Multiple ginsenosides ratios pattern — A pointer to identify Panax ginseng root extracts adulterated with other plant parts?

Fitoterapia 121 (2017) 64–75

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Multiple ginsenosides ratios pattern — A pointer to identify Panax ginseng root extracts adulterated with other plant parts?

MARK

Suresh Govindaraghavan Network Nutrition – IMCD Australia, Unit 9, 7 Meridian Place, Bella Vista, NSW 2153, Australia

1. Introduction

1.3. Species delineation and adulteration among medicinal ginsengs

1.1. Ginseng market demand

Morphological distinction between P. ginseng and P. quinquifolium is difficult due to close phenotypic similarities [7] and due to their genetic proximity in comparison to P. notoginseng [11]. Difficulty in identification is compounded once the plant parts (eg. root) are sorted. Lack of availability of quality herb material and increasing costs have led to intentional adulteration of one medicinal ginseng species with the other in the market place. Adulteration in medicinal ginsengs has been known for long [7]. Adulteration of P. quinquefolium roots with P. ginseng roots [12] and adulteration of P. notoginseng root extracts either with P. ginseng roots and/or leaf and flower extracts of P. notoginseng for economic gain ([38] c.f. [13]) have also been reported.

The medicinal ginseng species, P. ginseng, P. quinquefolium, P. notoginseng and P. japonicus totally account for ~80,000 tons of fresh ginseng supply world-wide with an estimated cost of US $1123 million [1]. The former two species alone account for over 90% of total fresh ginseng supply. In 2014, P. ginseng root prices in China stood between US$ 100–126 per kg depending on the quality grade specifications [2]. P. ginseng is the most produced world-wide followed by P. quinquifolium and P. notoginseng in decreasing order, with all three species presently cultivated in China. The USA and Canada mostly produce P. quinquifolium and South Korea cultivate only P. ginseng [1]. The slow growing nature of the perennial P. ginseng [3], stringent regulations world-wide on pesticide residues and the need to achieve optimal ginsenosides content in harvested dried ginseng root result in limited supply of quality material. An increase in Asian ginseng root prices is also attributed to large pharmaceutical companies competing for procurement for use as both food and medicine [2]. Wild-crafting and over harvesting of American ginseng (P. quinquifolium) throughout North America led to decline of wild material [4] and hence, till recently, was the most expensive in the ginseng market world-wide [5]; however, the prices declined due to increasing cultivation in China [6]. 1.2. Ginseng pharmacology and phytochemistry Panax ginseng C.A. Mey. (Araliaceae), is native to Asia-temperate and its traditional medicinal use has a long written history [7]. Asian ginseng is used as ‘tonic/adoptogen’ in traditional Chinese Medicine, and as an aphrodisiac, nourishing stimulant and for treating sexual dysfunction in men in modern herbal medicines [8]. P. quinquifolium is traditionally used for cardiovascular disease treatment [9]. Pharmacological effects of all the three ginseng species are attributed to their ginsenosides content and hence most commercial ginseng root extracts are standardised to ginsenosides. Ginsenosides mostly share a basic saturated 1,2-cyclopentano-perhydrophenanthrene nucleus, with the aglycone moieties placed into dammarane, oleanane and ocotillol triterpene skeletons. More than 150 naturally occurring ginsenosides from different parts of medicinal ginsengs have been reported [6,10].

E-mail address: [email protected]. http://dx.doi.org/10.1016/j.fitote.2017.06.024 Received 12 May 2017; Received in revised form 27 June 2017; Accepted 28 June 2017 Available online 29 June 2017 0367-326X/ © 2017 Elsevier B.V. All rights reserved.

1.4. Analytical methods for species delineation and for extract purity To delineate species among commercial root extracts of medicinal ginsengs, a number of analytical methods based on HPTLC and HPLC hyphenated with UV, ELSD, and MS detectors that target separation of major root ginsenosides have been employed in conjunction with metabolomics and chemometric approaches [13–21]. This led to the identification of species specific ginsenosides and specific ginsenoside ratios to establish the authenticity and purity of ginseng preparations. Species indicative markers include, ginsenoside Rf [22,23], 24(R)-pseudoginsenoside F11 [15,24,25], and notoginsenoside R1 [15]. Ginsenosides ratios used for testing extract purity include Rb1:Rg1, Rb2:Rb1, Rg1:Rb1 [19,26], ginsenoside Rf:24(R)pseudoginsenoside F11, protopanaxadiol ginsenosides (PPD) and protopanaxatriol ginsenosides (PPT) levels [24,25,27,28] and their ratios [23], to cite a few. Absence or trace level presence of ocotillol-type (e.g. 24(R)pseudoginsenoside F11) and oleanolic acid-type ginsenosides are also used to distinguish P. notoginseng from the other two medicinal ginsengs [10]. 1.5. Variations in ginsenoside profiles Ginsenosides content varies among medicinal ginsengs due to sub-specific differences, spatio-temporal, agro-climatic, cultural and phytogeographical influences [14,15,18,20,29–32]. Liu [32] and Li et al. [31] have reported that in cultivated P. ginseng, optimal ginsenosides levels is reached in roots of 4–5 years of growth which forms material of trade. Ginsenoside

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Table 1 A complete list of P. ginseng crude herb, commercial extracts and products used in this study. Sample code

Material

Source/mfg. by

Authenticated/ID by

Authentication/ID method

SMNNPG-20101106 SMNNPG-111020 SMNNPG-201001 SMNNPG111119 121586-201001 NNPG59G-12020103 NNPG59G-12070918 NNPG59G-12090505 NNPG59G-13032203 NNPG59G-1410201-10 NNPG59G-1503018-07 NNPG59G-1505039-19 NNPG59G-1611017-23 NNPG1015-12070512 NNPG1015-12020306 NNPG1015-1502015-06 NNPG1015-1501009-22 NNPG1015-1602012-05 NNPG1015-1605052-16 NNPG1015-1606064-10 NNPG1015-1608079-16 CH-M-1 EU sample 1/Germany EU sample 2/China EU sample 3/China EU sample 4/Australia EU sample 5/China EU sample 6/Germany EU sample 7/Spain EU sample 8/Germany EU sample 9/Swiss EU sample 10/S Korea EU sample 11/Italy EU sample 12/Swiss AUS sample 1 AUS sample 2 AUS sample 3 AUS sample 4

Root Root Root Root Leaf-stem Root extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Stem/leaf extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Root extract Berry extract Root extract Root extract Capsule/extract Capsule/extract Capsule/herb Tablet

Network Nutrition Network Nutrition Network Nutrition Network Nutrition NICPBP Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Network Nutrition Chinese Market European Market European Market European Market European Market European Market European Market European Market European Market European Market European Market European Market European Market Australian Market Australian Market Australian Market Australian Market

SCU Med Plant Herb SCU Med Plant Herb Internal-USPa Internal-USPa NICPBPb, China Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc Internal-USPc N/A BLINDED; Internal-USPc BLINDED; Internal-USPc BLINDED; Internal-USPc BLINDED; Internal-USPc BLINDED; Internal-USPc BLINDED; Internal-USPc BLINDED; Internal-USPc BLINDED; Internal-USPc BLINDED; Internal-USPc BLINDED; Internal-USPc BLINDED; Internal-USPc BLINDED; Internal-USPc N/A; monoherbal N/A; monoherbal N/A; monoherbal N/A; monoherbal

Taxonomic, macroscopic, microscopic, HPLC Taxonomic, macroscopic, microscopic, HPLC ID A HPTLC, ID-B HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-B HPLC, Rb2:Rb1 ratio N/A ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio N/A ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio ID A HPTLC, ID-C HPLC, Rb2:Rb1 ratio N/A N/A N/A N/A

As prescribed in USP Monograph for Asian Ginseng - Identification A HPTLC finger-printing as outlined by CAMAG Applicn Note F-31, Identification B HPLC, Rb2:Rb1 ratio. National Institute for the Control of Pharmaceutical and Biological Products, PR China. c as prescribed in USP Monograph for Powdered Asian Ginseng Extract - Identification A HPTLC finger-printing as outlined by CAMAG Applicn Note F-31, Identification C HPLC, Rb2:Rb1 ratio; BLINDED – samples sourced by IMCD-Europe from European market place and provided as blinded samples to check the utility of adulteration detection methodology employed. a

b

and Re and Rd in leaves [13]. Taking cue from such differential accumulation patterns of ginsenosides in different plant parts, this paper explores the utility of multiple ginsenoside ratios as possible identifiers of adulteration in P. ginseng root extracts with other plant part preparations.

profile patterns of commercial extracts are also influenced by post-harvest processing conditions [10]. Among all the plant parts, higher content of ginsenosides has been reported in the leaf and root hairs [13,29,31]. P. ginseng leaves/stem also produce similar major ginsenosides as the roots (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2). Maximum content of ginsenosides occur in P. ginseng leaves during the first year of growth [29,31] making it an attractive choice for adulteration of P. ginseng root extracts by unscrupulous traders. Purported addition of P. ginseng leaf extracts to root extracts to increase levels of ginsenosides in the market place has also been reported [7] increasing the suspicion of wide-spread adulteration of root extracts of the medicinal ginsengs with other plant parts for economic gains. Pharmacopoeias (USP, BP, EP, for example) clearly stipulate hydro-alcoholic solvents for extraction, extract ratios, the HPLC method for separation and assay of targeted ginsenosides in P. ginseng root extracts for use in herbal medicines. Extensive analysis on ginsenoside profiles in such pharmacopoeia compliant P. ginseng extracts resulted in specification of ginsenosides profile, their content and ratios to check extract purity. However, pharmacopoeial methods do not specifically identify plant-partspecific marker ginsenoside in their HPLC profile. Hence, detection of adulteration of root extracts of P. ginseng with other parts is difficult to decipher. Spatio-temporal distribution of major ginsenosides in P. ginseng [20], showed significant accumulation of Rb1, Rg1 and Rf in the main roots,

2. Materials and methods 2.1. Reference substances Individual ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2 were purchased from PhytoLab, Germany. All the solvents used are of HPLC grade. 2.2. Ginseng samples Table 1 provides a complete list of ginseng samples included in this study. Multiple batches of bulk crude P. ginseng dried root materials (with prefix of SMNNPG) that were used for commercial extract batches (with prefix NNPG) were sourced from China by Network Nutrition-IMCD. Multiple commercial batches of P. ginseng root extracts - NNPG 1015 (hydroalcoholic extract, extract ratio of 10:1, ≥15% total ginsenosides calculated as ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2) and NNPG59G (hydroalcoholic/butanol extract, extract ratio of 5:1, ≥10% total 65

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Fig. 1A. Structures of the ginsenosides used in this study. Glc – glucose; Rha – rhamnose; (p) and (f) indicate pyronose and furanose forms; numbers in superscript indicates linkage.

ginsenosides calculated as ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2) were manufactured under GMP in China by Network Nutrition – IMCD. Details on the authentication of the starting crude herbs and identification of commercial extracts and the methods employed are provided in Table 1. Selected crude, dried root materials (SMNNPG-111020 and SMNNPG 20101106) were also independently authenticated at Southern Cross University Medicinal Plant Herbarium. Authentic P. ginseng leaf and stem sample (121586-201001) was sourced from National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), China. Network Nutrition-IMCD sourced a number of blinded commercial P. ginseng extracts from the European market place. A number of P. ginseng (monoherbal) finished products (capsules, tablet) were also sourced from the shelf in Australia.

and filtered. The extract was concentrated to dryness. 100 mg of residue was redissolved in 1 mL absolute ethanol by sonication for 30 min, filtered and used. 1.0 g of commercial extract sample was dissolved in 10 mL absolute ethanol by sonication for 30 min, filtered and used. 3 μL of standard/crude herb extract/commercial extract samples were loaded as 10 mm bands and subjected to HPTLC analysis. A mixture of chloroform:ethyl acetate:methanol:water (15:40:22:9) was used as mobile phase. On completion of run, the plates were dried with a stream of cool air and sprayed with 10% sulphuric acid in methanol in a fume hood. The plates were dried and then heated at 100 °C for 5–10 min till the bands were clearly visible.

2.3. HPTLC analysis [21]

2.4.1. Standard preparation Stock solutions (500 μg/mL) of individual ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2) (Fig. 1A) were prepared by dissolving 5 mg of pure reference substance in 2 mL each of ethanol and acetonitrile and made up to 10 mL with water. The stock was diluted with a mixture of ethanol:acetonitrile:water (20:20:60) to have final standard

2.4. HPLC analysis

Merck HPTLC Silica gel 60 F254 (10 cm × 10 cm) plates were used. 1 mg of ginsenoside Rg1 dissolved in 2 mL methanol was used in the reference lane. 1 g of powdered crude herb sample was extracted in 10 mL 40:60 methanol:water under reflux conditions for 10 min, cooled 66

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Table 2 Individual and total ginsenoside content (%w/w) and Rb2:Rb1 ratios in P. ginseng crude herb, commercial extracts and products used in this study. Sample code

Rb1

Rb2

Rc

Rd

Re

Rf

Rg1

Rg2

Total

Rb2:Rb1

SMNNPG 20101106 SMNNPG 120201 SMNNPG 111020 SMNNPG 111119 NICPBP 121586-201001 NNPG59G 12020103 NNPG59G 12070918 NNPG59G 12090505 NNPG59G 13032203 NNPG59G 1410201-10 NNPG59G 1505039 NNPG59G 1503018 NNPG59G 1611017-23 NNPG1015 12070512 NNPG1015 12020306 NNPG1015 1501009-22 NNPG1015 1502015-06 NNPG1015 1602012-05 NNPG1015 1605052-16 NNPG1015 1606064-10 NNPG1015 1608079-16 EU sample 1 EU sample 2 EU sample 3 EU sample 4 EU sample 5 EU sample 6 EU sample 7 EU sample 8 EU sample 9 EU sample 10 EU sample 11 EU sample 12 CH-M-1a AUS sample 1b AUS sample 2b AUS sample 3b AUS sample 4c

0.54 0.15 0.29 0.39 0.41 2.25 2.08 2.18 1.88 3.3 2.91 2.96 3.34 3.41 3.63 4.72 6.12 5.23 4.65 5.47 5.83 0.45 1.53 6.75 5.12 5.21 1.81 1.47 4.17 1.35 1.05 2.77 1.32 1.82 1.53 2.26 4.1 2.28

0.28 0.11 0.21 0.3 0.42 1.29 1.23 1.41 1.14 1.48 1.06 1.23 1.56 2.53 2.2 2.03 1.41 2.12 1.82 2.24 2.3 1.19 0.76 0.31 2.17 2.46 4.49 0.99 2.59 0.63 0.26 1.12 0.66 NR 3.1 2.81 0.83 1.05

0.44 0.15 0.3 0.7 0.68 2.24 1.43 1.77 1.48 2.1 1.74 1.99 1.8 2.64 3.86 3.05 2.53 3.48 3.15 3.49 3.37 1.24 1.09 2.61 3.42 3.33 3.99 1.19 3.25 0.97 0.36 1.61 0.99 NR 2.39 6.99 1.17 1.14

0.2 0.033 0.12 0.28 0.42 1.01 0.92 0.86 0.85 0.26 0.73 0.58 0.82 1.91 1.88 0.88 1.52 0.53 0.35 0.55 0.53 1.02 0.67 0.52 0.58 0.49 3.42 0.19 0.57 0.61 0.09 0.21 0.17 NR 9.93 4.47 2.05 0.62

0.43 0.29 0.39 0.69 2.79 1.62 3.17 2.45 3.22 2.58 2.41 2.39 1.49 4.15 2.41 3.5 2.04 1.59 1.69 1.52 1.46 2.55 2.78 1.74 1.54 1.37 14.2 0.63 1.36 1.91 3.56 0.91 0.35 NR 11.32 18 3.75 1.21

0.17 0.058 0.12 0.12 0.13 0.32 0.31 0.24 0.25 0.22 0.14 0.2 0.31 0.44 0.45 0.3 0.3 0.29 0.32 0.4 0.38 0.05 0.07

0.39 0.14 0.31 0.32 0.52 0.95 0.68 0.61 0.68 0.49 0.64 0.4 0.7 1.1 0.82 0.65 0.32 2.54 2.84 2.77 2.86 4.23 4.74 0.89 1.98 4.48 21.9 1.23 2.99 2.83 1.02 3.4 0.71 1.29 4.85 4.53 0.93 0.96

0.08 0.025 0.05 0.015 0.21 0.19 0.19 0.16 0.19 0.35 0.33 0.25 0.14 0.26 0.31 0.45 1.25 0.47 0.3 0.45 0.38 0.38 0.23 0.13 0.54 0.7 2.81 0.13 0.3 0.24 0.3 0.12 0.07 NR 2.41 1.09 0.57 0.1

2.53 0.956 1.79 2.815 5.58 9.87 10.01 9.68 9.69 10.78 9.96 10 10.16 16.44 15.56 15.58 15.49 16.25 15.12 16.89 17.11 11.11 11.87 12.95 15.65 18.63 52.78 6.01 15.67 8.54 6.7 10.46 4.37 9.59 35.63 40.84 13.47 7.68

0.52 0.8 0.72 0.84 1.21 0.67 0.62 0.75 0.67 0.49 0.44 0.47 0.53 0.79 0.71 0.49 0.49 0.56 0.46 0.47 0.45 3.06 0.59 0.055 0.49 0.55 2.86 0.79 0.714 0.526 0.27 0.46 0.58 0.46 2.3 1.46 0.23 0.51

0.3 0.59 0.16 0.18 0.44 0.06 0.32 0.1 NR 0.1 0.69 0.07 0.32

NR: not reported. The data in bold indicate that the material fails Rb2:Rb1 ratio as stipulated by the Pharmacopoiea. a Values as reported in the manufacturer's certificate of analysis. b Values expressed as mg/capsule. c Values expressed as mg/tablet.

concentrations of 60, 6 and 3 μg/mL to generate a standard curve.

150 mm × 4.6 mm column. The column temperature was maintained at 25 °C. The mobile phase A was water and the mobile phase B was acetonitrile:water (4:1, v/v). The gradient program was as follows: 0–12 min, 76% A and 24% B; 12–28 min 76% to 65% A, 24% to 35% B; 28–51.5 min 65% to 56.5% A, 35% to 43.5% B; 51.5 to 52.5 min 56.5% to 0% A, 43.5% to 100% B; 56.5 min to 64.5 min 0% to 76% A and 100% to 24% B; 64.5 to 77 min 76% A and 24% B; Equilibration time between runs was 10 min. The injection volume was 10 μL, the mobile phase flow was 1.5 mL min and the detection wavelength was UV 203 nm.

2.4.2. Sample preparation Finely powdered 1.0 g of raw herb samples (ginseng roots, stem/ leaf) in a 100 mL RB flask fitted with a reflux condenser were extracted with 50 mL of 40:60 methanol:water under reflux for 60 min. Cooled sample was filtered. The marc was redissolved with 20 mL of the same solvent. The extracts were combined, evaporated to dryness under vacuum. 90 mg of the dry residue was redissolved in 25 mL of 50:50 ethanol:water and sonicated at 50 °C for 30 min in a 50 mL volumetric flask. Extract sample then was cooled after a clear homogeneous dispersion. 10 mL acetonitrile was added and made up to volume with water. The sample was shaken well and filtered thru 0.45 μ filter before analysis. Commercial powdered ginseng extracts (90 mg) were dissolved in 25 mL of 50:50 ethanol:water and sonicated at 50 °C for 30 min in a 50 mL volumetric flask. Extract sample then was cooled after a clear homogeneous dispersion. 10 mL acetonitrile was added and made up to volume with water. The sample was shaken well and filtered thru 0.45 μ filter before analysis.

2.4.4. Ginsenosides assay All the samples were analysed by HPLC in an Australian Government licensed (Therapeutic Goods Administration), independent contract testing laboratory in Australia as per the method provided above and results of individual ginsenosides and total ginsenosides content were reported as %w/w of dry powdered crude herb/dry extract. USP39 [26] (Powdered Asian Ginseng Extract Monograph) for system suitability was followed. For the Chinese market place extract sample (CH-M-1), the result was reported from the manufacturer's Certificate of Analysis. For AUS samples 1–3, content was expressed as mg/capsule and for AUS sample 4, content expressed as mg/tablet. For complete data refer Table 2.

2.4.3. Analysis [26] The chromatographic system consisted of Agilent HPLC 1100 system fitted with a diode array detector. Analytical separation of ginsenosides was carried out on a Phenomenex- Luna C18- 3μ- 100 Å67

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Fig. 1B. A typical HPLC chromatographic profile of NNPG1015 showing the elution sequence of the eight ginsenosides.

sample (Fig. 2B) showed unusually intense peaks due ginsenoside Rd and Re. A perusal of the HPLC profiles of our authentic P. ginseng root extract (Fig. 2A, Lane 1) as well as USP powdered Asian ginseng extract RS [33] showed ginsenoside Rb1 to be the most intense peak, followed by ginsenosides Re, Rc and Rb2, which are more or less, of similar intensity. A perusal of literature [13,29], revealed that the major ginsenosides of leaf and leaf-stem are ginsenosides Re and Rd and occurs at maximum levels in the first year foliage. This led to the suspicion that CH-M-1 might be adulterated/contaminated with substantial levels of leaf or leaf/stem biomass to have higher contents of ginsenosides Rd and Re. Further investigation revealed that it is common practice in Chinese market place to add a pre-determined amount of leaf-stem biomass to root material of low ginsenosides yields to increase total ginsenosides content. Another surprising observation was that addition of leaf or leaf/stem biomass did not substantially alter Rb2:Rb1 ratio in this sample (0.46), thereby passing the USP requirement. The USP and other pharmacopoeial HPLC methods primarily focus on the separation and assay of 6 to 8 neutral ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2), as well as provide a partial profile of other detectable (ginsenoside) constituents. The same method may also be applied to the analysis of different parts of P. ginseng due to the predominantly overlapping ginsenosides profiles. However, reports on the spatial differences in quantitative neutral ginsenosides accumulation in different plant parts of P. ginseng prompted us to explore the utility of multiple ginsenosides ratios to detect adulteration of root extracts with other plant parts. Measure of peak areas of the 8 targeted ginsenosides and ratio calculations required computation of 88 ratio values making data analysis more cumbersome. To avoid such complex analysis of data, we simplified by generating ratio of ginsenosides values based on the elution sequence in HPLC, totalling to 28 ginsenosides ratios. As a first step, we calculated the ginsenosides ratios of sixteen batches of two grades of Network Nutrition-IMCD P. ginseng commercial extracts, NNPG59G and NNPG1015. Mean of ratios ± SE are presented in Fig. 3. The mean values of all the ginsenosides ratios of the two grades were within expected SE of mean and presents excellent repeatability of data and ratio pattern. To check how the ratio values of commercial extract batches compare with authentic starting root materials, mean ginsenosides ratios data generated for four batches of authenticated P. ginseng starting (root) materials (SMNN) were computed. We also chose a literature report [31] that provided the assay data for all the 8 ginsenosides especially for 4 year old P. ginseng root material; the assay data (as the peak area was not available) was computed to generate ginsenosides ratios. Both these data were compared with NNPG59G and NNPG1015 in Fig. 4. SMNN and literature reported data demonstrated very similar ratio pattern; significant deviations in SMNN and literature data when compared to NNPG59G and NNPG1015 were noticed with Rg1:Rg2 (6.4 and 9.1 respectively

2.4.5. Ginsenoside ratio analysis Individual ginsenoside peak area averages of duplicate sample injections were calculated. Peak area ratios were considered based on sequence of elution of the eight ginsenosides in HPLC – Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2 and Rd (Fig. 1B). This is in addition to the requirement of Rb2:Rb1 by USP39 [26]. For comparison, we also calculated and included ratio data (ginsenoside content) from one literature report on root and leaf ginsenosides [31]; the choice was based on the report that considered all the 8 ginsenosides mentioned in this paper. Mean of replicates and batches ± SEM are presented. 3. Results and discussion Most commercial P. ginseng root extracts used in herbal medicines, dietary supplements and functional foods conform to pharmacopoeial specifications. With stipulated hydroalcholic solvent extraction to enrich only neutral ginsenosides content and cGMP in manufacture ensures more or less consistent qualitative and quantitative HPLC ginsenosides profile pattern in pharmacopoeia-stipulated, commercial P. ginseng dry root extracts. This is well illustrated by the use of ginsenosides ratios as a measure of extract purity. The other factors that may influence individual variability of ginsenosides content in the starting material are cultivation conditions (edaphic and climatic influences) and possible genotype/chemotype variabilities. Most published literature relate to the variability in total ginsenoside content only. However, Xiao et al. [39] provided accumulation patterns of some individual ginsenosides in P. ginseng sourced from three regions in China and concluded ‘although there were apparent discrepancies among contents of ginsenosides for sample of same age from three cultivation areas, most of the five kinds of ginsenosides showed an identical variation trend in content…’ Aforesaid, clearly illustrates the possibility of reduced variability in dynamic accumulation of individual ginsenosides, if the source, age and manufacturing processes are properly controlled. This formed the basis for generating ginsenosides ratios pattern for detection of adulteration. In our pursuit to evaluate both the purity of commercial extracts and the utility of existing pharmacopoeial method protocols to detect adulteration, we routinely sourced and investigated a number of commercial P. ginseng root extracts from the market place. One commercial P. ginseng root extract sample sourced from the Chinese market place (coded CH-M-1; claimed ginsenosides assay of 9.59%), when subjected to USP39 [26] identification tests A (HPTLC) and C (HPLC ginsenosides profile and Rb2:Rb1 ratio), was within the acceptance criteria and returned a ginsenoside Rb2:Rb1 ratio of 0.46 (NLT 0.4). The results showed that this sample is devoid of P. quinquefolium adulteration. However, a critical look at the HPTLC (in comparison with an authentic P. ginseng root extract) revealed an unusually high intensity of most ginsenosides (Fig. 2A, Lane 2). The more specific HPLC profile of the 68

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Fig. 2. A. HPTLC finger-printing of ginsenosides. B. HPLC chromatographic profile of the Chinese market extract sample (CH-M-1) showing the ginsenosides profile. Note the higher intensities of ginsenosides Rd, Re, Rg1 and Rb2 in Chinese market sample (CH-M-1) compared to the authentic root extract (NNPG59G). Individual ginsenosides are identified based on mobility relative to ginsenoside Rg1 [typical of leaf stem adulteration].

compared to 0.67 and 0.57), Rf:Rg2 (0.019 compared to 1.1 and 0.96) and Rb1:Rd (0.099 compared to 2.12 and 2.19). Interestingly, a similar trend was observed when authentic leaf stem sample (NICPBP) data was plotted against NNPG59G and NNPG1015 (Fig. 6) wherein the most significant deviations were again Rg1:Rf (7.16 compared to 2.5 and 2.3), Rg1:Rb1 (3.28 compared to 0.33 and 0.25), Re:Rf (13.42 compared to 5.2 and 5.4), Re:Rb1 (6.15 compared to 0.67 and 0.57), and Rb1:Rd (0.37 compared to 2.12 and 2.19). These results confirmed the utility of multiple ginsenoside ratios in detecting CH-M-1 as a material adulterated with leaf biomass. The differences in ratio values between CH-M-1 and the authentic leaf stem raw material can again be attributed to the commercial (large) scale extraction of mixed biomass and lab scale extraction of leaf stem raw material; however, the ratios were still at large significant variance from the mean data presented for

compared to 2.6 and 2.2) and Rg1:Rd (2.9, 1.9 compared to 0.7, 0.5) ratios. Literature report differed additionally with Rg2:Rb1 (0.05 compared to 0.13, 0.12), Rg2:Rc (0.07 compared to 0.22. 0.21), and Rb1:Rb2 (4.1 compared to 1.8, 1.9) ratios, with all other ratios are similar to NNPG commercial extracts. It is probable that differences in large scale commercial extraction conditions and lab scale extractions may partly influence variability of individual ginsensoides ratios between commercial extracts and starting root materials resulting in such minor deviations. The ginsenosides ratios of the Chinese market place sample, CH-M – 1, were plotted against NNPG59G and NNPG1015 data and found that 22 of the 28 ratios were significantly different (Fig. 5). Most significant were Rg1:Rf (36.8 compared to 2.5 and 2.3), Rg1:Rb1 (5.9 compared to 0.33 and 0.25), Re:Rf (82.4 compared to 5.2 and 5.4), Re:Rb1 (13.3 69

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Fig. 3. Ginsenosides ratios of source authenticated commercial Panax ginseng root extracts (NNPG59G, NNPG1015).

Fig. 4. Ginsenosides ratios of source authenticated Panax ginseng roots - SMNN*, commercial extracts NNPG59G, NNPG1015 and literature reported# [31] – a comparison. *Average peak area ratios of all the crude root materials with a prefix SMNN mentioned in Table 1; #ratio calculated from the actual content - mg/g DW.

Fig. 5. Ginsenosides ratios of source authenticated Panax ginseng root commercial extracts NNPG59G, NNPG1015 and CH-M-1 sample – a comparison.

blinded, commercial extract samples of P. ginseng obtained from Europe (Table 1). Individual and total assay of ginsenosides (expressed as %w/ w) and ginsenosides Rb2:Rb1 ratios of these samples are presented in Table 2. The ratio data of the European samples were plotted against

NNPG59G and NNPG1015. Once we established that multiple ginsenosides ratios provided clues to detection of adulteration of root extracts with other plant part extracts, we attempted to test the validity of this protocol using twelve, 70

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Fig. 6. Ginsenosides ratios of source authenticated Panax ginseng root commercial extracts NNPG59G, NNPG1015 and authentic leaf stem sample (NICPBP) – a comparison.

Fig. 7. Ginsenosides ratios of source authenticated Panax ginseng root commercial extracts NNPG59G, NNPG1015 and European commercial extract samples 1–4: a comparison.

Fig. 8. Ginsenosides ratios of source authenticated Panax ginseng root commercial extracts NNPG59G, NNPG1015 and European commercial extract samples 5–8: a comparison.

this sample which returned a value of 16.19 that confirmed (value ≥ 10) adulteration with P. quinquefolium. European samples 1, 2 and 4 passed the USP identification test A and C for P. ginseng root extract. When the individual ginsenoside ratios of European samples 1–4 were plotted (Fig. 7), the pattern clearly revealed that 19 and 14

NNPG59G and NNPG1015 are presented in Figs. 7–9. European Sample 3 failed USP–Identification test C due to be absence of ginsenoside Rf in the HPLC profile and returned Rb2:Rb1 ratio of 0.055 (requirement is ≥ 0.4) indicating possible adulteration with P. quinquefolium roots. Based on this information, Rb1:Rg1 peak ratio was also calculated for 71

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Fig. 9. Ginsenosides ratios of source authenticated Panax ginseng root commercial extracts NNPG59G, NNPG1015 and European commercial extract samples 9–12 — a comparison.

(1.7), Re:Rb1 (2.5), Re:Rc (4.03), Re:Rb2 (4.7), Rb1:Rd (0.54), Rc:Rd (0.33) and Rb2:Rd (0.28) also deviated compared to NNPG59G and NNPG1015 (Fig. 9). EU sample 10 failed the stipulated USP test of Rb2:Rb1 peak area ratio (0.27) and a number of ginsenosides ratios were significantly different (Rg1:Re, 0.08; Re:Rf, 66.7; Re:Rb1, 5.9; Re:Rc, 20.04; Re:Rb2, 21.89; Re:Rd, 16.7; Rf:Rg2 0.22; Rg2:Rc, 1.36; Rg2:Rb2, 1.49; Rg2:Rd 1.1) compared to NNPG59G and NNPG1015. Ginsenoside Re was the major constituent (3.56%) and hence Re related ratios patterns were significantly different in EU sample 10 suggesting adulteration with a different plant part other than leaf/stem. The blinded EU sample 10 was revealed to be a Korean ginseng berry extract powder, reiterating the utility of ginsenosides ratios patterns in identifying adulteration/substitution of ginseng root extract materials with other plant parts. Ginsenosides ratios of blinded EU samples 11 and 12 mostly fitted well with the ratio patterns of NNPG59G and NNPG1015 except with minor deviations (especially EU sample 11). As an additional exercise, we calculated %w/w of each ginsenoside in total ginsenosides content (Table 3). In both crude root (SMNNPG) and root extracts (NNPG), ginsenoside Rb1 was the highest (ranged from 14 to 40%) in content among all the ginsenosides. In authentic leaf and stem material (NICPBP 121586-201001), ginsenoside Re (50% was the highest followed by Rc (12%)). Interestingly, in EU sample 1 (leaf extract) ginsenoside Rg1 was the highest at 38% followed by 23% Re and 4% ginsenoside Rb1. EU samples 2 and 6 which were suspected to have been adulterated with other plant parts, recorded 40% and 41% Rg1 followed by 23% and 27% Re with just 13% and 3% Rb1. Interestingly, EU sample 10, a P. ginseng berry extract, recorded 53% Re, 15% Rg1 and 16% Rb1. These trends more or less are in line with the observations of Li et al. [31] wherein the leaves recorded higher ginsenosides Re, Rd, Rg1 and Rc, while the roots tend to accumulate more of ginsenoside Rb1. Similar observations on higher accumulation of Rb1 in roots and significant accumulation of Re and Rd leaves were reported by other authors [13,20]. These trends of % of individual ginsenoside of the total assay also provide interesting pointers to detect adulteration of P. ginseng root extracts with other plant parts. Based on the foregoing, an attempt was made to utilise generated patterns of individual ginsenosides ratios, their assay and % ginsenoside content of root, leaf/stem and commercial root extract materials as well as blinded EU extract samples to establish purity/adulteration in four commercial P. ginseng (monoherbal) formulations (3 capsules and 1 tablet) available in the Australian retail supplements market (Table 1). HPLC analytical data on individual ginsenosides content and their % in Australian commercial samples are provided in Tables 2 and 3. The individual ginsenosides ratios of the AUS samples 1–4 were plotted against the data generated for NNPG59G and NNPG1015 (Fig. 10). AUS

ratios of EU samples 1 and 2 respectively clearly deviated from the ratio patterns established for NNPG59G and NNPG1015. The significant deviations of EU samples 1 and 2 included Rg1:Rf (26.9, 19.6), Rg1:Rb1 (4.4, 1.5), Rg1:Rc (1.9, 2.4), Re:Rf (60.1, 42.5), Re:Rb1 (9.8, 3.1), Re:Rc (4.3, 5.3), Re:Rb2 (3.2, 5.5), Rf:Rg2 (0.14, 0.35), Rf:Rb2 (0.05, 0.13), Rb1:Rd (0.11, 0.56), Rc:Rd (0.25, 0.34) and Rb2:Rd (0.33, 0.33). Additionally, EU Sample1 also deviated in Rf:Rd (0.018), Rg2:Rb1 (1.2), Rb1:Rc (0.4), and Rb1:Rb2 (0.33) ratios. These ratio patterns suggested that EU Samples 1 and 2 may be adulterated with leaf biomass. EU sample 3 showed an unusual ratio pattern which did not fit either with NNPG59G and NNPG1015 or with EU samples 1 and 2. The ginsenosides ratios EU sample 3 that significantly deviated included Rg2:Rb1 (0.03), Rg2:Rc (0.09), Rb1:Rb2 (18.08), Rc:Rb2 (5.9) and Rb2:Rd (0.18). The ratios of Rg2:Rb1 and Rb1:Rb2 values along with the absence of ginsenoside Rf and high Rb1:Rg1 (16.19) provide evidence that this sample is adulterated with P. quinquefolium root biomass. EU sample 4 was within the specification provided and passed USP monograph for P. ginseng extract and its' ginsenosides ratios perfectly fitted with NNPG1015 and NNPG59G ratio patterns (Fig. 7). On completion and reporting back the data, it was revealed to us that EU sample 1 was a P. ginseng (leaf and stem) dry extract and EU samples 2 (10% ginsenosides) and 3 (15% ginsenosides) were claimed to be P. ginseng root extracts. The blinded EU sample 4 turned out to be a batch of NNPG1015 supplied to the European market. Ginsenosides ratios of EU samples 5–8 are provided in Fig. 8. Except for EU sample 6, ginsenosides ratios of all other samples (EU samples 5, 7 and 8) neatly fitted with the pattern for NNPG59G and NNPG1015 and samples 5,7 and 8 also passed USP ID test A and C clearly indicating that these samples are indeed genuine P. ginseng root extracts. EU sample 6 showed significant deviations of ginsenosides ratios that include Rg1:Rf (41.8), Rg1:Rb1 (5.7), Rg1:Rc (3.0), Rg1:Rb2 (1.99), Re:Rf (100.3) Re:Rb1 (13.7), Re:Rc (7.3), Re:Rb2 (4.8), Rf:Rg2 (0.06), Rf:Rb2 (0.05), Rf:Rd (0.02), Rg2:Rb1 (2.2), Rg2:Rc (1.2), Rb1:Rd (0.13), Rc:Rd (0.24) and Rb2:Rd (0.37). Interestingly, EU sample 6 returned an assay of 52.7%. It was revealed that this extract sample was claimed to have 80% ginsenosides; since the ginsenosides ratios of Rg1:Rf, Re:Rf and Re:Rb1 values highly deviated from NNPG59G, NNPG1015 and the leaf/stem authentic samples, we were not in a position to clearly indicate whether this sample was indeed a P. ginseng root extract adulterated largely with leaf/stem or a clear case of substitution or whether it is an extract sample fortified with pure ginsenosides to achieve such high level of assay and unusual ginsenosides ratios. EU sample 9 failed USP ID test C due to the absence of ginsenoside Rf in the HPLC chromatogram, though it passed the assay and Rb2:Rb1 ratio (0.526). A number of ginsenoside ratios of EU sample 9 (Rb1:Rb2 72

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sample 4, except for few minor deviations, more or less fit neatly with ginsenoside ratios of NNPG59G and NNPG1015 indicating the purity of the P. ginseng root extract material used in this tablet formulation. 18 of the 28 ginsenosides ratios of AUS sample 1 largely deviated from the set ratio pattern. The ginsenosides ratios that were most significantly different include Rg1:Rf (47.6), Rg1:Rb1 (4.6), Rg1:Rc (2.8), Rg1:Rb2 (3.4), Re:Rf (94.0), Re:Rb1 (9.0), Re:Rc (5.5), Re:Rb2 (6.7), Rf:Rg2 (0.04), Rf:Rb2 (0.07), Rf:Rd (0.01), Rg2:Rb1 (2.5), Rg2:Rc (1.5), Rg2:Rb2 (1.9), Rb1:Rc (0.6), Rb1:Rd (0.12), Rc:Rd (0.21) and Rb2:Rd (0.17). This commercial sample claimed to contain P. ginseng root extract is suspected to have been heavily adulterated or completely substituted with leaf/stem or other plant part extracts. Interestingly, this sample also returned 28% ginsenoside Rd, 32% ginsenoside Re, 14% ginsenoside Rg1, as against 4% ginsenoside Rb1, whilst the pure root extracts are known to contain maximum content of ginsenoside Rb1, reinforcing our suspicion that this material is heavily adulterated with leaf and/or other plant parts. Ginsenoside ratios of AUS sample 2, another batch of capsules of the same brand as AUS sample 1, also deviated from the set ratio pattern, but to a significantly lesser degree than AUS sample 1. At least 10 out of the 28 ginsenosides ratios showed significant variation, that include Rg1:Rf (6.7), Rg1:Rb1 (3.0), Rg1:Rb2 (2.0), Re:Rf (13.5), Re:Rb1 (6.0), Re:Rb2 (4.1), Rg2:Rb1 (0.77), Rb1:Rc (0.36), Rb1:Rd (0.19), and Rb2:Rd (0.28). In addition, AUS sample 2 returned high % of ginsenoside Re (44%), ginsenoside Rg1 (11%), and a very low % of ginsenoside Rb1 (6%), indicating leaf adulteration, probably to a lesser degree compared to AUS sample 1. AUS sample 3 showed substantial deviation in 9 ginsenosides ratios which included Rg1:Rf (13.3), Re:Rf (45.3), Re:Rc (3.9), Re:Rb2 (4.8), Rf:Rg2 (0.1), Rf:Rb2 (0.11), Rg2:Rc (0.76), Rc:Rd (0.46), and Rb2:Rd (0.38). Interestingly, this sample returned 30% of ginsenoside Rb1 in total ginsenosides content; also returned 15% ginsenoside Rd and 28% ginsenoside Re. However, the considerable deviation of ratios especially R1:Rf and Re:Rf points to probable adulteration with leaf material.

Table 3 Percent (%) of individual ginsenoside of the total ginsenoside content in P. ginseng crude herb, commercial extracts and products used in this study. Sample code

%Rb1

%Rb2

%Rc

%Rd

%Re

%Rf

%Rg1

%Rg2

SMNNPG 20101106 SMNNPG 120201 SMNNPG 111020 SMNNPG 111119 NICPBP 121586201001 NNPG59G 12020103 NNPG59G 12070918 NNPG59G 12090505 NNPG59G 13032203 NNPG59G 141020110 NNPG59G 1505039 NNPG59G 1503018 NNPG59G 161101723 NNPG1015 12070512 NNPG1015 12020306 NNPG1015 150100922 NNPG1015 150201506 NNPG1015 160201205 NNPG1015 160505216 NNPG1015 160606410 NNPG1015 160807916 EU sample 1 EU sample 2 EU sample 3 EU sample 4 EU sample 5 EU sample 6 EU sample 7 EU sample 8 EU sample 9 EU sample 10 EU sample 11 EU sample 12 AUS sample 1a AUS sample 2a AUS sample 3a AUS sample 4b

21% 16% 16% 14% 7%

11% 12% 12% 11% 8%

17% 16% 17% 25% 12%

8% 3% 7% 10% 8%

17% 30% 22% 25% 50%

7% 6% 7% 4% 2%

15% 15% 17% 11% 9%

3% 3% 3% 1% 4%

23% 21% 23% 19% 31%

13% 12% 15% 12% 14%

23% 14% 18% 15% 19%

10% 9% 9% 9% 2%

16% 32% 25% 33% 24%

3% 3% 2% 3% 2%

10% 7% 6% 7% 5%

2% 2% 2% 2% 3%

29% 30% 33%

11% 12% 15%

17% 20% 18%

7% 6% 8%

24% 24% 15%

1% 2% 3%

6% 4% 7%

3% 3% 1%

21% 23% 30%

15% 14% 13%

16% 25% 20%

12% 12% 6%

25% 15% 22%

3% 3% 2%

7% 5% 4%

2% 2% 3%

40%

9%

16%

10%

13%

2%

2%

8%

32%

13%

21%

3%

10%

2%

16%

3%

31%

12%

21%

2%

11%

2%

19%

2%

32%

13%

21%

3%

9%

2%

16%

3%

34%

13%

20%

3%

9%

2%

17%

2%

4% 13% 52% 33% 28% 3% 24% 27% 16% 16% 26% 30% 4% 6% 30% 30%

11% 6% 2% 14% 13% 9% 16% 17% 7% 4% 11% 15% 9% 7% 6% 14%

11% 9% 20% 22% 18% 8% 20% 21% 11% 5% 15% 23% 7% 17% 9% 15%

9% 6% 4% 4% 3% 6% 3% 4% 7% 1% 2% 4% 28% 11% 15% 8%

23% 23% 13% 10% 7% 27% 10% 9% 22% 53% 9% 8% 32% 44% 28% 16%

0% 1% 0% 2% 3% 0% 3% 3% 0% 1% 3% 2% 0% 2% 1% 4%

38% 40% 7% 13% 24% 41% 20% 19% 33% 15% 33% 16% 14% 11% 7% 13%

3% 2% 1% 3% 4% 5% 2% 2% 3% 4% 1% 2% 7% 3% 4% 1%

4. Conclusion Utility of species indicative marker ginsenosides and specific ginsenosides ratios to detect adulteration of one species of medicinal ginseng roots with another has been well documented [18]. Similarly, species markers and metabolomics approaches have been utilised to discriminate leaf extracts of the three medicinal ginsengs [34] and adulteration with one another [35]. However, detection of adulteration of P. ginseng root extracts with aerial (leaf) parts and to discriminate between different plant parts with predominantly overlapping ginsenosides profiles has been proved difficult. The present pharmacopoeial methods, though sufficient to detect adulteration with root extracts of other ginsengs, are not sufficient enough to detect adulteration of P.

The data in bold indicate that the material fails Rb2:Rb1 ratio as stipulated by the Pharmacopoiea. a Values expressed as mg/capsule. b Values expressed as mg/tablet.

Fig. 10. Ginsenosides ratios of source authenticated Panax ginseng root commercial extracts NNPG59G, NNPG1015 and Australian finished product samples AUS Samples 1–4: a comparison.

73

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Q-TOF-MS, J. Pharm. Biomed. Anal. 52 (2010) 66–72. [6] L. Jia, Y. Zhao, Current evaluation of the millennium phytomedicine — ginseng (I): etymology, pharmacognosy, phytochemistry, market and regulations, Curr. Med. Chem. 16 (19) (2009) 2475–2484. [7] S. Foster, Towards understanding of ginseng adulteration: the tangled web of names, history, trade and perception, HerbalGram 111 (2016) 37–57. [8] L. Jia, Y. Zhao, X.-J. Liang, Current evaluation of the millennium phytomedicine — ginseng (II): collected chemical entities, modern pharmacology, and clinical applications emanated from traditional Chinese medicine, Curr. Med. Chem. 16 (22) (2009) 2924–2942. [9] S. Cui, J. Wu, J. Wang, X. Wang, Discrimination of American ginseng and Asian ginseng using electronic nose and gas chromatography - mass spectrometry coupled with chemometrics, J. Ginseng Res. 41 (2017) 85–95. [10] L.P. Christensen, Ginsenosides: chemistry, biosynthesis, analysis, and potential health effects, Adv. Food Nutr. Res. 55 (2009) 1–99. [11] T.T.X. Dong, X.M. Cui, Z.H. Song, K.J. Zhao, Z.N. Ji, C.K. Lo, K.W.K. Tsim, Chemical assessment of roots of Panax notoginseng in China: regional and seasonal variations in its active constituents, J. Agric. Food Chem. 51 (2003) 4617–4623. [12] R.A. Shellie, P.J. Marriott, C.W. Huie, Comprehensive two-dimensional gas chromatography (GC X GC) and GC X GC-quadrupole MS analysis of Asian and American ginseng, J. Sep. Sci. 26 (2003) 1185–1192. [13] C.-Z. Wang, M. Ni, S. Sun, X.-L. Li, H. He, S.R. Mehendale, C.S. Yuan, Detection of adulteration of notoginseng root extract with other Panax species by quantitative HPLC coupled with PCA, J. Agric. Food Chem. 57 (2009) 2363–2367. [14] J.-B. Wan, S.-P. Li, J.-M. Chen, Y.-T. Wang, Chemical characteristics of three medicinal plants of the Panax genus determined by HPLC-ELSD, J. Sep. Sci. 30 (2007) 825–832. [15] K.S.-Y. Leung, K. Chan, A. Bensoussan, M.J. Munroe, Application of atmospheric pressure chemical ionisation mass spectrometry in the identification and differentiation of Panax species, Phytochem. Anal. 18 (2007) 146–150. [16] G. Xie, R. Plumb, M. Su, Z. Xu, A. Zhao, M. Qiu, X. Long, Z. Liu, W. Jia, Ultraperformance LC/TOF MS analysis of medicinal Panax herbs for metabolomics research, J. Sep. Sci. 31 (2008) 1015–1026. [17] H.-W. Park, G. In, S.-T. Han, M.-W. Lee, S.-Y. Kim, K.-T. Kim, B.-G. Cho, G.-H. Han, I.-M. Chang, Simultaneous determination of 30 ginsenosides in Panax ginseng preparations using ultra performance liquid chromatography, J. Ginseng Res. 37 (4) (2013) 457–467. [18] H.-W. Park, G. In, J.-H. Kim, B.-G. Cho, G.-H. Han, I.-M. Chang, Metabolomic approach for discrimination of processed ginseng genus (Panax ginseng and Panax quinquefolius) using UPLC-QTOF MS, J. Ginseng Res. 38 (2014) 59–65. [19] C. Yu, C.-Z. Wang, C.-J. Zhou, B. Wang, L. Han, C.-F. Zhang, X.-H. Wu, C.-S. Yuan, Adulteration and cultivation region identification of American ginseng using HPLC coupled with multivariate analysis, J. Pharm. Biomed. Anal. 99 (2014) 8–15. [20] J. Liu, Y. Liu, L. Zhao, Z.-H. Zhang, Z.-H. Tang, Profiling of ginsenosides in the two medicinal Panax herbs based on ultra-performance liquid chromatography-electrospray ionization-mass spectrometry, SpringerPlus 5 (2016) 1770. [21] CAMAG, Application Notes F-31. HPTLC Identification of Asian Ginseng, (2004) (3 pp.). [22] K.T. Choi, Botanical characteristics, pharmacological effects and medicinal components of Korean Panax ginseng C A Meyer, Acta Pharmacol. Sin. 29 (2008) 1109–1118. [23] H.-H. Kim, Chemical diversity of Panax ginseng, Panax quinquefolium, and Panax notoginseng, J. Ginseng Res. 36 (1) (2012) 1–15. [24] T.W.D. Chan, P.P.H. But, S.W. Cheng, I.M.Y. Kwok, F.W. Lau, H.X. Xu, Differentiation and authentication of Panax ginseng, Panax quinquefolius, and ginseng products by using HPLC/MS, Anal. Chem. 72 (2000) 1281–1287. [25] W. Li, C. Gu, H. Zhang, D.V.C. Awang, J.F. Fitzloff, H.H.S. Fong, R.B. van Breeman, Use of high-performance liquid-chromatography-tandem mass spectrometry to distinguish Panax ginseng C.A.Meyer (Asian ginseng) and Panax quinquefolium L. (North American ginseng), Anal. Chem. 72 (2000) 5417–5422. [26] USP39–NF34, US Pharmacopoeia – National Formulary, The United States Pharmacopoeial Convention, Rockville, 2016. [27] N. Fuzzati, Analysis methods of ginsenosides, J. Chromatogr. B 812 (2004) 112–133. [28] W. Li, J.F. Fitzloff, HPLC determination of ginsenosides content in ginseng dietary supplements using ultraviolet detection, J. Liq. Chromatogr. Relat. Technol. 25 (2002) 2485–2500. [29] W. Shi, Y. Wang, J. Li, H. Zhang, L. Ding, Investigation of ginsenosides in different parts and ages of Panax ginseng, Food Chem. 102 (2007) 664–668. [30] P. Chen, D. Luthria, P.B. Harrington, J.M. Harnly, Discrimination among Panax species using spectral fingerprinting, J. AOAC Int. 94 (5) (2011) 1411–1421. [31] Li X-g, Y.Z. Yan, X.-J. Jin, Y.K. Kim, M.D. Uddin, Y.B. Kim, H. Bae, Y.C. Kim, S.W. Lee, S.U. Park, Ginsenoside content in the leaves and roots of Panax ginseng at different ages, Life Sci. 9 (4) (2012) 679–683. [32] N.N. Liu, Cultural Methods of Ginseng (in Chinese), Wu-Chou Publishing Co, Taipei, Taiwan, 1988 (c.f. Li et al, 2012). [33] USP Dietary Supplements Compendium, A 130-B, The United States Pharmacopoeial Convention, Rockville, USA, 2009. [34] S.O. Yang, S.W. Lee, Y.O. Kim, S.H. Sohn, Y.C. Kim, D.Y. Hyun, Y.P. Hong, Y.S. Shin, HPLC-based metabolic profiling and quality control of leaves of different Panax species, J. Ginseng Res. 37 (2) (2013) 248–253. [35] Q. Mao, M. Bai, J.-D. Xu, M. Kong, L.-Y. Zhu, H. Zhu, Q. Wang, Discrimination of leaves of Panax ginseng and P. quinquefolius by ultra-high performance liquid

ginseng root extracts with other plant parts. Taking the cue from the reports on the differential accumulation patterns of neutral ginsenosides in different plant parts of P. ginseng, we explored the utility of multiple ginsenosides ratios pattern as an additional approach to detect adulteration. This approach does not require any additional testing and utilizes data generated from pharmacopoieal HPLC method stipulated for P. ginseng root extracts. The consistency in multiple ratios patterns provided a platform to test the extract purity and to detect adulteration with other plant parts used in the blinded market samples of P. ginseng (from Europe) as well as in four P. ginseng mono-herbal commercial products from Australian market place, wherein, three of them were shown to be adulterated with aerial parts. Based on the foregoing, the following are suggested for adulteration detection in commercial P. ginseng root extracts with other plant parts. 1. Ginsenoside Rb1 peak in the HPLC chromatographic profile (by BP/ USP) should be equal to or larger than ginsenosides Rc, Rd, Re and Rg1 peaks. 2. Significantly lower ginsenoside Rb1 content (%) with a concomitant higher Rd and/or Re and/or Rg1 content (%) is a pointer to adulteration. 3. Substantial deviation from the established ginsenoside ratios pattern (Fig. 3), especially higher Rg1:Rf, Rg1:Rb1, Re:Rf, Re:Rb1, Re:Rc, Re:Rb2, and Rg2:Rb1 ratios and lower Rf:Rg2, Rf:Rb2, Rf:Rd and Rb1:Rd ratios points to possible adulteration. 4. Minor deviations in ratios patterns may occur due to herb variability; however, substantial changes from pharmacopoeia-stipulated process conditions for dry extracts, may alter the ratio patterns. Although acidic (malonyl) root-specific (F1, F2, F3, F5 and notoginsenoside Fe) and leaf-specific ginsenosides marker ginsenosides are known in P. ginseng [35,36], identifying and quantitating them require additional analytical methods and tools (such as MS) that are cost prohibitive. Multiple ginsenosides ratio pattern proposed here is a simple alternative for use by the industry, utilising existing pharmacopoeial HPLC method with no additional costs. However, it should be emphasized that adulteration detection protocols complement a quality assurance paradigm that ensures source authentication of bulk starting material herb and a complete transparency in supply (value) chain to establish purity of commercial herbal extracts [37] and is not a standalone quality statement. Conflict of interest The author declares no conflict of interest. Acknowledgement Thanks are due to Ryan Gorman and Network Nutrition-IMCD Australia for thought provoking discussions, encouragement and support. References [1] I.-H. Baeg, S.-H. So, The world ginseng market and the ginseng (Korea), J. Ginseng Res. 37 (1) (2013) 1–7. [2] Market Insider, Higher Prices Reported for 2014 Ginseng Crops, American and Asian. http://www.intracen.org/blog/Higher-prices-reported-for-2014-ginsengcrops-American-and-Asian/ (accessed December, 2016). [3] M. Daniel, Medicinal Plants, Science Publishers, New Hampshire, USA, 2006 (230 pp.). [4] J.M. Searels, K.D. Keen, J.L. Horton, H.D. Clarke, J.R. Ward, Comparing ginsenoside production in leaves and roots of wild American ginseng (Panax quinquefolius), Amer. J. Plant Sci. 4 (2013) 1252–1259. [5] L. Li, G.-A. Luo, Q.-L. Liang, P. Hu, Y.-M. Wang, Rapid qualitative and quantitative analyses of Asian ginseng in adulterated American ginseng preparations by UPLC/

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medicines, Epilepsy Behav. 52 (Pt. B) (2015) 363–371. [38] Y. Qian, Identification of adulteration of Panax notoginseng, Shizen Guoyi Guoyao 8 (4) (1997) 61. [39] D. Xiao, H. Yue, Y. Xiu, X. Sun, Y. Wang, S. Liu, Accumulation characteristics and correlation analysis of five ginsenosides with different cultivation ages from different regions, J. Ginseng Res. 39 (2015) 338–344.

chromatography quadrupole/time-of-flight mass spectrometry based metabolomics approach, J. Pharm. Biomed. Anal. 97 (2014) 129–140. [36] G.-Y. Liu, H.-Y. Zhou, J. Lu, N. Zhu, M.-Y. Gui, Y.-R. Jin, Y.-H. Zhang, X. Wang, X.W. Li, Determination of saponins in leaf of Panax ginseng C.A.Mey. by high performance liquid chromatography, Chem. Res. Chin. Univ. 25 (3) (2009) 297–301. [37] S. Govindaraghavan, N. Sucher, Quality assessment of medicinal herbs and their extracts: criteria and prerequisites for consistent safety and efficacy of herbal

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