Food Chemistry 132 (2012) 1808–1813

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Food Chemistry

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The processing of Panax notoginseng and the transformationof its saponin components

Dong Wang, Peng-Ying Liao, Hong-Tao Zhu, Ke-Ke Chen, Min Xu, Ying-Jun Zhang ⇑, Chong-Ren YangState Key Laboratory of Phytochemistry and Plant Resources of West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, PR China

a r t i c l e i n f o

Article history:Received 6 April 2011Received in revised form 13 November 2011Accepted 6 December 2011Available online 13 December 2011

Keywords:Panax notoginsengProcessSaponinTransformation

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.12.010

⇑ Corresponding author. Tel.: +86 871 522 3235; faE-mail address: zhangyj@mail.kib.ac.cn (Y.-J. Zhan

a b s t r a c t

Notoginseng, the root of Panax notoginseng (Burk.) F. H. Chen (Araliaceae), a famous traditional Chinesemedicine, has been used in both raw and processed forms due to their different therapeutic functions.In this study, HPLC analyses on saponin composition of processed notoginseng were conducted, whichrevealed that, during the steaming process, the five main saponin constituents (ginsenosides Rg1, Rb1,Rd, and Re, and notoginsenoside R1) in raw notoginseng decreased gradually and some other new sapo-nins were formed. Among these, eight newly converted ginsenosides were identified as 20(S)-Rh1, 20(R)-Rh1, Rk3, Rh4, 20(S)-Rg3, 20(R)-Rg3, Rk1 and Rg5. Different processing methods, including steaming andbaking, were studied, along with the correlative dynamic curves of the transformation of saponins. Theresults indicated that the moisture content and temperature were the determinant effecting factors inthe preparing process, and water content was necessary and high temperature was helpful for saponintransformation.

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1. Introduction

The use of processed medicines and herbs (or prepared pieces)plays an important role in the application of traditional Chinesemedicines (TCM). Physicians of TCM believe that the processedforms of TCM, processed by different methods, are provided withproperties different from those of raw material. Generally, themain purpose of TCM processing is to transform the properties ofmedicines and generate new effects, or to increase the curative ef-fect and reduce or eliminate toxicity and side-effects. The process-ing of TCM has involved special crafts, such as baking, toasting,steaming, cooking, carbonization, calcination, fermentation andspecial processing methods with wine, honey, salt, vinegar, gingerjuice, minerals and other liquid or solid supplementary materials.Although ancient practitioners recognised these conversions fortherapeutic effects in processed TCM, the material essence of thesechanges has not been fully understood. In recent decades, manytraditional herbs have been investigated, chemically and pharma-cologically but, for the processed medicines, little relevant knowl-edge was acquired.

Notoginseng, the root of Panax notoginseng (Burk.) F. H. Chen,one of highly valued TCM, has been used medicinally for itshaemostatic and cardiovascular properties for more than400 years. It was included as a dietary supplement by the US

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x: +86 871 515 0124.g).

Dietary Supplement Health and Education Act in 1994 (Sun et al.,2010). The processed notoginseng is one important form of thismedical plant. Traditionally, raw notoginseng is used as a hemo-static, traumatic and cardiovascular medicine, while the processedform is used as a tonic for its blood cell-increasing function. In thepast 20 years, a great number of chemical and pharmacologicalstudies on P. notoginseng have been carried out. Dammarane-typesaponins are considered to be the main bioactive compounds. Bynow, more than 70 saponins have been isolated from this medicalplant, among which, ginsenosides Rg1, Re, Rb1, Rd and notoginseno-side R1 are five major components, while the others are in very lowconcentration (Wang, McEntee, Wicks, Wu, & Yuan, 2006; Wang,Wang, Ma, Zhang, & Yang, 2008). Compared with raw notoginseng,there is much less chemical study carried out on processed notogin-seng. Lau, Seo, Woo, and Koh (2004) and Lau, Woo, and Koh (2003)reported the HPLC and LC–MS analyses on steamed notoginsengand eight major saponins were identified as 20(S)-Rh1, 20(R)-Rh1,Rk3, Rh4, 20(S)-Rg3, 20(R)-Rg3, Rk1 and Rg5. In our previouswork, we carried out a detailed chemical study on the steamed not-oginseng, from which 27 dammarane-type triterpenoids, includingfour new glycosides, were isolated (Liao, Wang, Zhang, & Yang,2008).

Steaming and baking are two currently used processing meth-ods for P. notoginseng. In the present study, the different processingmethods for P. notoginseng were compared and the dynamic curvesof saponin transformation were investigated by HPLC analysis.The main effecting factors in the preparing process were alsodiscussed.

D. Wang et al. / Food Chemistry 132 (2012) 1808–1813 1809

2. Materials and methods

2.1. Solvents and chemicals

Methanol (MeOH) was purchased from Tianjing Chemical Ltd.,China. Acetonitrile (MeCN) was from Merck Darmstadt, Germany.Standard ginsenosides Rg1, Re, Rb1, Rd, 20(S)-Rh1, 20(R)-Rh1, Rk3,Rh4, 20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5, and notoginsenoside R1 wereisolated by our laboratory, and their chemical structures weredetermined by use of NMR and MS (Liao et al., 2008).

2.2. Plant material

Fresh roots of P. notoginseng were collected in the farm of Mia-oxiang Ltd., Wenshan county, Yunnan province, China, and chippedinto grains of the size about 2–3 mm.

2.3. Steaming of fresh notoginseng

The fresh notoginseng grains were wrapped with gauze (100 gfor each package) and steamed at 100 and 120 �C separately inan autoclave for 48 h. One package was collected as a sample foranalysis at intervals of 4 h. After drying to constant weight at

Fig. 1. The structur

45 �C, each sample was powdered with an electric grinder andsieved through a 40 mesh sieve for HPLC analysis.

2.4. Steaming of dried notoginseng

The fresh notoginseng grains were dried at 45 �C to constantweight and then the samples were wrapped with gauze (in pack-ages of 30 g for each), and steamed and sampled as described above.

2.5. Baking of notoginseng

The 45 �C dried notoginseng grains were powdered and siftedwith a 40 mesh sieve. The powder was baked at 100 and 120 �C,separately, in an oven for 48 h. Samplings were conducted every4 h for HPLC analysis.

2.6. Sample preparation for HPLC analysis

The powdered notoginseng sample (0.5 g), in a 10 ml volumet-ric flask, was extracted ultrasonically with 70% aq. MeOH at roomtemperature for 30 min, after cooling and made up to volume withmethanol; the sample solution was obtained by filtering the super-natant with a 0.45 lm nylon filter membrane.

es of saponins.

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2.7. HPLC analysis

HPLC analysis was performed on a Waters Alliance liquid chro-matograph (Milford, MA, USA) equipped with an Alliance separa-tion module 2695 and photodiode array detector 2996. Thereversed-phase column used was a Waters Symmetry C-18(3.9 � 150 mm, i.d., 5 lm) column. The gradient elution systemconsisted of water (A) and acetonitrile (B). Separation wasachieved by using the following gradient: 0–20 min: 20–22% B,20–45 min: 22–46% B, 45–55 min: 46–55% B, 55–70 min: 55% B.The column temperature was set at 25 �C. The flow rate was1 ml/min and the injection volume was 20 ll. The UV detectionwavelength was set at 203 nm. The peak areas of saponins werecalculated.

2.8. Calibration curves

Calibration solutions were prepared by dissolving Rg1

(2.60 mg), Rb1 (2.93 mg), Re (2.89 mg), Rd (2.96 mg), R1

(2.48 mg), 20(S)-Rh1 (4.94 mg), 20(R)-Rh1 (5.07 mg), Rk3

(5.11 mg), Rh4 (5.09 mg), 20(S)-Rg3 (3.33 mg), 20(R)-Rg3

(1.79 mg), Rk1 (3.20 mg) and Rg5 (5.19 mg) with methanol in5 ml volumetric flasks. The standard curves were generated byinjecting standard solutions in different volumes. Each calibrationcurve was performed with five different injecting volumes. Thepeak area for each analyte was determined and the calibrationcurves were constructed by Microsoft Office Excel 2003.

Fig. 2. HPLC chromatogram of raw notoginseng (A), fresh notoginseng steamed at120 �C for 8 h (B) and powdered dry notoginseng baked at 120 �C for 48 h (C). Peak1, 20-S Rh1; 2, 20-R Rh1; 3, Rk3; 4, Rh4; 5, 20-S Rg3; 6, 20-R Rg3; 7, Rk1; 8, Rg5.

3. Results and discussion

The results of HPLC analysis indicated a distinct difference insaponin composition between the raw and steamed P. notoginseng.During the steaming process, ginsenosides Rg1, Rb1, Rd, and Re, andnotoginsenoside R1, the five main saponin constituents in the rawnotoginseng, decreased gradually, while other new saponins wereformed (Fig. 1). Among these, by comparing the retention times onHPLC with standard compounds, eight newly converted saponinswere identified as 20(S)-Rh1, 20(R)-Rh1, Rk3, Rh4, 20(S)-Rg3,20(R)-Rg3, Rk1 and Rg5 (Fig. 2). This result is coincident with the re-ports by Lau et al. (2003, 2004) and Liao et al. (2008).

In the process of steaming notoginseng, the transformation ofsaponins began at an early stage. For instance, decreases of Rg1,Rb1, Rd, Re and R1 could be observed evidently after 2 h steaming.Then, the extent of transformation (both degradation and forma-tion) of saponins increased steadily during the steaming process.In the steaming process, Rg1 was the most rapidly declining sapo-nin while Rg5 and Rh4 were the two most abundant new-formedsaponins.

By comparing the steaming methods at different temperatures,it is easy to conclude that steaming temperature exerted an impor-tant influence on saponin transformation. For the fresh notogin-seng, after steaming for 8 h at 120 �C, Rg1, Re, Rb1, Rd, R1

decreased sharply and almost disappeared within 40 h, and thedegradation products, 20(S)-Rh1, 20(R)-Rh1, Rk3, Rh4, 20(S)-Rg3,20(R)-Rg3, Rk1, Rg5, increased rapidly in the first 8 h, and the con-centrations of these eight new saponins culminated after 40 hsteaming. However, in the case of steaming at 100 �C, the forma-tion of new products (as well as the degradation of Rg1, Re, Rb1,Rd, R1), was steady and much slower than at 120 �C. And, even after48 h steaming, the original saponins could be detected and thecontents of hydrolysis products were still increasing (Figs. 3 and 4).

Similar results were obtained in the case of steaming of driednotoginseng. When steamed at 120 �C, the five main saponins ofraw material decreased sharply in the first 12 h, while concentra-tions of the main degradation products were increasing rapidly.

However, when steamed at 100 �C, the conversions of saponinswere also much slower (Figs. 3 and 4).

The water content of raw material is another determinant effec-tive factor in the steaming process. At early stages of steaming, thespeeds of degradation of original saponins and formation of newsaponins in the dried samples were much lower than those inthe fresh ones when steamed at either 100 or 120 �C. It was obvi-ous that the moisture directly exerted an influence on saponin con-versions and the soaking of dried material by steam at the initialstage was beneficial to the transformation of saponins. But, afterthe notoginseng root was saturated with water, some saponin con-tents would be drained away with the surplus steam condensed onthe surface of root. This is the reason why the dried root gave high-er saponin contents than did the fresh one at later stages of thesteaming process. And this would explain why concentrations ofnewly-formed saponin dropped in the fresh root after 40 h steam-ing at 120 �C (Fig. 4).

When comparing the processing methods, steaming and baking,the importance of water contained in notoginseng for saponintransformations is more obvious. Baking only resulted in negligibledegradation of original saponins and, accordingly, very littleformation of new products. Whether at 100 or 120 �C, the propor-tion of saponins did not differ from that of the raw notoginseng,even if the baking process lasted for 48 h (Fig. 2C). This resultproved that the absence of water will impede the conversion ofsaponins.

In dammarane-type tetracyclic triterpenoid glycosides of noto-ginseng, the glycosyl group is prone to being hydrolyzed; some

Fig. 3. The degradation curves of ginsenosides Rg1, Rb1, Rd, Re and notoginsenoside R1 during the steaming process. (A) Dried notoginseng steamed at 100 �C; (B) Driednotoginseng steamed at 120 �C; (C) Fresh notoginseng steamed at 100 �C; (D) Fresh notoginseng steamed at 120 �C.

Fig. 4. The formation curves of ginsenosides 20S-Rh1, 20R-Rh1, Rk3, Rh4, 20S-Rg3, 20R-Rg3, Rk1 and Rg5 during the steaming process. (A) Dried notoginseng steamed at 100 �C;(B) Dried notoginseng steamed at 120 �C; (C) Fresh notoginseng steamed at 100 �C; (D) Fresh notoginseng steamed at 120 �C.

D. Wang et al. / Food Chemistry 132 (2012) 1808–1813 1811

Fig. 5. Proposed transformation of saponins in the process of steaming of notoginseng.

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reactions, such as oxidation, reduction and rearrangement, can oc-cur at the side chain to produce diverse products (Chen, Xie, & Luo,1988; Feng, Wang, Wang, Yang, & Zhou, 1987; Shibata, Ando, & Ta-naka, 1966; Wei et al., 1982, 1984). The mechanism of saponintransformations in the steaming process can be extrapolated fromthe chemical structures of saponins, especially the changes in theirsugar moiety. In the steaming condition, the hydrolysis of the xylo-syl residue attached to C-6 of notoginsenoside R1 and the hydroly-sis of the rhamnosyl residue at C-6 of ginsenoside Re formedginsenoside Rg1. Ginsenoside Rb1 was hydrolyzed at the glucosylof C-20 to yield ginsenoside Rd. Rg1 and Rd were likely to be theparent compounds of newly-formed saponins. The further hydroly-sis of the glucosyl at C-20 of Rg1 yielded Rh1 which then formedRh4 and Rk3 through dehydration at C-20. Similarly, the hydrolysa-tion of the glucosyl at C-20 of Rd produced Rg3, and dehydration ofRg3 at C-20 yielded Rg5 and Rk1 (Fig. 5). During this process, it wasevident that water content was necessary and high temperaturewas helpful for these reactions.

Although the raw and the processed notoginseng are used fordifferent therapeutic treatments in traditional Chinese iatrology,the relationship of the effects and the effective constituents haveremained unclear for a long time. Modern pharmacologicalresearch has found that notoginseng exerts various effects onthe blood, cardiocerebral vascular system, central nervoussystem and endocrine system inflammation (Chan, Thomas, &Tomlinson, 2002; Kwan, 1995). It is well known that the ginse-nosides deglycosylated at C-20, such as ginsenoside Rh1 and Rg3,have anticancer activity, and Rg3 has been developed as an anti-cancer medicine clinically. Rg5, Rh2 and Rh4 have also shownanti-tumor properties (Feng & Lin, 2005; Iishi et al., 1997; Popo-vich & Kitts, 2002, 2004a, 2004b; Wang et al., 2005; Wu, Hu, &Wang, 2005; Yun, 2003; Yun, Lee, Lee, Kim, & Yun, 2001). Inrecent years, the neuroprotective effects of Rg3 (Kim et al.,2007; Tian et al., 2005) and Rh1 (Jung, Kim, & Kim, 2010) havealso been reported. Obviously, the conversions of saponin inthe processed notoginseng account for a substantial part of thechange in its efficacy.

Our study elucidates the dynamic changes of saponin composi-tions and the main influential factors in notoginseng processing.The result implies a prospective feasibility to set up different pro-cessing techniques relevant to prepared notoginseng with differentsaponin compositions for special purposes. On the other hand, pro-cessed P. notoginseng will provide a potential access to certaineffective saponin components, such as Rh4 or Rg5, which are in verylow concentrations in plants.

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