Rebaudiosides R and S, Minor Diterpene Glycosides from the Leaves

Apr 27, 2016 - However, a few minor compounds with α-glucosyl linkages have been recently described.(9-11) Steviol glycosides with rhamnose, xylose, ...
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Rebaudiosides R and S, Minor Diterpene Glycosides from the Leaves of Stevia rebaudiana Mohamed A. Ibrahim,†,‡ Douglas L. Rodenburg,† Kamilla Alves,† Wilmer H. Perera,† Frank R. Fronczek,§ John Bowling,⊥ and James D. McChesney*,† †

Ironstone Separations, Inc., Etta, Mississippi 38627, United States Department of Chemistry of Natural Compounds, National Research Center, Dokki 12622, Cairo, Egypt § Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, United States ⊥ Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, United States ‡

S Supporting Information *

ABSTRACT: Two new diterpene glycosides have been isolated from a commercial extract of the leaves of Stevia rebaudiana. Compound 1 was shown to be 13-[(2-O-β-Dglucopyranosyl-3-O-β-D-glucopyranosyl-β-D-xylopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester (rebaudioside R), while compound 2 was determined to be 13[(2-O-α-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur16-en-19-oic acid 2-O-α-L-rhamnopyranosyl-β-D-glucopyranosyl ester (rebaudioside S). Six additional known compounds were identified, dulcoside B, 13-[(2-O-β-D-xylopyranosyl-β-Dglucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester, eugenol diglucoside, rebaudioside G, 13-[(2-O-6deoxy-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester, and rebaudioside D (3), respectively. The structures of 1 and 2 were determined based on comprehensive 1D and 2D NMR (COSY, HSQC, and HMBC) studies. A high-quality crystal of compound 3 allowed confirmation of its structure by X-ray diffraction.

As evidenced by the presence of numerous steviol glycosides, the plant has biosynthetic machinery capable of producing several different diterpene glycosides with potential sweet tastes. Cultivars of S. rebaudiana have been selected for enhanced production of selected glycosides and are now grown commercially in mainland China, Japan, Korea, India, and elsewhere and extracts prepared.5 The influence of geographic origin on the chemistry and, thus, the organoleptic properties of these products is being examined.6,7 Several diterpene glycosides containing from one sugar unit up to seven units linked to the ent-kaurene core have been isolated from different sources of S. rebaudiana.8,9 Glucose, rhamnose, xylose, 6-deoxyglucose, and fructose are the most common carbohydrate units attached to the aglycone, mainly at positions C-13 and C-19. Glucose appears frequently linked to the ent-kaurene skeleton through β-linkages. However, a few minor compounds with α-glucosyl linkages have been recently described.9−11 Steviol glycosides with rhamnose, xylose, 6deoxyglucose, and fructose have also been discovered and grouped in the rebaudioside C, rebaudioside F, fructose, and 6-

Stevia rebaudiana (Bertoni) Bertoni is a perennial shrub of the botanical family Asteraceae that grows up to 1 m tall. The plant is native to Brazil and Paraguay,1 where the leaves have been used by indigenous peoples of both countries for their sweet taste. It is now recognized that the diterpene glycosides with an ent-kaurene skeleton are the sweet principles and that rebaudioside A and stevioside are the major compounds of this subfamily of secondary metabolites. The United States Food and Drug Administration granted generally recognized as safe (GRAS) regulatory acceptance of rebaudioside A, commonly known as “reb A”, in 2008 and of steviol glycosides in 2010.2 The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has established a monograph for steviol glycosides, with the latest version issued in 2010.3 The European Union approved steviol glycosides for marketing in November 2011.4 Increased interest in noncaloric natural sweeteners has generated significant attention on glycosides from S. rebaudiana. The GRAS acceptance for rebaudioside A and stevioside has led to their utilization in foods and beverages. However, these two major glycosides have an undesirable lingering aftertaste that has raised interest for the characterization of new and/or minor glycosides from S. rebaudiana. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 18, 2016

A

DOI: 10.1021/acs.jnatprod.6b00048 J. Nat. Prod. XXXX, XXX, XXX−XXX

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deoxyglucose families.9 In all the diterpene glycosides previously isolated, glucose was attached to the main glycosylation sites, C-13 and C-19.9 However, our group has recently described a minor diterpene glycoside with the glycosylation site at position C-12 of the ent-kaurene skeleton.12 In almost all the steviol glycoside derivatives known until now, rhamnose, xylose, 6-deoxyglucose, and fructose are attached through the glucose moiety, mainly at position 2.9 In contrast, very few compounds have been described with a xylose and fructose linked at C-3 and/or C-6 of the glucose.13−15 In our continuing interest to characterize the diterpene glycoside pool from leaves of S. rebaudiana, two new compounds, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-xylopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-Dglucopyranosyl ester (1) and 13-[(2-O-α-D-glucopyranosyl-βD-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid 2-O-α-L-rhamnopyranosyl -β-D-glucopyranosyl ester (2), and six known glycosides were isolated and characterized. This is the first report of the isolation and structure elucidation of a steviol glycoside derivative with a xylose unit directly attached at C-13. We were fortunate to grow a high-quality crystal of compound 3, which allowed the acquisition of X-ray diffraction data and thus the confirmation of its structure through this procedure for the first time.

identified by comparison of the spectroscopic data with literature values. Many trials have been conducted to form high-quality crystals of this group of metabolites for X-ray diffraction processing. In the present investigation, the X-ray data of a crystal of rebaudioside D (3) was used to confirm its structure (Figure 1).

Figure 1. X-ray structure of compound 3, with ellipsoids at the 50% level (Mercury). Solvent molecules and H atoms are not shown.

The 1H and 13C NMR data for compound 1 (Table 1) as well as the high-resolution mass spectrum (m/z 935.4191 for [M − H]−) suggested a molecular formula of C43H68O22 with the presence of three glucose units and one xylose unit. The 3J HMBC correlation between C-13 (87.2 ppm) and H-1′ of Xyl (4.96 ppm) was used to establish the attachment of the xylose unit at C-13. An ESIMS/MS experiment showed an intense fragment at m/z 773 [M − H]− due to the loss of one hexose at C-19 portion (−162 amu). Other fragments were observed at m/z 611 (−162 amu), 449 (−162 amu), and 317 (−132 amu), corresponding to a sequential loss of two hexoses and a pentose unit. That the xylose is directly linked at C-13 was supported by the observation of product ion at m/z 449 (steviol-xylose) (Figure S16, Supporting Information). The 3J HMBC correlation between C-1″ of Glc-1 (105.0 ppm) and H-2′ of Xyl (4.26 ppm) established the attachment of the first glucose unit at C-2′ of Xyl, and the position of H-2′ was confirmed through a COSY correlation between H-2′ (4.26 ppm) and H1′ (4.96 ppm) (Figures S6 and S7, Supporting Information). The 3J HMBC correlation between C-1‴ of Glc-3 (105.1 ppm) and H-3′ of Xyl (4.11 ppm) was used to establish the attachment of the second glucose unit at C-3′ of Xyl, and the position of H-3′ was confirmed through a COSY correlation between H-3′ (4.11 ppm) and H-2′ (4.26 ppm). The 3J HMBC correlation between H-1⁗ of Glc⁗-3 (6.16 ppm) and C-19 (177.3 ppm) was used to establish the position of attachment of the third glucose unit at C-19. The three glucose units and the xylose unit were determined to be D-sugars through comparison with standards using the HPLC method of Tanaka

A continuing effort to isolate minor components from a commercial S. rebaudiana leaf extract has yielded two new glycosides (1 and 2) and the previously reported glycosides, dulcoside B, 13-[(2-O-β-D-xylopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester, eugenol diglucoside, rebaudioside G, 13-[(2-O-6-deoxy-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]entkaur-16-en-19-oic acid β-D-glucopyranosyl ester, and rebaudioside D (3), respectively.8,13,16−19 The 1H and 13C NMR data of the isolated compounds showed a high degree of similarity especially for the signals representing the diterpene core, confirming the presence of the common diterpene ent-kaur-13 hydroxy-16-en-19-oic acid.20 The six known compounds were B

DOI: 10.1021/acs.jnatprod.6b00048 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR and 13C NMR Spectroscopic Data (Pyridine-d5) for Compounds 1 and 2 1a δC, type

δH (J in Hz)

δC, type

0.75 m, 1.77 m

40.1, CH2

41.1, CH2

2

1.42 m, 2.22 m

19.9, CH2

3

1.89 m, 2.38 m

38.9, CH2

0.76 m, 1.66 m 1.70 m, 2.11 m 1.91 m, 2.15 m

4 5 6

1.05 m 1.98 m, 2.40 m

44.4, C 57.8, CH 22.6, CH2

7

1.36 m, 1.43 m

42.2, CH2

8 9 10 11 12 13 14 15 16 17 18 19 20 R1 Glc-1

δH (J in Hz)

2b

1

position

1.68 m 2.19 m, 1.89 m

44.7, 54.7, 40.2, 20.9, 38.4,

1.74 m, 2.55 m

87.2, C 44.7, CH2

0.91 m

2.06 m, 2.14 m 5.2 (br s), 5.60 (br s) 1.27 (s, 3H) 1.25 (s, 3H) 6.16 (d, 8 Hz)

C CH C CH2 CH2

48.5, CH2 154.7, C 105.4, CH2 29.0, CH3 177.3, CO 16.1, CH3 96.3, CH

Rha2-1 R2 Glc-1 Glc2-1 Xyl-1 Glc21(Xyl) Glc31(Xyl)

COSY correlation between H-2′ (4.07 ppm) and H-1′ (5.10 ppm). This confirmed the attachment of the third glucose unit at C-2′. The position of attachment of the rhamnose unit was established through the 3J HMBC correlation between C-1⁗ (102.0 ppm) and H-2″ (4.34 ppm), where the position of H-2″ was confirmed through the COSY correlation between H-2″ (4.34 ppm) and H-1″ (6.24 ppm) (Figure S15, Supporting Information). The three glucose units and rhamnose unit were determined to belong to the D-series and L-series, respectively, through the comparison with standards using the HPLC method reported by Tanaka et al.21 It is proposed to name compound 2 as rebaudioside S, and the structure is as shown.

1.00 m 1.86 m, 2.11 m 1.32 m, 1.62 m 0.91 m 1.70 m 2.68 m, 1.15 m 1.70 m, 2.50 m 2.10 m 5.10 m, 5.72 (s) 1.50 (s) 1.14 (s) 6.24 (d, 5.2 Hz) 6.40 (br s) 5.10 (br s) 5.22 (d, 4.8 Hz)

4.96 (d, 7.3 Hz) 5.54 (d, 7.7 Hz)

98.7, CH 105.0, CH

5.33 (d, 7.8 Hz)

105.1, CH

20.3, CH2 38.1, CH2 44.8, C 58.8, CH 22.5, CH2

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42.1, CH2 43.1, 54.4, 40.2, 21.1, 38.1,

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using an Autopol IV instrument at ambient temperature. NMR spectra were acquired on either a Bruker Avance III NMR spectrometer equipped with a DCH/F cryoprobe or a Bruker AV NMR spectrometer (Bruker Biospin, Bruker Inc.) with an RT DCH probe operating at 500 and 400 MHz (1H) and 125 and 100 MHz (13C), respectively. Chemical shift values are expressed in ppm. Multiplicity determinations (DEPT) and 2D NMR spectra (COSY, HMQC, HMBC, NOESY) were obtained using standard Bruker pulse programs. The mass spectrometric data were obtained on an Agilent series 1100 SL mass spectrometer. HRMS were obtained by direct injection using a Bruker Bioapex-FTMS with electrospray ionization (ESI) in negative mode. HPLC was performed with a Hewlett-Packard Agilent 1100 with a single-wavelength detector at 205 nm. The silica gel 60 F254 HPTLC plates and acetonitrile, methanol, and water for HPLC were purchased from EMD Millipore, flash silica gel was from Sorbent Technologies (Norcross, GA, USA), and acetic acid, ethyl acetate, methyl tert-butyl ether (MTBE), and isopropyl alcohol (IPA) were from Reagents (Nashville, TN, USA). Plant Material. The starting material was a partially processed commercially available extract of Stevia rebaudiana purchased from American Mercantile (Memphis, TN, USA). HPLC comparison of this extract with several other S. rebaudiana extracts purchased from various sources showed high similarities, differing only in the relative concentrations of specific glycosides but not their presence or absence. The isolated compounds obtained were byproducts from the isolation and purification of rebaudioside C and rebaudioside D (3). Approximately 1.5 kg of commercially available S. rebaudiana leaf extract was processed. The composition of the extract was approximately 40% rebaudioside A, 30% stevioside, 10% rebaudioside C, and 95%). A portion of pool 5 (2 g) was chromatographed using similar conditions, and 61 fractions were collected, from 2−57 × 25 mL and the other ones about 125 mL. Column analysis allowed pooling into six pools. Pool 3 [158 mg (80%)] was crystallized in methanol to produce 91.9 mg of 13-[(2O-β-D-xylopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester (>95%). The remainder of pool 5 was divided into three portions and submitted to normal-phase chromatographic separation in a 7.5 i.d. × 50 cm column with MTBE−IPA− water−acetic acid (62.5:25:12.5:0.1). Again, column analysis allowed combined pooling of the three chromatographies into pools rich in rebaudioside G. A portion of this pool was chromatographed over a reversed-phase column (2.5 i.d. × 45 cm) using a four-step gradient, each of 250 mL, of 0.1% acetic acid−water and acetonitrile−water− acetic acid (30:70:0.1; 40:70:0.1, and 50:50:0.1) to afford 243 mg (>95%) of eugenol diglucoside. All the pools containing rebaudioside G (>80%) were combined (6.4 g) and submitted to reversed-phase chromatography in a 7.5 × 50 cm column using acetonitrile−water− acetic acid (30:70:0.1) to afford 3.53 g (>95%) of this compound. The rebaudioside C pools were dried and dissolved in methanol, and stevioside was allowed to crystallize. The crystals of stevioside were filtered, and the mother liquors were dried and dissolved in water, with rebaudioside C allowed to crystallize and the rebaudioside C solids collected. The rebaudioside C mother liquors were then chromatographed again by three normal-phase chromatographic separations on a high-performance 7.5 i.d. × 50 cm column. The mobile phase was the same as used for the large column (“rebaudioside C mobile phase”). Pools were selected of rebaudioside C and rebaudioside F. The rebaudioside C pool was dissolved in water, and another crop of rebaudioside C crystals was harvested. The rebaudioside C mother liquor and the rebaudioside F pool were divided into six portions and then chromatographed in six reversedphase column runs. The column was 7.5 i.d. × 50 cm packed with 1.4 kg of 10 μm spherical C18 gel. The mobile phase was 65:35:0.1 v/v methanol−water−acetic acid. From the late eluting compounds (2.6 g of a mixture of 1 and other steviol glycoside mainly), pool 6, a 500 mg portion was rechromatographed using a reversed-phase column (2.5 i.d. × 45 cm) with acetonitrile−water−acetic acid (32:68:0.1). Pool 1 (fractions 1−11) yielded 108 mg (>95%) of 1 after crystallization in methanol, and pool 3 (fractions 19−26) yielded 187.1 mg (>95%) of 13-[(2-O-6-deoxy-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-Dglucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester. Regeneration pool (pool 6 of the large-scale rebaudioside C normalphase chromatography) was digested with ethanol, and the insoluble solids were crystallized in methanol−water to produce 25.8 g (>95%) of rebaudioside D. A portion of the mother liquors from the rebaudioside A pool (pool 5 of large-scale normal-phase chromatography) after the crystallization of this compound was subjected to reversed-phase chromatography (7.5 i.d. × 50 cm) using acetonitrile−water−acetic acid (26:74:0.1), with a 2.5 L forerun and 70 fractions collected of 50 mL, to yield fractions 15−20, 97 mg (>95%) of 2. Compound 1: amorphous, off-white solid; [α]25D −34.0 (c 0.1, MeOH); 1H and 13C NMR (Table 1); HRMS m/z 935.4190 (calcd for C43H67O22, 935.4124). Compound 2: amorphous, off-white solid; [α]25D −22.0 (c 0.1, MeOH); 1H and 13C NMR (Table 1); HRMS m/z 949.0173 (calcd for C44H69O22, 949.4280).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00048. Spectroscopic data and mass analysis of compounds 1 and 2 and the absolute configuration analysis of the carbohydrate units (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: (303) 808-4104. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dr. B. Avula for her help in obtaining HRESMS data. This work was funded through Ironstone Separations, Inc., Etta, MI, USA.

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REFERENCES

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