Enzymatic Formation of Novel Ginsenoside Rg1-α-Glucosides by Rat Intestinal Homogenates

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Applied Biochemistry andBiotechnologyPart A: Enzyme Engineering andBiotechnology ISSN 0273-2289 Appl Biochem BiotechnolDOI 10.1007/s12010-015-1847-0

Enzymatic Formation of Novel GinsenosideRg1-α-Glucosides by Rat IntestinalHomogenates

Ramya Mathiyalagan, Young-Hoi Kim,Yeon Ju Kim, Myung-Kon Kim, Min-JiKim & Deok Chun Yang

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Enzymatic Formation of Novel GinsenosideRg1-α-Glucosides by Rat Intestinal Homogenates

Ramya Mathiyalagan1 & Young-Hoi Kim2 &

Yeon Ju Kim3 & Myung-Kon Kim2 & Min-Ji Kim2 &

Deok Chun Yang1

Received: 18 March 2014 /Accepted: 9 September 2015# Springer Science+Business Media New York 2015

Abstract The variation of linkage positions in ginsenosides leads to diverse pharmacologicalefficiencies. The hydrolysis and transglycosylation properties of glycosyl hydrolase familyenzymes have a great impact on the synthesis of novel and structurally diversified compounds.In this study, six ginsenoside Rg1-α-glucosides were found to be synthesized from the reactionmixture of maltose as a donor and ginsenoside Rg1 as a sugar acceptor in the presence of ratsmall intestinal homogenates, which exhibit high α-glucosidase activities. The individualcompounds were purified and were identified by spectroscopy (HPLC-MS, 1H-NMR, and13C-NMR) as 6-O-[α-D-glcp-(1→4)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol, 6-O-β-D-glcp-20-O-[α-D-glcp-(1→6)-(β-D-glcp)]-20(S)-protopanaxatriol, 6-O-β-D-glcp-20-O-[α-D-glcp-(1→4)-(β-D-glcp)]-20(S)-protopanaxatriol, 6-O-[α-D-glcp-(1→6)-β-D-glcp]-20-O-(β-glcp)-20(S)-protopanaxatriol, 6-O-[α-D-glcp-(1→3)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol, and 6-O-β-D-glcp-20-O-[α-D-glcp-(1→3)-(β-D-glcp)]-20(S)-protopanaxatriol. Among these six, 6-O-β-D-glcp-20-O-α-D-glcp-(1→6)-(β-D-glcp)-20(S)-protopanaxatriol and 6-O-α-D-glcp-(1→6)-β-D-glcp-20-O-(β-D-glcp)-20(S)-protopanaxatriol

Appl Biochem BiotechnolDOI 10.1007/s12010-015-1847-0

Ramya Mathiyalagan and Young-Hoi Kim contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1847-0)contains supplementary material, which is available to authorized users.

* Yeon Ju [email protected]

* Deok Chun [email protected]

1 Graduate School of Biotechnology and Ginseng Bank, College of Life Science, Kyung HeeUniversity, Yongin 446-701, Republic of Korea

2 Department of Food Science and Biotechnology, Chonbuk National University, Iksan 570-752,Republic of Korea

3 Department of Oriental Medicinal Biotechnology, College of Life Science, Kyung Hee University,Yongin 449-701, Republic of Korea

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are considered to be novel compounds of alpha-ginsenosidal saponins which pharmacologicalactivities should be further characterized. This is the first report on the enzymatic elaborationof ginsenoside Rg1 derivatives using rat intestinal homogenates. To the best of our knowledge,it is also the first to reveal the sixth and 20th positions of an unusual α-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl sugar chain with 20(S)-protopanaxatriol saponins in Panax ginsengMayer.

Keywords Transglycosylation . Rat intestinal homogenates . Alpha glucosidase . Alphaglucosylginsenoside . Rg1 derivatives . Ginsenoside Rg1

Introduction

Korean ginseng (Panax ginseng Meyer) is a traditionally known medicinal plant highly usedin Oriental nations. It contains more than 124 dammarane-type triterpene glycosides, which arecalled ginsenosides. These ginsenosides are grouped into protopanaxadiol- (ppd) andprotopanaxatriol (ppt)-type saponins based on their sugar and linkage positions, which areresponsible for various efficacies [2, 19]. Even though β-linkage glucose is present in the ppd-type ginsenosides F2, CK, and Rh2 and the ppt-type ginsenosides Rg1, F1, and Rh1, variationin their types and linkage positions produces different pharmacological activities [12].Ginsenoside Rg1 is one of the major, abundant ginsenosides in Korean ginseng and has beenconfirmed as an active component in angiogenesis inducers [23]. It has been reported topossess various pharmacological efficacies, including improvement of memory and liverfunctions, as well as anti-fatigue and anti-stress properties [2].

Various structurally diversified compounds are effectively produced by glycosylation,which is nature’s way of extending the pharmacological effects of biomolecules. A highregioselective glucosylation is efficiently achieved using an enzymatic method with a simplereaction process rather than a complicated chemical method. Glycoside hydrolase (GH) andglycosyltransferase (GTs) enzymes are mainly utilized for the synthesis of complex oligosac-charides and glycoconjugates [3, 5].

The attractiveness of natural glycosylated saponins for drug development is due to theirpharmacological properties; however, the limitation acquiring pure compounds hindered thisresearch progress. An enzymatic method can be an efficient technique to overcome theseproblems [1]. The formation of linkage bonds by enzymatic transglycosylation (transfer of aglucose molecule from a donor to the acceptor molecule) of active substances to enhance theirphysiological functions and properties on phenolic compounds [22], vitamins [10], sugars, andnatural glycosides has gained special attention. Various reports have been published on theglycosylation of steroidal saponins and ginseng saponins by cyclodextrin glycosyltransferase(CGTase) [6], toruzyme [26], and glycosidase [4, 7].

It was reported that rat intestinal homogenates contain high alpha glucosidase activity,which has highly specific transglycosylation efficiency in forming stable L-ascorbic acid α-glucoside (AA-2G) molecules with maltose as a substrate. This transglycosylation is distinctfrom that of microbial α-glucosidase [11, 24, 25].

In this study, we aimed to synthesize unusual or alpha-glycosylated ginsenoside compoundsby enzymatic transglycosylation of rat intestinal homogenates, which have high α-glucosidaseactivity. In this study, six transglycosylated compounds from ginsenoside Rg1 were identified.The new Rg1 derivatives (compounds P1–P6) were purified by column chromatography. Their

Appl Biochem Biotechnol


molecular weights were identified by negative electrospray ionization/high-performance liquidchromatography-mass spectrometry (ESI-HPLC-MS), and structures of P-1 to P-6 werecharacterized by 1H nuclear magnetic resonance (NMR) and 13C NMR. The six α-D-glucosylated Rg1 derivatives were confirmed. To the best of our knowledge, this is the firstreport on the enzymatic elaboration of ginsenoside Rg1 derivatives using rat intestinalhomogenates.

Materials and Methods

Materials

Ginsenoside Rg1 was isolated from dried P. ginseng roots by the usual procedure [16], and thepurified compound was identified by comparing the spectral data and retention time of high-performance liquid chromatography (HPLC) with that of an authentic sample. Maltose,isomaltose, alpha cyclodextrin, dextrin, oyster glycogen, cellobiose, glucose, Folin phenolreagent, and glucose assay kits (GAHK20-1KT) were purchased from Sigma-Aldrich Co. (StLouis, MO, USA). Silica gel (70–230 mesh) and silica gel 60 F254 TLC plates, methanol-d4,and trimethylsilane were obtained from Merck Co. (Darmstadt, Germany). Acetonitrile anddistilled water for HPLC were purchased from J. T. Baker Co. (PA, USA). All other reagentswere analytical grade.

Preparation of Rat Intestinal Homogenates

Male Sprague-Dawley rats weighing 230–250 g were supplied from the animal laboratory ofthe KT&G Research Institute (Daejeon, Korea). The animals were sacrificed by cardiacbleeding under ether anesthesia. Small and large intestinal regions were separately removed,rinsed with cold saline, and homogenized in four volumes of 0.1 M potassium phosphatebuffer (pH 7.0). The homogenates were filtered through four layers of gauze to removeparticles, and the filtrates obtained were centrifuged at 4000g for 10 min; the obtainedsupernatants were used as enzyme sources.

α-Glucosidase Assay

α-Glucosidase activity was assayed with slight modifications [11]. Briefly, 1-ml reactionmixtures contained 500 μl of 4 % maltose solution in a 100 mM acetate buffer (pH 6.0),1.35 mM ethylene diamine tetraacetic acid (EDTA) solution and 40 μl of enzyme solution, anda 100 mM sodium acetate buffer (pH 6.0). The mixtures were incubated at 37 °C for 30 min.The amount of liberated glucose was determined by the glucose oxidase-peroxidase methodusing the glucose assay kit. One unit of α-glucosidase activity was defined as the amount ofenzyme that hydrolyzed 1 μmole of maltose per min. The protein content was assayed usingbovine serum albumin as standard [8].

Transglycosylation Reaction

The 0.3-mL reaction mixture was composed of 13.8 mg maltose, 13.8 mg G-Rgl, an enzymesolution containing α-glucosidase (0.52 units), and a 0.1 M acetate buffer (pH 5.3), and it was



incubated at 50 °C for various time intervals in the dark with gentle shaking. An aliquot(200 μl) was withdrawn and mixed with 800 μl of methanol to terminate the reaction, and theformation of G-Rgl-α-glucosides was monitored by thin-layer chromatography (TLC) andHPLC. The transglycosylation ratio was calculated by interpolating the peak area (%) of G-Rg1-α-glucosides after the enzyme reaction.

Thin-Layer Chromatography

TLC was performed on silica gel 60 F254 plates with a solvent system of chloroform-methanol-water (65:35:10, v/v, lower phase). Spots were detected by heating the solution at 110–120 °Cfor 10 min after spraying it with 10 % sulfuric acid in ethanol. HPLC was carried out with aWaters Nova-pak C18 (3.9 mm × 300 mm) column using a solvent system of acetonitrile-water (19:81, v/v) at a flow rate of 1.0 mL/min and detection at 203 nm.

Acid Hydrolysis of Newly Synthesized Compounds

G-Rg1 and its α-glucosyl compounds were hydrolyzed in 50 % aqueous acetic acid at 70 °Cfor 2 h, neutralized by the addition of Na2CO3 solution, and extracted with water-saturated n-butanol. The concentrated samples were subjected to TLC with a comparison of the Rf valueof the Rg1 standard.

Instrumental Analysis

A Fisher-Johns melting point apparatus was used to measure melting points. HPLC-MS(negative ESI mode) was performed using an Agilent model 1100 mass spectrometer. NMRspectra were measured with a Bruker AMX 400 (1H-NMR, 400 MHz; 13C-NMR, 100 MHz)with Pyridine-d5 as the solvent and tetramethylsilane as the internal standard.

Results

Transglucosylation is the formation of a glycosidic bond by the transfer of a glycosyl moleculefrom the donor molecule to the acceptor. In this study, we attempted to synthesize newtransglycosylation products of α-glucosidase with maltose and ginsenoside Rg1 using ratintestinal homogenates, which show high α-glucosidase activities (Supplementary Fig. S1).

Transglycosylation of Rg1 Using Rat Intestinal Homogenates

The ginsenoside Rg1 was used as an acceptor, while maltose was employed as a substrate forrat intestinal homogenates. The mixture was reacted for different time points and extractedwith n-butanol, and a series of new spots was detected with lower Rf values of Rg1 in TLC(Fig. 1). The new spots were not present in the control mixture, which included Rg1 andmaltose without enzyme homogenates. This result indicates that the new spots were related tothe transglycosylated compounds. The hydrolysis of maltose and the release of glucosegradually increased, and maltose completely disappeared after 20 h. The appearances of someof the new spots (G-Rgl-α-glucosyl compounds) gradually increased, whereas some of themdisappeared with the increase in incubation time (Fig. 1). This finding indicates that the newly



synthesized products were hydrolyzed again by rat intestinal α-glucosidase. Therefore, itseems that the available maltose and glucose concentrations were important factors intransglycosylation.

Isolation and Identification of Transglycosylated Compounds

For a semi-preparative synthesis of G-Rg1-α-glucosides, rat small intestinal homogenatescontaining α-glucosidase (92 units) were incubated with a mixture that consisted of 1.4 g ofmaltose, 1.4 g of (G)-Rg1, and a 0.1-M sodium acetate buffer (pH 5.3) to create a final volumeof 30 mL at 50 °C in a dark environment with gentle stirring for different times. Atcorresponding time intervals, the samples were withdrawn and heat treated in a boiling waterbath for 10 min. The reaction mixture was extracted with water-saturated n-butanol and thenconcentrated to dryness in a vacuum. The transglycosylated products were isolated using silicagel column chromatography with a solvent system of chloroform-methanol-water (65:35:10,v/v, lower phase), and further separation and purification were achieved by repeated prepHPLC using a Nova-pak C18 column. The four spots detected by TLC and HPLC showed sixindividual peaks (Peaks 1–6) (Fig. 2). The isolated compounds were further characterized byNMR.

Structure Determination of the Compounds

Compound P-1 is a white amorphous powder; mp 210–212 °C; m/z 961.7 [M–H]; m/z 1007.6[M + HCOO]. Using 1H NMR, three anomeric proton signals attributed to two β-glycosidiclinkages at 4.90 ppm (1H, d, J = 7.76 Hz) and 5.18 ppm (1H, d, J = 7.76 Hz) and one α-glucosidic linkage at 5.90 ppm (1H, d, J = 3.78 Hz) were identified, whereas ginsenoside Rg1showed two anomeric proton signals due to two β-glycosidic linkages at 5.03 ppm (1H, d,J = 7.81 Hz) and 5.18 ppm (1H, d, J = 7.71 Hz). The molecular structures of the glycosylationproducts were determined using 13C NMR. The 13C NMR chemical shifts of compound P-1were compared with ginsenoside Rg1. As shown in Table 1, two anomeric carbon signals

Fig. 1 TLC chromatogram oftransglycosylated productssynthesized from maltose andginsenoside Rg1 (G-Rg1) by ratsmall intestinal homogenate. S-Isynthesized product 1, S-II syn-thesized product 2



attributed to two β-glycosidic linkages were identified at 105.5 and 98 ppm, along with oneadded anomeric carbon signal at 102.9 ppm, indicating that the glycosidic linkage was in an α-configuration. The C4’ signal of the β-glucose moiety linked at the C6-hydroxyl group ofaglycone was moved downfield by 9.4 ppm, while that of C3’ was shifted upfield by 0.4 ppm.No other significant shifts were observed in the carbon signals of the β-glucose moiety linkedat the C20-hydroxyl group of aglycone. It is known that acetic acid treatment (50 % aqueous)of ginsenosides leads to rapid hydrolysis and is accompanied by the consequent epimerizationof the C20-hydroxyl group in the 20-O-glycosyl moiety of the aglycone, producing 20 (R &S)-prosapogenin andΔ20-prosapogenin [6]. The 50 % acetic acid hydrolysis leads to productswith lower Rf values than those from ginsenoside Rg1 (Fig. 3a). Figure 3b implies that α-glucosylation was bonded to the C-4’ position in the glucose moiety linked at the C6-hydroxylgroup of aglycone. Therefore, compound P-1 was identified as 6-O-[α-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl]-20-O-(β-D-glucopyranosyl)-20 (S)-protopanaxatriol. The proposedchemical structure is shown in Fig. 4.

Fig. 2 HPLC chromatograms of transglycosylated products (peaks 1–6) formed from maltose and G-Rgl by ratsmall intestinal homogenate. S-I synthesized product 1, S-II synthesized product 2



Table 1 13C-NMR chemical shifts of transglycosylated products from maltose and G-Rg1 by rat small intestinalhomogenates

Carbon number G-Rg1 P-1 P-2 P-3 P-4 P-5 P-6

Aglycone moiety

1 39.4 39.5 39.5 39.3 39.3 39.2 39.1

2 27.9 27.7 27.7 27.8 27.9 27.7 27.7

3 78.5 78.7 78.4 78.5 78.7 78.4 78.0

4 40.3 40.2 40.2 40.2 40.2 40.1 40.2

5 61.3 61.1 61.2 61.2 61.5 61.2 61.2

6 79.2 75.3 79.0 80.0 79.5 80.2 80.0

7 45.1 44.7 45.0 45.0 45.8 44.9 45.0

8 41.1 40.8 41.0 41.0 41.3 41.0 41.0

9 49.9 49.8 49.8 49.8 50.1 49.8 49.7

10 39.6 39.4 39.5 39.5 39.5 39.5 39.4

11 30.9 30.7 30.5 30.8 30.9 30.7 30.7

12 70.1 71.4 70.1 70.0 70.2 70.0 70.1

13 49.1 49.0 48.8 49.0 49.2 49.0 48.8

14 51.3 51.3 51.2 51.2 51.4 51.1 51.2

15 30.6 30.4 30.4 30.4 30.8 30.4 30.5

16 26.6 26.4 26.3 26.4 26.7 26.4 26.4

17 51.4 51.2 51.4 51.2 51.6 51.3 51.7

18 17.6 17.6 17.4 17.4 17.5 17.3 17.4

19 17.6 17.6 17.4 17.4 17.5 17.3 17.4

20 83.1 83.0 83.2 83.3 83.4 83.1 83.4

21 22.3 22.1 22.3 22.0 22.2 22.1 22.2

22 36.1 36.0 35.7 36.0 35.9 36.0 36.0

23 23.2 23.0 23.1 23.0 23.1 23.0 23.1

24 126.0 126.0 126.0 125.7 126.0 125.8 125.7

25 130.9 130.7 130.8 130.8 130.9 130.7 130.8

26 25.7 25.6 25.6 25.6 25.7 25.6 25.6

27 17.7 17.6 17.7 17.6 17.8 17.6 17.6

28 31.7 31.6 31.6 31.6 31.6 31.5 31.6

29 16.4 16.1 16.2 16.2 16.6 16.1 16.2

30 17.2 17.0 16.9 17.0 17.3 17.0 16.8

Carbon no G-Rg1 P-1 P-2 P-3 P-4 P-5 P-6

6-β-glc

1’ 106.0 105.5 105.6 105.8 106.1 105.5 105.0

2’ 75.3 75.3 75.3 75.3 75.4 73.5 75.4

3’ 79.5 79.1 79.5 71.6 79.3 89.1 79.5

4’ 71.7 81.1 71.8 71.7 71.6 70.7 71.8

5’ 80.0 78.7 80.2 78.0 76.5 77.4 78.0

6’ 62.7 62.7 62.3 63.0 69.3 62.6 63.0

6-α-glc(transferred)

1” – 102.9 – – 101.5 102.0 –

2” – 74.7 – – 74.1 74.5 –

3” – 75.1 – – 75.2 75.4 –



Compound P-2 is a white amorphous powder; mp 14–217 °C;m/z 961.7 [M-H];m/z 1007.3[M + HCOO]. Using 1H NMR, three anomeric proton signals due to two β-glycosidic linkagesat 5.03 ppm (1H, d, J = 7.75 Hz) and 5.10 ppm (1H, d, J = 7.62 Hz) and one α-glucosidiclinkage at 5.46 ppm (1H, d, J = 3.37 Hz) were identified. 13C NMR spectra of compound P-2were related to ginsenoside Rg1 (Table 1). 13C NMR spectrum showed an additional anomericcarbon signal at 100.3 ppm due to an α-glucosidic linkage together with two anomeric carbonsignals from two β-glycosidic linkages at 105.6 and 97.9 ppm. The C6’ signal of the β-glucosemoiety linked at the C20-hydroxyl group of aglycone was shifted downfield 5.2 ppm, whilethat of C5’ was shifted upfield by 2.8 ppm. The β-glucose moiety linked at the C6-hydroxylgroup of aglycone carbon signals was unaffected (Table 1). The TLC pattern of 50 % aceticacid hydrolysate of compound P-2 was different from that of ginsenoside Rg1 (Fig. 3a). Theseresults demonstrated that the α-glucosylation site was present at C-6’ in the glucose moietylinked at the C20-hydroxyl group of aglycone (Fig. 3b). Therefore, compound P-2 wasidentified as 6-O-β-D-glucopyranosyl-20-O-[α-D-glucopyranosyl-(1→6)-(β-D-glucopyranosyl)]-20(S)-protopanaxatriol. The structure of P-2 is shown in Fig. 4.

Compound P-3 is a white amorphous powder; mp 218–220 °C; m/z 961.4 [M-H]; m/z1007.3 [M + HCOO]. Three anomeric proton signals of two β-glycosidic linkages at5.03 ppm (1H, d, J = 7.78 Hz) and 5.18 ppm (1H, d, J = 7.72 Hz) and one α-glucosidiclinkage at 5.88 ppm (1H, d, J = 3.95 Hz) were predicted by 1H NMR. 13C NMR spectrawere compared with ginsenoside Rg1 (Table 1). The 13C NMR spectrum indicated anadditional anomeric carbon signal at 102.5 ppm due to an α-glucosidic linkage, togetherwith two anomeric carbon signals of two β-glycosidic linkages at 105.8 and 98.0 ppm.The C4’ signal of the β-glucose moiety linked at the C20-hydroxyl group of aglyconewas moved downfield 9.5 ppm, while that of C3’ was shifted upfield by 0.5 ppm. The

Table 1 (continued)

Carbon number G-Rg1 P-1 P-2 P-3 P-4 P-5 P-6

4” – 71.7 – – 72.2 71.7 –

5” – 75.0 – – 74.3 74.1 –

6” – 62.6 – – 62.9 62.3 –

20-β-Glc

1”’ 98.2 98.0 97.9 98.0 98.2 98.0 98.0

2”’ 75.0 75.2 75.1 75.1 75.1 75.0 73.3

3”’ 78.0 79.1 78.0 78.5 79.3 80.2 88.3

4”’ 71.5 71.5 71.6 81.0 71.6 71.4 70.5

5”’ 78.1 78.4 75.3 76.4 78.2 78.1 77.5

6’” 63.0 62.7 68.2 62.9 62.7 62.3 62.3

20-α-Glc(transferred)

1”” – – 100.3 102.5 – – 101.8

2”” – – 73.8 74.3 – – 74.5

3”” – – 75.3 75.1 – – 75.4

4”” – – 71.7 71.7 – – 71.6

5”” – – 74.0 74.2 – – 74.1

6’”’ – – 62.7 62.5 – – 62.3

G-Rg1 ginsenoside Rg1, β-glc β-glucopyranosyl, α-glc α-glucopyranosyl



β-glucose moiety linked at the C6-hydroxyl group of aglycone carbon signals wasunaffected (Table 1). Figure 3a shows that the TLC pattern of 50 % acetic acidhydrolysate of compound P-3 was similar to that of compound P-2. This findingindicates that an α-glucosylation site was present at C-4’ in the glucose moiety linkedat the C20-hydroxyl group of aglycone, as shown in Fig. 3b. Consequently, compound P-

Fig. 3 a Degradation of six transglycosylated products (HPLC peaks 1–6) under mildly acidic conditions. (a)St&ard (G-Rg1); (b) TLC spot S-I (mixture of HPLC peaks 1, 3, 5, and 6); (c), TLC spot S-II (mixture of HPLCpeaks 2 and 4); (e), G-Rh1; (f), G-Rg2. b Degradation of six transglycosylated products (HPLC peaks 1–6) undermildly acidic conditions

Fig. 4 Proposed structures of six G-Rg1-α-glucosides (HPLC peaks 1–6) formed from maltose and ginsenosideRg1 by rat intestinal α-glucosidases



3 was identified as 6-O-β-D-glucopyranosyl −20-O-[α-D-glucopyranosyl]-(1→4)-(β-D-glucopyranosyl)-20(S)-protopanaxatriol (Fig. 4).

Compound P-4 is a white amorphous powder; mp 199–201 °C; m/z 961.3 [M-H]; m/z10,073 [M + HCOO]. With 1H NMR, three anomeric proton signals due to two β-glycosidic linkages at 4.94 ppm (1H, d, J = 7.73 Hz) and 5.17 ppm (1H, d, J = 7.82 Hz)and one α-glucosidic linkage at 5.52 ppm (1H, d, J = 3.74 Hz) were identified. The 13CNMR chemical shifts of compound P-4 were compared with ginsenoside Rg1 (Table 1).The 13C NMR spectrum showed an additional anomeric carbon signal at 101.5 ppm,indicating that the glycosidic linkage was in an α-configuration. Along with this, the twoanomeric carbon signals were due to two β-glycosidic linkages at 106.1 and 98.2 ppm.The C6’ signal of the β-glucose moiety linked at the C6-hydroxyl group of aglycone wasshifted downfield 6.6 ppm, while that of C5’ was shifted upfield by 3.5 ppm. The β-glucose moiety linked at the C20-hydroxyl group of aglycone carbon signals wasunaffected (Table 1). The TLC pattern of 50 % acetic acid hydrolysate of compoundP-4 was similar to that of compound P-1, as shown in Fig. 3a. The α-glucosylation sitewas present at C-6’ in the glucose moiety linked at the C6-hydroxyl group of aglycone(Fig. 3b). Therefore, compound P-4 was identified as 6-O-[α-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]-20-O-(β-D-glucopyranosyl)-20(S)-protopanaxatriol, and itscorresponding structure is shown in Fig. 4.

Compound P-5 is a white amorphous powder; mp 208–210 °C; m/z 961.7 [M-H]; m/z1007.7 [M + HCOO]; three anomeric proton signals due to two β-glycosidic linkages at4.94 ppm (1H, d, J = 7.66 Hz) and 5.19 ppm (1H, d, J = 7.68 Hz) and one α-glucosidic linkageat 5.92 ppm (1 H, d, J = 3.80 Hz) were identified by 1H NMR. Chemical shifts of compoundP-5 were compared with ginsenoside Rg1 by 13C NMR (Table 1), which showed an additionalanomeric carbon signal at 102.0 ppm, a typical sign of an α-glucosidic linkage, along with twoanomeric carbon signals due to two β-glycosidic linkages at 105.5 and 98.2 ppm. The C3’signal of the β-glucose moiety bonded at the C6-hydroxyl group of aglycone was shifteddownfield 9.6 ppm. The β-glucose moiety linked at the C20-hydroxyl group of aglyconecarbon signals was unaffected (Table 1). Figure 3a demonstrates that the TLC pattern of theacid hydrolysate of compound P-5 with 50 % acetic acid was similar to that of compound P-1.The α-glucosylation site was present at C-3’ in the glucose moiety linked at the C6-hydroxylgroup of aglycone (Fig. 3b). Therefore, compound P-5 was identified as 6-O-[α-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl]-20-O-(β-D-glucopyranosyl)-20(S)-protopanaxatriol (Fig. 4).

Compound P-6 is a white amorphous powder; mp 220–223 °C; m/z 961.4 [M-H]; m/z1007.3 [M + HCOO]. 1H NMR spectra showed three anomeric proton signals: two β-glycosidic linkages at 5.02 ppm (1H, d, J = 7.73 Hz) and 5.13 ppm (1H, d, J = 7.72 Hz) andone α-glucosidic linkage at 5.92 ppm (1H, d, J = 3.86 Hz). 13C NMR chemical shifts ofcompound P-6 were compared with ginsenoside Rg1 (Table 1). The 13C NMR spectrumexhibited two anomeric carbon signals of two β-glycosidic linkages at 105 and 98.0 ppm.In addition, another anomeric carbon signal at 101.8 ppm was identified as a result of anα-glucosidic linkage. The C3’ signal of the β-glucose moiety linked at the C20-hydroxylgroup of aglycone was moved downfield 10.3 ppm. The β-glucose moiety linked at theC6-hydroxyl group of aglycone carbon signals remained unchanged (Table 1). The 50 %acetic acid hydrolysate of compound P-6 was the same as that of compound P-3, as shownin the TLC results of Fig. 3a, denoting that α-glucosylation occurred at the C-3’ positionin the glucose moiety linked at the C20-hydroxyl group of aglycone (Fig. 3b). As a result,



compound P-6 was recogn ized as 6-O -β -D -g lucopyranosy l -20-O - [α -D -glucopyranosyl-(1→3)-(β-D-glucopyranosyl]-20(S)-protopanaxatriol, the structure ofwhich is shown in Fig. 4.

Substrate Specificity of the Rat Intestinal Homogenates

The substrate specificity of rat intestinal homogenates for the formation of transglycosylatedproducts was analyzed by HPLC. Table 2 shows that maltose, dextrin, oyster glycogen, andisomaltose acted as a good substrate for these homogenates, whereas α-cyclodextrin, cellobi-ose, and glucose were not utilized as a substrate for the implementation of glycosylation. Inaddition, G-Rg1 was used as the acceptor for transglycosylation product formation. It has beenreported that rat small intestinal homogenates highly favor maltose over other substrates [11].Various reports have shown that α-glucosidase can form α-1, 4; α-1, 3; and α-1, 6 linkages byutilizing maltose as a substrate [13, 21]. The optimization of pH on the transglycosylationreaction was carried out with rat small intestinal homogenates. pH values from 4.8 to 6.8showed higher transglycosylation ratios, with pH 5.3 exhibiting the highest transglycosylationratio, as shown in Supplementary Fig. 2. The transglycosylation ratio was expressed as thecombined total peak area (%) of all newly formed peaks (transglycosylated products) deter-mined by HPLC.

Comparisons of Transglycosylation Efficiency of Small and Large IntestineHomogenates

The transglycosylation efficiency of the formation of newly synthesized individual compoundswas analyzed with small and large intestine homogenates, respectively. Digestive enzymes inboth small and large intestines were reported [9, 15]; the two homogenates were incubatedwith maltose at different time points and analyzed by HPLC. As shown in Fig. 5, a higherpercentage of transglycosylated products was formed by the small intestine (Fig. 5a) than thelarge intestine (Fig. 5b). In addition, the transglycosylated product formation increased withincubation time (h) and declined at 24 h in small intestine homogenates (Fig. 5a), whereas itgradually increased over time in the large intestine (Fig. 5b). Transglycosylation ratio wasexpressed as peak area (%) by HPLC.

Table 2 Effects of sugar donors on the formation of G-Rg1-α-glucosides by rat small intestinal homogenates

Sugar donors Transglycosylation ratio1 G-Rg1

P-1 P-2 P-3 P-4 P-5 P-6

Maltose 8.4 3.4 4.5 0.9 4.0 4.7 74.7

Dextrin 4.6 6.4 4.9 4.1 4.4 4.7 70.9

Oyster glycogen 4.4 6.8 4.7 4.5 4.1 4.3 71.2

Isomaltose 0 12.3 0 10.0 0 0 77.7

α-Cyclodextrin 0 0 0 0 0 0 100

Cellobiose 0 0 0 0 0 0 100

Glucose 0 0 0 0 0 0 100

1 The transglycosylation ratio was expressed as peak area (%) of each transglycosylation product by HPLC



Discussion

The enzymatic formation of glycosidic linkages by glucosylation is mainly accomplished byglycosyl hydrolase (GH) and glycosyltransferase (GTs) enzymes. Even though GT enzymesare used for synthetic purposes, the complex, expensive donation process and very stringentsubstrate specificity were the limitations for using GTs in this study, whereas GH-basedtransglycosylation has an advantage over GT with its easily available donors and relaxedsubstrate specificity. However, a limited number of transglycosylation products and producthydrolysis are the limitations of GH [23]. The GH enzyme catalyzes both hydrolysis andtransglycosylation reactions. The transglycosylation reaction is a mechanism for glycosidicbond formation in which a glycosyl molecule from a donor (substrate molecule) is transferredto a hydroxyl group of an acceptor molecule to form a new glycoside [21]. There are severalreports of transglycosylation reactions of α-glucosidase [13, 24, 25]. Our results also indicatedthat rat intestinal homogenates exhibited both hydrolytic (maltose was hydrolyzed into glucoseas in Fig. 1) and transferase activity (newly synthesized spots), similar to previous reportsabout α-glucosidase [13, 21].

The pharmacological efficacies of each dammarane triterpenoid (ginsenoside) vary accord-ing to their mono-, di-, or tri-glycosylation [12, 17]. However, the isolation and synthesis ofnatural and non-natural derivatives have received great attention in the attempt to understandand obtain better pharmacologically active drugs. In this study, we found that six new saponinswere synthesized from Rg1. Compounds 6-O-β-D-glcp-20-O-[α-D-glcp-(1→4)-β-D-glcp]-20(S)-protopanaxatriol (Re3), 6-O-[α-D-glcp-(1→3)-β-D-glcp]-20-O-β-D-glcp-20(S)-protopanaxatriol (Re2), and 6-O-β-D-glcp-20-O-[α-D-glcp-(1→3)-β-D-glcp]-20(S)-protopanaxatriol (Re1) were isolated from the root of Panax ginseng [27], and 6-O-[α-D-glcp-(1→4)-β-D-glcp]-20-O-β-D-glcp-20(S)-protopanaxatriol was synthesized by the enzy-matic transglycosylation of cyclomaltodextrin glucanotransferases [6], whereas compounds6-O-β-D-glcp-20-O-[α-D-glcp-(1→6)-β-D-glcp]-20(S)-protopanaxatriol and 6-O-[α-D-glcp-(1→6)-β-D-glcp]-20-O-β-D-glcp-20(S)-protopanaxatriol are considered to be novel

Fig. 5 Time course of G-Rg1-α-glucoside formation by transglycosylation of rat small and large intestinalhomogenates with maltose as the donor. a Small intestinal homogenate, b large intestinal homogenate. 6G-4: 6-O-〔α-D-glcp-(1→4)-β-D-glcp〕-20-O-(β-D-glcp)-20(S)-protopanaxatriol; 20G-6: 6-O-β-D-glcp-20-O-〔α-D-glcp-(1→6)-(β-D-glcp)〕-20(S)-protopanaxatriol; 20G-4: 6-O-β-D-glcp-20-O-〔α-D-glcp-(1→4)-(β-D-glcp)〕-20(S)-protopanaxatriol; 6G-6: 6-O-α-D-glcp-(1→6)-β-D-glcp〕-20-O-(β-D-glcp)-20(S)-protopanaxatriol; 6G-3:6-O-α-D-glcp-(1→3)-β-D-glcp〕-20-O-(β-D-glcp)-20(S)-protopanaxatriol; 20G-3: 6-O-β-D-glcp-20-O-〔α-D-glcp-(1→3)-(β-D-glcp)〕-20(S)-protopanaxatriol



ginsenosidal saponins with potential activity and should be further characterized. These newlysynthesized compounds were unique from β-glycosylated ginsenosides. Even though βisomers of glucose are common in saponins, α isomers of glucose also play a significant rolein the specific function of the saponins, such as the 100-fold increased solubility, decreasedbitter taste, and enhanced slight sweet taste of (+)-catechin 3’-O-α-D-glucopyranoside (α-C-G)compared to that of (+)-catechin [18], and the stronger inhibitory activity of 4-hydroxyphenyl-α-glucopyranoside (α-arbutin) on tyrosinase from human malignant melano-ma cells than 4-hydroxyphenyl-β-glucopyranoside (arbutin) [20].

Enzymatic galactosylation of ginsenoside Rg1 and its cellobiosyl derivatives with betalinkages have also been reported [4]. Transglycosylation of Rg1 by cyclomaltodextringlucanotransferases was specific at the C3’- and C4’-hydroxyl groups of the glucose moietylinked at the C6-hydroxyl group of aglycone [6], whereas rat intestinal homogenates producedglycosylation activity of the glucose moiety linked at the C20-hydroxyl group of aglycone inour study. In addition, α-D-glcp-(1→3)-β-D-glcp and α-D-glcp-(1→4)-β-D-glcp sugar chainson the C20-hydroxyl group in Rg1 were reported [27], whereas ours is the first report of theunusual α-D-glcp-(1→6)-β-D-glcp sugar chain.

The new six Rg1 derivatives presented here are distinct from each other by their linkagepositions. Although new conjugates of L-ascorbic acid (AA) were synthesized bytransglycosylation of α-glucosidase from Aspergillus niger (6-O-a-D-glucopyranosyl-L-ascor-bic acid; AA-6G) and rat intestinal homogenates (2-O-a-D-glucopyranosyl-L-ascorbic acid;AA-2G) [11], AA-2G was much more stable and non-reducible than AA-6G in aqueoussolutions [24]. Therefore, regioselective glucosylation with specific linkage and glucosylationpositions may determine the efficacy of new ginsenosides.

Glycosylation offers considerably enhanced solubility, increases sweetness [18], and de-creases the bitterness of ginsenosides [6]. Similarly, our newly synthesized compounds are alsoexpected to increase solubility and decrease bitterness compared with that of the source Rg1.

Many reports are available regarding the PPT-type saponins with the usual form of sugarmoieties, such as β-D-glucopyranosyl, α-L-rhamnopyranosyl, and β-D-xylopyranosyl [14].However, only a few reports have examined the unusual form of α-D-glucopyranosyl sugarlinked with β sugar in ginsenosides [6, 27]. The present study will provide additional supportfor the use of α-D-glucopyranosyl sugar in P. ginseng saponins. Also, future studies will needto consider di- and tri-transglycosylated molecules to verify our results.

Ginsenoside Rg1 is one of the major saponins. It is present in a comparatively higheramount in P. ginseng and is noted for various efficacies. Similarly, the newly synthesizedginsenoside Rg1-based derivatives are expected that they may have potential pharmacologicalefficacies, including angiogenesis and wound healing, skin cell healing, neuroprotectiveeffects, effects of inflammatory diseases, memory enhancing functions, liver functions, andanti-fatigue and anti-stress properties.

Conclusion

Ginsenosides have been extensively observed to convert into minor ginsenosides by thehydrolysis of glucose using glucosyl hydrolase enzyme [14]. In our study, we attempted toattach a specific α isomer of glucose with ginsenosides by a transglycosylation reaction of theglycosyl hydrolase enzyme. In our study, six alpha-glucopyranoside ginsenoside Rg1 deriva-tives were synthesized by rat intestinal homogenates and purified, and NMR was used to



identify these structures. The additional support of regioselective glucosylation with α-isomerwas achieved using an enzymatic method. Diverse novel saponins and higher yields of purifiedcompound may be achieved using this method. However, the pharmacokinetic behaviors ofthe newly synthesized compounds remain to be thoroughly addressed.

Acknowledgments This research was supported by Korea Institute of Planning & Evaluation for Technology inFood, Agriculture, Forestry & Fisheries (KIPET NO: 309019-03-3-SB010) and Next-Generation BioGreen 21Program (SSAC, grant#: PJ00952903), Republic of Korea.

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Enzymatic Formation of Novel Ginsenoside Rg1-α-Glucosides by Rat Intestinal Homogenates