( chrom tech , apple valley , mn , usa ) , into another tube .
inorganic arsenic in the chloroform phase was back - extracted into an aqueous phase with 10  ml of 1  n hcl and centrifuged at 1,000  g for 10  min .
the amount of inorganic arsenic in the combined acidic aqueous phase was quantified as described in section 2.4 , with the addition of 2.5  ml of the ashing suspension and 10  ml of 50% ( v / v ) hno3 .
a perkinelmer ( waltham , ma , usa ) aanalyst 300 atomic absorption spectrometer ( norwalk , ct , usa ) interfaced with an as-90 autosampler and a fias-400 flow injection system was used to determine total and inorganic arsenic concentrations in the final solutions .
the atomic absorption spectrophotometric conditions were as follows : wavelength , 193.7  nm ; slit width , 0.70  nm ; edl current , 380  ma ; loop sample , 0.5  ml .
the hydride generation conditions were as follows : quartz cell , 16  cm path length , 90.7  cm i.d .
, electrothermal heating , cell temperature , 900c , carrier gas ( argon ) flow rate , 50100  ml / min , reducing agent ( 0.2% ( w / v ) nabh4 in 0.05% ( w / v ) sodium hydroxide solution ) flow rate , 57 ml / min , and hcl flow rate , 911 ml / min . the q2b analytical procedure described by the us fda was used for determination of the limit of quantification ( loq ) .
mixtures of lyophilized samples of all six species with equal weights were used for the determination of the limit of quantification ( loq ) of the method . for the determination of the loq for total arsenic ,
samples were fortified with a standard arsenic mixture ( as(iii )  :  dimethylarsinic acid ( dma ) , 1  :  1 ( w / w ) ) equivalent to total arsenic at concentrations of 250 , 500 , 1,000 , and 2,500  ng / g ; blank samples were not fortified with arsenic .
a total of twelve regression lines ( six regression lines each for intraday and interday analyses ) were obtained by the least - square linear regression analyses of the residual peak heights of standard total arsenic versus fortified total arsenic concentrations .
the residual peak heights were peak heights of total arsenic - fortified samples minus that of blank sample .
the loq of the method was calculated using the equation loq = 10/s , where  is the standard deviation of y - intercepts and s is the slope of linear regression analysis . for the determination of the loq for inorganic arsenic
, samples were fortified with an inorganic arsenic mixture ( as(iii )  :  as(v ) 1  :  1 ( w / w ) ) at concentrations of 50 , 100 , 500 , and 1,000  ng / g ; blank samples were not fortified with inorganic arsenic .
a total of twelve regression lines ( six regression lines each for intraday and interday analyses ) were obtained by the least - square linear regression analyses of the residual peak heights of standard inorganic arsenic versus fortified inorganic arsenic concentrations .
the residual peak heights were the peak heights of fortified samples of inorganic arsenic minus the average peak height of blank sample .
the loq of the method was calculated using the equation loq = 10/s , where  is the standard deviation of y - intercepts and s is the slope of linear regression analysis .
the accuracy of determination of total arsenic was assessed by analyzing srm 2568a ( rice flour ) , because no commercial rice standard reference materials for inorganic arsenic are available .
the amount of inorganic arsenic in srm 1568a ( rice flour ) was determined and compared with the values previously reported .
one - way analysis of variance and tukey 's test using the spss statistics version 17.0 software were performed to determine differences in concentrations of total arsenic , inorganic arsenic ( both on wet weight and dry weight basis ) , and percentages of inorganic arsenic with respect to total arsenic of six rhizome types . a significance level of p < 0.05 was accepted for all comparisons .
the calculation for loqs was based on the standard deviation of y - intercepts of the linear regression analysis (  ) and the slope ( s ) by using the equation loq = 10/s .
the loqs for total and inorganic arsenic in rhizome samples were 19.7 and 15.7  ng / g , respectively .
concentrations of total arsenic and inorganic arsenic found in srm 1568a ( rice flour ) were 283  34  ng / g ( n = 10 , reference value of 290  30  ng / g ) and 102  3.7  ng / g ( n = 10 ) , respectively .
the concentration of inorganic arsenic was in agreement with previous reports of 111  6  ng / g   and 111  3  ng / g , which was analyzed by the same method .
the accuracy and precision for the determination of total and inorganic arsenic in rhizome samples fortified with arsenic mixture at four concentrations are shown in table 1 .
the accuracy was assessed as percent recovery from the analysis of fortified arsenic mixture in the rhizome samples .
the average recoveries across the four concentrations of fortified arsenic mixtures were 95.6% and 95.4% for total and inorganic arsenic , respectively .
the precision of the method expressed as percentage of relative standard deviation ( % rsd ) was calculated with the equation % rsd = 100sd/  x- , where sd is the standard deviation and x- is the mean of arsenic concentrations recovered from the arsenic - fortified samples .
the % rsd ranged from 1.7 to 5.0 for total arsenic and from 1.1 to 5.5 for inorganic arsenic . 

table 2 summarizes concentrations of total arsenic , inorganic arsenic , and percentages of inorganic arsenic with respect to total arsenic in rhizomes of six plants in zingiberaceae family collected from four regions of eight areas in thailand .
the highest content of inorganic arsenic was found in plai ( 71.2  11.6  ng / g , dry wt ) .
the inorganic arsenic content in six dried rhizomes was expressed within the range of 20.4 to 92.8  ng / g , total arsenic levels ranged from 42.5 to 145.1  ng / g , and the percentages of inorganic arsenic with respect to total arsenic ranged from 28.5 to 83.2  ng / g .
determined concentrations of total arsenic in dried ginger ( n = 3 ) from china .
average concentration was 77.9  ng / g , whereas the value in this study was 69.3  ng / g .
the indicated concentration may imply that the growth environments and soil condition for growing ginger in thailand ( 69.3  11.8  ng / g ) were safer than those in china ( 77.9  8.7  ng / g ) .
the study of the arsenic - affected area in india reported higher levels of arsenic in fresh cumin and turmeric powder than our study .
they reported that arsenic level ranged from 47.86 to 209.75  ng / g and from 297.33 to 280.9  ng / g , respectively .
our study found that the total arsenic levels in khamin - chan ( wet wt and dry wt ) ranged from 5.8 to 21.3  ng / g and from 42.5 to 87.2  ng / g , respectively .
these results clearly showed that the high amount of arsenic deposited in rhizomes is according to soil location . in six plants of zingiberaceae family in this study , the average concentrations of inorganic arsenic in the roots of c. longa and c. zedoaria were 4.3  1.5 and 6.2  1.5  ng / g ( wet wt ) , respectively .
the inorganic arsenic contents in two curcuma species were not statistically different ( p > 0.05 ) but they were significantly different ( p < 0.05 ) from zingiber genus .
the inorganic arsenic contents in z. cassumunar and z. officinale were 11.7  4.5 and 4.4  1.0  ng / g ( wet wt ) , respectively
. the well - known species of alpinia genus , a. galangal , showed the concentration ( wet wt ) of total arsenic ( 15.9  5.1  ng / g ) and inorganic arsenic ( 8.4  3.0  ng / g ) .
finally , kra - chaai in boesenbergia genus contained the total arsenic level of 20.8  4.6  ng / g , the inorganic arsenic level of 13.4  3.1  ng / g , and the percentage of inorganic arsenic of 64.4% .   in this study , the total arsenic concentrations in fresh rhizomes of kra - chaai , plai , khaa , khamin - oi , khamin - chan , and ginger ranged from 11.5 to 27.3 , 7.9 to 26.8 , 10.0 to 28.3 , 6.9 to 21.8 , 5.8 to 21.3 , and 4.8 to 13.7  ng / g , respectively .
these values are in accordance with the fresh vegetable arsenic concentration reported that ranged from 0 to 195  ng / g . other edible roots , such as carrot and potato
were reported high levels of arsenic at 195 and 103  ng / g , respectively .
reported that the total arsenic level for vegetable group ranged from 8 to 604  ng / g , and the total and inorganic arsenic concentrations in edible roots or rhizomes were higher than other organs of herbs .  
of the six medicinal rhizomes of zingiberaceae family analyzed in this study , both of kra - chaai and plai exhibited high mean levels ( wet weight ) of total arsenic ( 20.8  4.6 and 17.5  6.6  ng / g ) , inorganic arsenic ( 13.4  3.1 and 11.7  4.5  ng / g ) , and percentage of inorganic arsenic ( 64.4 and 67.4% ) , respectively .
a possible explanation is that these high levels were related to their arsenic accumulative capacity in the rhizosphere soils associated with each species .
in contrast , the low levels of inorganic arsenic in khamin - chan , khamin - oi , and ginger ( wet weight ) ranged from 2.6 to 8.2 , 2.5 to 8.4 , and 2.6 to 6.0  ng / g , respectively , whereas the average percentages of inorganic arsenic were 41.8 , 44.5 , and 56.9 , respectively . form the statistical analysis , comparative contents of total arsenic ( ng / g , dry  wt ) among six species , khaa , kra - chaai , khamin - oi , and plai were significantly different from khamin - chan and ginger .
from our study , the total arsenic level in ninety - six samples complied with thai regulatory limit ( 2  g / g ) and national limit for arsenic in herbal medicines ( 4  g / g ) [ 3032 ] .   table 3 shows a comparison of total arsenic level in some rhizomes or roots from various countries .
further investigations should be performed on other botanical rhizomes with incidence of high arsenic accumulation to crucially guarantee that plants are safe for dietary supplements for health and well - being .
all investigated amounts of total arsenic concentrations meet the requirements at a national level . among six species
we found that dried root of z. cassumunar showed a higher level of inorganic arsenic than other species .
arsenic accumulation in rhizomes may be an important risk factor that needed to be taken into account .
the results will be valuable for preliminary risk assessment in raw materials of natural products .
therefore , long - term consumption of herbal products that comprised some particular zingiberaceous rhizomes might cause adverse health effects .
 the arsenic accumulation in rhizomes of zingiberaceous plants was determined by atomic absorption spectrometry interfaced with hydride generation system ( hg - aas ) . 
 the raw herbal materials , rhizomes , were collected from different regions of thailand between december 2011 and january 2012 . 
 six well - known zingiberaceous plants , 16 samples from each and a total of 96 samples , were analyzed alpinia galanga ( khaa ) , boesenbergia rotunda ( kra - chaai ) , curcuma longa ( khamin - chan ) , curcuma zedoaria ( khamin - oi ) , zingiber cassumunar ( plai ) and zingiber officinale ( ginger ) . concentrations of total arsenic based on dry weight were 92.4  9.2 , 103.5  20.8 , 61.7  12.5 , 89.8  17.5 , 106.7  19.5 and 69.3  11.8  ng / g , respectively and inorganic arsenic were 48.8  7.0 , 66.3  12.7 , 25.5  5.0 , 38.7  4.7 , 71.2  11.6 , and 38.5  5.5  ng / g , respectively . among these , 
 plai and kra - chaai exhibited the highest levels of total arsenic and inorganic arsenic accumulation that remind consumers to be aware of excess consuming of these rhizomes . 
 on the contrary , the lowest value found in khamin - chan indicating natural dietary supplements and herbal medicines comprising kamin - chan are safe from arsenic poison . 
 all investigated amounts of total and inorganic arsenic were much lower than limits recommended by thai food and drug administration . 
1. Introduction 
2. Materials and Methods
3. Results and Discussion 
4. Conclusion