Panax notoginseng Root and Rhizome

Panax notoginseng Root and Rhizome

Proposed For Comment Version 0.2

Panax notoginseng Root and Rhizome


DEFINITION

The article consists of the dried roots, rootlets, and rhizomes of Panax notoginseng (Burk.) F. H. Chen (Family Araliaceae) collected before flowering in autumn. It contains NLT 5.0% of saponins calculated as the sum of notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd, on the dried basis.

 

SYNONYMS

Aralia quinquefolia var. notoginseng Burkill

Panax pseudoginseng var. notoginseng (Burkill) G. Hoo & C. L.Tseng

 

POTENTIAL CONFOUNDING MATERIALS

Panax ginseng roots and rhizomes

Panax notoginseng leaves

Panax notoginseng flowers

Panax quinquefolius roots and rhizomes

 

SELECTED COMMON NAMES

Chinese: 三七, 田七

Danish: Sydkinesisk ginseng

English: Notoginseng, tienchi ginseng, san-qi ginseng, sanchi ginseng, south China ginseng   

Japanese: サンシチニンジン

Korean: 삼칠삼, 삼칠인삼

Pinyin: San qi, sanqi, tian qi, sanchi

 

CONSTITUENTS OF INTEREST

Saponins: Notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic

Root: Subconical or cylindrical; 1–6 cm long, 1–4 cm in diameter; externally grayish-brown or grayish-yellow; showing interrupted longitudinal wrinkles, scars of rootlets, and scar of the stem at the apex surrounded by irregular-shaped protrusions; texture heavy and compact, fracture grayish-green, yellowish-green or grayish-white.

Rootlets: Cylindrical or conical, 2–6 cm long, the upper end 0.8 cm in diameter, the lower end 0.3 cm in diameter

Rhizomes: Irregular lumps; show several stem scars and annulations; fracture grayish-green or grayish-white in the center and deep green or gray at the margin

Microscopic

Transverse section: Cork consisting of several rows of radially arranged, thin-wall cells; layers of parenchyma cells, cluster crystals of calcium oxalate are rare and mostly distributed in parenchyma close to cork; phloem parenchyma showing resin canals containing yellow masses; cambium in a ring; and xylem broad, vessels 1–2 rows in groups, arranged radially.

B. Thin-Layer Chromatography

Standard solution A: 0.5 mg/mL of USP Ginsenoside Rg1 RS in methanol

Standard solution B: 10 mg/mL of USP Panax notoginseng Powdered Extract RS in alcohol. Sonicate for about 10 min, centrifuge, and use the supernatant.

Sample solution: Sonicate about 1.0 g of Panax notoginseng Root and Rhizome, finely powdered, in 10 mL of alcohol for 15 min, centrifuge, and use the supernatant.

[NoteSample solution is stable for 6 h at room temperature.]

Chromatographic system

(See Chromatography <621>, Thin-Layer Chromatography.)

Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)

Application volume: 2 µL, as 8-mm bands

Relative humidity: Condition the plate to a relative humidity of about 33% using a suitable device.

Temperature: 25°

Developing solvent system: Chloroform, alcohol, and water (60:45:6.5)

Developing distance: 6 cm

Derivatization reagent: A solution of 10% sulfuric acid in alcohol. [Note—Prepare fresh. Keep alcohol cold over ice, carefully and gradually add sulfuric acid.]

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Apply the Samples as bands to a suitable HPTLC plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry. Treat with Derivatization reagent, heat for 5–10 min at 105°, and examine immediately under visible light and UV light at 366 nm.

System suitability: Under visible light, the chromatogram of Standard solution B exhibits five reddish-violet bands in the following order with increasing RF: a band in the lower-third section of the chromatogram due to ginsenoside Rb1; three less intense bands clearly separated in the middle-third section of the chromatogram due to ginsenoside Rd, ginsenoside Re, and notoginsenoside R1, respectively; and a band at an RF corresponding to the ginsenoside Rg1 band in the chromatogram of Standard solution A. The two most intense bands in the chromatogram are those of ginsenoside Rb1 and ginsenoside Rg1.  

Under UV light at 366 nm, the chromatogram of Standard solution B exhibits bands in the following order with increasing RF: a blue fluorescent band in the lower-third section of the chromatogram due to ginsenoside Rb1; three less intense bands clearly separated in the middle-third section of the chromatogram due to ginsenoside Rd, ginsenoside Re, and notoginsenoside R1, respectively (the band due to ginsenoside Rd is blue fluorescent while the other two bands are pinkish-violet); and a pinkish-violet band at an RF corresponding to the ginsenoside Rg1 band in the chromatogram of Standard solution A. The two most intense bands in the chromatogram are those of ginsenoside Rb1 and ginsenoside Rg1.

Acceptance criteria: Under visible light, the chromatogram of the Sample solution exhibits five reddish-violet bands corresponding in RF to similar bands in the chromatogram of Standard solution B. These bands appear in the following order of increasing RF: a band in the lower-third section of the chromatogram due to ginsenoside Rb1; three less intense bands clearly separated in the middle-third section of the chromatogram due to ginsenoside Rd, ginsenoside Re, and notoginsenoside R1, respectively; and a band at an RF corresponding to the ginsenoside Rg1 band in the chromatogram of Standard solution A. The two most intense bands in the chromatogram are those of ginsenoside Rb1 and ginsenoside Rg1.

Under UV light at 366 nm, the chromatogram of the Sample solution exhibits the following bands, with increasing RF, corresponding to similar bands in the chromatogram of Standard solution B: a blue fluorescent band in the lower-third section of the chromatogram due to ginsenoside Rb1; three less intense bands clearly separated in the middle-third section of the chromatogram due to ginsenoside Rd, ginsenoside Re, and notoginsenoside R1, respectively (the band due to ginsenoside Rd is blue fluorescent while the other two bands are pinkish-violet); a pinkish-violet band at an RF corresponding to the ginsenoside Rg1 band in the chromatogram of Standard solution A; and two minor bands, due to ginsenoside Rg3 and ginsenoside Rg2, directly below the ginsenoside Rg1 band. The two most intense bands in the chromatogram are those of ginsenoside Rb1 and ginsenoside Rg1.

[Note—Under UV light at 366 nm and in distinction from Panax notoginseng Root and Rhizome, Panax notoginseng leaves and flowers chromatograms exhibit a red band due to chlorophyll at the solvent front and a minor or no bands at the RF corresponding to ginsenoside Rg1.]

[Note—Under UV light at 366 nm and in distinction from Panax notoginseng Root and Rhizome, the Panax ginseng Root and Rhizome chromatogram lacks the band at the RF corresponding to notoginsenoside R1 in the chromatogram of Standard solution B and it exhibits a pinkish-violet band at an RF slightly higher than that of notoginsenoside R1. The band due to ginsenoside Rg1 is not one of the most intense bands in the chromatogram, and the most intense band is detected in the lower-third section of the chromatogram and in the region where the RF of ginsenoside Rb1 is detected.]

[Note—Under UV light at 366 nm and in distinction from Panax notoginseng Root and Rhizome, the Panax quinquefolius Root and Rhizome chromatogram lacks the band at the RF corresponding to notoginsenoside R1 in the chromatogram of Standard solution B, exhibits a minor band due to ginsenoside Rg1, and the most intense band is detected in the lower-third section of the chromatogram and in the region where the RF of ginsenoside Rb1 is detected.]

C. UHPLC

Analysis: Proceed as directed in the Assay for Content of Saponins.

Acceptance criteria: The chromatogram of the Sample solution exhibits peaks at the retention times corresponding to the peaks due to notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd in the chromatogram of Standard solution B. The two most abundant peaks in the chromatogram are those of ginsenoside Rg1 and ginsenoside Rb1.

 

ASSAY

• Content of Saponins

Solution A: 0.03% Phosphoric acid in water

Solution B: Acetonitrile

Mobile phase: See Table 1.

 

Table 1

Time
(min)

Solution A
(%)

Solution B
(%)

0

83

17

2.4

80

20

3.5

70

30

4.2

69

31

5.0

58

42

5.1

0

100

6.0

0

100

6.1

83

17

7.5

83

17

 

Solvent: Methanol and water (7:3)

Standard solution A: 0.04 mg/mL of USP Ginsenoside Rg1 RS in methanol

Standard solution B: 3.0 mg/mL of USP Panax notoginseng Powdered Extract RS in Solvent. Sonicate for about 10 min, centrifuge, and use the supernatant. Before injection, pass through a PFTE filter of 0.2-μm pore size, and discard the first portion of the filtrate.

Sample solution: Transfer about 0.3 g of Panax notoginseng Root and Rhizome (capable of passing through a 250-μm sieve), accurately weighed, to a 50-mL centrifuge tube. Add 10 mL of Solvent and sonicate for 10 min (140W, 42 kHz). Centrifuge and transfer the supernatant to a 25-mL volumetric flask. Repeat the above extraction two more times, each with 5 mL of Solvent. Combine the extracts in the volumetric flask, dilute with Solvent to volume, and mix. Before injection, pass through a PFTE filter of 0.2-μm pore size, and discard the first portion of the filtrate. [NoteSample solution is stable for 24 h at room temperature.]

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: UPHLC

Detector: UV 203 nm

Column: 2.1-mm × 5-cm; 1.7-µm packing L1 (similar to Kinetex™ C18 100 Å)

Column temperature: 30±1°

Flow rate: 0.8 mL/min

Injection volume: 5 µL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

Chromatogram similarity: The chromatogram from Standard solution B is similar to the reference chromatogram provided with the lot of USP Panax notoginseng Powdered Extract RS being used.

Resolution: NLT 1.5 between the ginsenoside Rg1 and ginsenoside Re peaks, Standard solution B

Tailing factor: NMT 2.0 for the ginsenoside Rg1 peak, Standard solution A

Relative standard deviation: NMT 2.0%, determined from the ginsenoside Rg1 peak in repeated injections, Standard solution A

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Using the chromatograms of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Panax notoginseng Powdered Extract RS being used, identify the retention times of the peaks corresponding to different saponins in the Sample solution chromatogram. The approximate relative retention times of the different peaks are provided in Table 2.

Separately calculate the percentage of each of the saponins in the portion of Panax notoginseng Root and Rhizome taken.

 

      Table 2

Analyte

Relative Retention Times

Factor

Notoginsenoside R1

0.78

1.09

Ginsenoside Rg1

1.00

1.00

Ginsenoside Re

1.06

1.02

Ginsenoside Rb1

1.78

1.26

Ginsenoside Rd

2.04

1.03

  

Result = (rU/rS) × CS × (V/W) × F × 100

 

rU   = peak response of the analyte from the Sample solution

rS   = peak response for ginsenoside Rg1 from Standard solution A

CS  = concentration of ginsenoside Rg1 in Standard solution A (mg/mL)

V    = volume of the Sample solution (mL)

W   = weight of Panax notoginseng Root and Rhizome taken to prepare the Sample solution (mg)

F    = conversion factors for the analytes as provided in Table 2

 Calculate the content of saponins as the sum of the percentages of notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd.

Acceptance criteria: NLT 5.0% on the dried basis

 

CONTAMINANTS

• Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 1.0 µg/g

Lead: NMT 5.0 µg/g

Mercury: NMT 1.0 µg/g

• Articles of Botanical Origin, General Method for Pesticide Residues Analysis <561>: Meets the requirements

• Articles of Botanical Origin, Test for Aflatoxins <561>: Meets the requirements

• Microbial Enumeration Tests <61>: The total aerobic bacterial count does not exceed 105 cfu/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the bile-tolerant Gram-negative bacteria does not exceed 103 cfu/g.

• Tests for Specified Microorganisms <62>: Meets the requirements of the tests for the absence of Salmonella species and Escherichia coli.

 

SPECIFIC TESTS

• Articles of Botanical Origin, Foreign Organic Matter <561>: NMT 2.0%

• Loss on Drying <731>

Analysis: Dry 1.0 g of Panax notoginseng Root and Rhizome, finely powdered, at 105° for 5 h.

Acceptance criteria: NMT 14.0%

• Articles of Botanical Origin, Total Ash <561>

Analysis: 4.0 g of Panax notoginseng Root and Rhizome, finely powdered

Acceptance criteria: NMT 6.0%

• Articles of Botanical Origin, Acid-Insoluble Ash <561>

Analysis: 4.0 g of Panax notoginseng Root and Rhizome, finely powdered

Acceptance criteria: NMT 3.0%

 

ADDITIONAL REQUIREMENTS

• Packaging and Storage: Preserve in well-closed containers, protected from light and moisture, and store at room temperature.

• Labeling: The label states the Latin binomial and the part(s) of the plant contained in the article.

• USP Reference Standards <11>

USP Aflatoxins RS

USP Ginsenoside Rg1 RS

USP Panax notoginseng Powdered Extract RS

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1 comment

Anonymized Userposted Wednesday, August 28, 2013 - 11:05pm
Comments on HMC monograph Panax notoginseng Root and Rhizome 1. Comments on “Constituents of Interest” and quality control marker for “Assay”: In the proposed monograph, only the content of saponins was considered as the quantification marker for quality evaluation of Panax notoginseng Root (PNR), which is not good enough to control the quality of PNR comprehensively. Chinese herbal medicines were commonly used for treatment of more than one illness, attribute to their various kinds of bioactive components. For PNR, its two major kinds of bioactive effects, promoting blood circulation (Huo Xue in Chinese) and stopping bleeding (Zhi Xue in Chinese), are attribute to dammarane-type saponins (ginsenoside Rg1, Rb1, notoginsenoside R1, etc.) [1] and dencichine (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid) [2-3], respectively. Traditionally and clinically, PNR was widely used for hemostasis and it is the main raw material for famous Chinese traditional formulae such as “Yunnan Baiyao” for treatment of injury-induced trauma and bleeding [4-5]. The correlation analysis between hemostatic activity and commercial grades of PNR was investigated, and the results showed that the higher content of dencichine the better hemostaic activity [6]. Therefore, dencichine is an important pharmacological constituent in PNR. Traditionally and clinically, PNR after thermal processing such as steaming and roasting, was used for treatment of cardiovascular diseases but not used for hemostasis, which is different from the raw PNR [7]. The investigations [8-12] indicated dencichine is thermally unstable, and heat treatment like steaming and roasting causes its hydrolysis and isomerization in proper medium. It was found that the equilibrium concentration ratio of dencichine and its isomer (α-ODAP) is 3/2 at 55°C [8], then both dencichine and the isomer can be hydrolysis at high temperature and produce 2,3-diaminopropionic acid (DAP) [10] (Figure 1, Please See the attached file, same as below). After thermal processing (steaming), contents of dencichine in powdered PNR or Tablet preparation made from PNR were decreased from 59.3% to 91.7%, averagely decreased 68.1% (Table 1). It was also found that when the duration of steaming increased from 2 h to 9 h, the content decreased from 0.0283% to a negligible amount of 0.0003% (Table 2). Based on the determination of multiple batches of PNR (Table 3, Figure 2) in out laboratory, we conducted that the content of dencichine in PNR varied from 0.10% to 0.57%, and the variation was significant (RSD 41.5%). Furthermore, after summary of determinations from previous investigations [7,13-21], we conducted that the batch-to-batch content of dencichine in PNR varied significantly (Table 4, for total 123 samples, the relative standard derivation, RSD, was larger than 27.9%), investigations also indicated the content varies noticeable among different cultivation places (Table 4, 18.4% of RSD) [19], different harvest time (Table 1, 16.4% of RSD) [17] and different commercial grade (classified by number of heads or “tou”, 20.8% of RSD, Table 4) [15,17,19,20-21], and therefore influence quality of PNR. Also, the content was significantly different among plant parts (main root, rhizome, lateral root, bud, stem and leaf, 38.9% of RSD, Table 5) [17, 21], which means commingling of different parts during sampling could also causes variation of dencichine content. Additionally, dencichine content was found to be different among the similar species Asian ginseng, American ginseng and PNR (Table 6) and could be a marker for differentiation of the three species [13]. In summary, it can be conducted from the evidences that the dencichine, an important bioactive component responsible to PNR’s hemostatic effect, with significantly varied concentration in PNR, should be seriously considered as the quantitative marker for quality control of PNR. 2. Quantification method in “Assay-Content of saponins” For quantification of saponins by HPLC, the method used in monograph are Absolutely quantification of Rg1 and quantification other four saponins using Rg1 with calibration factors . However, calibration factor is not necessary for quantification, the reasons: 1) Calibration factor is no significance for quantification of other saponins, since the saponin content variation among PNR is much greater than the quantification error without calibration factor [22]. 2) The accuracy of quantification by using calibration factors can be affected by the chromatographic conditions, and the calibration factors are more prone to be affected in different laboratories [23]. 3. Analytical method in “Assay-Content of saponins” 1) Analytical column selection: The proposed monograph select a 2.1-mm×5-cm, 1.7 m L1 packing column (using Phenomenex Kinetex C18 column for demonstration) for LC analysis. However, since Chinese medicines like PNR are complex matrices, generally the analysis will cause ineluctable polution damage to the column, make the lifetime of columns with 1.7m of particle size for CM analysis much shorter than conventional ones. The proposed method employed a Phenomenex Kinetex C18 column for demonstrational analysis, but according to the column manual, only isocratic elution could be used for this type of column, therefore the lifetime of this column is questionable when performing gradient elution. Additionally, columns with extra-small particle size are difficult for application on conventional LC system which will increase the analysis cost. 2) Gradient program: The gradient elution program is complex, therefore slight change in composition of mobile phase may affect the separation of abutted analytes. 4. Acceptance criteria in “Assay-Content of saponins” The proposed monograph setup the criteria as “total saponins (NR1+Rg1+Re+Rb1+Rd): NLT 5.0%”. However notoginsenoside R1 (NR1) is a unique component in PNR, so make NR1 as an individual marker is better to retionally evaluate PNR’s quality, and to control its quality from comprehensive view. 5. “Specific Tests”: The proposed monograph has no item for Water-soluble extractives. Actually, Water-soluble extractives are a key index for quality control of bioactive polar components such as polysaccharides, etc. 6. “Identification-TLC method”: Th developing solvent system consisting of chloroform, alcohol, and water (60:45:6.5). However, chloroform can cause relative serious health and environmental hazards and were banned in some European laboratories. 7. “Definition”: The proposed monograph defined the medicinal part as “dried roots, rootlets and rhizomes”. This is debatable because 1) Traditionally, only roots of Panax notoginseng were used as San-qi; 2) Only roots could be found and used in traditional clinic; 3) Other important standards including both European Pharmacopeia (EP) and Hong Kong Chinese Materia Medica Standards (HKCMMS) define only roots as the medicinal part; 4) The rootlets (Jing tiao) and rhizomes (Jian kou) can be used as material for extraction of PNRsaponins or substitute of San qi but were not considered as authentic San qi. 8. Sample preparation ( for Assay of content of saponins by HPLC): The proposed extraction method is “Sonication (10 min, three times)” . Sonication is less effective on extraction of PNR than other advanced methods such as Pressurized Liquid Extraction (PLE), which was proved to perform highest extraction yield, and provides high automation and good reproducibility[24]. Also, sonication thrice processing is complex for routine analysis. Reference [1] Ng TB. Pharmacological activity of sanchi ginseng (Panax notoginseng). J Pharm Pharmacol. 58(2006) :1007-1019. [2] Y. P. Zhang, Q. Yu. Effect of dencichine on hemostasis and neurotoxicity. Shan Dong Zhong Yi Yao, 29(2010) 43-45 [3] Kozuga T, Yokota M, Aono S, Ito Y, Kato H (1980) L-N-b- Oxalyl-a,b-diaminopropionic acid. Jpn Kokai Tokyo Koho, JP 55043045 19800326 Showa 4 pp [4] T. Kosuge, M. Yokota, A. Ochiai, [Studies on antihemorrhagic principles in the crude drugs for hemostatics. II. On antihemorrhagic principle in Sanchi Ginseng Radix (author's transl)], Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan, 101 (1981) 629-632. [5] T.B. Ng, Pharmacological activity of sanchi ginseng (Panax notoginseng), The Journal of pharmacy and pharmacology, 58 (2006) 1007-1019. [6] L.M. Shan, Y.L. Zhao, L. Hong, et al. Correlation analysis between hemostatic activity and commercial grades of Panax notoginseng. Chinese Traditional Herbal Drugs. 42(2011) 1779-1782 [7] H.L. Koh, A.J. Lau, E.C.Y. Chan, Hydrophilic interaction liquid chromatography with tandem mass spectrometry for the determination of underivatized dencichine (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid) in Panax medicinal plant species, Rapid Communications in Mass Spectrometry, 19 (2005) 1237-1244. [8] B.M. Abegaz, P.B. Nunn, A. Debruyn, F. Lambein, Thermal-Isomerization of N-Oxalyl Derivatives of Diamino Acids, Phytochemistry, 33 (1993) 1121-1123. [9] G. Akalu, G. Johansson, B.M. Nair, Effect of processing on the content of beta-N-oxalyl-alpha, beta-diaminopropionic acid (beta-ODAP) in grass pea (Lathyrus sativus) seeds and flour as determined by flow injection analysis, Food Chemistry, 62 (1998) 233-237. [10] A. De Bruyn, D. Van Haver, F. Lambein, B.M. Abegaz, Chemical properties of the natural neurotoxin of Lathyrus sativus 3-N-oxalyl-2,3-diamino-propanoic acid (beta-ODAP), its nontoxic 2-N-oxalyl isomer, and its hydrolysis product 2,3-diamino-propanoic acid (DAPRO) by 1H- and 13C-NMR spectroscopy, Nat Toxins, 1 (1993) 328-340. [11] V. Padmajaprasad, M. Kaladhar, R.V. Bhat, Thermal isomerisation of beta-N-oxalyl-L-alpha, beta-diaminopropionic acid, the neurotoxin in Lathyrus sativus, during cooking, Food Chemistry, 59 (1997) 77-80. [12] L. Zhao, X. Chen, Z. Hu, Q. Li, Q. Chen, Z. Li, Analysis of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid and homoarginine in Lathyrus sativus by capillary zone electrophoresis, J Chromatogr A, 857 (1999) 295-302. [13] C.F. Qiao, X.M. Liu, X.M. Cui, D.J. Hu, Y.W. Chen, J. Zhao, S.P. Li, High-performance anion-exchange chromatography coupled with diode array detection for the determination of dencichine in Panax notoginseng and related species, J Sep Sci, (2013) (in press, doi: 10.1002/jssc.201300334). [14] L.L. Song, Y.P. Zhang, Determination of dencichine in Panax notoginseng by HPLC-ELSD, Beijing Zhong Yi Yao, 29(2010) 216-217. [15] C.M. Fu, S.K. Liu, Z.W. Li, Determination of Dencichine in Sanchi Using LiquidChromatography on Porous Graphitic Carbon Column, Se Pu, 25 (2007) 834-837. [16] P. Zheng, Determination of dencichine in Panax notoginseng by capillary zone electrophoresis. Hua Xi Yao Xue Za Zhi, 27(2012) 453. [17] X.M. Cui, L. S. Xu, Q. Wang, Z. J. Chen, T. X. Dong, W. K. Tsim, Determination of dencichine in Radix Notoginseng, Zhong Guo Yao Xue Za Zhi, 40 (2005) 1017-1019. [18] G.X. Xie, Y.P. Qiu, M.F. Qiu, X.F. Gao, Y.M. Liu, W. Jia, Analysis of dencichine in Panax notoginseng by gas chromatography-mass spectrometry with ethyl chloroformate derivatization, J Pharm Biomed Anal, 43 (2007) 920-925. [19] T.T.X. Dong, X.M. Cui, Z.H. Song, K.J. Zhao, Z.N. Ji, C.K. Lo, K.W.K. Tsim, Chemical assessment of roots of Panax notoginseng in China: Regional and seasonal variations in its active constituents, Journal of Agricultural and Food Chemistry, 51 (2003) 4617-4623. [20] C. M. Fu, J. Zhu, S. K. Liu, Z.W. Li, Determination of dencichine in Panax notoginseng and its preparations by HPLC, Zhong Cao Yao, 37(2006): 68-69. [21] J. Zhu, S. K. Liu, C. M. Fu, Z. W. Li, Analysis of dencichine by HPLC with pre-column derivatization, Zhongguo Zhong Yao Za Zhi, 31(2006) 1865-1867. [22] S.P. Li, C.F. Qiao, Y.W. Chen, J. Zhao, X. M. Cui, Q.W. Zhang, X. M. Liu, D. J. Hu. A novel strategy with standardized reference extract qualification and single compound quantitative evaluation for quality control of Panax notoginseng used as a functional food. J Chromatogr A (2013), http://dx.doi.org/10.1016/j.chroma.2013.07.025 [23] J.J. Hou, W.Y. Wu, J. Da, S. Yao, H.L. Long, Z. Yang, L.Y. Cai, M. Yang, X. Liu, B. H. Jiang, D. A. Guo, Ruggedness and robustness of conversion factors in method of simultaneous determination of multi-components with single reference standard. J Chromatogr A. 1218 (2011) 5618-5627. [24] J.B. Wan, C. M. Lai, S. P. Li, M.Y. Lee, L. Y. Kong, Y. T. Wang, Simultaneous determination of nine saponins from Panax notoginseng using HPLC and pressurized liquid extraction. J Pharm Biomed Anal. 41 (2006) 274-279.

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