Phytochemical analysis of Panax species: a review.

Panax species have gained numerous attentions because of their various biological effects on cardiovascular, kidney, reproductive diseases known for a long time. Recently, advanced analytical methods including thin layer chromatography, high-performance thin layer chromatography, gas chromatography, high-performance liquid chromatography, ultra-high performance liquid chromatography with tandem ultraviolet, diode array detector, evaporative light scattering detector, and mass detector, two-dimensional high-performance liquid chromatography, high speed counter-current chromatography, high speed centrifugal partition chromatography, micellar electrokinetic chromatography, high-performance anion-exchange chromatography, ambient ionization mass spectrometry, molecularly imprinted polymer, enzyme immunoassay, 1H-NMR, and infrared spectroscopy have been used to identify and evaluate chemical constituents in Panax species. Moreover, Soxhlet extraction, heat reflux extraction, ultrasonic extraction, solid phase extraction, microwave-assisted extraction, pressurized liquid extraction, enzyme-assisted extraction, acceleration solvent extraction, matrix solid phase dispersion extraction, and pulsed electric field are discussed. In this review, a total of 219 articles published from 1980 to 2018 are investigated. Panax species including P. notoginseng, P. quinquefolius, sand P. ginseng in the raw and processed forms from different parts, geographical origins, and growing times are studied. Furthermore, the potential biomarkers are screened through the previous articles. It is expected that the review can provide a fundamental for further studies.

Introduction

Genus Panax belonging to Family Araliaceae contains eleven species (three varieties) namely P. trifolius, P. notoginseng, P. quinquefolius, P. ginseng, P. pseudoginseng, P. zingiberensis, P. stipuleanatus, P. japonicus, P. japonicus var. angustifolius, P. japonicus var. major, and P. japonicus var. bipinnatifidus, which are mainly distributed in the Eastern Asia and Northern America [1]. Among them, most of the investigations have been conducted on P. notoginseng, P. quinquefolius, and P. ginseng for their pharmacological activity. Their use to treat cardiovascular, kidney, and reproductive diseases has a long history [2]. Various bioactive constituents including ginsenosides, polysaccharides, alkaloids, glucosides, and phenolic acids have been identified in P. ginseng in a previous study [3]. The main ginsenosides isolated from Panax species are shown in Fig. 1. They contain protopanaxadiol, protopanaxatriol, ocotillol, oleanolic acid, and C-17 side chain type [4,5]. Protopanaxadiol has a glucose moiety attached to C-20 and C-3, and protopanaxatriol has glycosylation sites at C-20, C-3, and C-6. The cleavage of glucose bond at C-20 is hydrolyzed before bond at C-3 and C-6 in processed condition [6]. The amount of isomer pairs is detected, and 20(S)-ginsenosides are always eluted more easily than 20(R)-ginsenosides [6]. Moreover, Δ20(21) ginsenosides are eluted before their Δ20(22) derivatives. Ocotillol-type and oleanane-type have a side chain at C-20. Yao et al have identified 945 ginsenosides from P. notoginseng leaves and 662 potentially novel ginsenosides [7]. Various species, parts, processings, regions, and growing times have a great influence on the chemical compounds of herbal medicines.

Fig. 1

The main ginsenosides of Panax species (protopanaxadiol, protopanaxatriol, ocotillol, oleanane, and C-17 side chain type).

In the previous review, chemical and pharmacological diversity of ginsenosides of genus Panax L. was summarized [4,8,9]. Wang et al (2015) reviewed analytical techniques that were used in the evaluation of P. quinquefolius, while some advanced methods such as 2D-HPLC, micellar electrokinetic chromatography, and high-performance anion-exchange chromatography (HPAEC) were not investigated. In addition, P. ginseng and P. notoginseng with phenolic acids, dencichines, trilinoleins, flavonoids, and vitamins were not described [10]. Qi et al (2011) reviewed preparation, analytical advance, and applications of ginseng from January 2000 to September 2010 [11]. However, there are only few investigations in which analytical methods were applied to evaluate Panax species. Some advanced techniques such as ambient ionization mass spectrometry are hardly described in previous studies. In this review, we analyzed the published phytochemical analysis of Panax based on the keywords “Panax, ginseng” from Pubmed and Google Scholar. A total of 219 articles from 1980 to 2019 in the analytical methods of Panax species were investigated. As shown in Fig. 2, it is found that few researches are conducted during 1980 and 2000. The number of papers gradually grows with the time. It increased rapidly after 2011. Different sample preparations have significant influence on analysis of the bioactive compounds. The different analytical methods have different performances on the analysis of constituents of Panax species. Analytical methods including thin layer chromatography (TLC), high-performance thin layer chromatography (HPTLC), gas chromatography (GC), high-performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UHPLC) with tandem ultraviolet (UV) detector, diode array detector (DAD), evaporative light scattering detector (ELSD), and mass detector, two-dimensional high-performance liquid chromatography (2D-HPLC), ambient ionization mass spectrometry, high speed counter-current chromatography (HSCCC), and high speed centrifugal partition chromatography (HPCPC) are investigated. Furthermore, the methods have been applied to raw and processed ginseng of different species, from different parts, regions, growing ages, and biochemical analysis. The application in various fields is to screen the potential biomarkers for evaluating and quality control of Panax species. It is expected that the current review would have a solid fundamental for the future investigation.

Fig. 2

The number of papers published during 1980 and 2019.

Sample preparations

During isolation and purification of bioactive components from natural products, extraction is the first and essential step [12]. A method with short extraction time, less extraction solvent, simple operation, low cost, and high extraction efficiency could be accepted. Sometimes many of factors are not satisfied because of the chemical profile of medicinal plants. In this review, the factors of sample preparations for Panax species are discussed (Table 1). As a traditional method, heat reflux extraction is used to extract ginsenosides, while it has the disadvantages of chemical transformation, wasting extraction solvent, and complicate operation [13]. Owing to convenient, simple, and high-efficient extraction, various extraction solvents (different concentrations of ethanol and methanol) and times have been used to extract ginsenosides, polyacetylenes, phenolic acids, flavonoids, and so on [[14], [15], [16]]. The operation time of microwave-assisted extraction is 60 times more efficient than that of Soxhlet extraction and 20 times more efficient than that of ultrasonic extraction [17]. Moreover, malonyl-ginsenosides Rb1, Rc, Rb2, and Rd can transform into corresponding neutral ginsenosides Rb1, Rc, Rb2, and Rd under high pressure microwave-assisted extraction at 400 kPa in 70% ethanol–water and at 600 kPa in methanol [18]. Compared with Soxhlet extraction, heat reflux extraction, ultrasonic extraction, and microwave-assisted extraction, pressurized liquid extraction has the highest extraction efficiency in the shortest time for P. quinquefolius, P. notoginseng, and red ginseng [12,19,20]. The amount of total ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) increased with ultra-high-pressure extraction, whereas pressuring level and time have no influence on the content of ginsenosides [21]. The extraction time of pulsed electric field is less than 1 s, which is much less than that of the heat extraction method (6 h) [22]. In addition, matrix solid phase dispersion extraction has the advantages of short extraction time and less solvent usage, when compared with reflux extraction [23].

Table 1

Various factors of sample preparation of Panax genus

Technology	Extraction Time	Extraction Solvent	Extraction Efficiency	Operation	Cost	Reference
Soxhlet extraction	Long	More	High	Moderate	Low	[13]
Heat reflux extraction	Long	More	High	Moderate	Low	[125]
Ultrasonic extraction	Moderate	Moderate	High	Simple	Moderate	[126]
Solid phase extraction	Long	Moderate	Moderate	Simple	Moderate	[127]
Microwave-assisted extraction	Short	Less	High	Simple	High	[17]
Pressurized liquid extraction	Short	Less	High	Simple	High	[128]
Enzyme-assisted extraction	Long	Less	Low	Complex	Low	[113]
Accelerated solvent extraction	Short	Less	High	Simple	High	[129]
Matrix solid phase dispersion extraction	Short	Less	High	Simple	Moderate	[23]
Pulsed electric field	Short	More	High	Simple	Moderate	[22]

Analytical methods

In the previous study, chromatographic methods including TLC/HPTLC, GC, HPLC, UHPLC (UV detector, DAD, ELSD, and MS detector), 2D-HPLC, HSCCC/HPCPC, and spectroscopic analysis, e.g., near infrared (NIR) spectroscopy and NMR, have been used to evaluate Panax species. Moreover, some advanced techniques such as ambient ionization mass spectrometry are applied to Panax. It is obvious that different techniques show different advantages and shortcomings. Detailed comparisons are provided in Table 2.

Table 2

The advantages and shortcomings of technique analysis for Panax species

Technique		Advantages	Shortcomings	Reference
TLC/HPTLC		Rapid analysisConvenient operationHigh sensitivity and specificityLow cost	Bad efficiency in separationBad stabilityNeed volatile organic solventsLow accuracy in quantification	[[24], [25], [26]]
GC		Rapid analysisLess solvent consumingHigh sensitivityLess time analysis	Limited to volatile compoundsOperation with the derivationHigh cost	[76,130]
HPLC/UHPLC	UV/DAD	Convenient operationHigh specificityHigh repeatabilityLow costCombining with multiple detector	Long analysis timeLarge solvent consumingAnalytes with ultraviolet absorptionLow sensitivity	[[131], [132], [133]]
	ELSD	High specificityLow cost	Long analysis timeLarge solvent consumingLow sensitivity	[52,77,104]
	MS	Convenient operationHigh sensitivityLess solvent consumingHigh resolution	High costBad stability	[93,134,135]
2D-LC		Wide coverageGood orthogonalityHigh efficiency in separation	Complicated operationLong analysis timeLarge solvent consuming	[55,56]
Ambient ionization mass spectrometry		Rapid analysisConvenient operationLess solvent consuming	Bad stabilityHigh costLow sensitivitySome compound with the derivation	[59]
HSCCC/HPCCC		High efficiency in separation	More solvent consumingLow sensitivity	[62,136]
1H NMR		Fast analysisLess solvent consumingEasy operation	High costLow accuracy in quantification	[65,66]
Near infrared		Fast analysisNo solvent consumingNo sample preparationLow cost	Low accuracy in quantificationLow specificity	[137,138]

TLC/HPTLC

As a rapid qualitative and quantitative analysis technology, TLC is recorded by Chinese Pharmacopoeia. Some scholars have applied TLC to evaluate Panax species (Table 3). In P. ginseng, ginsenosides Rb1, Rb2, Rc, Rd, Re, and Rg1 are determined simultaneously by HPTLC at an absorption of 275 nm. The method consists of a quaternary-solvents system (1,2-dichloroethane–100% ethanol–methanol–water, 56.8:19.2:19.2:4.8) to have an efficient saponins recovery and selective separation [24]. Different species with free mono- and oligo-saccharides are identified by HPTLC [25]. Moreover, to determine ginsenosides in P. trifolius, 2D-TLC with eluent A (chloroform–methanol–ethyl acetate–butanol–water, 4:4:8:1:2), eluent B (chloroform–butanol–methanol–water, 4:8:3:4), and eluent C (chloroform–methanol–water, 13:7:2) were used [26]. The TLC technology has some advantages of rapid, convenient, and sensitive characteristics to target compounds, whereas it always needs standards and there is a lack of uniqueness for bioactive compounds. In recent years, HPTLC-MS with rapid and accurate profile will hope for evaluating Panax species [27]. Two-dimensional HPTLC showed an efficient performance and good isolation profiles for Panax species in another study [28].

Table 3

Chemical analysis of Panax species by TLC/HPTLC

Method	Species	Part	Analytes	Reference
HPTLC	P. ginseng	Root	Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rg1	[24]
HPTLC	P. ginseng, P. quinquefolius, P. notoginseng	Root	Glycome	[25]
2D-TLC	P. trifolius	Root	Ginsenosides Ro, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2	[26]

Gas chromatography

Gas chromatography is employed to determine volatile organics, ginsenosides, and phenolic acids from Panax species (Table 4). Different derivatizations for chemical components were selected. For volatile organics, the GC–MS method can determine bioactive compounds of headspace without sample preparation for discriminating Panax species [29]. When determining ginsenosides in P. ginseng, it is applied to high-molecular-weight saponins after derivatization with trimethylsilylation [30]. Sample is subjected to trimethylsilane derivatization for evaluating phenolic acids in white and red ginsengs [31]. After derivatization with ethyl chloroformate, dencichine or other amino acids of P. notoginseng are determined [32]. GC–MS for volatile components can take some advantage with simple, fast, and effective characters, whereas for some non-volatile components, a complex operation is required. 2D-GC with high peak capacity, orthometric characteristic can be used to evaluate volatile components of samples, which is necessary to be discussed for the further study.

Table 4

Chemical analysis of Panax species by GC–MS

Method	Species	Part	Analytes	Reference
GC–MS	P. ginseng	Root	Ginsenosides Rg1, Re, Rd, Rc, Rb2, Rb1, F1	[30]
GC–MS	Panax genus	Root	Panaxynol and panaxydol	[139]
GC–MS	P. ginseng	Root	Phenolic acids	[31]
GC–MS	P. notoginseng	Root	Dencichine	[32]
GC–MS	P. ginseng	Root	Volatile organic compounds	[76]
GC–MS	P. ginseng	Root	Volatile organic compounds	[130]
GC–MS	P. ginseng, P. notoginseng, P. quinquefolius	Root	Volatile organic compositions	[29]
GC–MS	P. ginseng, P. quinquefolius, P. notoginseng	Root	Volatile organic compounds	[140]

HPLC/UHPLC

HPLC/UHPLC is the most frequently used method for Panax species in the qualitative and quantitative analysis. In this review, it is found that stationary phases including C18 column (250 × 4.6 mm, 5 μm) with different brands are used for ginsenosides, OV-170 (500 × 0.25 mm), LiChrosorb for polyacetylenes, polymer C18 column (250 × 4 mm, 10 μm) for trilinoleins, Waters Atlantis HILIC (hydrophilic interaction liquid chromatography) silica (50 × 2.1 mm, 3 μm) [33] for dencichine, and Zorbax SB-Aq column (150 × 4.6 mm, 5 μm) for nucleobases and nucleosides. Moreover, the small particle size ACQUITY UHPLC BEH C18 (2.1 × 100 mm, 1.7 μm) is used in UHPLC. Two-phase solvent systems contain water or buffer solution in water (formic acid, acetic acid, phosphoric acid, ammonium formate, or ammonium acetate) and acetonitrile or methanol. Formic acid in water improves resolution and eliminates peak tailing [[34], [35], [36]]. The solvent range of 1% to 100% is changed to obtain the appropriate gradient elution grogram. Ginsenosides could be eluted by the solvent range of 30–50% as observed in the literature. UHPLC with less analytical time has the better performance than HPLC.

UV/DAD and ELSD detector

UV detector is the traditional detector for the qualitative and quantitative analysis of chemical compounds in the Panax species (Table 5, Table 6). The detector with its low cost and simple operation has become the most commonly used analytical method in the laboratory. Therefore, it has been widely employed to determine the ginsenosides (malonyl ginsenoside, protopanaxadiol, protopanaxatriol, ocotillol, and oleanane), trilinoleins, polyacetylenes [37], phenolics [38], phytosterols [39], flavonoids, and vitamins [40]. The detection wavelengths for different types of biochemical compounds are various. It is reported that ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2, F2, gypenoside XVII, and notoginsenoside R1 could be detected in the wavelength of 203 and 198 nm [[41], [42], [43], [44]]. The detection wavelength is set at 205 nm for trilinoleins [45], 254 nm for polyacetylenes [37], 260 nm for nucleobases and nucleosides [46], and 280 nm for phenolic compounds [38]. However, oleanane ginsenosides (ginsenoside Ro) are poor chromophores with weak UV absorption and are disturbed by solvents (the cut-off wavelength of methanol is 205 nm) that have low sensitivity with UV detection. DAD has the better recognition than conventional UV detection (Table 7). It is widely used to determine polar and non-polar [47], neutral and malonyl ginsenosides [48] in P. ginseng, P. quinquefolius, and P. notoginseng. As a mass detection, ELSD is mainly used for analysis of biological compounds that lack appropriate chromophores (Table 8). It can be used to identify and quantify neutral and acidic ginsenosides Rg1, Rg2, Ro, Rb1, Rb2, Rc, and Rd in P. ginseng, while the sensitivity of ELSD is five times lower than that with UV detection [49].

Table 5

Ginsenosides analysis of Panax species by HPLC-UV

Method	Species	Part	Analytes	Reference
HPLC-UV	P. ginseng	Root	Ginsenosides Rb1, Rb2, Rc, Rd, Rg1, Re, Rf	[141]
HPLC-UV	P. ginseng	Different parts and ages	Ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rb3, Rd	[102]
HPLC-UV	P. ginseng	Root	Ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rd	[22]
HPLC-UV	P. ginseng	Leaf	Ginsenosides F1, F2, F3, Re, Rg1, Rd, Rc, Rb2	[23]
HPLC-UV	P. ginseng	Root	Ginsenosides Rg2, Rg3, Rg5, Rg6, Rh1, Rh4, Rk1, Rk3, F1, R4	[73]
HPLC-UV	P. ginseng	Root	Ginsenosides Rg1, Re, Rb1, Rd	[142]
HPLC-UV	P. ginseng	Root	Ginsenosides Rb1, Rb2, Rc, Rd, Rf, Rg1, Rg2, Rg3, Rg5, Rg6, Rh1, Rh4, Rk1, Rk3, F1, F4	[131]
HPLC-UV	P. ginseng	Root	Ginsenosides Rg1, Re, Ro	[143]
HPLC-UV	P. ginseng	Root	Malonyl ginsenosides	[144]
HPLC-UV	P. ginseng	Root	Ginsenosides and phenolic	[145]
HPLC-UV	P. quinquefolius	Root	Ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rd	[132]
HPLC-UV	P. quinquefolius	Leaf, stem, root	Ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rd	[125]
HPLC-UV	P. quinquefolius	Root	Ginsenosides Rb1, Rc, Rd, Re, Rg1 and F2, gypenoside XVII	[43]
HPLC-UV	P. quinquefolius	Root	Ginsenosides Rb1, Rc, Rd	[17]
HPLC-UV	P. quinquefolius	Root	Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1	[146]
HPLC-UV	P. quinquefolius	Root	Ginsenosides Rg1, Re, Rb1, Rc, Rd	[147]
HPLC-UV	P. quinquefolius	Root	Ginsenosides Rg1, Re, Rb1, Rb2, Rc, Rd	[12]
HPLC-UV	P. quinquefolius	Root	Ginsenosides Rb1, Rb2, Rc, Rd, Rg1, Rg2	[113]
HPLC-UV	P. quinquefolius	Different parts and ages	Ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rb3, Rd	[42]
HPLC-UV	P. quinquefolius	Root	Rare ginsenosides 20(S/R)-Rh1, Rg6, F4, Rk3, 20(S/R)-Rg3, Rk1, Rg5	[148]
HPLC-UV	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Rb1, Rd	[133]
HPLC-UV	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Rb1, Rd,	[127]
HPLC-UV	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Rb1, Rd,	[119]
HPLC-UV	P. notoginseng	Rat tissue	Ginsenosides Rg1, Rb1, Rd	[149]
HPLC-UV	P. notoginseng	Flower bud	Notoginsenoside R1, ginsenosides Rg1, Re, Rb1, Rb2, Rb3, Rd, F2	[150]
HPLC-UV	P. notoginseng	Different parts	Notoginsenoside R1, ginsenosides Rb1, Rb2, Rd, Re, Rg1, Rb3, Rg2, Rg3, Rh1	[110]
HPLC-UV	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Re, Rg1, Rb1, Rd	[151]
HPLC-UV	P. notoginseng	Root	Ginsenosides Rg1, Re, Rb1, 20(S/R)-Rh1, Rk3, Rh4, 20(S/R)-Rg3, notoginsenoside R1	[152]
HPLC-UV	P. notoginseng	Root	Ginsenosides Rg1, Re, Rb1, Rd, notoginsenoside R1	[153]
HPLC-UV	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Re, Rb1, Rd	[154]
HPLC-UV	P. notoginseng	Root, leaf, stem	Ginsenosides Rg1, Re, Rb1, Rd, notoginsenoside R1	[155]
HPLC-UV	P. notoginseng	Root, rhizome	Notoginsenoside R1, R2, R3, ginsenosides Rg1, Rg2, Rg3, Rb1, Rd, Rh1, Re, quercetin	[13,156]
HPLC-UV	P. ginseng, P. quinquefolius, and ginseng drug preparations	Different parts	Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2	[41]
HPLC-UV	P. sokpayensis, P. bipinnatifidus	Rhizomes	Ginsenosides Rg1, Rg2, Rf, Re, Rd, Rc, Rb1, Rb2	[95]

Table 6

Chemical analysis of Panax species by HPLC-UV

Method	Species	Part	Analytes	Reference
HPLC-UV	P. ginseng	Root	Ginsenosides and total phenolic	[157]
UHPLC-UV	P. ginseng	Fruit, leaf, root	Phenolic compounds	[38]
HPLC-UV	P. ginseng	Root	Phytosterols	[39]
HPLC-UV	P. ginseng	Main root, root hair, and leaf	Phenolic, flavonoid, vitamin	[14]
HPLC-UV	P. ginseng	Root, rhizome, and root hair	Trilinolein, 1,2-dilinoleoyl-3-oleoyl-glycerol	[45]
HPLC-UV	P. pseudoginseng	Root	Trilinolein	[45]
HPLC-UV	P. ginseng, P. quinquefolius, P. japonicus, P. notoginseng	Root	Polyacetylenes, ginsenosides	[37]
UHPLC-UV	P. notoginseng	Root	Fingerprinting analysis	[158]
HPLC-UV	P. notoginseng	Root	Fingerprinting analysis	[115]
HPLC-UV	P. ginseng, P. quinquefolius	Leaf	Metabolic profiling	[100]

Table 7

Chemical analysis of Panax species by HPLC-DAD

Method	Species	Part	Analytes	Reference
HPLC-DAD	P. ginseng	Root	Polar and non-polar ginsenosides	[47]
UHPLC-DAD	P. ginseng	Root	Panaxfuraynes A and B	[101]
HPLC-DAD	P. ginseng	Root	Spectrum-efficacy relationship	[159]
HPLC-DAD	P. quinquefolius	Root	Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rg1, Ro, gypenoside XVII, pseudoginsenoside-F11	[160]
HPLC-DAD	P. quinquefolius	Root	Neutral and malonyl ginsenosides	[48]
HPLC-DAD	P. quinquefolius	Root	Ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rd	[114]
HPLC-DAD	P. quinquefolius	Root	Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rg1	[126]
HPLC-DAD	P. quinquefolius	Fresh root	Ginsenosides and polyacetylenes	[161]
HPLC-DAD	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3, Rd	[19]
HPLC-DAD	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Re, Rb1, Rc, Rd	[44]
HPLC-DAD	P. notoginseng	Root	Ginsenosides Rb1, Rc, Rd, Re, Rg1, Rg5, Rk1, 20(R/S)-Rg3, 20(R/S)-Rh1, notoginsenosides R1	[162]
HPLC-DAD	P. notoginseng	Root	Saponins	[79]
HPLC-DAD	P. notoginseng	Root	Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rg1	[21]
HPLC-DAD	P. notoginseng	Main root, rhizome, fibrous root	Notoginsenosides R1, R4, Fa, and K, ginsenosides Rg1, Rb1, Rd, Re, Rf, Rg2, Rh1	[107]
HPLC-DAD	P. notoginseng	Root	Notoginsensides R1, ginsenosides Rg1, Re, Rb1, Rd	[163]
HPLC-DAD	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Rb1, Rd, Re	[164]
UHPLC-DAD	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Re, Rf, Rb1, Rg2, Rb3, Rd, Rg3	[165]
UHPLC-DAD	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Re, Rb1, Rd	[166]
HPLC-DAD	P. notoginseng	Different parts	Fingerprint analysis	[109]
HPLC-DAD	P. notoginseng	Flower	Fingerprint analysis	[167]
HPLC-DAD	P. notoginseng, P. vietnamensis, P. stipuleanatus	Root	Fingerprint analysis	[97]
UPLC-PDA	P. ginseng	Root	Ginsenosides Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rd, Ro	[168]

Table 8

Chemical analysis of Panax species by HPLC-ELSD

Method	Species	Part	Analytes	Reference
HPLC-ELSD	P. ginseng	Root	Ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rd	[49]
HPLC-ELSD	Red ginseng	Root	Ginsenosides Rg1, Re, Rf, Rh1, Rg2, Rb1, Rc, Rb2, Rb3, Rd, Rg3, Rk1, Rg5, Rh2	[77]
HPLC-ELSD	Black ginseng	Root	Less polar ginsenosides	[78]
HPLC-ELSD	P. ginseng	Root	Ginsenosides Rh1, Rg2, Rg3, Rg1, Rf, Re, Rd, Rb2, Rc, Rd	[169]
HPLC-ELSD	P. quinquefolius	Different parts	Ginsenosides Rg1, Re, F11, Rf, Rg2, Rh1, Rb1, Rc, Rb2, Rb3, Rd, Rh2	[104]
HPLC-ELSD	P. quinquefolius	Different parts	20(R)-dammarane-3β,6α,12β,20,25-pentol, 25(R)-ocotillol, 20(R)-protopanaxatriol, 20(S)-panaxatriol and 20(R)-dammarane-3β,12β,20,25-tetrol	[105]
HPLC-ELSD	P. ginseng, P. quinquefolius	Root	Ginsenoside Rf, 24(R)-pseudoginsenoside F11	[90]
HPLC-ELSD	P. notoginseng	Root	Ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rd	[170]
HPLC-ELSD	P. notoginseng	Different parts	Ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3, Rd	[108]
HPLC-ELSD	P. notoginseng	Root	Ginsenosides Re, Rg1, Rb1, Rb2, Rc, Rd, notoginsenoside R1	[52]
HPLC-ELSD	P. notoginseng, P. quinquefolius, P. ginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Re, Rf, Rg2, Rc, Rb2, Rb3, Rd, Rg3	[94,128]

MS detector

Modern analytical techniques based on MS with chromatographic separation have the sensitivity and specificity characteristic when compared with traditional detection analysis of Panax species (Table 9) [10]. Ion sources including atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are used. The APCI can be applied to low molecule and polar compounds, such as 24(R)-pseudoginsenoside F11, ginsenoside Rf, and polyacetylenes [16,50,51]. The most of bioactive constituents of Panax species in the ESI mode has the better performance than that in the APCI mode, especially for the large and moderate polar compounds. Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, and notoginsenoside R1 have been analyzed with ESI mode in previous studies [52,53]. Dencichine, triterpenoid saponins, nucleobases, nucleosides, and polyacetylenes could be conducted by HPLC-MS as well (Table 10). In addition, MS hyphenations with Q-TOF, IT-TOF, Q-Trap, and Q-Orbitrap have been used to determine ginsenosides accurately and sensitively (Table 11). A total of 234 ginsenosides including 67 potential new ones were isolated tentatively by HPLC–QTOF-MS [54]. It is found that 646 ginsenosides were identified from stems and leaves of P. ginseng using linear ion-trap/Orbitrap mass spectrometry [55]. In the qualitative analysis, full scan, parent scan, daughter scan, and neutral loss scan have been employed. Selective ion monitoring and multiple reaction monitoring have been used to quantify bioactive compounds.

Table 9

Ginsenosides analysis of Panax species using HPLC-MS

Method	Species	Part	Analytes	Reference
HPLC-MS	P. ginseng	Root	Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2	[118]
HPLC-ESI-MS	P. ginseng	Root	Ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rd	[18]
HPLC-FD-MS	P. ginseng	Ginseng extract	Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2	[134]
HPLC-ESI-MS/MSn	P. ginseng	Root	Ginsenosides Rg1, 20(S)-Rg2, Rb1, Rc, Rh2, malonyl-ginsenoside Rb2 and Rc	[75]
HPLC-ESI-MS/MS	P. ginseng	Root	Low-polar ginsenosides	[80]
UHPLC-MS	P. ginseng	Root	Ginsenosides Rb1, Rb2, Rg1, Rg2, Rg3, Rc, Rd, Re, Rf	[171]
HPLC-MS/MS	P. ginseng	Root	Ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rd, Rg2, Rh1, F1, F2, Rg3, PPT	[122]
HPLC-MS/MS	P. ginseng	Fresh root	Ginsenosides Rg1, Re, Rf, Rb1, Rb2, Rd, 20(S)-Rg2, Rc, 20(S)-Rh1, F1, F2, 20(S)-Rg3, 20(S)-protopanaxatriol, compound K, 20(S)-Rh2	[172]
HPLC-Qtrap-MS	P. ginseng	Root	Ginsenosides	[173]
HPLC-MS	P. ginseng	Root	Notoginsenoside R1, ginsenoside Rb2, Re, Rb1, Rc, Rg1, Rb3, Rf, F1, Rd, Rh1, Rg2, F2, Rg3, Rh2, compound K	[174]
LC-MS/MS	P. ginseng	Root	15 ginsenosides	[175]
UHPLC-HRMS	P. quinquefolius	Root	Ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, Rg3, Rh1, Rh2, Ro, F1, F2, F3, pseudoginsenoside F11, notoginsenosides R1, R2	[93]
HPLC-APCI-MS	P. quinquefolius	Root	24(R)-pseudoginsenoside F11	[50]
UPLC-MS/MS	P. quinquefolius	Different parts	22 ginsenosides	[176]
HPLC-MS	P. ginseng, P. quinquefolius	Root	Ginsenosides Rb1, Rb2, Rc, Ro, Rd, Re, Rf, Rg1, pseudoginsenoside F11	[88]
HPLC-MS	P. ginseng, P. quinquefolius	Root	Ginsenoside Rf, 24(R)-pseudoginsenoside F11	[89]
UHPLC-ESI-MS	P. notoginseng	Different parts	Metabolite profiling	[112]
HPLC-MS	P. notoginseng	extraction	Ginsenosides Rg1, Rb1, notoginsenoside R1	[177,178]
UHPLC-MS/MS	P. notoginseng	Extract	Notoginsenoside R1, ginsenosides Rg1, Rb1, Re, Rd	[120]
UPLC-MS/MS	P. notoginseng	Compounds	Notoginsenoside R1, ginsenosides Rg3, Rd, Rg2, Rb2, Rf, Rg1, Rb1, Re	[179]
HPLC-MS	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Rb1, Rd, F2, Re	[180]
LC-Q-Trap-MS	P. notoginseng	Extraction	Notoginseng total saponins	[181]
LC-MS/MS	Steamed notoginseng	Rat plasma	23 triterpenoids	[182]
UHPLC-MS	P. japonicus	Leaf	Chikusetsusaponins V, Ib, IV, IVa, IV ethyl ester	[183]
HPLC-MS	P. ginseng, P. quinquefolius, P. notoginseng	Root	Ginsenosides Ro, Ra2, Ra3, Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, 20(S)-Rg3, Rf, notoginsenosides R1, R2, R4 and 24(R)-pseudoginsenoside F11	[91]
HPLC-APCI-MS	P. quinquefolius, P. ginseng, P. notoginseng	Root	Ginsenosides Rf, F11, notoginsenoside R1	[51]

Table 10

Other chemical constituents of Panax species using HPLC-MS

Method	Species	Part	Analytes	Reference
HPLC-MS	Panax	Root	Dencichine	[33]
HPLC-ESI-MS	P. notoginseng	Root	Triterpenoid saponins	[184]
HPLC-MS	P. notoginseng	Root	Nucleobases, nucleosides, and saponins	[46]
HPLC-APCI-MS	P. ginseng	Root	Polyacetylenes	[16]
NanoESI-MS	P. ginseng	Different parts	Lipidomics	[185]
UPLC-MS/MS	P. quinquefolius	Root	Zoxamide	[186]
LC-Q-TOF-MS	P. ginseng	Root	Malonyl ginsenoside, amino acids, polysaccharides	[187]

Table 11

Qualitative analysis of Panax species by HPLC-MS, HPLC-QTOF-MS, LC-IT-TOFMS

Method	Species	Part	Analytes	Reference
HPLC-ESI-MS/MSn	P. ginseng	Root	Multicomponent quantification fingerprint	[188]
UHPLC-QTOF-MS	P. ginseng	Different parts	Qualitative analysis	[189]
LC-QTOF/MS	P. ginseng	Root	Fingerprint analysis	[190]
LC-QTOF-MS/MS	P. ginseng	Root	Ginsenosides Rc, Rb2, Rb3, malonyl-ginsenosides	[191]
UHPLC-QTOF MS	P. ginseng	Root	Metabolomics analysis	[117]
UHPLC-QTOF-MS	P. ginseng	Hairy root	Metabolomics analysis	[116]
LC-QTOF/MS	P. ginseng	Root	Metabolite profiling	[35]
UPLC-QTOF-MS	P. ginseng	Ginseng extract	22 ginsenosides	[6]
UPLC-QTOF-MS	P. ginseng	Rhizome and root	59 ginsenosides	[103]
UHPLC-QTOF-MS	P. ginseng	Root	Original neutral, malonyl, and chemically transformed ginsenosides	[192]
UPLC-DAD-QTOF-MS/MS	P. ginseng	Root	Qualitative and quantitative analysis	[193]
UPLC-QTOF-MS	P. ginseng	Root	Metabolomics analysis	[121]
UHPLC-Q-TOF MS	P. ginseng	Root	Metabolite profiling	[194]
UPLC-QTOF-MS	P. ginseng	Root	Metabolite profiling	[195]
UHPLC/QTOF-MS	P. ginseng	Leaf	Metabolite profiling	[196]
UPLC-QTOF-MS	P. ginseng	Root	Ginsenosides	[197]
UPLC-QTOF-MS	P. ginseng	Root	Metabolite profiling	[198]
UPLC-QTOF-MS	Red ginseng	Root	Metabolite profiling	[199]
UPLC-QTOF-MS	P. ginseng	Root	44 ginsenosides	[200]
UPLC-QTOF-MS	P. ginseng	Different parts	58 ginsenosides	[201]
UPLC-QTOF-MS	P. ginseng	Root	Cell-based neuroactivity screening	[202]
UPLC-QTOF-MS	P. ginseng	Flower	Transformation of ginsenosides	[203]
UPLC-QTOF-MS	P. ginseng (different processed)	Root	Metabolite profiling	[204]
UHPLC QTOF-MS	P. ginseng (different age)	Root	Metabolite profiling	[205]
UHPLC-QTOF-MS	White and red ginseng	Root	Fingerprint analysis	[206]
UPLC-QTOF-MS	P. ginseng (different age)	Root	Metabolomics analysis	[207]
LC-TOF-MS	P. quinquefolius	Root	Ginsenosides	[208]
UPLC-QTOF-MS	P. quinquefolius	Root	Metabolomics analysis	[209]
LC–MS	P. quinquefolius	Root	Fingerprint analysis	[210]
HPLC-ESI-MS	P. quinquefolius	Root	Metabolomics analysis	[211]
HPLC-MSn	P. quinquefolius	Root	59 ginsenosides of protopanaxadiol, protopanaxatriol, oleanane and ocotillol types	[81]
UHPLC-QTOF-MS/MS	P. quinquefolius	Root	Metabolite profiling	[74]
UHPLC-QTOF-MS	P. ginseng, P. quinquefolius	Leaf	Metabolomics analysis	[36]
UHPLC-QTOF MS	P. ginseng, P. quinquefolius	Root	Metabolite profiling	[99]
HPLC-ESI-MS	P. notoginseng	Different parts	Metabolomics analysis	[111]
LC-MS	P. notoginseng	Root	Metabolite profiling	[15]
UHPLC-QTOF-MS	P. notoginseng	Root	Metabolite profiling	[53]
UHPLC-QTOF-MS	P. notoginseng	Root	Metabolite profiling	[72]
LC-QTOF-MS	P. notoginseng	Extract	Metabolomics analysis	[212]
LC-QTOF-MS	P. notoginseng	Leaf	Metabolite profiling	[34]
LC-IT-MS and UHPLC-QTOF-MS	P. notoginseng	Flower bud	Metabolite profiling	[70]
UPLC-ESI-QTOF-MS	P. notoginseng	Root	Fingerprint analysis	[213]
HPLC-QTOF-MS	P. notoginseng	Root	Metabolite profiling	[54]
LC-triple-TOF/MS	P. notoginseng	Extraction	Ginsenosides Rb1, Rb2, Rd, Re, Rf, Rg1 and notoginsenoside R1	[214]
UPLC/Q-TOF MS	P. notoginseng	Leaf	Ginsenosides Rb1, Rc, Rb2, Rb3, notoginsenosides Fc, Fe, Fd	[215]
HPLC-QTOF-MS	P. ginseng, P. notoginseng, P. japonicus, P. quinquefolius	Root	Metabolite profiling	[98]
LC-MS-IT-QTOF	P. ginseng, P. quinquefolius, P. notoginseng	Root	Qualitative analysis	[87]
UHPLC-IMC-NLF	P. ginseng, P. quinquefolius, P. notoginseng	Root	Malonyl-ginsenosides	[216]
UPLC-LTQ-Qrbitrap-MS	P. ginseng, P. quinquefolius, P. notoginseng	Different parts	Malonyl-ginsenosides	[217]
UHPLC-QE-HRMS	P. ginseng, P. quinquefolius, P. notoginseng	Root	101 compounds	[135]

2D-HPLC

The traditional methods for comprehensive chemical analysis of Panax species are of low-efficiency and incomplete. Recently, two-dimensional liquid chromatography has been used to analyze the complicated bioactive constituents (Table 12). Online and offline systems are constructed to obtain a high orthogonality and peak capacity. On offline 2D LC system, the first dimensional HILIC analysis for separation of polar compounds and the second dimensional ACQUITY UPLC BEH C18 are used to determine ginsenosides in P. notoginseng; the results indicated that orthogonality could be up to 81%, and the peak capacity is found to be 10200 [56]. It is similar that two-dimensional liquid chromatography, hybrid linear ion-trap/Orbitrap mass spectrometry could discover the new natural molecules, and some even trace amount in P. ginseng [55]. Online 2D LC systems have a simpler operation than offline ones. For instance, a quick, reproducible, and fast method for separation of saponins from P. notoginseng is established by using an online two-dimensional chromatography [57].

Table 12

2D-LC applied to Panax species

Method	Species	Part	Analytes	Reference
2D LC/LTQ-Orbitrap-MS/NMR	P. ginseng	Stems and leaves	A total of 646 ginsenosides were characterized, and 427 have not been isolated from the genus of Panax L.	[55]
2D LC-ESI	P. ginseng	Extraction	Triterpenoid saponins	[218]
2DLC-MS	P. ginseng	Extraction	Ginsenosides Rd, Rc, Rb2, Rb1, Re	[219]
2D chromatographic method	P. notoginseng	Root	Ginsenosides Rb1, Rg1, Rg2, Rh1, Rh4, Rd, 20(S)-Rg3, notoginsenosides R1, T5	[57]
HILIC × RPLC	P. notoginseng	Root	Metabolomics analysis	[56]
2D LC-QTOF-MS	P. notoginseng	Extraction	Total saponins	[220]

Ambient ionization mass spectrometry

Recently, the developed ambient ionization mass spectrometry such as DART-MS and MALDI TOF-MSI are used to evaluate Panax (Table 13) [40,58]. For these methods, direct sampling and ionization are conducted in the open air with no or minimal sample preparation [59]. The most of ginsenosides need derivatization, whereas pseudoginsenoside F11, compound K, protopanaxatriol, and protopanaxadiol are detected without derivatization [59]. In addition, notoginsenoside R1, ginsenosides Rb1, Rg1, and Re from P. ginseng, and P. notoginseng are simultaneously determined by DART-MS [58,60].

Table 13

Ambient ionization mass spectrometry applied to Panax species

Method	Species	Part	Analytes	Reference
DART-MS	P. ginseng	Root	Ginsenosides	[59]
DART-MS	P. ginseng	Root	Ginsenosides Rb1, Re, Rg1	[58]
DART-MS	P. notoginseng	Root	Notoginsenoside R1, ginsenoside Rb1, Rg1	[60]
MALDI-TOF-MSI	P. ginseng	Root	Ginsenosides	[40]

HSCCC/HPCPC

As shown in Table 14, the similar techniques including HSCCC and HPCPC are liquid–liquid partition chromatography. The appropriate solvent systems composed of n-hexane, n-butanol, methylene chloride, methanol, isopropanol, ethyl acetate, and water are employed to isolate the bioactive compounds. In addition, ammonium acetate could reduce the separation time and eliminate emulsification [61]. Ginsenosides Rb1, Re, Rg1, Rb2, Rd, Rf, Rh1, and notoginsenoside R1 could be isolated by HSCCC, and the purity of ginsenosides are more than 95% [62].

Table 14

HPCCC and HSCCC applied to Panax species

Method	Species	Part	Analytes	Reference
HSCCC-ELSD	P. ginseng	Root	Ginsenosides Rg3, Rk1, Rg5, F4	[62]
HSCCC-DAD	P. ginseng	Leaf	Ginsenosides Rk1, Rg5, Rs5, 20(R)-Rg3, Rs4	[129]
HPCCC-ESLD	P. ginseng	Root	Ginsenosides Rf, Rd, Re, Rb1	[61]
HSCCC-ELSD	P. ginseng	Root	Ginsenosides Rg1, Re, Rf, Rh1, Rb1, Rc, Rb2, Rd	[221]
HPCCC	P. ginseng	Root	Ginsenosides Re, Rb1, Rc, Rb2	[222]
HSCCC-MCI gel column	P. ginseng	Root	Ginsenosides Re, Rg1	[223]
CPC-ELSD	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Re, Rb1	[224]
HSCCC	P. ginseng	Root	Ginsenosides Rg1, Re, Rf, Rg2, Rb1, Rb2, Rd, Rg3, Rk1, Rg5, Rg6, and F4	[225]
HPCCC	P. notoginseng	Root	Notoginsenosides R6, R1, Spt1, ginsenosides Rb1, F4, Rh3, Rg3, Rs3, Rk1	[136]
HSCCC	P. notoginseng	Root	Ginsenosides Rg1, Rd, R1, Re, Rb1	[68]

Others

Micellar electrokinetic chromatography could measure the ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3, Rd, Rf, Rh1, Rg1, and notoginsenoside R1 in high separation efficiency without any organic solvent and with shorter run time when compared to chromatographic analysis (Table 15) [63]. It can extract dencichine from P. notoginseng with a purity of 98.5% [64]. Moreover, NMR technique in the qualitative analysis is used to discriminate geographical origins of P. ginseng and to obtain the potential markers [65]. It also quantifies malonyl-ginsenosides Re, Rb1, Rb2, Rc, and Rd [66]. HPAEC-PAD could analyze amadori compounds in processed ginseng within 15 min of single chromatographic run and eliminate the complex derivatization [67]. Enzyme immunoassay by anti-Rf antiserum quantifies ginsenosides Rg2 and Rf in P. ginseng [68]. Dencichine is measured by HPAEC for discrimination of P. notoginseng, P. ginseng, and P. quinquefolius [69]. In addition, MCI gel column chromatography combining with LC-MS could analyze metabolic profiling qualitatively [70]. To determine the various constituents of Panax species, multiple techniques have been used (Table 16). HPLC-UV coupled with GC-MS has been used to evaluate ginsenosides and volatile compounds [20]. Zhu et al using HPLC, CE, and NIR discriminated different parts of P. notoginseng [71].

Table 15

Other analysis of Panax species

Method	Species	Part	Analytes	Reference
Micellar electrokinetic chromatography	P. ginseng	Root	Ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rd	[226]
Micellar electrokinetic chromatography	P. notoginseng	Root	Ginsenosides Rd, Rc, Rb3, Rb1, Rh1, Rg2, Rf, Rg1, Re, notoginosides R1	[227]
High-performance anion-exchange chromatography	P. ginseng	Extraction and rat plasma	Arginyl-fructose, arginyl-fructosyl-glucose	[67]
High-performance anion-exchange chromatography	P. notoginseng, P. ginseng, P. quinquefolius	Root	Dencichine	[69]
Molecularly imprinted polymer	P. notoginseng	Root	Dencichine	[64]
Enzyme immunoassay	P. ginseng	Root	Ginsenosides Rf and Rg2	[63]
1H NMR	P. ginseng	Root	Qualitative analysis	[65]
1H NMR	P. ginseng	Flower bud	Malonyl-ginsenosides Re, Rb1, Rb2, Rc, Rd	[66]
1H NMR	P. quinquefolius	Root	Qualitative analysis	[228]
SFC-MS	P. ginseng, P. quinquefolius	Root	Nucleobases, nucleosides, ginsenosides	[229]
UHPSFC-QTOF-MS	P. ginseng, P. quinquefolius, P. notoginseng		Lipids	[230]
FT-IR spectroscopy	P. notoginseng	Root	Protein	[231]
Near-infrared spectroscopy	P. ginseng	Root	Ginsenosides Rg1, Rb1, Re, Rf, Rc, Rb2, Rg2, Rb3, Rd	[137]
Near-infrared spectroscopy	P. notoginseng	Root	Fingerprint analysis	[232]
Infrared and ultraviolet spectroscopy	P. notoginseng	Root	Notoginsenoside R1, ginsenosides Rg1, Re, Rb1, Rd	[138]
FT-IR spectroscopy	P. ginseng	Different parts	Fingerprint analysis	[233]

Table 16

Multiple techniques applied to Panax species

Method	Species	Part	Analytes	Reference
HPLC-UV, UHPLC-PDA, CE-UV, IR	P. notoginseng	Main root, rhizome	Fingerprint analysis	[71]
HPLC-UV, GC-MS	P. ginseng	Root	Ginsenosides Rg1, Re, Rf, Rh1, Rg2, Rb1, Rc, Rb2, Rg3, F2, compound K, Rk1, Rg5, Rh2	[20]
HPLC-UV, HPLC-MS	P. notoginseng	Extract	Fingerprinting and quantitative analysis	[234]
HPLC-UV, HPLC-MS	P. ginseng	Rhizome	Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2	[235]
HPLC-DAD, LC-ESI-MSn	P. notoginseng	Leaf	Chemical profiles and anticancer	[236]
GC–MS, LC–MS	P. ginseng, P. quinquefolius	Different parts	Primary and second metabolites	[106]
LC-ELSD, LC-Q-TOF-MS	P. vietnamensis	Radix and rhizome	Ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rd, majonoside R1, R2 and vina-ginsenoside R2	[96]

Analytical methods applied to Panax species

As we all know, the different processing methods, species, parts, regions, and ages have different chemical information. To display the chemical markers of different conditions, we have reviewed the advanced techniques evaluating samples of Panax. In addition, the mechanisms of chemical compounds changing for Panax are illustrated.

Raw and processed ginseng

Processing Panax species leads to various bioactive characteristics, which have been used in the treatment of different diseases when compared to raw ginseng. In the Chinese medicine, “Sheng Da Shu Bu” and “Sheng Leng Shu Wen” with regard to raw P. notoginseng are used for hemostasis and cardiovascular diseases, whereas the steamed form is used to “nourish” blood [10]. Those theories suggested that raw and processing have the opposite effect on some illness. Different chemical profiles in the processing have been investigated in the previous study. As a formal method, from raw to processed material steaming with different temperatures and times has been used. P. ginseng is steamed at 98°C and 120°C at 2 h, 6 h, and 9 h, which shows the various bioactive constituents. Time-dependent profiling of raw and steamed Panax species is studied [[72], [73], [74]]. “Red ginseng” is formed at two- or three-time steaming and “black ginseng” is formed with cyclic nine-time steaming at 98°C for 3 h. Therefore, phytochemical components including saponins and volatile oils are reviewed in this investigation. It is found that chemical constituents with polar ginsenosides can be transformed to low polar ginsenosides by hydrolysis, isomerization, and dehydration [75]. The concentration of polar ginsenosides, notoginsenoside R1, ginsenosides Rg1, Re, Rb1, Rc, Rb1, Rc, Rb2, Rb3, and Rd, decreased by steaming, wheras that of low polar ginsenosides, Rh1, Rg2, Rg3, Rh2, Rs3, Rk1, Rs5, and Rs4, increased, and ginsenosides Rg3, Rg5, and Rk1 are the unique compounds from steamed ginseng [44,[76], [77], [78], [79], [80]].

Usually, the types of saponins in the Panax species are mainly protopanaxadiol, protopanaxatriol, ocotillol, and oleanane. As shown in Scheme 1, protopanaxadiol including ginsenosides Rb1, Rb2, Rb3, and Rc converted to Rd by hydrolysis of a glycosylation moiety at C-20. Then, the loss of glycosylation moiety at C-20 and C-3 of Rd through hydrolysis generated ginsenosides 20(R/S)-Rg3 and F2. 20(R/S)-Rh2, Rk1, and Rg5 under the reaction conditions gradually increased [44,74,75,[77], [78], [79], [80], [81]]. Interestingly, ginsenosides Rk1 and Rg5 were deduced to 20(R/S)-Rg3 by Δ20(21) and Δ20(22) dehydration at C-20. Ginsenosides Rk1 and Rg5 are hydrolyzed to generate Rk2 and Rh3 by loss of a glycosylation moiety at C-20 [74,75,80,81]. Protopanaxatriol including ginsenosides Re and Rg1 produced 20(R/S)-Rg2, F1, Rg6, 20(R/S)-Rh1, and Rg4 through hydrolysis of a glycosylation moiety at C-20 and C-6 when the creaming with high-temperature and long-time shown in Scheme 2 [74,75,77,80,81]. Specifically, ginsenosides 20(R/S)-Rf2 was deduced by C-24 and C-25 hydration of Rg2 [81]. In addition, malonyl and acetyl ginsenosides could convert to the corresponding neutral ginsenosides through demalonylation and deacetylation reaction shown in Scheme 3 [74,75]. Such as acetyl-ginsenosides 20(R/S)-Rs3, Rs4, and Rs5 were deduced to be generated from malonyl-ginsenosides Rb1, Rb2, and Rc through hydrolysis, decarboxylation, and dehydration [74,75]. For oleanane type, the chemical transformations have not been studied up to now. The possible transformation pathways deduced are shown in Scheme 4 [81].

Scheme 1

The potential transformation pathway of protopanaxadiol ginsenosides after processing.

Scheme 2

The potential transformation pathway of protopanaxatriol ginsenosides after processing.

Scheme 3

The potential transformation pathway of malonyl and acetyl ginsenosides after processing.

Scheme 4

The potential transformation pathway of oleanane ginsenosides after processing.

Different species

Different species of Panax have different effects on diseases. P. ginseng is used for its anticancer effect [82]. While P. quinquefolius has a good performance on antidiabetic, anti-inflammatory, and neuroprotective effects [[83], [84], [85]], P. notoginseng always have effects on the cardiovascular system, hemostatic, and antioxidant activities [86]. P. japonicus, P. vietnamensis, P. stipuleanatus, P. sokpayensis, and P. bipinnatifidus are also used to protect and treat diseases all over the world. Usually, ginsenosides are the main bioactive components for the Panax species. Yao et al have reported that 623 ginsenosides in the ethanol extract of P. ginseng, P. quinquefolius, and P. notoginseng are discovered, and among those, 437 are potentially novel ginsenosides [87]. Polysaccharides, essential oils, phenolic acids, alkaloids, and others were also investigated in a previous study [3]. The similar morphological characteristics especially medicinal power and its extraction are hard to evaluate them in the markets. The fake and inferior goods may arise owing to price difference for Panax species largely. It is therefore necessary to select some quality markers for distinguishing Panax.

For saponins, ginsenoside Rf is only detected in P. ginseng, whereas 24(R)-pseudoginsenoside F11 is mainly detected in P. quinquefolius [[88], [89], [90]]. Ginsenoside Rs1 is used to differentiate P. ginseng and P. quinquefolius also [91]. Furthermore, the higher amount of Rg1 group (Rf, Rg1) is in P. ginseng and that of the Rb1 group is in the P. quinquefolius [92]. A higher protopanaxadiol/protopanaxatriol ratio for P. quinquefolius is about 3, while the value is between 1 and 3 for P. ginseng [93]. When P. notoginseng and P. quinquefolius are compared, the former has the highest ginsenoside content (9.176%), and the latter has the highest polyacetylene content (0.08%) [37]. Notoginsenoside R1 is detected in both P. notoginseng and P. ginseng [51], whereas ginsenoside Rg3 is observed in the red ginseng [94]. Ginsenoside Rc was not detected in P. sokpayensis, and ginsenosides Rf, Rc, and Rb2 are not detected in P. bipinnatifidus [95]. P. vietnamensis mainly has ocotillol type of ginsenosides [96]. To describe the more chemical information, metabolic components combined with multivariate statistical analysis, hierarchical clustering analysis, principal component analysis, and partial least squares discriminant analysis have been applied to evaluate different species and to select the appropriate chemomarkers [97]. The results indicated that ginsenoside Rf, 20(S)-pseudoginsenoside F11, malonyl-ginsenoside Rb1, and ginsenoside Rb2 could be used to differentiate P. ginseng, P. notoginseng, P. japonicus, and P. quinquefolius [98]. 24(R)-Pseudoginsenoside F11, ginsenoside Rf, Ra1, F2, and 20-glucoginsenoside Rf can differentiate processed P. ginseng and P. quinquefolius [99]. The metabolic constituents of leaves to avoid damaging the roots can separate Panax species [100]. Pseudoginsenoside F11, Rb3, malonyl-notoginsenoside Fd, malonyl-ginsenosides F2, Rb3, Re, F3, R2, and F1 are selected as the chemical markers for leaves of P. ginseng and P. quinquefolius [36]. For essential oil, hexanal, 2-pyrrolidnone, (E)-2-heptenal, (E)-2-octenal, heptanal, isospathulenol, (E, E)-2,4-decadienal, 3-octen-2-one, benzaldehyde, 2-pentylfuran, and (E)-2-nonenal can discriminate P. ginseng and P. notoginseng [29]. Mono- and oligo-saccharide are similar in the different regions and Panax species [25]. However, dencichine varied in Panax species, the highest (0.36 ± 0.02%) is in P. notoginseng, then P. ginseng (0.31 ± 0.06%) and P. quinquefolium (0.1 ± 0.01%), and the lowest (0.03 ± 0.07%) was in steamed P. ginseng. The contents of panaxfuraynes A and B are less than 3 and 2 ng/g in the roots of P. quinquefolius, P. japonicum, P. notoginseng, and P. ginseng, whereas they were not found in P. japonicum [101].

Different parts

Different parts include aerial parts (flower, leaf, and stem) and underground parts (rhizome, main root, lateral root, and root hair) in Panax species, which have been used for medicinal purposes. As a medicinal tea, flower and leaf are used to prevent disease for the human in the eastern world, especially in China. An official herbal medicine, leaf of P. ginseng is recorded in Chinese Pharmacopoeia. Different parts of Panax species have long been used. For instance, rhizomes of P. notoginseng and P. ginseng are called as “Jinkou” and “Lutou” in the traditional medicine, respectively. Different parts have various pharmacological activities [86]. The chemical profile for different parts of Panax species is significant.

In P. ginseng, the content of ginsenosides is higher in the leaf and root hair and lower in stem and other parts. The content of ginsenosides in the root and root hair increases with age from one to five years [102]. More kinds of ginsenosides are found in cork than those in cortex, phloem, xylem, and resin canals; the content of ginsenosides of phloem, xylem, and resin canals from branch root is high than that from main root [103]. The content of total phenols in fruit and leaf is higher than in roots, including major phenolic compounds chlorogenic acid, gentisic acid, p- and m-coumaric acid, and rutin [38]. Moreover, the order for triacylglycerol content is rhizome > main root > root hair. Ginsenosides in P. quinquefolius follow this order leaf > root hair > rhizome > stem [104]. Sapogenins are found more in stem and leaves than other parts of P. quinquefolius [105]. Both P. ginseng and P. quinquefolius mainly have ginsenosides Rg1, Re, and Rd for leaves, and ginsenosides Re, Rb1, and Rc for root hair [41]. The reason for ginsenosides accumulation in P. ginseng main root and P. quinquefolius lateral roots may be high rates of C assimilation to C accumulation [106]. In P. notoginseng, different parts can be identified based on saponin content difference [107]. The type of 20(S)-protopanaxatriol is mainly distributed in the underground parts, whereas 20(S)-protopanaxadiol is mainly distributed in the aerial parts [108,109]. Different parts could be identified by metabolomic combined with principal component analysis [71,110,111]. Notoginsenosides R4, Fa, Q, S, Fc, R1, H, A, B, ginsenosides Rb1, Rb2, Rb3, Rc, Rd, F2, Rh2, Rg1, Re, Rf, Rg2, malonyl-ginsenoside-Rb1, and 20-O-glucoginsenoside-Rf contribute to up- or down-regulation of different parts of P. notoginseng [112]. The main roots have 31% higher ginsenosides content than rhizome [96].

Different region and age

P. ginseng is mainly distributed in Korea, North Korea, and Northeastern China, P. quinquefolius in America and Canada, and P. notoginseng in Southwestern China. Geographical origin is a major influential factor for quality control [35]. Metabolomics combined with OPLS-DA could be used to discriminate P. ginseng of different regions [65]. The contents of 1,2-dilinoleoyl-3-oleoyl-glycerol of P. ginseng from Korea, Japan, and China are 0.41 ± 0.009 mg/g, 0.45 ± 0.01 mg/g, and 0.22 ± 0.008 mg/g, and those of trilinolein are 0.37 ± 0.009 mg/g, 0.39 ± 0.016 mg/g, and 0.27 ± 0.009 mg/g . Furthermore, P. quinquefolius roots cultivated in Jilin Province are similar to those cultivated in Canada in the compositions [113], whereas those grown in China and North America showed no major difference [93]. Ginsenosides Rb1, Rc, Rb2, Rg1, and Rd are influenced by location [114]. The highest polyacetylene content is distributed in Nagano, Japan [37]. Chemical constituents of rhizome and main roots of P. notoginseng from Wenshan, Honghe, and Kunming have no significant difference [115]. Different growing years may lead to different chemical profiles. For P. ginseng, seven ginsenosides show age-dependent variations [116]. Metabolites combining with multivariate statistical methods could classify different ages, especially for 4, 5, and 6 years [117]. The total contents of ginsenosides for main root and fibrous root in four years are highest [118]. The highest concentrations of stigmasterol and β-sitosterol are found in 6-year-old P. ginseng cultivated in Jinan, Korea [39]. For notoginseng, different growth years can be identified by the saponin content, the content of most and total saponins in the order is 3 > 2>1-year-old in the main root samples [107]. The best season for harvesting is September to October [13].

Biochemical analysis

Metabolism of Panax species in the different tissues could obtain a better understanding of biological effects. Ginsenosides Rg1, Rb1, and Rd of P. notoginseng in rat tissues (kidney, liver, heart, spleen, and lung) are determined. The highest concentrations of three saponins were at 90 min except for spleen after oral dose, whereas after intravenous administration, they could not detect in all tissues after 8 h [119]. After nasal administration, notoginsenoside R1, ginsenosides Rg1, Rb1, Rd, and Re from P. notoginseng have been determined in brain [120]. The metabolites in the urine after being administered orally ginseng decoction were used to distinguish normal control group, deficiency of vital energy model group, and ginseng treatment group and to find potential biomarkers [121]. Biotransformation of P. ginseng in the rat intestinal microflora indicated that protopanaxadiol-type ginsenosides were more easily metabolized than protopanaxatriol-type ginsenosides [122].

Conclusion

In this review, different sample preparations including Soxhlet extraction, heat reflux extraction, ultrasonic extraction, solid phase extraction, microwave-assisted extraction, pressurized liquid extraction, enzyme-assisted extraction, accelerated solvent extraction, matrix solid phase dispersion extraction, and pulsed electric field were compared. The TLC technique has been used to quantify and identify Panax species quickly, although it always needs standards and lacks uniqueness for bioactive compounds. GC–MS could be used to determine ginsenosides, phenolic acids, dencichine, pesticide residues, and volatile components, although for some non-volatile components complex operation is required. UHPLC with less analytical time has the better performance than HPLC, and DAD has the better recognition than conventional UV detection. HPLC tandem MS has the sensitivity and specificity characteristic when compared with traditional detection. In the liquid–liquid partition chromatography (HSCCC and HPCPC), ammonium acetate could reduce the separation time and eliminate emulsification. After processing ginseng, chemical constituents with polar ginsenosides can be transformed to low polar ginsenosides by hydrolysis, isomerization, and dehydration. Ginsenoside Rf is only detected in P. ginseng, whereas 24(R)-pseudoginsenoside F11 is mainly detected in P. quinquefolius. When P. notoginseng and P. quinquefolius are compared, the former has the highest ginsenoside content (9.176%) and the latter has the highest polyacetylene content (0.08%). The content of ginsenosides in the leaf and root hair is higher, and it is lower in stem and other parts of P. ginseng. In addition, the content of total phenols in fruit and leaf is higher than in roots. For P. notoginseng, the type of 20(S)-protopanaxatriol is mainly distributed in the underground parts, whereas 20(S)-protopanaxadiol is mainly distributed in the aerial parts. P. ginseng is mainly distributed in Korea, North Korea, and Northeastern China, P. quinquefolius in America and Canada, and P. notoginseng in Southwestern China. Protopanaxadiol-type ginsenosides were more easily metabolized than protopanaxatriol-type ginsenosides in the rat intestinal microflora.

From the current review, the present analysis of Panax species is not sufficient. The following aspects need to be investigated.

According to previous studies, the different sample preparations and analytical methods have been used to evaluate ginsenosides of Panax species. It is necessary that the harmonious and practical standard criteria method is established for determining ginsenosides of different species, parts, and ages quickly and accurately.

As we all know, ginseng has been widely used for prevention and treatment of diseases all over the world. Meanwhile, the criteria of Chinese Pharmacopoeia, United States Pharmacopeia, Japanese Pharmacopoeia, and South Korean Pharmacopoeia for P. ginseng have been developed. Different countries have different criteria. It is expected that the uniform criteria for ginseng should be established for development of the ginseng industry.

As an oleanane type, ginsenoside Ro was only detected in the P. ginseng and P. quinquefolius, which could be used to inhibit testosterone 5α-reductase and for testosterone-treated disease [123]. Both Ro and its transformation products in red ginseng are the bioactive constituents [124]. The chemical transformation pathway and the metabolism in vitro and in vivo are the key research in the further investigation. Furthermore, in Chinese Pharmacopoeia, quality markers for P. ginseng and red ginseng are ginsenosides Rg1, Re, and Rb1, although they have the various pharmacological effects. It is reported that red ginseng has the better performance biological activity than fresh ginseng [92]. What has not been investigated until now is the different bioactive components. The condition of ginseng from raw to processed, temperature, time, and pressure are necessary to be optimized for future studies.

Notoginsenoside R1 and ginsenoside Rg3 are discovered in P. notoginseng and red ginseng, although they are not unique. Several biomarkers have been selected to discriminate Panax species by metabolite coupled to chemometrics. The possible biomarkers need to be verified through large number of samples. In Chinese Pharmacopoeia, Rg1+Re + Rb1≥2% for P. quinquefolius, Rg1+Re ≥ 0.3% and Rb1 ≥ 0.2% for P. ginseng, Rg1+Re ≥ 2.25% for leaves of P. ginseng, and Rg1+Rb1+R1 ≥ 5% for P. notoginseng are quality control. Obviously, the biomarkers are unique for each one. The comprehensive evaluation of quality control for Panax species needs further investigation.

Rhizome and main root of Panax species with different chemical profiles are recorded in official documents. Most of the time, main root is used and rhizome is not, such as “Qulu” (cutting out rhizome) in traditional medicine. Up to now, the differences between rhizome and main root have not been investigated. A comprehensive, accurate, and convenient method is necessary to establish in the further study.

Conflicts of interest

All authors declare that they have no conflict of interest.