AU2014100715A4 - Method for authenticating panax notoginseng plant material - Google Patents

Abstract

The present invention provides metabolite profile of various parts of the root of P. notoginseng and method of identifying and determining specific part(s) to which a plant material of notoginseng belongs using the metabolite profile. The present invention is useful for screening different plant materials susceptible to be one part of the root of notoginseng or for quantitatively identifying a specific part of notoginseng root. A,20 b1~ oe C OLIP * Iv oxa NO - N 0in (5/ftn) uoiIwJ.IuQOuoD OATILwp)

Description

METHOD FOR AUTHENTICATING PANAX NOTOGINSENG PLANT MATERIAL CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application 61/979,529 filed on April 15, 2014, the disclosures of which are incorporated by reference herein. FIELD OF INVENTION [0001] The present invention relates to methods of identifying and determining different parts of plant material belonging to Panax notoginseng. In particular, the present invention relates to identification and determination of different parts of the root of P. notoginseng. BACKGROUND OF INVENTION [0002] The root of Panax notoginseng (Burk) F. H. Chen, commonly referred to notoginseng and "Sanqi ", is a well-known medicinal herb in traditional Chinese medicine. It has been historically used as a tonic and haemostatic agent for the treatment of cardiovascular diseases, inflammation, different body pains, trauma, and internal or external bleeding caused by injury. Substantial studies have demonstrated haemeostatic, antioxidant, hypolipidaemic, hepatoprotective, renoprotective and estrogen-like activities of notoginseng. Moreover, chemopreventive and anti-tumor activities of this herb and its major constituents have been increasingly reported. [0003] Owing to the time-honored application in traditional Chinese medicine, notoginseng is quite frequently used as a major ingredient in numerous health products, herbal preparations and traditional Chinese proprietary medicines, including a large number of well-known preparations such as "Xueshuantong Injection", "Yunnan Baiyao", "Pien Tze Huang", etc. Beyond the medicinal application, notoginseng is also known as a distinguished functional food owing to its health benefits. In China and other Asian countries, notoginseng is widely consumed in the form of tea, powder, capsule, and functional foods. In U. S. A., a variety of notoginseng products are available as Over the Counter (OTC) dietary supplements in the health food market. All these applications made notoginseng as the most largely consumed medicinal herb in the world. The estimated demand of notoginseng was about 7000 tons every year, much higher than that for Asian ginseng (Radix Ginseng). 1 [0004] Although both rhizome and root (Notoginseng Radix et Rhizoma) are officially described as notoginseng in 'Chinese Pharmacopoeia' since 1985, individual parts of the root, i.e., rhizome, main root, branch root and fibrous root, are separately used in the manufacturing of products and their market values are largely different. Rhizome, commonly known as "Jiankou", is usually used for the manufacturing of ginsenosides and related preparations. The main root is generally utilized in Chinese medicine clinics and traditional Chinese proprietary medicines, while branch root (known as "Jintiao") and fibrous root are often grounded to yield notoginseng powder as health food. The market price of different parts ranked in the order of main root, followed by rhizome and branch root, and then fibrous root. [0005] Due to the differential utilization of individual parts of notoginseng, chemical differences among them are of big significance for both authentication and medicinal usage of different parts of notoginseng. Although numerous studies have been carried out to assess the chemical differences among notogingseng, e.g. American ginseng (the root of Panax quinquefolium L.) and Asian ginseng (the root of Panax ginseng C. A. Mey.). Only several studies have been carried out to assess the chemical characteristics of individual parts of notoginseng. However, limitations still exist. (1) More than 80 dammarane-type saponins with various bioactivities have been isolated from notoginseng and steamed notoginseng, but only major ginsenosides, i.e., R 1 , Rgi, Re, Rbi and Rd, have been examined. Several recent studies quantitatively determined both high and low-abundance ginsenosides, but the total number of quantified saponins is still limited. (2) Metabolite profiling with UPLC-ESI-MS has been applied for evaluating chemical characteristics of different parts of P. notoginseng, i.e., flower, leaf, seed, rhizome and main root, or notoginseng and steamed notoginseng. However, the examination of different underground parts of P. notoginseng has not been done. Especially, comparison among the four parts of notoginseng (rhizome, main root, branch root and fibrous root) using metabolomics approach is absent. (3) Comparison is often made among the different parts sampled from the root of diverse origin. However, the different origin itself might introduce significant variation in the content of chemical constituents, leading to a mask of the variation resulting from the different portions of the root. [0006] Therefore, a quantitative analysis and metabolite profiling on the different parts of the whole root of P. notoginseng collected from the major origin of notoginseng is required. Such combinative quantitative analysis and profiling of different parts of root of P. notoginseng will 2 provide a comprehensive understanding of chemical composition of notoginseng and developing a reliable means to identify and distinguish different specific parts of plant material from notoginseng or a plant material. SUMMARY OF INVENTION [0007] The present invention relates to metabolite profile of root of P. notoginseng. Root of P. notoginseng refers herein to notoginseng. In particular, the present invention provides metabolite profile of individual parts of notoginseng, namely, rhizome, main root, branch root and fibrous root of notoginseng. The present invention also provides qualitative and quantitative profiles of saponins of the four aforesaid parts of notoginseng. [0008] The present invention also relates to a method of identifying and determining a specific part of notoginseng for a plant material of notoginseng. Said method comprises obtaining a metabolite profile of the plant material of notoginseng to be identified and comparing the metabolite profile of the plant material to be identified with predetermined metabolite profile of each of the four different parts of notoginseng. The plant material of notoginseng to be identified and determined may comprise more than one specific parts of notoginseng if the metabolite profile of the plant material of notoginseng matches more than one predetermined metabolite profiles. BRIEF DESCRIPTION OF DRAWINGS [0009] FIG. 1 shows total ion chromatogram (ITC) of rhizome (A), main root (B), branch root (C) and fibrous root (D) of P. notoginseng. [0010] FIG. 2 shows concentration of notoginsenoside (R 1 ), four ginsenosides (Rgi, Re, Rbi and Rd) (A), three ginsenosides (Rc, Rb 2 and Rb 3 ) (B), concentration of total 20(S)-protopanaxatriol type saponins (PPT-type), total 20(S)-protopanaxadiol type saponins (PPD-type) and total saponins (C) in four known parts including rhizome, main root, branch root and fibrous root of P. notoginseng. Each bar represents mean :SD (n=12) *p<0.05, one way ANOVA (compared with main root). [0011] FIG. 3 shows concentration of notoginsenoside (R 1 ), four ginsenosides (Rgi, Re, Rbi and Rd) (A), three ginsenosides (Re, Rb 2 and Rb 3 ) in commercial samples of rhizome (n=4), main root 3 (n=2), branch root (n=4), fibrous root (n=6) of P. notoginseng. *p,0.05, **p <0.01, ***p<0.001, one way ANOVA (compared with main root) [0012] FIG. 4 shows PLS-DA score plot (FIG. 4A), OPLS-DA score plot (FIG. 4B) and loading plot (FIG. 4C) of ions in rhizome (1), main root (2), branch root (3) and fibrous root (4) analysed by UPLC-Q-TOF MS. [0013] FIG. 5 shows targeted MS/MS data for malonyl ginsenoside Rbi (FIG. 5A) and notoginsenoside Rwi (FIG. 5B). Nomenclature of the fragments is assigned according to a systematic nomenclature for carbohydrate fragmentations. [0014] FIG. 6 shows the relative concentration of twenty marker saponins in the total amount saponins in notoginseng. [0015] FIG. 7 shows picture of notoginseng and individual parts DETAILED DESCRIPTION OF INVENTION [0016] The present invention is not to be limited in scope by any of the specific embodiments described herein. The following embodiments are presented for exemplification only. [0017] In one aspect, the present invention provides a method of identifying and determining a specific part of notoginseng to which a plant material of notoginseng belongs. The method comprises obtaining a metabolite profile of the plant material to be identified, comparing the metabolite profile of the plant material to be identified with a predetermined metabolite profile of each of the four different parts of notoginseng and determining the specific part of notoginseng to which the plant material to be identified belongs or one of more specific parts of notoginseng that the plant material to be identified comprises. The specific parts of notoginseng are selected from rhizome, main root, branch root and fibrous root of notoginseng (FIG. 7). [0018] In one embodiment of the present invention, said obtaining a metabolite profile of the plant material to be identified comprises quantifying concentration of at least eight saponins comprising notoginsenoside (R 1 ), ginsenosides (Rgi, Re, Rbi, Rb 2 , Rb 3 , Re and Rd) in the plant 4 material to be identified. In another embodiment, said obtaining a metabolite profile comprises quantifying concentration of saponins selected from (1) Ginsenoside Rbi, (2) Malonyl-ginsenoside Rbi, (3) Ginsenoside Rd, (4) Ginsenoside Rgi, (5) Notoginsenoside K, (6) Notoginsenoside R 1 , (7) Notoginsenoside Rwi, (8) Ginsenoside Rb 2 , (9) Malonyl-ginsenoside Rd, (10) Ginsenoside Re, (11) Notoginsenoside Rg 3 (12) Notoginsenoside Fa, (13) Notoginsenoside I, (14) Notoginsenoside R 4 , (15) Notoginsenoside Fc, (16) Ginsenoside Rf, (17) Notoginsenoside R 2 , (18) Notoginsenoside A, (19) Ginsenoside Rg 2 and (20) Ginsenoside Rhi. In one embodiment, said obtaining the metabolite profile of the plant material to be identified comprises quantifying concentration of notoginsenoside Fc, notoginsenoside Rwi, malonyl-ginsenoside Rd and ginsenoside Rb 2 in the plant material. The concentration may be represented as weight percentage of individual saponins in the plant material of notoginseng. [0019] In another embodiment of the present invention, the predetermined metabolite profile of each of rhizome, main root, branch root and fibrous root of notoginseng comprises concentration and/or weight percentage of the following saponins (1)-(20) in the respective parts of notoginseng: (1) Ginsenoside Rbi, (2) Malonyl-ginsenoside Rbi, (3) Ginsenoside Rd, (4) Ginsenoside Rgi, (5) Notoginsenoside K, (6) Notoginsenoside R 1 , 7) Notoginsenoside Rwi, (8) Ginsenoside Rb 2 , (9) Malonyl-ginsenoside Rd, (10) Ginsenoside Re, (11) Notoginsenoside Rg 3 (12) Notoginsenoside Fa, (13) Notoginsenoside I, (14) Notoginsenoside R 4 , (15) Notoginsenoside Fc, (16) Ginsenoside Rf, (17) Notoginsenoside R 2 , (18) Notoginsenoside A, (19) Ginsenoside Rg 2 and (20) Ginsenoside Rhi. In yet another embodiment, the predetermined metabolite profile of the rhizome, main root, branch root and fibrous root of notoginseng further comprises a predetermined OPLS-DA score plot and loading plot of saponins (l)-(20), as shown in FIG. 4B and 4C. [0020] In one embodiment of the present invention, the plant material is identified as or comprises rhizome part of notoginseng wherein the plant material to be identified comprises at least one of the followings: (i) at least 0.30% or at least 0.5mg/g of Ginsenoside Rb 2 ; (ii) at least 0.15% of notoginsenosides Fc; (iii) at least 0.61% of notoginsenoside Rw 1 ; and (iv) at least 0.40% of malonyl ginsenoside Rd. In another embodiment of the present invention, the plant material is identified as or comprises one or more of main root, branch root and fibrous root of notoginseng wherein the plant material to be identified comprises (i) less than 0.30% or less than 0.5mg/g of Ginsenoside Rb 2 ; (ii) 5 less than 0.15% of notoginsenosides Fe; (iii) less than 0.61% of notoginsenoside Rwi; and (iv) less than 0.40% of malonyl-ginsenoside Rd. [0021] In another embodiment of the present invention, one or more specific parts of notoginseng to which a plant material of notoginseng belongs is determined by comparing the OPLS-DA score plot and loading plot of the plant material to be identified with the predetermined OPLS-DA score plot and loading plot of the specific parts of notoginseng (FIG. 4B and 4C). The one or more specific parts of notoginseng that can be identified by the present method are selected from rhizome, main root, branch root and fibrous root of notoginseng. [0022] In another aspect, the present invention provides a metabolite profile of each of the rhizome, main root, branch root and fibrous root portions of notoginseng. The metabolite profile provides qualitative and quantitative data of the metabolite constituents in notoginseng. [0023] Metabolite profiles of four different parts (rhizome, main root, branch root and fibrous root) of notoginseng have been obtained, as detailed below in the examples of the present application. Mass spectrometry is used to analyse the metabolite constituents of respective parts of notoginseng and the metabolite constituents are then analysed by non-targeted metabolomics approach to obtain a metabolite profile for each of the respective parts of notoginseng. It is shown that different parts of notoginseng vary in their saponin content, leading to an unique saponin profile for each of the parts that is distinct for any one of the specific parts of notoginseng. UPLC-TOF MS is used to quantitatively compare eight dammarane-type saponins in different parts of notoginseng. Commercial un-assorted samples of individual parts of notoginseng are also quantitatively analysed to verify the characteristic chemical composition. [0024] Metabolite profiles of different parts of notoginseng are obtained by the following methods. One skilled in the art would readily appreciate metabolite constituents of plant material may be obtained by other analytical means. [0025] Twelve batches of the whole root of P. notoginseng (3 years) are collected from the wholesale markets of Wenshan, Yunnan Province of China in December, 2008. For each batch of the root, 3-5 whole roots are selected and divided into rhizome, main root, branch root and fibrous 6 root. The individual parts of each batch are then mixed to yield the representative portion of each batch. Commercial un-assorted samples of individual parts of notoginseng are also collected from the markets of Wenshan during 2006 to 2008, including 4 batches of rhizome, main roots with growing year of 3 years (4 batches), 4 years (2 batches), 5 years (2 batches), 4 batches of main root without growing year information, 4 batches of branch root and 6 batches of fibrous root. [0026] Notoginsenoside R 1 and ginsenoside Rgi (Purity > 98%) are purchased from Kunming Zhongzheng Biotech Co., Ltd (Kunming, China), ginsenosides Re, Rbi, Rb 2 , Rb 3 , Re and Rd (each purity > 98%) are purchased from Chengdu Scholar Biotech Co., Ltd (Chengdu, China). HPLC grade methanol and acetonitrile are purchased from Anaqua Chemicals Supply Inc., Ltd (Houston, USA) and distilled water are purified by Milli-Q system (Millipore). [0027] Stock solutions of notoginsenoside R 1 , ginsenosides Rgi, Re, Rbi, Rb 2 and Rd in 70% methanol are diluted to a series of working solutions (0.125, 0.625, 1.25, 2.5, 5 and 10ptg/ml), while that of ginsenosides Rb 3 and Re are diluted to yield working solutions of 0.05, 0.25, 0.5, 1, 2 and 4ptg/ml. [0028] Sample solutions are prepared following the procedure as described in Hu et al. Arch Pharma Res 2009, 32 (5), pp 66 7

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7 6 and Wang et al., Chem Biodivers 2010, 7 (4), pp975-83, disclosures thereof are incorporated herein by reference by their entity. Briefly, 200 mg of powdered samples of each representative part is accurately weighed into a PTFE-stopped tube and sonicated with 10ml of 70% methanol for lhr. The mixture is centrifuged and the supernatant is collected followed by dilution of 200 folds and 4 folds for the quantitation of major ginsenosides (R 1 , Rgi, Re, Rbi, Rd, Rb 2 ) and minor ginsenosides (Rb 3 and Rc) respectively. All samples were filtered through a 0.2pm filter before analysis. [0029] UPLC-Q-TOF MS is performed using a Waters ACQUITY UPLC system (Waters, Milford, MA, USA) which is equipped with a binary solvent delivery system and a sample manager coupled to Bruker MicroTOF mass spectrometer with an ESI source (Bruker, Bremen, Germany). All the operation, acquisition and data analysis are operated by Hystar software (Bruker). 7 [0030] Chromatographic separation is performed on an Acquity UPLC BEH C 18 column (2.1 mm x 100 mm, 1.7 Im). Mobile phase consisted of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B), with a flow rate of 0.35ml/min. The following gradient was used: 0 - 23 min, 10 - 40% B; 23 - 27 min, 40 - 85% B; 27-29 min, 85 - 100% B; 29 - 32 min, 100% B; 32-36 min, 10% B. The column is maintained at 40'C, and the injection volume of each sample is 1 ptl. [0031] The mass spectrometer is operated in the negative mode with a scanning range of m/z 100 3000. The optimized mass conditions are as follows: drying gas (N 2 ) flow-rate, 8 L/min; drying gas temperature, 180 'C; capillary voltage, 4500 V; nebulizing gas (N 2 ) pressure, 2.5 bar; end plate offset, -500 V. The following ions are extracted for the quantitative analysis of different compounds: m/z 977.5 ([M+HCOO]-, notoginsenoside RI), ni/z 845.5 ([M+HCOO]-, ginsenoside Rgi), m/z 991.5 ([M+HCOO]~, ginsenoside Re or Rd), m/z 1153.5 ([M+HCOO]~, ginsenoside Rbl), m/z 1123.5 ([M+HCOO]~, ginsenoside Rb 2 , Rb 3 and Rc). [0032] The calibration curve (y = a + bx) for each ginsenoside is constructed by plotting the peak area against concentration and the linearity was verified by correlation coefficients (r 2 ). The lower limit of detection (LOD) and lower limit of quantification (LOQ) are determined on the basis of response at a signal-to-noise ratio (S/N) of 3 and 10 respectively. The precision is determined by intra- and inter-day variations. The intra-day precision is performed by 6 replicate analyses of a mixed standard solution within a day, and the inter-day precision is carried out by analyzing the same mixed standard solution on 5 consecutive days. The repeatability is carried out by six replicate analyses of the same sample solution. The recovery test is performed by spiking a sample with the mixed standards, and the recovery rate is calculated by the following formula: recovery rate (%) = (observed amount-original amount) / spiked amount x 100%. Variations are expressed as the relative standard deviations (RSD%). [0033] The UPLC-Q-TOF MS data of different parts of notoginseng is first exported as edf format. The peak finding, alignment and filtering of raw data are preprocessed by MassLynx V4.1 (Waters, Milford, MA, USA). The parameters were set as following conditions: retention time of 2 27 min, mass range of 100 to 1,500 Da, mass tolerance of 0.05 Da. The resulting three-dimensional matrix using retention time, mass to charge ratio and intensities of all detected peaks are tabulated and exported for subsequent analysis. Principal Component Analysis (PCA), Partial Least Squares 8 Discriminate Analysis (PLS-DA) and Orthogonal Partial Least Squares Discriminate Analysis (OPLS-DA) are performed by using SIMCA-P 13.0 software (Umetrics, Umea, Sweden). Saponins that are important in differentiating different parts of notoginseng are selected according to the parameter VIP (Variable Importance in the Projection). [0034] Eight dammarane-type saponins have been identified and quantified by UPLC-Q-TOF MS. All calibration curves show good linearity (R2 > 0.999). The LOD ranges from 0.0038-0.0146 pig/ml and the LOQ ranges from 0.0171-0.0541ptg/ml. The calibration curves, correlation coefficients, LOD and LOQ of the eight saponin compounds are given in Table 1. The UPLC-Q-TOF MS method shows good precision for quantifying the eight saponins with intra- and inter-day variations of 1.28-3.00% and 1.22-3.44%, respectively, and repeatability variations of 1.08-3.84%. The overall recoveries range from 98.65-122.19%, with variations of 1.60-3.46%. Table 1. Validation data of eight saponins in notoginseng by UPLC-Q-TOF MS Marker Regression equation Linearity r2 Precision (RSD%) (n = 6) Recovery LOD LOQ (Mg/m) Intra-day Inter-day Repeatability Rate (%) (jIg/ml) (pg/ml)

R

1 y = 132995x + 4969.8 0.125-10 0.9998 1.99 1.25 2.25 98.66 (1.81)a 0.005 0.021 Rgi y = 125416x - 6776.7 0.125-10 0.9998 1.28 1.99 1.08 99.10 (2.90) 0.004 0.024 Re y = 148540x + 15054 0.125-10 0.9995 2.67 2.18 2.11 103.03 (2.22) 0.005 0.017 Rbi y = 64969x -4164.7 0.125-10 0.9997 2.31 2.60 1.72 99.65 (3.29) 0.015 0.054 Rb 2 y = 116356x - 5883 0.125-10 0.9997 2.87 1.68 3.84 99.06 (3.43) 0.011 0.029 Rb 3 y = 134365x - 851 0.05-4 0.9999 3.00 3.44 1.61 122.19 (2.26) 0.008 0.025 Re y = 122449x - 500.33 0.05-4 0.9999 2.18 2.21 2.79 99.62 (2.70) 0.008 0.026 Rd y = 152720x - 6790.6 0.125-10 0.9999 2.41 1.22 2.97 101.65 (1.60) 0.006 0.021 a The values in the brackets are the RSDs of 6 replicated determinations of recovery rate. [0035] These results demonstrate that UPLC-Q-TOF MS method is sensitive, accurate and precise, and thus it is appropriate for quantitative analysis of the notoginsenoside R 1 , ginsenosides Rgi, Re, RbI, Rb 2 , Rb 3 , Rc and Rd in different parts of notoginseng. [0036] In order to evaluate the variation of ginsenosides inherently associated with different parts of the notoginseng root, the total amount of the ginsenosides in each respective parts of notoginseng are analysed. FIG. 1 and Table 2 show saponin compositions of four different notoginseng parts. Ginsenoside Rbi and Rgi are shown to be the most abundant ginsenosides in all parts of notoginseng, both accounting for more than 20% of the total saponins in respective parts. Concentrations of notoginsenoside R 1 , ginsenoside Re and Rd, which are less abundant than Rgi and Rbi, vary greatly 9 among individual samples in respective parts in both absolute content and relative composition of the total amount of saponins. The percentage of ginsenoside Re and Rd in the total amount of saponins in respective parts are independently kept at about 1-10% in all portions of the root. Ginsenosides Rb 2 , Re and Rb 3 are shown to be relatively minor constituents in all parts of the root. Similar results are obtained in commercial samples of individual parts of notoginseng (FIG. 3) 10 ' t - M N N Os0 C .0 I- r 0 Os 00 m 00 W) i N It ol1 O n o) 0 N s tf 0O 0 = N mn 0C 00 0 -) '1 en 0= "': 'I 00 n n *": 'I - C - - N = 00 'r- 00- 0o N W) t- NN C> t ' 00 Cl 0 CNo o O m C w co ~ W o - t-- C o o N - o Co o os ~os t eo O tO ee o 0 .0o0 Co en0 in oo o on m o C tn o o.N - - No Cn - NO It to ENo os o C 'o to C) o in ~ o~~~~~~~~~~ to n e o m e - m oc w e o o o s- 00 -t en , 00 To e 0 oW Mo ~ n - '. "C W o - t o o - o m oo 0 0 o Cl - 4 to ' en et = , en 00 ~tf '.0 _; 0000 C~0 -i -6 H o ~ ~ n o to e O . N C o 00 o a t o -0 e n n. C e o Cl Cl e n m0 o a 0 - - N o ' o 0 .o o o- N o o in o- C - l 0 o. on N o. o~ o. o o o~ o ot o 0 N o Cl o o. to o o- N o. o. o e o o e o o - o o e o o e o- - o 0 0 C ) C) Cl C) C- Cl C) CD C:) C) Cn CD CD ':0 Ct e> en C Cl en - C= en 0 00000 00000 66 0 06 6 6 660 00060 0 O ~ .o to C t - - - -m NO - w 0 ' e - 0 - m Wn m en - Cc C C' C' C' C' C' C' C' 'C C 'C C' C' C C' ' C' C' C' C' C' 'C' C m o N t o N 00 N 0 in 0 m l N e r n - m t N Ce .0000 N -00 N 000 ') C 00 m -00 oC o oo666 66666 -6666o 6o oo 0 o en Cl e 0 C t Cl 00 Cl . o N e '. e Cl '0 e to C' C e Ct o N o 00 o - , 00 ot 0t N V 0 m '0-0t e00 0o d 0000-000 00-666600 66 0006 4C4 C' C' ' C C' ' ' C' C' C' C' C' C' C' C C C) eD Co n C) 00 o1 C e o ) en e C - Co o e-:- Cl Cl CD kf) ~ CD oo o) C o o oo k o N - m m - W) oo~~~~\ tn "Co o o e e o - o 'n o -W )o i 00 o- o ~ o0 0 - - - o o os C, 0 00 0 0 0 0 0 o n m S l o C- Ne ent 00 0 i Cl Cl ,I: . o m C oo H 00 0 0 0 o - m - 6 6 6 66 6 6 6 6 o C' q' "It C C' C' C' C' q' C' C' C' C' In C' C' C' C' C' C' w - WN - o oo o oo - 0 oo 0 C - o m N 00 o C e n N 5 cn M M m mr so zi zi -M c d Cd-'4i4 o r o; E) -- t C m0 m m o) C) 0 C oc N t- o N o m 11 C o 000 C m N to W) t- C: n o 0 o N M - - 00 N '. 0 N o W) A m r Oto m r- o) Ctr om m 0 W)~~~ m w rn m oot -\o m C k -'T t m w w 0 0 Cl Cl r q w n t - n0N 000 qo "~ 0 0 c! 0 Cl - = C - o oon0 oo'00 Cl '.oc0 o~~e ~~' o C r t-- V-) -- o- o 0 C M en m V) 0 N. Nl m.0 '0 Cl w 't to tO N - 00 \ Cl Cl m~Q~'. Cl) C0 0 len 0 Ci C O ,: r' :' eenoen ~toN0 000to0i 0O en Cl N N o o o 0 o o o e N o o 0 e Cl eo o o It 0 o 0 - 0 lq 0 0- -) m Nt t- 0 Cn - eN It \C W).0 Cl N 10 Cl CD m. 0 en NU e =i lt t0 o = O to '.0 en ,I ' t0 't 00 0 l q (= I - r 004 o00 Cl NZ Co r- m NO C t - rn k N ) m~ "t Cl D Mn N. -) Cl 0Cl00 m. mo eN en I. t It m N- It m- mn t-- W) m t r- '.0 c t n C l r 00 r'- C' 0 c ' 00C 400 C') - k00 "C to N .0 rn r.0 to N- \c Wn V000 0 0 _t CN* N - mn It 0n ClNt-000 = = C-4 Cl ,4 0 0 t-- - 0 - Cl 4f 't0 t 00 r- N C l '. . 0 '.0 - r0- ~ 0 c 00 Cl 00 - Cl '.0 00 w4 4 '.c N Cl-C) r Cl 4 NM o O '0 0 0 - -o - 4- C '00 ent O H~ C '000e 0 0~ N '0 e 00 0 N N to 0 n 4 ' 0 0 lt 0 U0 ND 4 0 ~ 0 ' N N '0 N Cl ) en 0 '0 N . 0en e 0 '. to n 00 C '.0 en enC4 4 e 4 CA En N toe -e 4 0 t 4 0 N en ;I - 000 en NN o 0 . - 0 > '0 N N C N '.0 to 40Cl ~ O00~ 4C -- C - q m '.k. - . 0 4 0 0 0N 0 .

t-N C s N - o00 W) m 00 k ) c C) o N ~ O\ ~ N tl tr) C) N C' C) 0,, tN C It t 00 -~- 00 C I C tC W rrt ts ~ cm 00 o f 4 tri C 0o ot k No Co vn C; t-: N o r ' C \~~O 0 4 e so o Q & NO - N t 'O C W m O= O N= o N C N4 N - - ~~ - ~ ~ C 00 N ' N C n t O C - NI C m Co C C0 ' o N ~ ~ ' O O n t 110 m N "T \CCo N NI CD oo Vn x r- N' 0 N I, r, cr; Cl; 00 I C r, 6 Cr, Nr 0 C=; l Cl 0r 0 '-N f C m - - - - l N a os oo tN- 0 m C) m 't o or o- o N Cl N Cl CD N N W) - N C' '.0 tf C oIt N C en io o t MT t If, '.0 '.0 C; kt, - 0 N- Cl Cl 00 If, 00 N- 4t C' '.0 c, O 4 00 t-- N f, mr l 00 - '.0 kr, tf, 00 If, Wf, mr tN I, Wf, m C' 00 N m m N m m m Co on 00 C n n I m C' ' C' 'C' C' C' C'C C' C' C' C' C' C' C' 'C' C' C' C' C' C' C' C' C C)C' C'C C) CD D 'C'C'C) C CCC 6666)() )C)C C' C')C')C: '.0 Vf, V) N f, I, Cr, Nl CD 0 t C') N- '.0 r- N C m. mr C' C C' CC' C CC' C' C' C' C ' C' =: C' C' C' C' C' C' C' C' C'C C' C') C' CD C' CD 6: ~ClC) C' C' C' C' C' C' C' C' C' C' Cr N N C' t - O os ' Cl N O It C' N m m -C' C' N ' C' C' "t - C' V If C' C' ci C' C' Cr, C' C' C' C C' C')C :)'CCD'CD' DC C D C 666 66 ) 6D C) C' C''C) m in ' -k as ' 0 C or N C C, C' 0 o0 m ~ m O o o' CCo O O' o O O' - O O C' O oC'o oo' C' C' C' C C' ' o C oC C- C ~ 'C C 6666 oc C; Z C) o C5 cm C:, N C) CD C C) C Co -- C' C C' C CC' C: C C' C C C' C' C' C C' C' C' C' C' C' C' 6Z C' C' C' C' C' C' C' C' C' C' C' C' C' C' C' C' C' C' C' CrC C Cr, C r Cr, '. Cr, Cl Cl N - Ct -CC Cl C- C= C' C' C'> C C' C' C' C'> C' C' C' C' C' C' C' CD C' C' C' C' mr Cr, f 00 C' C N -Cl C' If, It Cl ') Cl4 'I Cl CD't .0 '.0 r-, ClC NN N I, - C' .)r 00 t- 00 Z if, Mr Mr '.0 C' C' 0 Q Vf, 000w ~ - C 00 00 mr Cr't 00 N - NC O s in 00C )C o ot ot C: 00 Nl '0 ND C:' Cr C' N~C =~~ 0 :) C' C'-4 ~~ '0 Cl C - - - tn m'Cm N If, N ~0 ~C Ifoo N 00 &~ .0 C r cr ClN . r '.0 Cl C' 00 nf If, r, '.1'0 00 C' C' . N- 00 "': N- C' C 0 -- 00 C m C' C, N~ O > Cl C' - C' N Cr C '0 t 0 0 0', %I Z!, ~ ' t t Z! ZZ:,Cl Cl Nl Cl k, Il kf, Z! 'n 'n Vf, N Cl C ClC C' O Cl N r C' Icf, C' Cl C' mr Cl c It co - crl O C' InI If, N N 00 0 Cr, N I 00 I: Cl 0) '.0 ,- .0 Cl -! ,~,0 Wt"'t cq It o - C Cr Cr 0 O m 0 C~ o N o' NC C' o o 00 l 00 oo Cl Co4 ID t o tC m t o o) tn N ' o 00- C) Cl C o t m Cn m It0 C'4 00 CN M ' . . C ' Cl l r C ) O- () " . t-! r I-! C S e t00 ' N 0 C) N N 0 Cl C 0 00 ) e t Cl 00 0 o o - o C-) Cl 00 '.0 If, V1> w. m. t 0 C C' mr mtCr 00 C':) C' N '.0 C' 00 '.0 00 llt '-0N- C> '.0 0 C' C> C' C' C> I- C> If, N- 00 N- 0 00 C> r N mCr 00 Cl I. t It Cr, It C' If, kf, tf, \ i m m c s N NO C 4 - ~ Cl Cr, N m o l Cl o m - m N C, 0 C/ Ln t m 0 00 C l /-N /l N /- C,0 W 0 Cl 00 Cl'0 N D tN t-- C> C'C> C' N w 00 r C> Cr, N Cr, N N mr f m C o00 -,I-t CDn'.0If 6Cl000> C ' 0 - C t- N N ts t r ao 0o r- rs t- 00 cO m "D "D Nq r- Nq c, m - - kn It Os M a n Ci C- od o0s a n O 0) O I: t': s es -C IT V to o e w oo osc w oo n a) e oo W) o N N os so M m oo m - oC Co C ' o o n o m ~ m o o ~r tf W) Ci O -o 0 0 0 C N C C -'0 >C 0 I,0 0 00 0tC S'N .~ - 00 NN N t ~ ~f, C Cl C Cl > C> > -'CrCCr r N - '. 00~ f r 0 C . 0 '' > '0 0 f ' C l C . r - [0037] Rhizome are found to contain the highest concentration of each of the eight saponins in Table 2 as compared to the other three notoginseng parts, followed by main root and branch root, and then fibrous root (FIG. 2). Except for notoginsenoside R 1 , concentrations of the other seven saponins in rhizome are significantly higher than that in main root. The higher content of each saponin in rhizome accumulatively leads to the significantly higher content of sub-total PPD-type and PPT-type ginsenosides, as well as the higher content of total saponins in rhizome than that in main root. On average, the content of total ginsenosides in rhizome is about 20-60% higher than that in main root and about 50-90% higher than that in branch root. The higher saponin content, together with the considerable biomass of the rhizome (about 15% of the whole root), support the importance of distinguishing rhizome from other parts of notoginseng and utilizing rhizome as the main source for manufacturing of ginsenoside-based products. [0038] While concentration of Rb 2 in rhizome accounts for a small percentage in the total amount of saponins, concentration of Rb 2 in rhizome is distinctively higher than those found in the other three parts. As evidenced in FIG. 2, Rb 2 concentration in rhizome is by up to 8 folds the concentration of that in main root. This distinctively high content of Rb 2 in rhizome is useful to distinguish rhizome from other parts of notoginseng. It is shown that Rb 2 concentration accounts for at least 0.30% of total saponins in rhizome (lower limit), whereas 0.09% or less of Rb 2 (upper limit) is shown in the other three parts. [0039] The quantitative results further show quite similar content of both PPT- and PPD-type ginsenosides in main root and branch root, thus these two parts can be equivalently utilized. Although main root is the traditional medicinal part, and believed to possess the highest quality and medicinal efficacy whereas branch root are thought to have lower quality, the results reveal that the saponin quantity of branch root is comparable to main root. [0040] In commercial un-assorted samples, concentration of individual saponins in the rank order of rhizome > main root > = branch root > fibrous root is also observed, demonstrating this is an overall pattern of saponin distribution in different parts of notoginseng. Consistent with that revealed in whole root-derived samples, significant differences in the content of ginsenosides Rgi, Rbi, Rd, Rc and Rb 2 between rhizome and main root are observed, despite the great intra-group variation (FIG. 3). In addition, significant difference in the content of R 1 , Rgi, Re and Rbi between 13 main root and fibrous root are observed. Most notably is the intervallic content of Rb 2 in rhizome and other parts (FIG. 3). Cumulative results obtained from the whole root-derived samples and commercial un-assorted sample reveal that the content of Rb2 in rhizome is unlimitedly higher than 0.5 mg/g, whereas the content in other parts is lower than 0.5 mg/g, and mostly lower than 0.2 mg/g. Based on these evidences, 0.5 mg/g of Rb 2 can be a threshold value for differentiation of rhizome from other parts of notoginseng. [0041] The present invention further provides untargeted metabolomics to profile diverse classes of metabolites to compare the overall metabolite composition between different parts of notoginseng. UPLC-Q-TOF MS is used for profiling metabolites due to its superiority in high resolution mass, precision and sensitivity. More than 15,000 ions achieved in the UPLC-TOF MS data of notoginseng are provided in the present application. Analysis results of these ions are exported for multivariate analysis by SIMCA-P 13.0. Score plot and loading plot are the important result generated from SIMCA-P. Score plot is a projection of data onto subspace, which is used to interpret relationships between observations. Loading plot is a plot of the relationship between the original variables and the subspace dimension, which is used to interpret relationships between variables. As shown in FIG. 4A, clear separation of rhizome (1), fibrous root (4), main root (2), and branch root (3) can be observed in the PLS-DA plot (R 2 Y = 0.912, Q 2 = 0.535), demonstrating significant difference in the metabolite profile of the different parts. To minimize any effects of non relevant metabolite variability, OPLS-DA analyses are also carried out to identify the differences between groups (FIG. 4B). As in FIG. 4B, OPLS-DA plot shows that individual parts of notoginseng are associated with distinctive metabolite profile. Consistent with the results obtained from quantitative analysis, branch root (3) and main root (2) are very close in the OPLS-DA plot, indicating high similarity between the two parts. In a loading plot (FIG. 4C), the ions farthest away from the main cluster of ions possess the higher VIP value and play more important role in the differentiation. As shown in FIG. 4C, a number of ions which is distant from the main cluster is identified as the marker ions. These marker ions are identified according to the accurate mass, retention time and targeted MS/MS data. FIG. 5 represents MS/MS data of markers including malonyl ginsenoside Rbi (FIG. 5A) and notoginsenodie Rwi (FIG. 5B). A list of the identified marker ions is shown in Table 3, which includes the eight saponins identified in notoginseng. This result identifies 20 saponins with high VIP value and relatively high content, including ginsenosides Rf, Rgi, Re, Rg 2 , Rhi, Rbi, Rb 2 , Rd and Rg 3 , and notoginsenosides A, Fa, Fe, I, K, R 1 , R 2 , R 4 , Rwi, 14 as well as malonyl-ginsenoside Rbi and Rd, as useful marker compounds responsible in differentiation of rhizome, main root, branch root and fibrous root of Panax notoginseng. 15 - t- q Z; I C)- 000 u -7 Xn Crl 0Q 90 ~ ~ 0 n- C'. l 'Z 3~ y, -, - 7 C, O On o ~ OOlooZ ~ ~' _ 0'~e 0 ' n~ o 0 rt~ :)o On C') 00~e -M0 00 m oo 0qI 00 r- m r)N nA C ;7 90- On cn . c M O e e , O rCO ln L90 ' ~ - e - N' I C~ C~~C C~09'~ ~ L orN

C

o Cl~.On~ .-. 0--%~- cc IN ~0N n ~ n n 00. O '0Ot\ 0 U 90 '\ ~ 0 O N O C lfcNd~~On 0. O N (0 0 ' n C n 0 l-N 0 c~ cq 0 00kr s-M 00 0 0 0 00 C0 ~~ClC) 2l ~ e~Q~l ClU C w~- -n~>'e, O ~~ 7X 7,$C 'C7 -c'y (D 0 CXC ~~CX, 'C C N m -- - n 0~~' 00X ~ ' - ~ 0 kQ~4 ClN A0 00' 'Xe0 'C 0r ' cc CX InIn r-I ON I0re n 7771C4k)C . .0 - ~ .. -'' Na.Xl ND C)I0 cqNI In 0- CA en) k) 0l In 00 0 0r- c , Q~ Q" uJ Y) u 6 KL c + 0 00c.) 0 0. 0 00 a,\ In 'In In C r- 0- rN 't Nn Cl 00 l C 00 00T '0 C'I 0 c I 'IX N-1 ' 0 00 Nl Oc r-- =0~ n0 ' C 0 In 00 t-fl - r0>l X ~ 'X,' rX N . e N0 'C 0 :n l00 0iX 00 C00 IN0 N XN 0 C 00 -0I 'IX~~~I 0100 Ni '0 'C -l I CfN n 00~~~~F- CX N rCX N -l C X C IVfX N C00 l C C' '0 Cfl 'I 'X C- 00 rn X '-X'X -l C- 'I Cc In ' - (i 0 C 00N' ct N 0 C 0~ ~~~~ ~~~~~~~ tI I 00 l lcX 0 -C X 0 C-l 0 C r ~ ~~~~~~~~~ 'N N C C0CCf l ' ~-l l e 0 0 X fC-l C-7 -f C-f4 Cl 711~ NC14 (q l Cl)l m [0042] The relative concentrations of the 20 identified marker compounds in Table 3 in each part of notoginseng are compared based on their peak area. The concentration of each marker compound is normalized as a relative concentration which is calculated by Relative concentration= concentration of a marker compound in an individual part/ total concentration of the marker compound in all four parts x100. As shown in FIG. 6, notoginsenosides Fc (at least 0.15%) and Rwi (at least 0.61%), malonyl-ginsenoside Rd (at least 0.40%) and ginsenoside Rb 2 (at least 0.30%) are found to be present in rhizome with significantly higher concentration than in other parts. Both quantitative and multivariate analyses show that the content of ginsenoside Rb 2 is much higher in rhizome than in other parts of notoginseng. This reveals that the reliability of the marker compounds identified from the multivariate analysis. [0043] The present invention provides quantitative comparison using validated UPLC-MS method and metabolite profiling approach for evaluating the similarities and differences in metabolites (e.g. saponins) among rhizome, main root, branch root and fibrous root of Panax notoginseng. Both quantitative analysis and metabolite profiling study show that rhizome has a chemical composition different from the other parts. Rhizome contains the highest content of different individual saponins and total saponins, clearly illustrating the suitability of this part as the main source for the manufacturing of ginsenoside-based products. The chemical composition of main root and branch root are quite similar and thereby can be utilized equivalently. The fibrous root is also showed to be a clear separation from the other parts and has the lowest saponins contents (both individual and total saponins). Moreover, the multivariate study shows that notoginsenosides Fe and Rwi, malonyl ginsenoside Rd and ginsenoside Rb 2 as the most useful chemical markers for differentiation of rhizome from other parts of notoginseng. INDUSTRIAL APPLICABILITY [0044] Study of the metabolite profiles of different parts of notoginseng aids the understanding of chemical characteristics of different parts of notoginseng. The study also reveals that individual parts of notoginseng are associated with distinct metabolite profiles. These distinct metabolite profiles are useful to identify and determine the specific part of notoginseng and facilitate the selection of individual parts of notoginseng for various applications. 18 [0045] While the foregoing invention has been described with respect to various embodiments and examples, it is understood that other embodiments are within the scope of the present invention as expressed in the following claims and their equivalents. Moreover, the above specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety. 19

Claims (5)

1. A method of identifying and determining a specific part of root of Panax notoginseng (notoginseng) for a plant material, comprising: obtaining a metabolite profile of said plant material; comparing the metabolite profile of said plant material against predetermined metabolite profile of each of rhizome, main root, branch root and fibrous root parts of notoginseng; and determining to which part of notoginseng the plant material belongs or whether the plant material comprises one or more rhizome, main root, branch root or fibrous root part of notoginseng based on said comparison.

2. The method of claim 1, wherein said obtaining a metabolite profile of said plant material comprises quantifying concentrations of saponins selected from Ginsenoside Rbi, Malonyl ginsenoside Rbi, Ginsenoside Rd, Ginsenoside Rgi, Notoginsenoside K, Notoginsenoside R 1 , Notoginsenoside Rwi, Ginsenoside Rb 2 , Malonyl-ginsenoside Rd, Ginsenoside Re, Notoginsenoside Rg 3 Notoginsenoside Fa, Notoginsenoside I, Notoginsenoside R 4 , Notoginsenoside Fe, Ginsenoside Rf, Notoginsenoside R 2 , Notoginsenoside A, Ginsenoside Rg 2 and Ginsenoside Rhi.

3. The method of claim 1, wherein the plant material is identified and determined to be or comprises the rhizome part of notoginseng, and the metabolite profile of said plant material comprises at least 0.30% or 0.5mg/g of Ginsenoside Rb 2 in said plant material.

4. The method of claim 2, wherein the plant material is identified and determined to be or comprises rhizome part of notoginseng, the metabolite profile of said plant material further comprises (i) at least 0.15% of notoginsenosides Fc; (ii) at least 0.61% of notoginsenoside Rwi; and (iii) at least 0.4% of malonyl-ginsenoside Rd.

5. The method of claim 1, wherein said comparing the metabolite profile of said plant material against the predetermined metabolite profile of each of rhizome, main root, branch root and fibrous root parts of notoginseng comprises comparing OPLS-DA score plot of the plant material to be identified with a predetermined OPLS-DA score plot and loading plot of the rhizome, main root, branch root and fibrous root parts of notoginseng. 20