Plant Foods Hum Nutr (2017) 72:13–19 DOI 10.1007/s11130-016-0589-7

ORIGINAL PAPER

Identification and Characterization of Diploid and Tetraploid in Platycodon grandiflorum

Jeoung-Hwa Shin1 & Yun Gyong Ahn2 & Ju-Hee Jung1 & Sun-Hee Woo3 & Hag-Hyun Kim4 & Shela Gorinstein5 & Hee-Ock Boo6

Published online: 28 December 2016 # Springer Science+Business Media New York 2016

Abstract Platycodon grandiflorum (PG), a of herba- amounts of metabolites than with other solvents. Principal ceous flowering perennial of the family , component analysis (PCA) and partial least-squares discrimi- has been used as a traditional oriental medicine for bronchitis, nant analysis (PLS-DA) plots showed significant differences asthma, pulmonary tuberculosis, diabetes, hepatic fibrosis, between the diploid and tetraploid metabolite profiles. bone disorders and many others similar diseases and as a food Extracts of tetraploid showed higher amounts of amino acids, supplement. For the primary profiling of PG gas chromatog- while extracts of diploid contained more organic acids and raphy coupled with high resolution – time of flight mass spec- sugars. trometry (GC/HR-TOF MS) was used as an analytical tool. A comparison of optimal extraction of metabolites was carried Keywords Gas chromatography/high resolution time of flight out with a number of solvents [hexane, methylene chloride, mass spectrometry (GC/HR-TOF MS) . Diploid . Tetraploid . methanol, ethanol, methanol: ethanol (70:30, v:v)]. In extracts Platycodon grandiflorum (PG) . Partial least-squares with methanol: ethanol (70:30 v:v) were detected higher discriminant analysis (OPLS-DA)

Electronic supplementary material The online version of this article (doi:10.1007/s11130-016-0589-7) contains supplementary material, Introduction which is available to authorized users. Platycodon grandiflorum (PG) has been used as a wild vege- Jeoung-Hwa Shin and Yun Gyong Ahn contributed equally to this work. table and traditional oriental medicine in and the * Shela Gorinstein major components are saponins such as platycodin A, C and [email protected] D of the triterpenoid group [1]. The roots of PG have been * Hee-Ock Boo used as an herbal medicine for bronchial diseases such as [email protected] drainage, discharge of phlegm, bronchitis and asthma in ori- ental medicine [2, 3]. In particular, the saponin component of 1 Seoul Center, Basic Science Institute, Anam-ro, Seongbuk-gu, PG has been found to reinforce the inhibitory action of central Seoul 02855, Republic of Korea nervous, acute chronic inflammation, antiulcer and gastric se- 2 Western Seoul Center, Korea Basic Science Institute, Bugahyeon-ro, cretion, hypoglycemic effect, cholesterol metabolism im- Seodaemun-gu, Seoul 03760, provement, antioxidant, anticancer effects and hepatoprotec- 3 Department of Crop Science, Chungbuk National University, tive properties in vitro and in vivo experiments [4–7]. Another Cheongju 28644, South Korea study also showed that the extracts of PG were effective for 4 Faculty of Food Nutrition and Cookery, Woosong Information the improvement of blood glucose, lipid compositions in se- College, Daejeon 34606, South Korea rum and liver [8]. The hypoglycemic effects of PG aqueous- 5 Institute for Drug Research, School of Pharmacy, The Hebrew ethanol extract (PGE) in streptozotocin (STZ) -induced dia- University, Hadassah Medical School, 91120 Jerusalem, Israel betic ICR mice were investigated and the results indicated that 6 PHYTO M&F Co. Ltd., BI Center, GIST, Gwangju 61005, South PGE could induce hypoglycemic effects without stimulating Korea insulin secretion [9]. For this reason, PG is well known for 14 Plant Foods Hum Nutr (2017) 72:13–19 various pharmacological effects on human health and its con- determine the antioxidant enzyme activity of diploid and tetra- sumption is increasing in two directions as food supplement and ploid in PG and their metabolite profiles. Tetraploid mutants herbal medicine [10]. A mass production by the breeding of su- were induced by the similar method according to the previous perior varieties is the basis of new development of functional study [14]. The colchicine treatment on seedlings was performed products using the physiological functionality of PG. The creating at the time of cotyledon emergence. The detailed treatment is of giant PG by the polyploidy breeding method could be maxi- described in our previous study [11]. The samples were crushed, mized in aspect of its effects. The polyploidy breeding method in extracted and derivatized for the measurements of metabolite is a way to increase radically the emergence of new useful profiles. traits and the quantity by polyploidy obtained through quantitative doubling of the genome, which is a set of chromosomes [11]. The Sample Extraction and Derivatization The extraction was polyploidy breeding is an effective approach of germplasm de- performed by sonicating twice for 10 min with 100 mL of sol- velopment for medicinal plants [12]. The numbers and structure vents [hexane, methylene chroride, methanol, ethanol, methanol: of chromosomes in plants are species dependant, change of which ethanol (70:30, v:v)]. Derivatization is the process of chemically causes an alteration of the cell and plant body characters. modifying a compound to produce a new one which has prop- Particularly, tetraploid plants have a large cell volume than diploid erties that are suitable for analysis using a GC. The dried residue plants, and therefore, tetraploid generally have large organs or was redissolved and two steps of derivatization were applied as stems [13]. Tetraploid of PG has higher contents of pharmaceuti- in the previous study [20]. Methoximation was performed first to cal constituents as well as higher yield than diploid. Some reports inhibit the ring formation of reducing sugars. During this step the showed that the tetraploid inductions of PG by colchicine treat- ring structure was opened and aldehyde and ketone groups were ment have been also available for breeding [14–17]. Recently, gas protected. A solution of 40 mg/mL of N-methylhydroxylamine chromatography coupled with high-resolution time-of flight mass hydrochloride in pyridine was prepared and 10 μL were added in spectrometry (GC/ HR-TOF MS) has been demonstrated as a the dried sample. Then the sample was mixed for 90 min at powerful and highly effective analytical tool in various fields such 30 °C. In the second step of derivatization 90 μlof as analysis of food and environmental contaminants, flavor com- MSTFA.1% TMCS were added for trimethylsilylation of acidic ponents, drugs screening, petrochemical analysis, or metabolo- protons and shaken at 37 °C for 30 min (Fig. 1,insert). mics studies [18–20]. The analytical method enables a good sta- tistical separation and facilitates the identification of differences or Instrumental Analysis by GC/HR-TOF MS GC/HR-TOF similarities between groups. In this study, the comparative analy- MS analysis was performed on a 7890A gas chromatograph sis of metabolites between diploid and tetraploid of PG was per- (Agilent Technologies, Palo Alto, CA, USA) equipped with a formed using GC/ HR-TOF MS. From the detected results dis- GCT premier TOF mass spectrometer (Waters, Manchester, crimination between two groups and identification of the different UK). The DB-5 MS capillary column (30 m × 0.25-mm i.d., metabolites were obtained. 0.25-μm film thickness, 5% diphenyl-95% dimethylsiloxane phase) was obtained from J&W Scientific (Folsom, CA, USA). Samples were injected in split ratio of 1:5 at an injector temper- Materials and Methods ature of 250 °C and an initial column temperature of 60 °C for 2 min and then was ramped to 320 °C at a rate of 10 °C/min. The Chemicals and Reagents Hexane, ethylene chloride, ethanol final temperature (320 °C) was held for 10 min. The ion source and methanol (HPLC grade) were purchased from Burdick & was operated at 250 °C, the electron energy was 70 eV, trap Jackson (Muskegon, MI, USA). Distilled water was fil- current of 200 μA, interface temp. 280 °C, detector voltage tered using a Mille-Q Reagent Water System (Millipore, 2650 V and acquisition range m/z 50–800. To obtain exact mass Billerica, MA, USA). Sodium sulfate, N-methyl hydrox- measurement, use a lock mass as well as a calibration file. The ylamine hydrochloride and pyridine were purchased calibration reference was heptacosa (perfluorotributylamine, from Sigma-Aldrich (St. Louis, MO, USA). N-Methyl- mass spectrometry grade, Apollo Scientific LTD, Bradbury, N-(trimethylsilyl)trifluoroacetamide with 1% UK). The heptacosa was continuously infused into the ion source trimethylchlorosilane for silylation reagent was also sup- as a lock mass. All data were acquired using Waters GCT plied by Sigma-Aldrich (St. Louis, MO, USA). Premier Software v. 4.1 and processed using ChromaLynx and MarkerLynx Application Managers. ChromaLynx XS Software Samples The diploid plants of PG was grown in Geumsan offers rapid detection, identification and semi quantitative deter- county, Chungcheong province, Korea and tetraploid plants were mination of components in complex matrices. MarkerLynx is provided by the cooperative research laboratory, Woosong used for comprehensive data processing for chemometric appli- Information College. Their root samples were freeze-dried, and cations, including multivariates statistical analysis and visuali- then ground. Each sample powder was stored at -20 °C for the zation. The mass spectra of all detected compounds were com- extraction. Two focusing experiments were performed to pared with the mass spectra in the National Institute of Plant Foods Hum Nutr (2017) 72:13–19 15

Fig. 1 Obtained chromatograms of each solvent extraction according to the applied analytical method, insert: All steps of analytical procedure

Standards and Technology mass spectral library (National was chosen for extraction from tetraploid and diploid samples. Institute of Standards and Technology (NIST) MS search 2.0). Figure 2 showed the different peak pattern on the chromato- All the samples were run in triplicate for reproducibility and gram compared with tetraploid (Fig. 2a) and diploid (Fig. 2b) stability assessment of the GC/TOF-MS system throughout the samples. In tetraploid sample, alanine, valine, glycerol, serine, experiment. threonine, malic acid, aspartic acid, pyroglutamic acid, glutamic acid, asparagines, phenylalanine, xylitol, glutamine, d-fructose, isocitric acid, butanal, o-methyloxime, n- Results and Discussion methylgluconamide, galactitol, tyrosine, glucopyranose, hexadecanoic acid, inositol, oleic acid, d-turanose, Comparison of Extraction Solvents On the comparison of monostearin, alpha-L.-allopyranose were found as shown in extraction solvent, more metabolites were extracted with Table 1. In the case of diploid sample, lactic acid, alanine, methanol:ethanol (70:30, v:v) than the other solvents as shown valine, phosphoric acid, butanedioic acid, threonine, malic ac- in Fig. 1 according to the procedure of the method. The mix- id, pyroglutamic acid, d-fructose, oleic acid, butanal, o- ture of methanol:ethanol (70:30, v:v) as an extraction solvent methyloxime, mannose, galactitol, glucopyranose, 16 Plant Foods Hum Nutr (2017) 72:13–19

24 a

22* 26

16 17* 25 18 15 21 12* 4* 14 3* 19* 7* 8* 10* 13 23 11* 5* 9* 20 1* 6 2

18 b 9 12 14 11 16

20 10* 15

4* 13 19* 7*

17* 2 5* 8 6* 1* 3

Fig. 2 Chromatograms of the extracts for tetraploid (a) and diploid (b)

hexadecanoic acid, inositol, 10′-apo-.beta.-carotenoic ac- integrates and aligns chemical and biological MS data id, 5,6-dihydro-5,6-dihydroxy-, methyl ester, d-turanose, points and converts them into Exact Mass Retention à-d-glucopyranosyl-d-glucose, monostearin were found Time (EMRT) pairs. Those EMRT pairs can then be as shown in Table 2. Most metabolites of tetraploid used for multivariate statistical analysis, such as princi- and diploid samples were amino acids, fatty acids and ple component analysis (PCA-X), partial least-squares sugars. This is in agreement with others that to latent structures data analysis (PLS-DA), and orthog- platycosides from the roots of PG are characterized by onal PLS data analysis (OPLS-DA) to visualize and a structure containing a tri-terpenoid aglycon and two interpret the information rich and complex MS data. sugar chains [1]. In some other reports [18]three PCA score plots of GC/HR-TOF MS spectra of tetra- known monodesmosidic saponins and two known non- ploid and diploid show the separation in Fig. 3a. saponin compounds were isolated together with another Figure 3b showed the S-plot of OPLS-DA model’sfor seven known compounds. the same data sets. The points are EMRTs plotted by covariance (X-axis) and correlation (Y-axis) values. The Comparison of Diploid and Tetraploid For the pattern rec- upper right quadrant of the S-plot shows those EMRT ognition analysis between diploid and tetraploid, PCA pairs that are elevated in the dosed group, while the and OPLS-DA were applied with the spectrum data set lower left quadrant shows EMRT pairs elevated in the of GC/HR-TOF MSusing the MarkerLynx software vehicle group. The farther along the X-axis the greater (Waters, Manchester, UK). Using MarkerLynx XS pro- the contribution to the variance between the groups, vides more rigorous mining of the data, which not only while the further along the y-axis the higher the reli- includes these major features but also helps discover ability of the analytical result. The lower portion of minor features that may be of greater biological signif- Fig. 3b illustrates a plot for the same EMRT pair point icance despite their low abundance. MarkerLynx XS (m/z 371.2983 at RT 18.17 in this case) from both the Plant Foods Hum Nutr (2017) 72:13–19 17 vehicle and lower-dosed samples. The difference of Table 2 Metabolites identified from diploid using GC/HR-TOF MS EMRT pair (point) is significant between the vehicle R. T(min) Identified metabolite Formula m/z samples and the lower-dose samples. The point’smeans a exact mass/retention time pairs (EMRTs) plotted by co- 17.10Lacticacid C3H6O3 90 variance (X-axis) and correlation (Y-axis) values. Those 27.79Alanine C3H7NO2 89 plots simplify the process of extraction and pin-pointing 3 9.50 Valine C5H11NO2 117 a significant markers from very complex datasets. 4 10.29 Phosphoric acid H3PO4 98 a Figure 3b shows plotted comparison columns of the 5 10.93 Butanedioic acid C4H6O4 118 most important EMRTs from those two groups of sam- 6 11.87 Threonine C4H9NO2 119 a ples. Those plots simplify the process of extraction and 7 13.20 Malic acid C4H6O5 134 pin-pointing significant markers from very complex 8 13.65 Pyroglutamic acid C5H7NO3 129 datasets. The metabolites contributing to the variance 9 16.96 D-Fructose C6H12O6 180 a between the groups were marked with an asterisk in 10 17.39 Oleic acid C18H34O2 282

Tables 1 and 2. The asterisk markings showed p corre- 11 17.51 Butanal, o-methyloxime C5H11NO 101 lation coefficients | ± 0.8| and | ± 0.0005|. In extracts of 12 17.76 Mannose C6H12O6 180 diploid sample, lactic acid, phosphoric acid, butanedioic 13 18.18 Galactitol C6H14O6 182 acid, malic acid, oleic acid, 10′-apo-.beta.-carotenoic 14 18.59 Glucopyranose C6H12O6 180 acid, 5,6-dihydro-5,6-dihydroxy-, methyl ester and à-d- 15 19.36 Hexadecanoic acid C16H32O2 256

16 19.69 Inositol C6H12O6 180

17 21.35 10′-Apo-.beta.-carotenoic acid, C3H6O3 133 Table 1 Metabolites identified from tetraploid using GC/HR-TOF MS 5,6-dihydro-5,6-dihydroxy-, methyl ester a R. T(min) Identified metabolites Formula m/z 18 24.22 D-Turanose C12H22O11 342 a a 19 25.02 à-D-glucopyranosyl-D-Glucose C3H6O3 129 17.79Alanine C3H7NO2 89 a 20 25.36 Monostearin C21H42O4 358 2 9.50 Valine C5H11NO2 117 a 3 10.33 Glycerol C3H8O3 92 High abundance metabolites; based on correlation (p(corr)) and contri- a bution coefficients (p) (| ± 0.8| and | ± 0.0005|), the different metabolites 4 11.50 Serine C3H7NO3 105 a were discriminated 5 11.87 Threonine C4H9NO2 119

613.20Malicacid C4H6O5 134 a 7 13.61 Aspartic acid C4H7NO4 133 glucopyranosyl-d-glucose were in larger quantity than a 8 13.65 Pyroglutamic acid C5H7NO3 129 in the tetraploid sample. In the case of tetraploid sam- a 9 14.81 glutamic acid C5H9NO4 149 ples, alanine, valine, serine, threonine, aspartic acid, a 10 14.88 Asparagine C4H8N2O3 132 pyroglutamic acid, glutamic acid, asparagine, phenylal- a 11 14.91 Phenylalanine C9H11NO2 165 anine, xylitol, n-methylgluconamide, tyrosine and inosi- a 12 15.90 Xylitol C5H12O5 152 tol were in larger quantity than in the diploid sample. 13 16.60 Glutamine C5H10N2O3 146 The extracts of tetraploid sample, amino acids were in 14 16.96 D-Fructose C6H12O6 180 larger amount than in the diploid sample in comparison 15 17.00 Isocitric acid C6H8O7 192 with organic acids and sugars. Previous studies have 16 17.51 Butanal, o-methyloxime C5H11NO 101 shown that amino acids play a role antioxidant effects a 17 17.78 N-methylgluconamide C7H15NO6 209 and enhance the immune function [21–23]. In addition, 18 18.18 Galactitol C6H14O6 182 the high antioxidant enzyme activity was investigated in a 19 18.26 Tyrosine C9H11NO3 181 the tetraploid root of PG [5, 7]. These results reflect the

20 18.59 Glucopyranose C6H12O6 180 assessment of antioxidant enzyme activity in diploid

21 19.36 Hexadecanoic acid C16H32O2 256 and tetraploid of PG [11, 12]. a 22 19.69 Inositol C6H12O6 180

23 21.15 Oleic acid C18H34O2 282 24 24.22 D-Turanose C12H22O11 342 Conclusions 25 25.36 Monostearin C21H42O4 358 26 29.43 Alpha-L.-allopyranose C6H12O6 180 In summary, we conducted metabolite profiling of tetra- ploid and diploid samples in PG using GC/HR-TOF MS a High abundance metabolites; based on correlation (p(corr)) and contri- bution coefficients (p) (| ± 0.8| and | ± 0.0005|), the different metabolites to examine differences. In the experiment, multivariate were discriminated analysis showed that important markers were shown for 18 Plant Foods Hum Nutr (2017) 72:13–19

a

Diploid Tetraploid

2

2

4 4 4 4 4

2 2

2

b Diploid

Tetraploid

Fig. 3 a, OPLS-DA score plot from GC/ HR-TOF MS, which mainly included primary metabolites. The symbols show tetraploid and diploid. b, Representative OPLS-DA S-plot showing relative contribution of bins to clustering of tetraploid and diploid

characterizing between two tetraploid and diploid species. The Acknowledgement This research was supported by High Value- approach showed significant differences in tetraploid and dip- added Food Technology Development Program of IPET, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. And loid samples in PG and indicated that the different species this research was supported by the Korea Basic Science Institute which may exhibit different biological properties (S1). (C36917). Plant Foods Hum Nutr (2017) 72:13–19 19

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