# Chemical Differentiation of Genetically IdentifiedAtractylodes japonica,A. macrocephala, andA. chinensis Rhizomes Using High-Performance Liquid Chromatography with Chemometric Analysis

Authors: Jung-Hoon Kim; Eui-Jeong Doh; Guemsan Lee
Journal: Evidence-Based Complementary and Alternative Medicine
 (2018)

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## Abstract

The rhizome ofAtractylodes japonica, which is a herbal medicine used for gastrointestinal therapeutics, has been categorized withA. macrocephala rhizome orA. chinensis rhizome based on different therapeutic criteria in Korea, China, and Japan. In the present study, 61A. japonica,A. macrocephala, andA. chinensisrhizomes were collected from Korea and China and were genetically identified by internal transcribed spacer sequencing analysis. Chromatographic profiles were obtained from high-performance liquid chromatography analysis of the methanol and hot-water extracts ofAtractylodes rhizomes and chemical differentiation of the rhizomes was carried out using chemometric statistical analyses such as principal component analysis, hierarchical clustering analysis, and Pearson’s correlation coefficient analysis. The results from chromatographic profiles and chemometric analyses demonstrate thatA. japonica rhizomes showed apparent chemical differences fromA. macrocephala andA. chinensis rhizomes in the methanol extracts. In contrast, no clear distinction was apparent for the hot-water extracts ofAtractylodes rhizomes, especiallyA. chinensis rhizomes. These results indicate that there is a clear chemical difference betweenA. japonica andA. macrocephala rhizomes; however, the chemical diversity ofA. chinensis rhizome shows different chemical relationships withA. japonica orA. macrocephala rhizome, dependent on the chemical features.


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## Body

## 1. Introduction

The rhizomes ofAtractylodes japonica,A. macrocephala, andA. chinensis (Asteraceae) have been therapeutically used to treat gastrointestinal disorders in Korea, China, and Japan. The medicinal classification of these rhizomes differs in the pharmacopeia of each country: the rhizomes ofA. japonica andA. macrocephala are categorized together in Korea, while the rhizome ofA. japonica is unofficial in the pharmacopeia but is categorized with that ofA. chinensis in China [1–3]. This inconsistency, especially with respect toA. japonica rhizome, causes confusion in the use of theAtractylodes rhizomes between countries with different categorization of such medicinal herbs.For the criteria of the use of Atractylodes rhizomes, we reported in our previous study that the rhizome ofA. japonica showed a closer genetic relationship and higher chemical similarity withA. chinensis rhizome than withA. macrocephala rhizome, and more consistent therapeutic effects would be expected when similarly categorized. Therefore, we proposed thatA. japonica rhizome is genetically and chemically distinct fromA. macrocephala rhizome and that the former should be used under the same medicinal categorization asA. chinensis rhizome [4]. However, there were a number of limitations at study, such as insufficient sample numbers, a lack of detailed sample information, and single solvent extraction. Hence, genetic and chemical relationships amongAtractylodes rhizomes should be evaluated under improved conditions.In the present study, a total of 61 Atractylodes samples were collected and their original species were identified using internal transcribed spacer (ITS) sequences from nuclear ribosomal DNA (nrDNA), which is the identification method mostly used for genetic confirmation of plant species [5–7]. Genetically identified samples were extracted with either organic or aqueous solvents, and the extracts were analyzed using a high-performance liquid chromatography-diode array detector (HPLC-DAD) instrument to compare their chromatographic profiles [8]. The chemical relevance of the rhizomes ofA. japonica,A. macrocephala, andA. chinensis was determined based on principal component analysis (PCA), hierarchical clustering analysis (HCA), and Pearson’s correlation analysis.


## 2. Materials and Methods

### 2.1. Plant Materials and Reagents

Methanol, water, and acetonitrile (HPLC grade) were purchased from J.T.Baker (Phillipsburg, NJ, USA). Trifluoroacetic acid (TFA) was purchased from Sigma-Aldrich (St Louis, MO, USA). Forty-one samples ofA. japonica rhizomes, 11 samples ofA. macrocephala rhizomes, and 9 samples ofA. chinensis rhizomes were obtained from the wild, agricultural fields, or local herbal markets in Korea and China in 2016. The samples were authenticated by their morphological characteristics and by ITS sequencing analysis. The samples were coded as AJ forA. japonicarhizomes, AM forA. macrocephalarhizomes, and AC forA. chinensis rhizomes (Table 1). Voucher specimens have been deposited at the School of Korean Medicine, Pusan National University.Table 1
Genetically original species and collecting area of Atractylodes samples.
Code Location Country Date of collection Code Location Country Date of collection AJ1 Euiseong, Gyeongbuk Korea Jan, 2016 AJ32 Yeongdeok, Gyeongbuk Korea Feb, 2016 AJ2 – Korea Feb, 2016 AJ33 Yeongcheon, Gyeongbuk Korea Feb, 2016 AJ3 Euiseong, Gyeongbuk Korea Feb, 2016 AJ34 Yangpyeong, Gyeonggi Korea Feb, 2016 AJ4 – Korea Jan, 2016 AJ35 Hongcheon, Gangwon Korea Feb, 2016 AJ5 Yeongdeok, Gyeongbuk Korea Feb, 2016 AJ36 Andong, Gyeongbuk Korea Feb, 2016 AJ6 – Korea Jan, 2016 AJ37 Yeongcheon, Gyeongbuk Korea Feb, 2016 AJ7 Bonghwa, Gyeongbuk Korea Feb, 2016 AJ38 Cheongsong, Gyeongbuk Korea Feb, 2016 AJ8 Yeongcheon, Gyeongbuk Korea Feb, 2016 AJ39 Neimengu China Apr, 2016 AJ9 Yeongcheon, Gyeongbuk Korea Feb, 2016 AJ40 Hebei China Jul, 2016 AJ10 – Korea Mar, 2016 AJ41 – China Aug, 2016 AJ11 – China Jan, 2016 AM1 – China Jan, 2016 AJ12 – China Jan, 2016 AM2 Anhui China Jul, 2016 AJ13 Yeongwol, Gangwon Korea Feb, 2016 AM3 – China Jan, 2016 AJ14 Hwasun, Jeonnam Korea Jan, 2016 AM4 Zhejiang China Mar, 2016 AJ15 Heilongjiang China Mar, 2016 AM5 Zhejiang China Feb, 2014 AJ16 – Korea (North) Jan, 2016 AM6 – China Jan, 2016 AJ17 – Korea Jan, 2016 AM7 – China Jan, 2016 AJ18 Yeongdeok, Gyeongbuk Korea Jan, 2016 AM8 Shanghai China Jul, 2016 AJ19 – Korea Jan, 2016 AM9 Panan, Zhejiang China Jul, 2016 AJ20 – China Jan, 2016 AM10 Panan, Zhejiang China Jul, 2016 AJ21 – China Jan, 2016 AM11 – China Aug, 2016 AJ22 Heilongjiang China Jan, 2016 AC1 Hubei China Mar, 2016 AJ23 Suwon, Gyeonggi Korea Jan, 2016 AC2 – China Jan, 2016 AJ24 Jangsu, Jeonbuk Korea Jan, 2016 AC3 Chengde, Hebei China Jul, 2016 AJ25 Yangsan, Gyeongnam Korea Oct, 2016 AC4 – China Jan, 2016 AJ26 Cheongyang, Chungnam Korea May, 2016 AC5 Chengde, Hebei China Jul, 2016 AJ27 Heilongjiang China Aug, 2016 AC6 Hebei China Jul, 2016 AJ28 Yeongcheon, Gyeongbuk Korea Feb, 2016 AC7 Neimengu China Mar, 2014 AJ29 Yeongcheon, Gyeongbuk Korea Feb, 2016 AC8 – China Jan, 2016 AJ30 – Korea Feb, 2016 AC9 – China Jan, 2016 AJ31 Jecheon, Chungbuk Korea Feb, 2016 AJ,Atractylodes japonica; AM, A. macrocephala; AC, A. chinensis. “–”: unclear location.


### 2.2. Preparation of Genomic DNA

Genomic DNA was extracted from the crude drugs of Atractylodes rhizomes according to the modified manuals of NucleoSpin® Plant II kit (Macherey-Nagel, Dueren, Germany). For some samples, 10% cetyltrimethyl ammonium bromide and 0.7 M NaCl were used to remove the phenolic compounds and polysaccharides.


### 2.3. PCR Amplification

For ITS amplification, PCR was performed using a T-personal cycler (Biometra; Göttingen, Germany). Briefly, 600 nM of the primer set of ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [9], AccuPower® GoldHotstart Taq PCR PreMix (Bioneer, Daejeon, Korea), and 50 ng of genomic DNA were used for PCR amplification. PCR cycling conditions, which were followed by predenaturation process (95°C, 5 min), were as follows: denaturation process (95°C, 30 s); annealing progress (52°C, 30 s); extension process (72°C, 40 s) × 36 cycles; final extension process (72°C, 5 min). The amplified PCR product was separated from other gradients by using 1.5% agarose gel electrophoresis after staining by the addition of SafeView™ (abm; Vancouver, Canada). Amplified products were analyzed using MyImage (Seoulin Biotechnology; Seongnam, Gyeonggi-do, Korea).


### 2.4. Determination of DNA Sequence of PCR Product

PCR product separated from agarose gel was cloned by using a TOPcloner™ TA-Blunt kit (Enzynomics, Daejeon, Korea) and the DNA sequence of the cloned PCR product was determined after interpretation performed by Bioneer (Daejeon, Korea).


### 2.5. Analysis of DNA Sequence and Preparation of Dendrogram

DNA sequences were analyzed using the ClustalW multiple sequence alignment (BioEdit, v7.0.9; available athttp://www.mbio.ncsu.edu/BioEdit/page2.html) and the phylogenetic tree was created using DNADist (BioEdit). To study the relationships amongAtractylodes rhizomes, the nucleotide sequences of the genera Atractylis and Carlina deposited in NCBI GenBank were used. The genera Brachylaena, Cardopatium, Cirsium, Echinops, Phonus, and Tarchonanthus were used as outgroups in the phylogenetic analyses, based on previous studies [10, 11]. ITS sequences of these taxa were collected from NCBI GenBank. The ITS sequences of nine specimens (coded with Arabic numerals) ofAtractylodes plants were collected as species reference samples for identification ofAtractylodes rhizomes in previous research [4], and these were used as a reference for species.


### 2.6. Preparation of Samples for HPLC Analysis

For the methanol extract, dried powder of the rhizomes (1.0 g) was extracted with 10 mL of methanol using an ultrasonic extractor (Power Sonic 520; Hwashin Tech, Daegu, Korea) for 30 min. The extract was centrifuged at 10,000 rpm for 5 min. The supernatant was transferred to a 1.5 mL microtube and evaporated using a nitrogen gas blowing concentrator (MGS–2200; Eyela, Miyagi, Japan). Residue was dissolved in methanol to a concentration of 10,000μg/mL and the extract solution was filtered through a 0.2 μm syringe filter (BioFact; Daejeon, Korea) prior to HPLC injection.For the hot-water extract, dried powder of the rhizome (0.5 g) was extracted in a 100 mL glass bottle with 20 mL of distilled water using a microwave oven (BP-111-RS; Microwave Research & Applications, Inc., IL, USA) at 90°C for 20 min. The water extract was transferred to a 15 mL conical tube and centrifuged at 3000 rpm for 10 min. The supernatant was lyophilized using a freeze-dryer (IlShinBioBase; Dongducheon, Gyeonggi-do, Korea). The powder of dried extract (20 mg) was dissolved in 1 mL of HPLC grade water and then the solution was centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.2μm syringe filter (BioFact) prior to HPLC injection.


### 2.7. HPLC Conditions for Chromatographic Fingerprinting

An Agilent 1260 liquid chromatography system (Agilent Technologies; Palo Alto, CA, USA) equipped with an autosampler, degasser, quaternary solvent pump, and diode array detector (DAD) was used for chromatographic fingerprinting. The data were processed with Chemstation software (Agilent Technologies). The separation of compounds was carried out on a Capcell Pak Mg II C18 column (4.6 mm × 250 mm, 5 μm; Shiseido, Tokyo, Japan) at 35°C. The flow rate was 1 mL/min and the injection volume was 10 μL.For analysis of the methanol extract, the mobile phase consisted of water (A) and acetonitrile (B), with the following gradient elution: 55% (B) over 0–1 min, 55–60% (B) over 1–35 min, 60% (B) over 35–36 min, 60–90% (B) over 36–51 min, 90% (B) over 51–52 min, and then reequilibration to 55% (B) until the end of the analysis. Detection was performed with a UV detector at wavelengths of 230, 255, 275, 315, and 340 nm.For analysis of the hot-water extract, the mobile phase consisted of 0.1% TFA in water (A) and acetonitrile (B), with the following gradient elution: 5% (B) over 0–5 min, 5–40% (B) over 5–35 min, 40% (B) over 35–36 min, 40–70% (B) over 36–51 min, 70% (B) over 51–52 min, and then reequilibration to 5% (B) until the end of the analysis. Detection was performed with a UV detector at wavelengths of 225, 255, 275, 295, and 325 nm.


### 2.8. Chemometric Statistical Analysis

The 61 samples that were genetically identified and recoded were used for PCA, HCA, and Pearson’s correlation analysis. In total, 45 and 31 peaks were selected as reference peaks (> 1.0% of total peak area) for the methanol extract and water extract, respectively, at their optimal UV absorption, and their absolute areas were calculated by peak area integration for chromatographic fingerprinting. A matrix composed of the rows (Atractylodes sample) and columns (absolute area of each reference peak) was used to construct the PC plot and dendrogram and for Pearson’s correlation analysis, using the open source software R (v. 3.4.3; The R Foundation for Statistical Computing).


## 2.1. Plant Materials and Reagents

Methanol, water, and acetonitrile (HPLC grade) were purchased from J.T.Baker (Phillipsburg, NJ, USA). Trifluoroacetic acid (TFA) was purchased from Sigma-Aldrich (St Louis, MO, USA). Forty-one samples ofA. japonica rhizomes, 11 samples ofA. macrocephala rhizomes, and 9 samples ofA. chinensis rhizomes were obtained from the wild, agricultural fields, or local herbal markets in Korea and China in 2016. The samples were authenticated by their morphological characteristics and by ITS sequencing analysis. The samples were coded as AJ forA. japonicarhizomes, AM forA. macrocephalarhizomes, and AC forA. chinensis rhizomes (Table 1). Voucher specimens have been deposited at the School of Korean Medicine, Pusan National University.Table 1
Genetically original species and collecting area of Atractylodes samples.
Code Location Country Date of collection Code Location Country Date of collection AJ1 Euiseong, Gyeongbuk Korea Jan, 2016 AJ32 Yeongdeok, Gyeongbuk Korea Feb, 2016 AJ2 – Korea Feb, 2016 AJ33 Yeongcheon, Gyeongbuk Korea Feb, 2016 AJ3 Euiseong, Gyeongbuk Korea Feb, 2016 AJ34 Yangpyeong, Gyeonggi Korea Feb, 2016 AJ4 – Korea Jan, 2016 AJ35 Hongcheon, Gangwon Korea Feb, 2016 AJ5 Yeongdeok, Gyeongbuk Korea Feb, 2016 AJ36 Andong, Gyeongbuk Korea Feb, 2016 AJ6 – Korea Jan, 2016 AJ37 Yeongcheon, Gyeongbuk Korea Feb, 2016 AJ7 Bonghwa, Gyeongbuk Korea Feb, 2016 AJ38 Cheongsong, Gyeongbuk Korea Feb, 2016 AJ8 Yeongcheon, Gyeongbuk Korea Feb, 2016 AJ39 Neimengu China Apr, 2016 AJ9 Yeongcheon, Gyeongbuk Korea Feb, 2016 AJ40 Hebei China Jul, 2016 AJ10 – Korea Mar, 2016 AJ41 – China Aug, 2016 AJ11 – China Jan, 2016 AM1 – China Jan, 2016 AJ12 – China Jan, 2016 AM2 Anhui China Jul, 2016 AJ13 Yeongwol, Gangwon Korea Feb, 2016 AM3 – China Jan, 2016 AJ14 Hwasun, Jeonnam Korea Jan, 2016 AM4 Zhejiang China Mar, 2016 AJ15 Heilongjiang China Mar, 2016 AM5 Zhejiang China Feb, 2014 AJ16 – Korea (North) Jan, 2016 AM6 – China Jan, 2016 AJ17 – Korea Jan, 2016 AM7 – China Jan, 2016 AJ18 Yeongdeok, Gyeongbuk Korea Jan, 2016 AM8 Shanghai China Jul, 2016 AJ19 – Korea Jan, 2016 AM9 Panan, Zhejiang China Jul, 2016 AJ20 – China Jan, 2016 AM10 Panan, Zhejiang China Jul, 2016 AJ21 – China Jan, 2016 AM11 – China Aug, 2016 AJ22 Heilongjiang China Jan, 2016 AC1 Hubei China Mar, 2016 AJ23 Suwon, Gyeonggi Korea Jan, 2016 AC2 – China Jan, 2016 AJ24 Jangsu, Jeonbuk Korea Jan, 2016 AC3 Chengde, Hebei China Jul, 2016 AJ25 Yangsan, Gyeongnam Korea Oct, 2016 AC4 – China Jan, 2016 AJ26 Cheongyang, Chungnam Korea May, 2016 AC5 Chengde, Hebei China Jul, 2016 AJ27 Heilongjiang China Aug, 2016 AC6 Hebei China Jul, 2016 AJ28 Yeongcheon, Gyeongbuk Korea Feb, 2016 AC7 Neimengu China Mar, 2014 AJ29 Yeongcheon, Gyeongbuk Korea Feb, 2016 AC8 – China Jan, 2016 AJ30 – Korea Feb, 2016 AC9 – China Jan, 2016 AJ31 Jecheon, Chungbuk Korea Feb, 2016 AJ,Atractylodes japonica; AM, A. macrocephala; AC, A. chinensis. “–”: unclear location.


## 2.2. Preparation of Genomic DNA

Genomic DNA was extracted from the crude drugs of Atractylodes rhizomes according to the modified manuals of NucleoSpin® Plant II kit (Macherey-Nagel, Dueren, Germany). For some samples, 10% cetyltrimethyl ammonium bromide and 0.7 M NaCl were used to remove the phenolic compounds and polysaccharides.


## 2.3. PCR Amplification

For ITS amplification, PCR was performed using a T-personal cycler (Biometra; Göttingen, Germany). Briefly, 600 nM of the primer set of ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [9], AccuPower® GoldHotstart Taq PCR PreMix (Bioneer, Daejeon, Korea), and 50 ng of genomic DNA were used for PCR amplification. PCR cycling conditions, which were followed by predenaturation process (95°C, 5 min), were as follows: denaturation process (95°C, 30 s); annealing progress (52°C, 30 s); extension process (72°C, 40 s) × 36 cycles; final extension process (72°C, 5 min). The amplified PCR product was separated from other gradients by using 1.5% agarose gel electrophoresis after staining by the addition of SafeView™ (abm; Vancouver, Canada). Amplified products were analyzed using MyImage (Seoulin Biotechnology; Seongnam, Gyeonggi-do, Korea).


## 2.4. Determination of DNA Sequence of PCR Product

PCR product separated from agarose gel was cloned by using a TOPcloner™ TA-Blunt kit (Enzynomics, Daejeon, Korea) and the DNA sequence of the cloned PCR product was determined after interpretation performed by Bioneer (Daejeon, Korea).


## 2.5. Analysis of DNA Sequence and Preparation of Dendrogram

DNA sequences were analyzed using the ClustalW multiple sequence alignment (BioEdit, v7.0.9; available athttp://www.mbio.ncsu.edu/BioEdit/page2.html) and the phylogenetic tree was created using DNADist (BioEdit). To study the relationships amongAtractylodes rhizomes, the nucleotide sequences of the genera Atractylis and Carlina deposited in NCBI GenBank were used. The genera Brachylaena, Cardopatium, Cirsium, Echinops, Phonus, and Tarchonanthus were used as outgroups in the phylogenetic analyses, based on previous studies [10, 11]. ITS sequences of these taxa were collected from NCBI GenBank. The ITS sequences of nine specimens (coded with Arabic numerals) ofAtractylodes plants were collected as species reference samples for identification ofAtractylodes rhizomes in previous research [4], and these were used as a reference for species.


## 2.6. Preparation of Samples for HPLC Analysis

For the methanol extract, dried powder of the rhizomes (1.0 g) was extracted with 10 mL of methanol using an ultrasonic extractor (Power Sonic 520; Hwashin Tech, Daegu, Korea) for 30 min. The extract was centrifuged at 10,000 rpm for 5 min. The supernatant was transferred to a 1.5 mL microtube and evaporated using a nitrogen gas blowing concentrator (MGS–2200; Eyela, Miyagi, Japan). Residue was dissolved in methanol to a concentration of 10,000μg/mL and the extract solution was filtered through a 0.2 μm syringe filter (BioFact; Daejeon, Korea) prior to HPLC injection.For the hot-water extract, dried powder of the rhizome (0.5 g) was extracted in a 100 mL glass bottle with 20 mL of distilled water using a microwave oven (BP-111-RS; Microwave Research & Applications, Inc., IL, USA) at 90°C for 20 min. The water extract was transferred to a 15 mL conical tube and centrifuged at 3000 rpm for 10 min. The supernatant was lyophilized using a freeze-dryer (IlShinBioBase; Dongducheon, Gyeonggi-do, Korea). The powder of dried extract (20 mg) was dissolved in 1 mL of HPLC grade water and then the solution was centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.2μm syringe filter (BioFact) prior to HPLC injection.


## 2.7. HPLC Conditions for Chromatographic Fingerprinting

An Agilent 1260 liquid chromatography system (Agilent Technologies; Palo Alto, CA, USA) equipped with an autosampler, degasser, quaternary solvent pump, and diode array detector (DAD) was used for chromatographic fingerprinting. The data were processed with Chemstation software (Agilent Technologies). The separation of compounds was carried out on a Capcell Pak Mg II C18 column (4.6 mm × 250 mm, 5 μm; Shiseido, Tokyo, Japan) at 35°C. The flow rate was 1 mL/min and the injection volume was 10 μL.For analysis of the methanol extract, the mobile phase consisted of water (A) and acetonitrile (B), with the following gradient elution: 55% (B) over 0–1 min, 55–60% (B) over 1–35 min, 60% (B) over 35–36 min, 60–90% (B) over 36–51 min, 90% (B) over 51–52 min, and then reequilibration to 55% (B) until the end of the analysis. Detection was performed with a UV detector at wavelengths of 230, 255, 275, 315, and 340 nm.For analysis of the hot-water extract, the mobile phase consisted of 0.1% TFA in water (A) and acetonitrile (B), with the following gradient elution: 5% (B) over 0–5 min, 5–40% (B) over 5–35 min, 40% (B) over 35–36 min, 40–70% (B) over 36–51 min, 70% (B) over 51–52 min, and then reequilibration to 5% (B) until the end of the analysis. Detection was performed with a UV detector at wavelengths of 225, 255, 275, 295, and 325 nm.


## 2.8. Chemometric Statistical Analysis

The 61 samples that were genetically identified and recoded were used for PCA, HCA, and Pearson’s correlation analysis. In total, 45 and 31 peaks were selected as reference peaks (> 1.0% of total peak area) for the methanol extract and water extract, respectively, at their optimal UV absorption, and their absolute areas were calculated by peak area integration for chromatographic fingerprinting. A matrix composed of the rows (Atractylodes sample) and columns (absolute area of each reference peak) was used to construct the PC plot and dendrogram and for Pearson’s correlation analysis, using the open source software R (v. 3.4.3; The R Foundation for Statistical Computing).


## 3. Results

### 3.1. ITS Genotype and Genetic Identification of Atractylodes Rhizomes

Amplification of the ITS region produced overall 733 bp of nucleotide sequences from 61 samples listed in Table1 and from nine dried voucher specimens [4]. The ITS sequences of samples were determined by comparing DNA sequences registered in NCBI GenBank as well as in [12], with the following accession numbers: forA. japonica AB219405, forA. macrocephala AB219406, forA. lancea AB219407, forA. chinensis AB219408, and forA. koreana AB219409. As shown in previous research [4], nucleotide substitutions were observed in 37 sites on the ITS regions of Atractylodes samples. Type 1, the ITS sequence ofA. japonica, showed multiple sequences compared with the other species; Types 2 and 3 were the genotypes ofA. macrocephala andA. lancea, respectively. Type 4, the genotype ofA. chinensis, was identical to Type 5, the genotype ofA. koreana.All 11 samples labeled AM, were determined asA. macrocephala; no difference of DNA sequence between samples was observed. Among the nine samples labeled AC, determined asA. chinensis, AC4, AC7, and AC8 showed differences in nucleotide sequence. The sequence of AC4, AC7, and AC8 differed from that of Type 4 by 1 bp (A → G) at nucleotide position 128 bp, which indicates intraspecific variation. All 41 samples labeled AJ were determined asA. japonica. Unlike in other species, some intraspecific variation was observed among the 41 AJ samples. Most of the differences were multisequences, which have already been shown to be Type 1 [4].


### 3.2. Genetic Relationship of the Atractylodes Rhizomes

Sixty-one samples of Atractylodes rhizomes were identified asA. chinensis,A. japonica, andA. macrocephala. Phylogenetic classifications based on the ITS region were made and the inferred evolutionary relationships amongAtractylodes rhizomes are represented as a phylogenetic tree. The genusAtractylodes is well separated from other close genera and outgroups. The samples ofA. japonica,A. lancea, andA. chinensis formedA. lancea complex, whereas those ofA. macrocephala formed their ownA. macrocephala complex (Figure 1).Figure 1
Phylogenic tree from DNADist (neighbor phylogenetic tree) analysis of the ITS nucleotide sequences. The ITS sequences of taxa with Atractylis, Carlina, and Outgroup were downloaded from NCBI GenBank. The samples with Arabic numerals were dried voucher specimens deposited at the Korea Institute of Oriental Medicine.


### 3.3. Chromatographic Profiling of the Methanol and Hot-Water Extracts of Atractylodes Samples

Chromatographic profiles of representative samples (AJ1, AC1, and AM1) at all UV wavelengths were compared; a total of 45 peaks and 31 peaks for the methanol and water extracts, respectively, were selected for comparison by macroscopic observation and further chemometric analysis (Figures2 and 3 and Table S1). There were distinct differences in chromatographic patterns of the methanol extracts of AJ, AC, and AM, and 28 out of 45 reference peaks were shared among the methanol extracts ofAtractylodessamples (Figure 4). In contrast, chromatographic differences among the water extracts were less distinctive, with 18 out of 31 references peaks being shared among the chromatograms from the hot-water extracts (Figure 5).Figure 2
Representative chromatograms of methanol extracts ofAtractylodes japonica (AJ1, (a)),A. chinensis (AC1, (b)), andA. macrocephala (AM1, (c)) at UV wavelengths of 230, 255, 275, 315, and 340 nm.
(a) (b) (c)Figure 3
Representative chromatograms of hot-water extracts ofA. japonica (AJ1, (a)),A. chinensis (AC1, (b)), andA. macrocephala (AM1, (c)) at UV wavelengths of 225, 255, 275, 295, and 325 nm.
(a) (b) (c)Figure 4
Representative chromatograms of methanol extracts ofA. japonica (AJ1–9, (a)),A. chinensis (AC1–9, (b)), andA. macrocephala (AM1–9, (c)) at UV wavelength of 315 nm.
(a) (b) (c)Figure 5
Representative chromatograms of hot-water extracts ofA. japonica (AJ1–9, (a)),A. chinensis (AC1–9, (b)), andA. macrocephala (AM1–9, (c)) at UV wavelength of 325 nm.
(a) (b) (c)


### 3.4. Clustering Analysis of Atractylodes Samples Using Chemometric Statistical Methods

Chromatographic profiles of the methanol and water extracts from Atractylodes samples were further analyzed by principal component analysis (PCA), hierarchical clustering analysis (HCA), and of Pearson’s correlation coefficient to estimate the relationship between theAtractylodes samples.In the principal component (PC) plot of the methanol extracts of the samples, three distinct clusters were observed: an AJ group (AJ1–41 including AC4), an AM group (AM1–10), and an AC group (AC1–9 except AC4). The distribution of PC1 score showed that the samples in the AJ group were plotted closer to those in the AM group than those in the AC group; in contrast, in the PC2 score, the samples in the AC group were located closer to those in the AJ group than those in the AM group (Figure6). However, the PC plot of the hot-water extracts of the samples differed from that of the methanol extracts. As the samples were distributed in a wide range of PC1 and PC2 scores, no distinct clusters were observed. AC4 and AC6 were plotted closer to the location of AJ samples, whereas AC5 and AC8 overlapped into the plots of AM samples (Figure 7).Figure 6
Score plot of principal components (PC1 versus PC2) on the variables (absolute area of reference peaks) withAtractylodes samples from the methanol extracts. PC1 and PC2 represent 40% and 21% of the total variance, respectively. AC:A. chinensis Koidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.Figure 7
Score plot of principal components (PC1 versus PC2) on the variables (absolute area of reference peaks) withAtractylodes samples from the hot-water extracts. PC1 and PC2 represent 40% and 14% of the total variance, respectively. AC:A. chinensis Koidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.The dendrogram of methanol extracts from HCA showed similar groups as obtained from PCA; namely, AJ, AC, and AM samples formed their own groups (groups II, III, and IV) except for AC4 in the AJ group. However, several AJ samples were divided into a separate group below a height of 250000 and clustered in group I below a height of 100000 (Figure8). For the dendrogram of the methanol extracts, the water extracts of AJ samples were also divided into two distinct groups (groups I and II), whereas those of AC and AM samples were not clearly distinguished within their own groups because they were not separated in the subgroup of group II, below a height of 4000 (Figure 9).Figure 8
Hierarchical clustering analysis of Atractylodes samples from methanol extracts. AC:A. chinensis Koidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.Figure 9
Hierarchical clustering analysis of Atractylodes samples from hot-water extracts. AC:A. chinensis Koidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.


### 3.5. Evaluation of the Correlation between A. japonica, A. chinensis, and A. macrocephala Samples by Pearson’s Correlation Coefficient

In the methanol extracts, the mean value of the Pearson’s correlation coefficient (r) of each AJ sample ranged from 0.01 to 0.31 (except for AJ41;r = –0.01) for the correlation of AM samples and from 0.01 to 0.1 for the correlation of AC samples (Figure 10(a)). The mean correlation coefficient of each AC sample ranged from –0.08 to 0.1 (except for AC4;r = 0.95) for the correlation of AJ samples and from –0.07 to 0.1 for the correlation of AM samples (Figure 10(b)). The mean correlation coefficient of each AM sample ranged from –0.05 to –0.01 for the correlation of AC samples and from –0.03 to 0.22 for the correlation of AJ samples (Figure 10(c)).Figure 10
Average coefficients of Pearson’s correlation coefficient ofAtractylodes samples from methanol extracts. (a) AJ–AC and AJ–AM, (b) AC–AJ and AC–AM, and (c) AM–AJ and AM–AC. AC:A. chinensisKoidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.
(a) (b) (c)In the hot-water extracts, the mean correlation coefficient of each AJ sample ranged from 0.25 to 0.80 for the correlation of AM samples and from 0.30 to 0.81 for the correlation of AC samples (Figure11(a)). The mean correlation coefficient of each AC sample ranged from 0.55 to 0.72 for the correlation of AJ samples and from 0.29 to 0.87 for the correlation of AM samples (Figure 11(b)). The mean correlation coefficient of each AM sample ranged from 0.49 to 0.79 for the correlation of AC samples and from 0.45 to 0.65 for the correlation of AJ samples (Figure 11(c)). The mean and median values of Pearson’s correlation coefficient fromAtractylodes samples are shown in Table 2.Table 2
Mean and median value of Pearson’s correlation coefficient (r) among AJ, AM, and AC samples.
Extraction Sample Parameter Correlation coefficient (r) AJ AM AC Methanol extract AJ Mean – 0.131 0.069 Median – 0.146 –0.054 AM Mean 0.131 – –0.028 Median 0.146 – –0.048 AC Mean 0.069 –0.028 – Median –0.054 –0.048 –  Water extract AJ Mean – 0.567 0.609 Median – 0.584 0.626 AM Mean 0.567 – 0.733 Median 0.584 – 0.800 AC Mean 0.609 0.733 – Median 0.626 0.800 – AJ,A. japonica; AM, A. macrocephala; AC, A. chinensis.Figure 11
Average coefficients of Pearson’s correlation coefficient ofAtractylodes samples from hot-water extracts. (a) AJ–AC and AJ–AM, (b) AC–AJ and AC–AM, and (c) AM–AJ and AM–AC. AC:A. chinensisKoidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.
(a) (b) (c)


## 3.1. ITS Genotype and Genetic Identification of Atractylodes Rhizomes

Amplification of the ITS region produced overall 733 bp of nucleotide sequences from 61 samples listed in Table1 and from nine dried voucher specimens [4]. The ITS sequences of samples were determined by comparing DNA sequences registered in NCBI GenBank as well as in [12], with the following accession numbers: forA. japonica AB219405, forA. macrocephala AB219406, forA. lancea AB219407, forA. chinensis AB219408, and forA. koreana AB219409. As shown in previous research [4], nucleotide substitutions were observed in 37 sites on the ITS regions of Atractylodes samples. Type 1, the ITS sequence ofA. japonica, showed multiple sequences compared with the other species; Types 2 and 3 were the genotypes ofA. macrocephala andA. lancea, respectively. Type 4, the genotype ofA. chinensis, was identical to Type 5, the genotype ofA. koreana.All 11 samples labeled AM, were determined asA. macrocephala; no difference of DNA sequence between samples was observed. Among the nine samples labeled AC, determined asA. chinensis, AC4, AC7, and AC8 showed differences in nucleotide sequence. The sequence of AC4, AC7, and AC8 differed from that of Type 4 by 1 bp (A → G) at nucleotide position 128 bp, which indicates intraspecific variation. All 41 samples labeled AJ were determined asA. japonica. Unlike in other species, some intraspecific variation was observed among the 41 AJ samples. Most of the differences were multisequences, which have already been shown to be Type 1 [4].


## 3.2. Genetic Relationship of the Atractylodes Rhizomes

Sixty-one samples of Atractylodes rhizomes were identified asA. chinensis,A. japonica, andA. macrocephala. Phylogenetic classifications based on the ITS region were made and the inferred evolutionary relationships amongAtractylodes rhizomes are represented as a phylogenetic tree. The genusAtractylodes is well separated from other close genera and outgroups. The samples ofA. japonica,A. lancea, andA. chinensis formedA. lancea complex, whereas those ofA. macrocephala formed their ownA. macrocephala complex (Figure 1).Figure 1
Phylogenic tree from DNADist (neighbor phylogenetic tree) analysis of the ITS nucleotide sequences. The ITS sequences of taxa with Atractylis, Carlina, and Outgroup were downloaded from NCBI GenBank. The samples with Arabic numerals were dried voucher specimens deposited at the Korea Institute of Oriental Medicine.


## 3.3. Chromatographic Profiling of the Methanol and Hot-Water Extracts of Atractylodes Samples

Chromatographic profiles of representative samples (AJ1, AC1, and AM1) at all UV wavelengths were compared; a total of 45 peaks and 31 peaks for the methanol and water extracts, respectively, were selected for comparison by macroscopic observation and further chemometric analysis (Figures2 and 3 and Table S1). There were distinct differences in chromatographic patterns of the methanol extracts of AJ, AC, and AM, and 28 out of 45 reference peaks were shared among the methanol extracts ofAtractylodessamples (Figure 4). In contrast, chromatographic differences among the water extracts were less distinctive, with 18 out of 31 references peaks being shared among the chromatograms from the hot-water extracts (Figure 5).Figure 2
Representative chromatograms of methanol extracts ofAtractylodes japonica (AJ1, (a)),A. chinensis (AC1, (b)), andA. macrocephala (AM1, (c)) at UV wavelengths of 230, 255, 275, 315, and 340 nm.
(a) (b) (c)Figure 3
Representative chromatograms of hot-water extracts ofA. japonica (AJ1, (a)),A. chinensis (AC1, (b)), andA. macrocephala (AM1, (c)) at UV wavelengths of 225, 255, 275, 295, and 325 nm.
(a) (b) (c)Figure 4
Representative chromatograms of methanol extracts ofA. japonica (AJ1–9, (a)),A. chinensis (AC1–9, (b)), andA. macrocephala (AM1–9, (c)) at UV wavelength of 315 nm.
(a) (b) (c)Figure 5
Representative chromatograms of hot-water extracts ofA. japonica (AJ1–9, (a)),A. chinensis (AC1–9, (b)), andA. macrocephala (AM1–9, (c)) at UV wavelength of 325 nm.
(a) (b) (c)


## 3.4. Clustering Analysis of Atractylodes Samples Using Chemometric Statistical Methods

Chromatographic profiles of the methanol and water extracts from Atractylodes samples were further analyzed by principal component analysis (PCA), hierarchical clustering analysis (HCA), and of Pearson’s correlation coefficient to estimate the relationship between theAtractylodes samples.In the principal component (PC) plot of the methanol extracts of the samples, three distinct clusters were observed: an AJ group (AJ1–41 including AC4), an AM group (AM1–10), and an AC group (AC1–9 except AC4). The distribution of PC1 score showed that the samples in the AJ group were plotted closer to those in the AM group than those in the AC group; in contrast, in the PC2 score, the samples in the AC group were located closer to those in the AJ group than those in the AM group (Figure6). However, the PC plot of the hot-water extracts of the samples differed from that of the methanol extracts. As the samples were distributed in a wide range of PC1 and PC2 scores, no distinct clusters were observed. AC4 and AC6 were plotted closer to the location of AJ samples, whereas AC5 and AC8 overlapped into the plots of AM samples (Figure 7).Figure 6
Score plot of principal components (PC1 versus PC2) on the variables (absolute area of reference peaks) withAtractylodes samples from the methanol extracts. PC1 and PC2 represent 40% and 21% of the total variance, respectively. AC:A. chinensis Koidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.Figure 7
Score plot of principal components (PC1 versus PC2) on the variables (absolute area of reference peaks) withAtractylodes samples from the hot-water extracts. PC1 and PC2 represent 40% and 14% of the total variance, respectively. AC:A. chinensis Koidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.The dendrogram of methanol extracts from HCA showed similar groups as obtained from PCA; namely, AJ, AC, and AM samples formed their own groups (groups II, III, and IV) except for AC4 in the AJ group. However, several AJ samples were divided into a separate group below a height of 250000 and clustered in group I below a height of 100000 (Figure8). For the dendrogram of the methanol extracts, the water extracts of AJ samples were also divided into two distinct groups (groups I and II), whereas those of AC and AM samples were not clearly distinguished within their own groups because they were not separated in the subgroup of group II, below a height of 4000 (Figure 9).Figure 8
Hierarchical clustering analysis of Atractylodes samples from methanol extracts. AC:A. chinensis Koidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.Figure 9
Hierarchical clustering analysis of Atractylodes samples from hot-water extracts. AC:A. chinensis Koidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.


## 3.5. Evaluation of the Correlation between A. japonica, A. chinensis, and A. macrocephala Samples by Pearson’s Correlation Coefficient

In the methanol extracts, the mean value of the Pearson’s correlation coefficient (r) of each AJ sample ranged from 0.01 to 0.31 (except for AJ41;r = –0.01) for the correlation of AM samples and from 0.01 to 0.1 for the correlation of AC samples (Figure 10(a)). The mean correlation coefficient of each AC sample ranged from –0.08 to 0.1 (except for AC4;r = 0.95) for the correlation of AJ samples and from –0.07 to 0.1 for the correlation of AM samples (Figure 10(b)). The mean correlation coefficient of each AM sample ranged from –0.05 to –0.01 for the correlation of AC samples and from –0.03 to 0.22 for the correlation of AJ samples (Figure 10(c)).Figure 10
Average coefficients of Pearson’s correlation coefficient ofAtractylodes samples from methanol extracts. (a) AJ–AC and AJ–AM, (b) AC–AJ and AC–AM, and (c) AM–AJ and AM–AC. AC:A. chinensisKoidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.
(a) (b) (c)In the hot-water extracts, the mean correlation coefficient of each AJ sample ranged from 0.25 to 0.80 for the correlation of AM samples and from 0.30 to 0.81 for the correlation of AC samples (Figure11(a)). The mean correlation coefficient of each AC sample ranged from 0.55 to 0.72 for the correlation of AJ samples and from 0.29 to 0.87 for the correlation of AM samples (Figure 11(b)). The mean correlation coefficient of each AM sample ranged from 0.49 to 0.79 for the correlation of AC samples and from 0.45 to 0.65 for the correlation of AJ samples (Figure 11(c)). The mean and median values of Pearson’s correlation coefficient fromAtractylodes samples are shown in Table 2.Table 2
Mean and median value of Pearson’s correlation coefficient (r) among AJ, AM, and AC samples.
Extraction Sample Parameter Correlation coefficient (r) AJ AM AC Methanol extract AJ Mean – 0.131 0.069 Median – 0.146 –0.054 AM Mean 0.131 – –0.028 Median 0.146 – –0.048 AC Mean 0.069 –0.028 – Median –0.054 –0.048 –  Water extract AJ Mean – 0.567 0.609 Median – 0.584 0.626 AM Mean 0.567 – 0.733 Median 0.584 – 0.800 AC Mean 0.609 0.733 – Median 0.626 0.800 – AJ,A. japonica; AM, A. macrocephala; AC, A. chinensis.Figure 11
Average coefficients of Pearson’s correlation coefficient ofAtractylodes samples from hot-water extracts. (a) AJ–AC and AJ–AM, (b) AC–AJ and AC–AM, and (c) AM–AJ and AM–AC. AC:A. chinensisKoidz.; AJ:A. japonica Koidz.; AM:A. macrocephala Koidz.
(a) (b) (c)


## 4. Discussion

We previously proposed that the rhizome ofA. japonica (AJ) should be considered for medicinal use as being in the same medicinal category as that ofA. chinensis (AC) because of their genetic and chemical similarity to that ofA. macrocephala (AM) [4]. However, an unresolved issue is whether the rhizome ofA. japonica should be medicinally used in parallel with the rhizome ofA. macrocephala or with the rhizome ofA. chinensis. Therefore, building on our previous work, in this study, 61 Atractylodes rhizomes that were collected from Korea and China in 2016 were genetically identified by their original species and their chemical differentiation was carried out by chromatographic profiling and chemometric statistical analysis.In the methanol extracts, three apparent clusters ofAtractylodes samples in the PC plot (AJ samples + AC4, AC samples, and AM samples) showed different proximity among each group. PC1 and PC2 scores, the most and next most influential factors of clustering in the PC plot, provided the group of AJ samples with more proximity to the group of AM samples than that of AC samples which results in better chemical relevance, as the PC scores of the AJ samples were closer to those of AM samples [13, 14]. The proximity among the groups of AJ, AC, and AM samples was also confirmed by the dendrogram from HCA, which produces clusters to classify samples by measuring the distance between samples [15, 16]. The dendrogram also indicated apparent groups of AJ, AC, and AM samples, and samples in the AJ group (cluster IV) showed closer chemical similarity to those in the AM group (cluster III) than to those in the AC group (cluster II) below a height of 100000, as samples with a similar distance were involved in the same group [17].Pearson’s correlation coefficient (r) analysis among the samples also supports the clusters obtained from PCA and HCA. The overall correlation between differentAtractylodes species was not strong and was even negative, indicating that the samples of AJ, AM, and AC groups represented a weak correlation and, hence, were separated from each other [18, 19]. Stronger correlation was observed between samples from the AJ–AM group, with positive and higher r values, than between samples from the AJ–AC group, with negative and lower r values (close to 0) [20, 21].In the hot-water extracts, the clusters of the samples according to their original species were not distinct in the PC plot, in which the samples from the same species were mostly gathered together. The AJ samples exhibited higher chemical similarity to a number of AC samples from the most influential PC1 scores, whereas the PC2 scores of AJ samples indicated a closer relationship to AM samples. Rather, AC and AM samples showed higher proximity to each other. These relationships between samples were also observed in the dendrogram: the AJ samples formed separate groups, whereas the AC and AM samples were consolidated in subgroups of cluster II, which makes the apparent delineation between AC and AM samples more difficult, as evaluated by the higherr values between two species. Moreover, AJ samples indicated a stronger correlation with AC samples than with AM samples overall.Unlike the distribution of the samples in the PC plot, about half of the AJ samples formed a separate AJ group (each cluster I in the dendrograms from the methanol and water extracts, respectively) in the dendrogram from HCA, showing least similarity with AC, AM, and the remaining AJ samples. In contrast, but similar to the results from PCA, each AJ sample was strongly correlated with other AJ samples from the methanol and hot-water extracts, with anr value close to 1 [19] (Supplementary Figures S1 and S2). These results might be ascribed to the differences in mathematical and statistical methods between HCA and PCA [22–24].In contrast to our previous study [4], the chemometric results of the present study demonstrate that the chemical correlation and relevance of AJ samples, particularly in the methanol extracts (organic extract), were weaker with AC samples than with AM samples, although AJ samples have a closer genetic relationship to AC samples than to AM samples. Regarding these results, an important difference from the previous work that should be considered, however, is that there was always a distance between AJ and AM samples in the previous and present study, which means that AJ samples were not chemically analogous to AM samples; that is, they had their own clusters of samples. Another point is that the location of AC samples changed from “inside” to “outside” the cluster of AJ samples, indicating that AC samples in the present work showed less chemical homogeneity to AJ samples, as reported previously [25, 26]. This might be explained by the fact that geographical diversity can lead to a wide range of variation in the content of chemical components from the rhizome ofA. chinensis [27, 28]. This is because environmental factors, such as climate, temperature, humidity, soil, and altitude, and the corresponding adaptation of plants are crucial for the biosynthesis and production of chemical components and secondary metabolites [29, 30], and such environmental differences can also influence the chemical variation in the plants of genusAtractylodes [31–34].Based on the results from chromatographic profiles and chemometric analyses,A. japonica rhizomes could apparently be differentiated fromA. macrocephala andA. chinensis rhizomes by their methanol-soluble components, whereasA. chinensis rhizomes were not clearly distinguishable fromA. japonica andA. macrocephala rhizomes by their hot-water-soluble components. These results indicate that the chemical difference betweenA. japonica andA. macrocephala was distinct in both extracts, as also reported previously [4]. However, the difference betweenA. japonica andA. chinensis was dependent on the chemical features that were identified despite their closer genetic relationship. Moreover, aqueous components fromA. chinensis rhizome were chemically closer to those fromA. macrocephala rhizome, although they are categorized as different therapeutic agents. Further pharmacological and clinical evidence is necessary to confirm the chemical correlation betweenAtractylodes rhizomes.


## 5. Conclusion

In this study, 61 Atractylodes rhizomes that were collected from Korea and China in 2016 were genetically identified by their original species by ITS DNA sequencing analysis:A. japonica,A. macrocephala, andA. chinensis. Chemical differentiation was carried out by chromatographic profiling and chemometric statistical analysis, namely, PCA, HCA, and Pearson’s correlation analysis, using the methanol and hot-water extracts ofAtractylodes samples. The results from chemical fingerprinting and statistical analyses demonstrated thatA. japonica rhizomes were chemically distinct fromA. macrocephala rhizomes. However,A. chinensis rhizomes represented diverse chemical variation showing a wide range of relationships toA. japonica andA. macrocephalarhizomes, presumably arising from their environmental differences.



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*Source: 4860371-2018-08-02.xml*