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Discrimination of ginseng cultivation regions using light stable isotope analysis
Forensic Science International 255 (2015) 43–49
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Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Discrimination of ginseng cultivation regions using light stable isotope analysis Kiwook Kim a, Joo-Hyun Song a, Sang-Cheol Heo a, Jin-Hee Lee a,b, In-Woo Jung a, Ji-Sook Min a,* a b
Chemical Analysis Division, National Forensic Service, Wonju-si 26460, Gangwon-do, Republic of Korea Department of Chemistry and Medical Chemistry, College of Science and Technology, Yonsei University, Wonju-si 26493, Gangwon-do, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Available online 26 July 2015
Korean ginseng is considered to be a precious health food in Asia. Today, thieves frequently compromise ginseng farms by pervasive theft. Thus, studies regarding the characteristics of ginseng according to growth region are required in order to deter ginseng thieves and prevent theft. In this study, 6 regions were selected on the basis of Korea regional criteria (si, gun, gu), and two ginseng-farms were randomly selected from each of the 6 regions. Then 4–6 samples of ginseng were acquired from each ginseng farm. The stable isotopic compositions of H, O, C, and N of the collected ginseng samples were analyzed. As a result, differences in the hydrogen isotope ratios could be used to distinguish regional differences, and differences in the nitrogen isotope ratios yielded characteristic information regarding the farms from which the samples were obtained. Thus, stable isotope values could be used to differentiate samples according to regional differences. Therefore, stable isotope analysis serves as a powerful tool to discriminate the regional origin of Korean ginseng samples from across Korea. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Stable isotope ratio Elemental analyzer-isotope ratio mass spectrometer (EA-IRMS) Ginseng Discrimination Region
1. Introduction In recent decades, stable isotope ratio analysis has been utilized in product-tracking and to determine the origins of products in forensic science. The use of stable isotope analysis to trace the origin of agricultural products is a stronger discrimination tool as compared to other forensic analysis methods [1–4]. Collaborative research for the TRACE program was implemented by various research institutes and universities for five years (2005–2009) to trace the production origin of various products. The technique most frequently used in this program to identify the origin of products was stable isotope analysis. Similarly, developed countries often take advantage of stable isotope analysis to identify the origin of foods [5–9]. In the Republic of Korea, research regarding the identification of the origin of agricultural products is underdeveloped. At the end of 2008, the Ministry of Agriculture Food and Rural Affairs (MAFRA) began to provide funding for research that investigated the origin of pork and cabbage. At that time, stable isotope ratio analysis of the origin of agricultural products depended on the results obtained by other countries,
* Corresponding author. Tel.: +82 33 902 5510; fax: +82 33 902 5933. E-mail address: [email protected] (J.-S. Min). http://dx.doi.org/10.1016/j.forsciint.2015.07.030 0379-0738/ß 2015 Elsevier Ireland Ltd. All rights reserved.
because database construction was not complete in Korea. After that, the Korea Basic Science Institute (KBSI) carried out carbon, nitrogen, and oxygen stable isotope analysis to determine the origins of Korean and imported beef provided by the central customs laboratory and scientific service (CCLS) [10]. Numerous origin tracking studies for food and agricultural and livestock products are ongoing; however, studies regarding the origin identification of ginseng do not exist currently. In 2008, Choi et al. [11] first reported that origin discrimination between Korean and Chinese ginseng could be carried out using Inducted Coupled Plasma-Mass Spectroscopy (ICP-MS). In this study, significant differences in the strontium stable isotope ratio were noted between Korean and Chinese ginseng [12]. However, the results were questionable, because an unsuitable analysis was performed [13,14]. In another study, Yu et al. [15] investigated the discrimination of Korean and Chinese ginseng using liquid chromatography, but the results were not obvious. Moreover, in collaboration with the Austrian Institute of Technology [16], we conducted a study regarding discrimination of Korean and Chinese ginseng using light stable isotope analysis. Specifically, Korean and Chinese ginseng were differentiated according to their hydrogen stable isotope values. However, regional differences among samples from Korea could not be determined in this study. Since the value of ginseng has been recognized in Asia as well as
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throughout the world, the demand for ginseng has increased recently. Although ginseng was traditionally grown in uninhabited environments (i.e., mountains), the number of ginseng farms has increased rapidly due to the wide demand, and ginseng thieves frequently compromise the farms. Accordingly, studies regarding regional differences in the isotopic characteristics of ginseng are required in order to deter ginseng thieves and prevent theft. In this study, we carried out stable isotope analysis of ginseng collected from various regions in Korea in order to identify potential differences. The objective of this study was to obtain data regarding the H, O, C, and N stable isotope ratios in ginseng samples from different regions in Korea and conduct a statistical analysis to determine potential factors that could be used to differentiate ginseng cultivation regions and farms. 2. Materials and methods 2.1. Ginseng collection and pre-processing of ginseng roots Fig. 1 shows the locations of the ginseng farms in the Republic of Korea. Six regions (Ganghwa-gun, Paju-si, Gimpo-si, Yeongju-si, Hwacheon-gun, and Cheolwon-gun) were selected on the basis of Korea regional criteria (si, gun, gu). These six areas are well-known ginseng cultivation regions in the Republic of Korea. Additionally, ginseng theft was prevalent in these regions just before the study
was initiated. Two ginseng-farms were randomly selected from each of the 6 regions and 4–6 ginseng samples were acquired from each ginseng farm. The adhered soil was removed from the ginseng roots, and the roots were placed in an oven set to 60 8C and dried until the moisture was completely removed. Using a ball-milling machine, the samples were subsequently processed in the form of a powder using only the main root portion of the dried ginseng roots, shown in Fig. 2. The powdered ginseng roots were wrapped five times using a silver capsule for hydrogen and oxygen stable isotope analysis and were wrapped three times using a tin capsule for carbon and nitrogen stable isotope analysis. 2.2. Analysis conditions The pretreated samples were analyzed using an elemental analyzer linked to an isotope mass spectrometer (EA-IRMS, Elemental Analysis-Isotope Ratio Mass Spectrometer; EURO EA 3000, EURO VECTOR, Italy) as well as a GV Instrument Isoprime (GV instrument Ltd., UK). Two calibration points were determined using the following standard samples in order to determine the accuracy of the measurements: hydrogen stable isotope ratios: Tibetan hair (IAEA-USGS-42, International Atomic Energy Agency, dD certified value: 78.5 2.3% VSMOW), polyethylene (IAEA-CH7, International Atomic Energy Agency, dD certified value: 100.3 2.0% VSMOW), and house cellulose; oxygen stable isotope ratios:
Fig. 1. Locations of ginseng farms across the Republic of Korea.
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coastal and inland regions could be distinguished, as noted in the figure with circles. 3.2. T-test analysis Proximity to the sea, growth conditions, differences in elevation, proximity to downtown, and the type of fertilizer etc. can affect dD, d18O, d13C, and d15N values. In this section, we describe significant differences in such factors determined using an independent sample t-test analysis.
Fig. 2. Parts of the ginseng root.
benzoic acid (IAEA-601, International Atomic Energy Agency, d18O certified value: 23.30 0.30% VSMOW) and (IAEA-USGS-42, d18O certified value: 8.56 0.10% VSMOW); carbon stable isotope ratios: cellulose standard samples (IAEA-CH-3, International Atomic Energy Agency, d13C certified values: 24.724 0.041% VPDB) and polyethylene (IAEA-CH-7, International Atomic Energy Agency, d13C certified values: 32.151 0.050% VPDB); nitrogen stable isotope ratios: ammonium sulfate (IAEA-N-1, International Atomic Energy Agency, d15N certified value: 0.4 0.2% Air) and potassium nitrate (IAEA-NO3, International Atomic Energy Agency, d15N certified value: 4.7 0.2% Air). The maximum standard deviations for the international standard samples (CH-7, 601, CH-3, and N-1) were dD: 3.1%, d18O: 1.5%, d13C: 0.5%, and d15N: 0.3% from our measurement system. Further, in consideration of the exchangeable hydrogen problem [17,18], all measurement samples including International standards (USGS-42, CH-7), the in-house cellulose standard, and ginseng samples were placed in a desiccator for two days before analysis. Additionally, the auto-sampler was purged with helium. Using the aforementioned procedure, the H-exchange problem was avoided. 3. Results The average, standard deviation, minimum, and maximum values of H, O, C, and N stable isotope ratios from samples from each farm are shown in Table 1. The dD, d18O, d13C, and d15N values were between 69.16 and 30.59%, 28.11 and 41.38%, 28.96 and 22.67%, and 2.33 and 11.81%, respectively, and the maximum standard deviations of one sample were dD: 4.5%, d18O: 2.2%, d13C: 0.9%, and d15N: 0.5%. The sample from Samdongarmri in Ganghwa-gun exhibited the greatest dD value, while the lowest value was observed in the sample from Sanbeob-ri in Yeongju-gun. The highest d18O value was observed in the sample from Geumwarl-ri in Ganghwa-gun, while the lowest value was observed from the sample from Baek-ri in Yeongju-si. The greatest d13C value was observed in the sample from Samdongarm-ri in Ganghwa-gun, while the lowest value was observed in the sample from Chowonji-ri in Gimpo-si. The greatest d15N value was observed in the sample from Juwarl-ri in Paju-si, while the lowest value was observed in the sample from Daei-ri in Hwacheon-gun. 3.1. The matrix scattered plot for dD, d18O, d13C, and d15N Matrix scattered plots were generated for the data obtained from samples from the farms shown in Fig. 3. The ginseng from Ganghwa-gun exhibited a notable difference compared to that from other regions in regard to the dD versus d18O value. Additionally, even though the values of a few samples overlapped,
3.2.1. The t-test analysis of samples from coastal and inland regions The t-test was performed to determine regional differences between coastal regions {Ganghwa (4), Gimpo (5), Paju (6)}, and inland regions {Yeongju (1), Chungju (2), Hwacheon (3)}, and the results are shown in Table 2. The p-value of dD was 0.000 and the p value of d18O was 0.000. Therefore, the hydrogen and oxygen stable isotope ratios in samples from coastal and inland regions were significantly different at the 1% level of significance. Notably, since sufficient amounts of the samples were not analyzed, the confidence level was set at 99%. 3.2.2. The t-test analysis of samples from different farms within the same region The t-test was carried out to determine differences in the stable isotope values between samples from different farms within same region, and the results are shown in Table 3. Differences in the nitrogen stable isotope value enabled the discrimination of ginseng samples from Baek-ri and Sanbeob-ri in Yeongju-si, between Daeri and Pungsan-ri in Hwacheon-gun, between Samdongarm-ri and Geumwarl-ri in Ganghwa-gun, and between Chowonji-ri and Tongarm-ri in Gimpo-si. Significant differences in the p-value were determined at the 1% level of significance. 4. Discussion 4.1. Hydrogen and oxygen stable isotope ratio The dD and d18O values of samples decreased in the following order: dD: Ganghwa (4), Gimpo (5), Paju (6), Hwacheon (3), Chungju (2), and Yeongju (1); d18O: Ganghwa (4), Paju (6), Gimpo (5) Chungju (2), Hwacheon (3), and Yeongju (1). Accordingly, the hydrogen and oxygen stable isotope ratios in samples obtained from coastal areas were higher than those obtained from inland areas. In particular, the hydrogen and oxygen stable isotope ratios in samples from Ganghwa island (4), which is closest to the yellowsea, were much higher than those from others. Samples from Yeongju (1) and Chungju (2), which are located near each other inland, showed similar low values. Therefore, according to the natural environment of the area, these values could be similar or different for each region. The matrix scattered plot (Fig. 3) showed differences between coastal and inland regions, and a t-test was conducted in order to verify the significance of statistical differences between values from samples obtained from coastal and inland regions. The t-test suggested that the differences in the hydrogen and oxygen stable isotope values between samples from coastal and inland regions were significant at the 1% level of significance. The potential reasons for the differences are as follows: hydrogen and oxygen stable isotopes in sap or plant tissue are affected by metabolic changes within the agricultural product, but exhibit a very close relationship with the isotopic composition of precipitation in the region in which they were produced [5]. The water used to grow agricultural products is supplied from soil water or groundwater, which is regenerated from precipitation [19]. The hydrogen and oxygen stable isotope ratios in precipitation are mainly affected by location; seawater contains a large
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Table 1 Average, standard deviation, minimum, and maximum values of dD, d18O, d13C, and d15N values (%) for each farm. Region (#)
Farm (# of farm)
Values
Yeongju-si (1)
Baek-ri (1-1)
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
56.27 4 6.32 65.24 50.72 67.42 4 1.29 69.16 66.06
28.50 4 0.42 28.11 29.07 33.59 4 1.14 32.60 34.96
25.76 4 0.94 26.88 24.59 27.24 4 0.89 28.54 26.62
6.75 4 1.15 5.39 8.02 0.82 4 0.27 1.12 0.47
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
58.64 5 1.60 60.97 56.64 64.53 5 2.07 66.38 60.98
31.40 5 0.45 30.94 32.14 31.88 5 1.13 30.33 33.20
25.68 5 1.78 27.89 23.63 26.90 5 1.43 28.96 25.23
7.62 5 1.17 5.74 8.78 6.40 5 0.91 5.01 7.48
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
54.32 5 0.71 55.04 53.27 54.69 6 2.59 57.63 50.43
32.26 5 1.57 29.83 34.19 30.10 6 1.25 28.70 31.96
26.07 5 0.71 26.79 25.03 24.93 6 1.47 27.08 22.99
1.64 5 0.70 2.33 0.71 7.65 6 2.26 4.64 11.01
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
40.07 5 6.83 46.58 30.59 42.92 5 2.64 45.92 39.64
38.38 5 0.79 37.41 39.45 39.69 5 1.75 37.31 41.38
23.43 5 1.38 25.88 22.67 24.34 5 0.61 24.79 23.42
9.04 5 1.21 7.09 10.38 4.90 5 1.32 2.76 6.26
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
52.74 6 2.32 55.95 50.03 52.66 4 1.88 55.16 50.81
31.46 6 0.71 30.32 32.07 32.06 4 1.40 30.90 33.93
27.79 6 1.00 28.90 26.44 24.78 4 0.32 25.05 24.32
8.77 6 1.85 6.48 11.61 2.57 4 1.66 0.81 4.79
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
49.75 5 4.93 54.97 44.22 56.21 6 1.73 58.33 54.52
31.50 5 2.05 28.81 34.06 34.64 6 1.37 33.13 36.69
25.28 5 1.32 26.66 23.19 23.64 6 1.04 24.97 22.78
6.92 5 3.37 2.44 11.81 5.94 6 0.87 4.71 7.21
Sanbeob-ri (1-2)
Chungju-si (2)
Saejeong-ri (2-1)
Hyangsan-ri (2-2)
Hwacheon-gun (3)
Daei-ri (3-1)
Pungsan-ri (3-2)
Ganghwa-gun (4)
Samdongarm-ri (4-1)
Geumwarl-ri (4-2)
Gimpo-si (5)
Chowonji-ri (5-1)
Tongarm-ri (5-2)
Paju-si (6)
Juwarl-ri (6-1)
Jeomwon-ri (6-2)
#
dD (%)
d18O (%)
d13C (%)
d15N (%)
Number; N: Number of samples; SD: Standard deviation.
amount of hydrogen stable isotopes, and seawater produces clouds. As the clouds move, heavy isotopes (deuterium) preferentially drop in coastal areas and light isotopes (hydrogen) drop successively in inland areas [20]. The isotopic composition can also be affected by temperature; changes in the altitude and latitude induce a change in temperature, which influences the isotopic composition of the precipitation.
4.2. Carbon stable isotope ratio Plants are widely classified into C3, C4, and CAM species depending on their metabolic activity (metabolism). The metabolism of C3 plants follows the Calvin cycle. Most flowering plants that can produce honey live in temperate regions and have carbon stable isotope compositions of about 30 to 22%. C4 plants live
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Fig. 3. Matrix scattered plot for each farm.
Table 2 The t-test results of regional differences between coastal and inland regions. Elements
Sig. (2-tailed)
dD(%)_Ave. d18O(%)_Ave. d13C(%)_Ave. d15N(%)_Ave.
0.000 0.000 0.012 0.034
in tropical and temperate regions and exhibit Hatch–Slack metabolism; their carbon stable isotope composition is typically about 14 to 8%. The carbon stable isotope composition of CAM plant species growing in arid regions is in between that of C3 and C4 plants [21]. Anthropogenic effects, such as CO2 emission, can lead to decreases in carbon stable isotope ratios. However, differences in isotopic compositions are known to be influenced more by the metabolic activity of the plant than by the growth environment [22,23]. Thus, variations in the carbon stable isotope content due to anthropogenic effects are typical changes within the range. Korean ginseng is known to be a C3-type species. The carbon stable isotope ratios of collected ginseng samples were
between 28.96 and 22.67%, which falls within the expected carbon stable isotope ratio range of C3 plants. In particular, the carbon stable isotope ratios in samples from Ganghwa island were the highest average value. Because the population density (163.39 people/km2) is low and the strong sea wind make circulate quickly emitted CO2 through the sea. Consequently, the d13C value could be higher than that in any other region. However, samples from Gimpo showed the lowest average value. The population density of Gimpo (1202.55 people/km2) is the highest and there are a lot of plant facilities forms. Thus, these effects could deplete the carbon stable isotope ratio. As a result, the carbon stable isotope ratio would be depleted by anthropogenic effects, but the value did not exceed the range of C3 plants; additionally it might serve as an auxiliary role for discrimination of the origin of ginseng. 4.3. Nitrogen stable isotope ratio The average nitrogen stable isotope ratios (Table 1) in samples obtained from farms in Yeongju, Hwacheon, Ganghwa, and Gimpo were significantly different. In addition to comparing the average values, a t-test was also performed to compare the samples from
Table 3 The t-test results of regional differences between different farms in the same region. Elements region
dD(%)_Ave. d18O(%)_Ave. d13C(%)_Ave. d15N(%)_Ave.
Sig. (2-tailed) Yeongju-si (1)
Chunju-si (2)
Hwacheon-gun (3)
Ganhwa-gun (4)
Gimpo-si (5)
Paju-si (6)
0.036 0.001 0.062 0.001
0.001 0.416 0.266 0.105
0.751 0.039 0.134 0.000
0.423 0.180 0.230 0.001
0.958 0.476 0.000 0.001
0.040 0.023 0.056 0.555
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various farms; the p values of samples from Yeongju, Hwacheon, Ganghwa, and Gimpo were all under 0.01. As such, the differences in nitrogen stable isotope ratios of samples from these farms were determined to be significant at the 1% level of significance. Differences in the nitrogen stable isotope values were attributed to the type of fertilizer (organic or chemical) that was used for plant growth. The nitrogen stable isotope composition in plants treated with organic fertilizer is greater than that in plants treated with chemical fertilizers [24]. In addition, the use of livestock manure as fertilizer yields plants with the highest nitrogen stable isotope values [5]. Farms Baek-ri (1-1), Saejeong-ri (2-1), Hyangsan-ri (22), Pungsan-ri (3-2), Samdongarm-ri (4-1), Chowonji-ri (5-1), Juwarl-ri (6-1), and Jeomwon-ri (6-2) seemed to use organic fertilizers, and farms Sanbeob-ri (1-2), Daei-ri (3-1), Guemwon-ri (4-2), and Tongarm-ri (5-2) seemed to use chemical fertilizers. Additionally, in regard to farms in the same area, farms 2-1 and 2-2, and farms 6-1 and 6-2 seem to have used organic fertilizers. In cases when different farms within the same area used the same type of fertilizer, it was difficult to distinguish the origin of the samples based on the nitrogen stable isotope ratio. Nevertheless, since farms in four of the six regions could be distinguished by comparing the nitrogen stable isotope ratio, the nitrogen stable isotope ratio was considered to be an effective identifying factor.
considered, because all regions were located in rural areas far from downtown, and differences in isotopic composition are more influenced by the metabolic activity of plants than the growth environment. The nitrogen stable isotope ratio does not reflect geographic factors, but can be used to discriminate farms using different fertilizers within the same area. Additionally, with reference to the carbon stable isotope ratio could be improving the distinguish ability of the farms. If these results are compiled in a database, the database could be used to determine potential regions from where stolen ginseng was obtained. Furthermore, if the region is revealed through further investigation, the farm from which the sample was obtained could also potentially be determined. To summarize, the possibility to distinguish different farms by using gaps in the isotope ratio was determined. However, isotope ratio variations within each farm makes it difficult to distinguish the farm, but it is natural and very important for forensic decisions. In future research, more ginseng samples, soil, and water from the periphery of ginseng farms could be analyzed to improve the statistical reliability; sulfur and strontium stable isotope as well as multi-element XRF (X-ray fluorescence spectroscopy) analysis could be performed to enhance the discrimination according to regional differences. Acknowledgements
4.4. Isotope ratio variation within same farm Ginseng is usually cultivated in large quantities, which means that the cultivation area is extremely large. Therefore, it is also important to check the isotope ratio within large farms. The range of dD values was larger than that of d15N, d18O, and d13C values. Broad scale ranges makes small differences look like big differences, but this can be a great forensic tool, because we can decide confidently whether or not the origin of each sample is different. The d18O values seemed to follow the dD patterns, but the standard deviation was smaller than that of the dD values because the scale range of the d18O value was smaller than that of the dD values. Notably, Bateman et al. [26], reported that the standard deviation of d15N values is about 1%, even when the same type of fertilizer and same quantity is used for various agricultural products (carrot, tomato, lettuce). Ginseng is usually cultivated for 4–6 years and the type of fertilizer used may be varied during this time. Thus, the d15N of ginseng may vary more for each ginseng from same farm. Carbon isotope ratios slightly depend on individual characteristics (e.g. degree of photosynthesis, nutrients, and water absorption [22,23]). Therefore, a 2% variation in the carbon isotope ratio within each farm is considered normal. As mentioned above, the isotope ratios in animal and plant samples may be changed, even if ginseng is grown in the same environment. However, variations within a single farm are important in forensic science. Therefore, different analytical methods (multi-elements analysis, isotope ratio of soil and water) should be used in combination in the future. 5. Conclusion The purpose of this study was to determine whether stable isotope ratios could be used to discriminate Korean ginseng according to regional differences. Even though this was a pilot study, reasonable results were obtained. The hydrogen and oxygen stable isotope ratios were affected by precipitation. Even though the total area of the Republic of Korea is small, ginseng samples from coastal regions were generally heavier than those from inland areas, indicating that regional differences could be noted. The carbon stable isotope ratio of all ginseng samples ranged from 30 to 22%; therefore, ginseng was confirmed to be a C3 plant. Environmental factors caused by anthropogenic effects were not
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2012-0009832), and this work was supported by a grant (2014Chemistry-01) from the National Forensic Service (NFS) received in 2014.
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Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Discrimination of ginseng cultivation regions using light stable isotope analysis Kiwook Kim a, Joo-Hyun Song a, Sang-Cheol Heo a, Jin-Hee Lee a,b, In-Woo Jung a, Ji-Sook Min a,* a b
Chemical Analysis Division, National Forensic Service, Wonju-si 26460, Gangwon-do, Republic of Korea Department of Chemistry and Medical Chemistry, College of Science and Technology, Yonsei University, Wonju-si 26493, Gangwon-do, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Available online 26 July 2015
Korean ginseng is considered to be a precious health food in Asia. Today, thieves frequently compromise ginseng farms by pervasive theft. Thus, studies regarding the characteristics of ginseng according to growth region are required in order to deter ginseng thieves and prevent theft. In this study, 6 regions were selected on the basis of Korea regional criteria (si, gun, gu), and two ginseng-farms were randomly selected from each of the 6 regions. Then 4–6 samples of ginseng were acquired from each ginseng farm. The stable isotopic compositions of H, O, C, and N of the collected ginseng samples were analyzed. As a result, differences in the hydrogen isotope ratios could be used to distinguish regional differences, and differences in the nitrogen isotope ratios yielded characteristic information regarding the farms from which the samples were obtained. Thus, stable isotope values could be used to differentiate samples according to regional differences. Therefore, stable isotope analysis serves as a powerful tool to discriminate the regional origin of Korean ginseng samples from across Korea. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Stable isotope ratio Elemental analyzer-isotope ratio mass spectrometer (EA-IRMS) Ginseng Discrimination Region
1. Introduction In recent decades, stable isotope ratio analysis has been utilized in product-tracking and to determine the origins of products in forensic science. The use of stable isotope analysis to trace the origin of agricultural products is a stronger discrimination tool as compared to other forensic analysis methods [1–4]. Collaborative research for the TRACE program was implemented by various research institutes and universities for five years (2005–2009) to trace the production origin of various products. The technique most frequently used in this program to identify the origin of products was stable isotope analysis. Similarly, developed countries often take advantage of stable isotope analysis to identify the origin of foods [5–9]. In the Republic of Korea, research regarding the identification of the origin of agricultural products is underdeveloped. At the end of 2008, the Ministry of Agriculture Food and Rural Affairs (MAFRA) began to provide funding for research that investigated the origin of pork and cabbage. At that time, stable isotope ratio analysis of the origin of agricultural products depended on the results obtained by other countries,
* Corresponding author. Tel.: +82 33 902 5510; fax: +82 33 902 5933. E-mail address: [email protected] (J.-S. Min). http://dx.doi.org/10.1016/j.forsciint.2015.07.030 0379-0738/ß 2015 Elsevier Ireland Ltd. All rights reserved.
because database construction was not complete in Korea. After that, the Korea Basic Science Institute (KBSI) carried out carbon, nitrogen, and oxygen stable isotope analysis to determine the origins of Korean and imported beef provided by the central customs laboratory and scientific service (CCLS) [10]. Numerous origin tracking studies for food and agricultural and livestock products are ongoing; however, studies regarding the origin identification of ginseng do not exist currently. In 2008, Choi et al. [11] first reported that origin discrimination between Korean and Chinese ginseng could be carried out using Inducted Coupled Plasma-Mass Spectroscopy (ICP-MS). In this study, significant differences in the strontium stable isotope ratio were noted between Korean and Chinese ginseng [12]. However, the results were questionable, because an unsuitable analysis was performed [13,14]. In another study, Yu et al. [15] investigated the discrimination of Korean and Chinese ginseng using liquid chromatography, but the results were not obvious. Moreover, in collaboration with the Austrian Institute of Technology [16], we conducted a study regarding discrimination of Korean and Chinese ginseng using light stable isotope analysis. Specifically, Korean and Chinese ginseng were differentiated according to their hydrogen stable isotope values. However, regional differences among samples from Korea could not be determined in this study. Since the value of ginseng has been recognized in Asia as well as
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throughout the world, the demand for ginseng has increased recently. Although ginseng was traditionally grown in uninhabited environments (i.e., mountains), the number of ginseng farms has increased rapidly due to the wide demand, and ginseng thieves frequently compromise the farms. Accordingly, studies regarding regional differences in the isotopic characteristics of ginseng are required in order to deter ginseng thieves and prevent theft. In this study, we carried out stable isotope analysis of ginseng collected from various regions in Korea in order to identify potential differences. The objective of this study was to obtain data regarding the H, O, C, and N stable isotope ratios in ginseng samples from different regions in Korea and conduct a statistical analysis to determine potential factors that could be used to differentiate ginseng cultivation regions and farms. 2. Materials and methods 2.1. Ginseng collection and pre-processing of ginseng roots Fig. 1 shows the locations of the ginseng farms in the Republic of Korea. Six regions (Ganghwa-gun, Paju-si, Gimpo-si, Yeongju-si, Hwacheon-gun, and Cheolwon-gun) were selected on the basis of Korea regional criteria (si, gun, gu). These six areas are well-known ginseng cultivation regions in the Republic of Korea. Additionally, ginseng theft was prevalent in these regions just before the study
was initiated. Two ginseng-farms were randomly selected from each of the 6 regions and 4–6 ginseng samples were acquired from each ginseng farm. The adhered soil was removed from the ginseng roots, and the roots were placed in an oven set to 60 8C and dried until the moisture was completely removed. Using a ball-milling machine, the samples were subsequently processed in the form of a powder using only the main root portion of the dried ginseng roots, shown in Fig. 2. The powdered ginseng roots were wrapped five times using a silver capsule for hydrogen and oxygen stable isotope analysis and were wrapped three times using a tin capsule for carbon and nitrogen stable isotope analysis. 2.2. Analysis conditions The pretreated samples were analyzed using an elemental analyzer linked to an isotope mass spectrometer (EA-IRMS, Elemental Analysis-Isotope Ratio Mass Spectrometer; EURO EA 3000, EURO VECTOR, Italy) as well as a GV Instrument Isoprime (GV instrument Ltd., UK). Two calibration points were determined using the following standard samples in order to determine the accuracy of the measurements: hydrogen stable isotope ratios: Tibetan hair (IAEA-USGS-42, International Atomic Energy Agency, dD certified value: 78.5 2.3% VSMOW), polyethylene (IAEA-CH7, International Atomic Energy Agency, dD certified value: 100.3 2.0% VSMOW), and house cellulose; oxygen stable isotope ratios:
Fig. 1. Locations of ginseng farms across the Republic of Korea.
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coastal and inland regions could be distinguished, as noted in the figure with circles. 3.2. T-test analysis Proximity to the sea, growth conditions, differences in elevation, proximity to downtown, and the type of fertilizer etc. can affect dD, d18O, d13C, and d15N values. In this section, we describe significant differences in such factors determined using an independent sample t-test analysis.
Fig. 2. Parts of the ginseng root.
benzoic acid (IAEA-601, International Atomic Energy Agency, d18O certified value: 23.30 0.30% VSMOW) and (IAEA-USGS-42, d18O certified value: 8.56 0.10% VSMOW); carbon stable isotope ratios: cellulose standard samples (IAEA-CH-3, International Atomic Energy Agency, d13C certified values: 24.724 0.041% VPDB) and polyethylene (IAEA-CH-7, International Atomic Energy Agency, d13C certified values: 32.151 0.050% VPDB); nitrogen stable isotope ratios: ammonium sulfate (IAEA-N-1, International Atomic Energy Agency, d15N certified value: 0.4 0.2% Air) and potassium nitrate (IAEA-NO3, International Atomic Energy Agency, d15N certified value: 4.7 0.2% Air). The maximum standard deviations for the international standard samples (CH-7, 601, CH-3, and N-1) were dD: 3.1%, d18O: 1.5%, d13C: 0.5%, and d15N: 0.3% from our measurement system. Further, in consideration of the exchangeable hydrogen problem [17,18], all measurement samples including International standards (USGS-42, CH-7), the in-house cellulose standard, and ginseng samples were placed in a desiccator for two days before analysis. Additionally, the auto-sampler was purged with helium. Using the aforementioned procedure, the H-exchange problem was avoided. 3. Results The average, standard deviation, minimum, and maximum values of H, O, C, and N stable isotope ratios from samples from each farm are shown in Table 1. The dD, d18O, d13C, and d15N values were between 69.16 and 30.59%, 28.11 and 41.38%, 28.96 and 22.67%, and 2.33 and 11.81%, respectively, and the maximum standard deviations of one sample were dD: 4.5%, d18O: 2.2%, d13C: 0.9%, and d15N: 0.5%. The sample from Samdongarmri in Ganghwa-gun exhibited the greatest dD value, while the lowest value was observed in the sample from Sanbeob-ri in Yeongju-gun. The highest d18O value was observed in the sample from Geumwarl-ri in Ganghwa-gun, while the lowest value was observed from the sample from Baek-ri in Yeongju-si. The greatest d13C value was observed in the sample from Samdongarm-ri in Ganghwa-gun, while the lowest value was observed in the sample from Chowonji-ri in Gimpo-si. The greatest d15N value was observed in the sample from Juwarl-ri in Paju-si, while the lowest value was observed in the sample from Daei-ri in Hwacheon-gun. 3.1. The matrix scattered plot for dD, d18O, d13C, and d15N Matrix scattered plots were generated for the data obtained from samples from the farms shown in Fig. 3. The ginseng from Ganghwa-gun exhibited a notable difference compared to that from other regions in regard to the dD versus d18O value. Additionally, even though the values of a few samples overlapped,
3.2.1. The t-test analysis of samples from coastal and inland regions The t-test was performed to determine regional differences between coastal regions {Ganghwa (4), Gimpo (5), Paju (6)}, and inland regions {Yeongju (1), Chungju (2), Hwacheon (3)}, and the results are shown in Table 2. The p-value of dD was 0.000 and the p value of d18O was 0.000. Therefore, the hydrogen and oxygen stable isotope ratios in samples from coastal and inland regions were significantly different at the 1% level of significance. Notably, since sufficient amounts of the samples were not analyzed, the confidence level was set at 99%. 3.2.2. The t-test analysis of samples from different farms within the same region The t-test was carried out to determine differences in the stable isotope values between samples from different farms within same region, and the results are shown in Table 3. Differences in the nitrogen stable isotope value enabled the discrimination of ginseng samples from Baek-ri and Sanbeob-ri in Yeongju-si, between Daeri and Pungsan-ri in Hwacheon-gun, between Samdongarm-ri and Geumwarl-ri in Ganghwa-gun, and between Chowonji-ri and Tongarm-ri in Gimpo-si. Significant differences in the p-value were determined at the 1% level of significance. 4. Discussion 4.1. Hydrogen and oxygen stable isotope ratio The dD and d18O values of samples decreased in the following order: dD: Ganghwa (4), Gimpo (5), Paju (6), Hwacheon (3), Chungju (2), and Yeongju (1); d18O: Ganghwa (4), Paju (6), Gimpo (5) Chungju (2), Hwacheon (3), and Yeongju (1). Accordingly, the hydrogen and oxygen stable isotope ratios in samples obtained from coastal areas were higher than those obtained from inland areas. In particular, the hydrogen and oxygen stable isotope ratios in samples from Ganghwa island (4), which is closest to the yellowsea, were much higher than those from others. Samples from Yeongju (1) and Chungju (2), which are located near each other inland, showed similar low values. Therefore, according to the natural environment of the area, these values could be similar or different for each region. The matrix scattered plot (Fig. 3) showed differences between coastal and inland regions, and a t-test was conducted in order to verify the significance of statistical differences between values from samples obtained from coastal and inland regions. The t-test suggested that the differences in the hydrogen and oxygen stable isotope values between samples from coastal and inland regions were significant at the 1% level of significance. The potential reasons for the differences are as follows: hydrogen and oxygen stable isotopes in sap or plant tissue are affected by metabolic changes within the agricultural product, but exhibit a very close relationship with the isotopic composition of precipitation in the region in which they were produced [5]. The water used to grow agricultural products is supplied from soil water or groundwater, which is regenerated from precipitation [19]. The hydrogen and oxygen stable isotope ratios in precipitation are mainly affected by location; seawater contains a large
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Table 1 Average, standard deviation, minimum, and maximum values of dD, d18O, d13C, and d15N values (%) for each farm. Region (#)
Farm (# of farm)
Values
Yeongju-si (1)
Baek-ri (1-1)
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
56.27 4 6.32 65.24 50.72 67.42 4 1.29 69.16 66.06
28.50 4 0.42 28.11 29.07 33.59 4 1.14 32.60 34.96
25.76 4 0.94 26.88 24.59 27.24 4 0.89 28.54 26.62
6.75 4 1.15 5.39 8.02 0.82 4 0.27 1.12 0.47
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
58.64 5 1.60 60.97 56.64 64.53 5 2.07 66.38 60.98
31.40 5 0.45 30.94 32.14 31.88 5 1.13 30.33 33.20
25.68 5 1.78 27.89 23.63 26.90 5 1.43 28.96 25.23
7.62 5 1.17 5.74 8.78 6.40 5 0.91 5.01 7.48
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
54.32 5 0.71 55.04 53.27 54.69 6 2.59 57.63 50.43
32.26 5 1.57 29.83 34.19 30.10 6 1.25 28.70 31.96
26.07 5 0.71 26.79 25.03 24.93 6 1.47 27.08 22.99
1.64 5 0.70 2.33 0.71 7.65 6 2.26 4.64 11.01
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
40.07 5 6.83 46.58 30.59 42.92 5 2.64 45.92 39.64
38.38 5 0.79 37.41 39.45 39.69 5 1.75 37.31 41.38
23.43 5 1.38 25.88 22.67 24.34 5 0.61 24.79 23.42
9.04 5 1.21 7.09 10.38 4.90 5 1.32 2.76 6.26
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
52.74 6 2.32 55.95 50.03 52.66 4 1.88 55.16 50.81
31.46 6 0.71 30.32 32.07 32.06 4 1.40 30.90 33.93
27.79 6 1.00 28.90 26.44 24.78 4 0.32 25.05 24.32
8.77 6 1.85 6.48 11.61 2.57 4 1.66 0.81 4.79
Average value N SD Minimum value Maximum value Average value N SD Minimum value Maximum value
49.75 5 4.93 54.97 44.22 56.21 6 1.73 58.33 54.52
31.50 5 2.05 28.81 34.06 34.64 6 1.37 33.13 36.69
25.28 5 1.32 26.66 23.19 23.64 6 1.04 24.97 22.78
6.92 5 3.37 2.44 11.81 5.94 6 0.87 4.71 7.21
Sanbeob-ri (1-2)
Chungju-si (2)
Saejeong-ri (2-1)
Hyangsan-ri (2-2)
Hwacheon-gun (3)
Daei-ri (3-1)
Pungsan-ri (3-2)
Ganghwa-gun (4)
Samdongarm-ri (4-1)
Geumwarl-ri (4-2)
Gimpo-si (5)
Chowonji-ri (5-1)
Tongarm-ri (5-2)
Paju-si (6)
Juwarl-ri (6-1)
Jeomwon-ri (6-2)
#
dD (%)
d18O (%)
d13C (%)
d15N (%)
Number; N: Number of samples; SD: Standard deviation.
amount of hydrogen stable isotopes, and seawater produces clouds. As the clouds move, heavy isotopes (deuterium) preferentially drop in coastal areas and light isotopes (hydrogen) drop successively in inland areas [20]. The isotopic composition can also be affected by temperature; changes in the altitude and latitude induce a change in temperature, which influences the isotopic composition of the precipitation.
4.2. Carbon stable isotope ratio Plants are widely classified into C3, C4, and CAM species depending on their metabolic activity (metabolism). The metabolism of C3 plants follows the Calvin cycle. Most flowering plants that can produce honey live in temperate regions and have carbon stable isotope compositions of about 30 to 22%. C4 plants live
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Fig. 3. Matrix scattered plot for each farm.
Table 2 The t-test results of regional differences between coastal and inland regions. Elements
Sig. (2-tailed)
dD(%)_Ave. d18O(%)_Ave. d13C(%)_Ave. d15N(%)_Ave.
0.000 0.000 0.012 0.034
in tropical and temperate regions and exhibit Hatch–Slack metabolism; their carbon stable isotope composition is typically about 14 to 8%. The carbon stable isotope composition of CAM plant species growing in arid regions is in between that of C3 and C4 plants [21]. Anthropogenic effects, such as CO2 emission, can lead to decreases in carbon stable isotope ratios. However, differences in isotopic compositions are known to be influenced more by the metabolic activity of the plant than by the growth environment [22,23]. Thus, variations in the carbon stable isotope content due to anthropogenic effects are typical changes within the range. Korean ginseng is known to be a C3-type species. The carbon stable isotope ratios of collected ginseng samples were
between 28.96 and 22.67%, which falls within the expected carbon stable isotope ratio range of C3 plants. In particular, the carbon stable isotope ratios in samples from Ganghwa island were the highest average value. Because the population density (163.39 people/km2) is low and the strong sea wind make circulate quickly emitted CO2 through the sea. Consequently, the d13C value could be higher than that in any other region. However, samples from Gimpo showed the lowest average value. The population density of Gimpo (1202.55 people/km2) is the highest and there are a lot of plant facilities forms. Thus, these effects could deplete the carbon stable isotope ratio. As a result, the carbon stable isotope ratio would be depleted by anthropogenic effects, but the value did not exceed the range of C3 plants; additionally it might serve as an auxiliary role for discrimination of the origin of ginseng. 4.3. Nitrogen stable isotope ratio The average nitrogen stable isotope ratios (Table 1) in samples obtained from farms in Yeongju, Hwacheon, Ganghwa, and Gimpo were significantly different. In addition to comparing the average values, a t-test was also performed to compare the samples from
Table 3 The t-test results of regional differences between different farms in the same region. Elements region
dD(%)_Ave. d18O(%)_Ave. d13C(%)_Ave. d15N(%)_Ave.
Sig. (2-tailed) Yeongju-si (1)
Chunju-si (2)
Hwacheon-gun (3)
Ganhwa-gun (4)
Gimpo-si (5)
Paju-si (6)
0.036 0.001 0.062 0.001
0.001 0.416 0.266 0.105
0.751 0.039 0.134 0.000
0.423 0.180 0.230 0.001
0.958 0.476 0.000 0.001
0.040 0.023 0.056 0.555
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various farms; the p values of samples from Yeongju, Hwacheon, Ganghwa, and Gimpo were all under 0.01. As such, the differences in nitrogen stable isotope ratios of samples from these farms were determined to be significant at the 1% level of significance. Differences in the nitrogen stable isotope values were attributed to the type of fertilizer (organic or chemical) that was used for plant growth. The nitrogen stable isotope composition in plants treated with organic fertilizer is greater than that in plants treated with chemical fertilizers [24]. In addition, the use of livestock manure as fertilizer yields plants with the highest nitrogen stable isotope values [5]. Farms Baek-ri (1-1), Saejeong-ri (2-1), Hyangsan-ri (22), Pungsan-ri (3-2), Samdongarm-ri (4-1), Chowonji-ri (5-1), Juwarl-ri (6-1), and Jeomwon-ri (6-2) seemed to use organic fertilizers, and farms Sanbeob-ri (1-2), Daei-ri (3-1), Guemwon-ri (4-2), and Tongarm-ri (5-2) seemed to use chemical fertilizers. Additionally, in regard to farms in the same area, farms 2-1 and 2-2, and farms 6-1 and 6-2 seem to have used organic fertilizers. In cases when different farms within the same area used the same type of fertilizer, it was difficult to distinguish the origin of the samples based on the nitrogen stable isotope ratio. Nevertheless, since farms in four of the six regions could be distinguished by comparing the nitrogen stable isotope ratio, the nitrogen stable isotope ratio was considered to be an effective identifying factor.
considered, because all regions were located in rural areas far from downtown, and differences in isotopic composition are more influenced by the metabolic activity of plants than the growth environment. The nitrogen stable isotope ratio does not reflect geographic factors, but can be used to discriminate farms using different fertilizers within the same area. Additionally, with reference to the carbon stable isotope ratio could be improving the distinguish ability of the farms. If these results are compiled in a database, the database could be used to determine potential regions from where stolen ginseng was obtained. Furthermore, if the region is revealed through further investigation, the farm from which the sample was obtained could also potentially be determined. To summarize, the possibility to distinguish different farms by using gaps in the isotope ratio was determined. However, isotope ratio variations within each farm makes it difficult to distinguish the farm, but it is natural and very important for forensic decisions. In future research, more ginseng samples, soil, and water from the periphery of ginseng farms could be analyzed to improve the statistical reliability; sulfur and strontium stable isotope as well as multi-element XRF (X-ray fluorescence spectroscopy) analysis could be performed to enhance the discrimination according to regional differences. Acknowledgements
4.4. Isotope ratio variation within same farm Ginseng is usually cultivated in large quantities, which means that the cultivation area is extremely large. Therefore, it is also important to check the isotope ratio within large farms. The range of dD values was larger than that of d15N, d18O, and d13C values. Broad scale ranges makes small differences look like big differences, but this can be a great forensic tool, because we can decide confidently whether or not the origin of each sample is different. The d18O values seemed to follow the dD patterns, but the standard deviation was smaller than that of the dD values because the scale range of the d18O value was smaller than that of the dD values. Notably, Bateman et al. [26], reported that the standard deviation of d15N values is about 1%, even when the same type of fertilizer and same quantity is used for various agricultural products (carrot, tomato, lettuce). Ginseng is usually cultivated for 4–6 years and the type of fertilizer used may be varied during this time. Thus, the d15N of ginseng may vary more for each ginseng from same farm. Carbon isotope ratios slightly depend on individual characteristics (e.g. degree of photosynthesis, nutrients, and water absorption [22,23]). Therefore, a 2% variation in the carbon isotope ratio within each farm is considered normal. As mentioned above, the isotope ratios in animal and plant samples may be changed, even if ginseng is grown in the same environment. However, variations within a single farm are important in forensic science. Therefore, different analytical methods (multi-elements analysis, isotope ratio of soil and water) should be used in combination in the future. 5. Conclusion The purpose of this study was to determine whether stable isotope ratios could be used to discriminate Korean ginseng according to regional differences. Even though this was a pilot study, reasonable results were obtained. The hydrogen and oxygen stable isotope ratios were affected by precipitation. Even though the total area of the Republic of Korea is small, ginseng samples from coastal regions were generally heavier than those from inland areas, indicating that regional differences could be noted. The carbon stable isotope ratio of all ginseng samples ranged from 30 to 22%; therefore, ginseng was confirmed to be a C3 plant. Environmental factors caused by anthropogenic effects were not
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2012-0009832), and this work was supported by a grant (2014Chemistry-01) from the National Forensic Service (NFS) received in 2014.
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