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Venkata Sai Prakash Chaturvedula et al / IJRAP 2011, 2 (4) 1395-1401

International Journal of Research in Ayurveda & Pharmacy, 2(4), 2011 1395-1401

Research Article Available online through www.ijrap.net ISSN 2229-3566

ISOLATION AND NMR SPECTRAL ASSIGNMENTS OF STEVIOLBIOSIDE

AND STEVIOSIDE Venkata Sai Prakash Chaturvedula*, Indra Prakash

The Coca-Cola Company, Organic Chemistry Department, Research and Technology, One Coca-Cola Plaza, Atlanta, GA 30313, USA

Received on: 02/08/2011 Revised on: 19/08/2011 Accepted on: 28/08/2011

ABSTRACT The complete 1H and 13C NMR assignments of the two diterpene glycosides, 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid (steviolbioside) and 3-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester (stevioside) isolated from Stevia rebaudiana were achieved on the basis of extensive NMR (1H and 13C, COSY, HMQC, HMBC) and MS spectral data. The structures of steviolbioside and stevioside were further supported by acid and enzymatic hydrolysis studies by identifying their corresponding aglycone and sugar residues. Key words: Steviolbioside, Stevioside, Diterpenoid glycosides, 1D and 2D NMR spectral data, HRMS data, Chemical studies *Address for Correspondence Venkata Sai Prakash Chaturvedula, The Coca-Cola Company, Organic Chemistry Department, Research and Technology, One Coca-Cola Plaza, Atlanta, GA 30313, USA, E-mail: [email protected], INTRODUCTION Stevia rebaudiana (Bertoni) is a perennial shrub belonging to the family of Asteraceae (Compositae) native to Brazil and Paraguay, but now grown commercially in a number of countries, particularly in Japan, Taiwan, Korea, Thailand and Indonesia 1-2. Extracts of the leaves of S. rebaudiana have been used for decades to sweeten food and beverages in Japan, South America and China. The major constituents in the leaves of S. rebaudiana are the potently sweet diterpenoid glycosides namely stevioside, rebaudiosides A and D, and dulcoside A; of which stevioside and rebaudioside-A are the major compounds. Stevioside is about 150-200 times sweeter to sucrose. These compounds, which are known as Stevia sweeteners are the glycosides of the diterpene ent-13-hydroxykaur-16-en-19-oic acid (steviol)3. In our continuing research to discover natural sweeteners, we have collected commercial extracts of S. rebaudiana from various suppliers all over the World and isolated several novel diterpene glycosides4-10. Apart from isolating novel compounds from S. rebaudiana and utilizing them as possible natural sweeteners or sweetness enhancers, we are also engaged in understanding the stability of the steviol glycosides in

various systems of interest and identification as well as the complete NMR analysis of the identified degradation products11. In this article, we are describing the complete 1H and 13C NMR spectral assignments for the two diterpene glycosides 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid (steviolbioside, 1) and 3-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester (stevioside, 2) that were identified from S. rebaudiana (Figure 1). The complete NMR assignments were achieved on the basis of 1D and 2D NMR as well as HRMS spectroscopic data and chemical studies. MATERIAL AND METHODS General Melting points were measured using a SRS Optimelt MPA 100 instrument and are uncorrected. HPLC analysis was performed using an Agilent (Wilmington, DE) 1200 system, including a quaternary pump, a temperature controlled column compartment with additional 6-port switching valve, an autosampler and a UV absorbance detector. The reversed phase (RP) HPLC was employed using a Phenomenex (Torrance, CA) Synergi-Hydro column (250 mm x 4.6 mm, 4 µm) with a Phenomenex Security guard C18 cartridge and a



tertiary solvent mobile phase (A: 0.040% NH4OAc/AcOH buffer, B: MeCN and C: 0.040% AcOH). The column was maintained at a temperature of 55°C and the flow rate was 1.0 ml/minute. The injection volume of each sample was 100 µl, which were kept at ambient temperature while in the autosampler. Charged Aerosol Detector (CAD) was used for the analysis of all steviol glycosides with a total run time of 43 min (Table 1). Analytical HPLC was carried out with a Waters 600E multisolvent delivery system using a Phenomenex Luna C18 (150 x 4.6 mm, 5 mm) column. NMR spectra were acquired on Bruker Avance DRX 500 MHz and Varian Unity Plus 600 MHz instruments using standard pulse sequences. The spectra were referenced to the residual solvent signal (dH 3.30, dC 49.0 for CD3OD), chemical shifts are given in d (ppm), and coupling constants are reported in Hz. HRMS and MS/MS data were generated with a Waters Premier Quadrupole Time-of-Flight (Q-TOF) mass spectrometer equipped with an electrospray ionization source operated in the positive-ion mode and Thermo Fisher Discovery OrbiTrap in the electrospray positive mode. Samples were diluted with water: acetonitrile (1:1) containing 0.1% formic acid and introduced via infusion using the onboard syringe pump. Material SG95, the commercial aqueous extract consisting of a mixture of diterpenoid glycosides of the leaves of S. rebaudiana was obtained from the Pure Circle (Kuala Lumpur, Malaysia). The authenticity of the crude extract was confirmed by performing its retention time (tR) comparison with the internal standard compounds of known steviol glycosides namely rebaudioside A-D, and dulcoside A isolated from S. rebaudiana using the preparative HPLC method as reported earlier 12. A voucher specimen is deposited at The Coca-Cola Company, No. VSPC-3166-002. Isolation Compounds 1 and 2 were purified by using an Agilent HPLC 1200 system equipped with a Phenomenex Synergi-Hydro column (250 mm x 4.6 mm, 4 µm) with a Phenomenex Security guard C18 cartridge. Using the above mentioned HPLC method, collected the peaks eluting at tR 26.87 and 19.07 min; and dried the corresponding solutions under nitrogen yielded 1 and 2 respectively. 13-[(2-O-bbbb-D-glucopyranosyl-bbbb-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid (steviolbioside, 1) White powder; mp 195-200 °C; IR νmax: 3307, 2937, 1605, 1053, 970 cm-1; 1H-NMR (500 MHz, CD3OD, δ ppm) and 13C-NMR (125 MHz, CD3OD, δ ppm)

spectroscopic data see Table 2; HRMS (M+NH4)+ m/z 660.3589 (calcd. for C32H54O13N: 660.3595); (M+Na)+ m/z 665.3140 (calcd. for C32H50O13Na: 665.3149). 3-[(2-O-bbbb-D-glucopyranosyl-bbbb-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester (stevioside, 2) White powder; mp 196-198 °C; IR νmax: 3352, 2928, 1726, 1034, 891 cm-1; 1H-NMR (500 MHz, CD3OD, δ ppm) and 13C-NMR (125 MHz, CD3OD, δ ppm) spectroscopic data see Table 2; HRMS (M+NH4)+ m/z 822.4109 (calcd. for C38H64O18N: 822.4123); (M+Na)+ m/z 827.3661 (calcd. for C38H60O18Na: 827.3677). General procedure for acid hydrolysis and determination of sugar configuration in 1 and 2 Each compound (500 mg) was hydrolyzed with 0.5 M HCl (0.5 mL) for 1.5 h. After cooling, the mixture was passed through an Amberlite IRA400 column and the eluate was lyophilized. The residue was dissolved in pyridine (0.25 mL) and heated with L-cysteine methyl ester HCl (2.5 mg) at 60ºC for 1.5 h, and then O-tolyl isothiocyanate (12.5 uL) was added to the mixture and heated at 60ºC for an additional 1.5 h. The reaction mixture was analyzed by HPLC: column Phenomenex Luna C18, 150 x 4.6 mm (5 u); 25% acetonitrile-0.2% TFA water, 1 mL/min; UV detection at 250 nm. The sugar was identified as D-glucose in each experiment (tR, 12.16 to 12.32 min) [authentic samples, D-glucose (tR, 12.34) and L-glucose (tR, 11.12 min)13. Enzymatic hydrolysis of 1 and 2 Each compound (250 mg) was dissolved in 2.5 ml of 0.1 M sodium acetate buffer, pH 4.5 and crude pectinase from Aspergillus niger (50 uL, Sigma-Aldrich, P2736) was added. The mixture was stirred at 50o C for 48 hr. The product precipitated out during the reaction and was filtered and then crystallized. The resulting product obtained from the hydrolysis of 1 and 2 was identified as steviol by comparison of its co-TLC with standard compound and 1H NMR spectral data14. RESULTS AND DISCUSSION Compound 1 was isolated as white powder and its molecular formula has been deduced as C32H50O13 on the basis of its HRMS data which showed the presence of an [M+NH4]+ ion at m/z 660.3589 together with [M+Na]+ adduct at m/z 665.3140, this composition was supported by the 13C NMR spectral data. The 1H NMR spectrum of 1 showed the presence of two methyl singlets at δ 1.00 and 1.19, two olefinic protons as singlets at δ 4.86 and 5.24 of an exocyclic double bond, nine methylene and two methine protons between δ 0.86-2.27 characteristic for the diterpenes belongs to the class of ent-kaurenes isolated earlier from the genus Stevia 3-10. The basic skeleton of ent-kaurene diterpenoids was supported by



the COSY (H-1/H-2; H-2/H-3; H-5/H-6; H-6/H-7; H-9/H-11; H-11/H-12) and HMBC (H-1/C-2, C-10; H-3/C-1, C-2, C-4, C-5, C-18, C-19; H-5/C-4, C-6, C-7, C-9, C-10, C-18, C-19, C-20; H-9/C-8, C-10, C-11, C-12, C-14, C-15; H-14/C-8, C-9, C-13, C-15, C-16 and H-17/C-13, C-15, C-16) correlations. The 1H NMR spectrum of 1 also indicated the presence of two anomeric protons at δ 4.59, and 4.61 suggesting the presence of two sugar units in its structure. Enzymatic hydrolysis of 1 furnished an aglycone which was identified as ent-13-hydroxykaur-16-en-19-oic acid (steviol) by comparison of TLC and 1H NMR spectral data14. Acid hydrolysis of 1 afforded D-glucose which was identified by preparing the corresponding thiocarbamoyl-thiazolidine carboxylate derivatives with L-cysteine methyl ester and O-tolyl isothiocyanate, and in comparison of its retention times with the standard sugars as described in the literature.13 The 13C NMR values for all the carbons were assigned on the basis of COSY, HMQC and HMBC correlations and are given in Table 2. The carbonyl group resonating at δ 180.4 in the 13C NMR spectrum of 1 did not show correlation to either of the two anomeric protons in its HMBC spectrum suggested the presence of an acid group at C-19 position. On the basis of above observation, the placement of one β-D-glucosyl unit was assigned at C-13 position leaving the identification of an additional β-D-glucosyl unit. The fact that both H-2′ and C-2′ were shifted downfield in the NMR spectral data of 1 to δ 3.46 and δ 82.3 respectively suggested that the second sugar is attached at C-2 of sugar I. This was confirmed by the key HMBC correlations: H-2′/C-1′, C-3′, C-1′′ and H-1′′/C-2′, C-2′′, C-3′′ (Figure 2), supporting the presence of a 2-branched-β-D-glucobiosyl substituent at C-13. The 1H- and 13C-NMR values for all the protons and carbons were assigned on the basis of COSY, HMQC and HMBC correlations (Table 2). The large coupling constants observed for the two anomeric protons of the glucose moieties at δ 4.59 (d, J = 8.2 Hz), and 4.61 (d, J = 8.2 Hz), suggested the β-orientation as reported for steviol glycosides 4-10. Based on the results from chemical and spectral studies, 1 was assigned as 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid (steviolbioside). The molecular formula of compound 2 was deduced as C38H60O18 from its HRMS data which showed [M+NH4]+

and [M+Na]+ adduct ions at m/z 822.4109 and 827.3661 respectively. The 1H NMR spectrum of 2 also showed the presence of two methyl singlets at δ 0.98, and 1.21, nine methylene and two methine protons, similar to 1 (Table 2). Enzymatic hydrolysis of 2 furnished a compound which was found identical to steviol,

confirming the identical aglycone present as in 1. The 1H NMR of 2 showed the presence of three anomeric protons as doublets at δ 4.57 (J=7.8, 1H), 4.59 (J=7.8, 1H), and 5.38 (J=8.2, 1H), suggesting the presence three sugar residues in its structure which was supported from its MS/MS spectrum which showed sequential loss of three hexose moieties from its (M+H)+ ion at m/z 805 to yield fragment ions at m/z 643, 481, and 319. Acid hydrolysis of 2 afforded D-glucose, together with the coupling constants of the anomeric protons confirming the presence of three β-D-glucosyl moieties in 2. The 1H and 13C NMR values for all the protons and carbons were assigned on the basis of COSY, HMQC and HMBC correlations and are given in Table 2. A close comparison of the 1H and 13C NMR values of 2 with 1 together with the key COSY and HMBC correlations shown in Figure 3 suggested the presence of 2- branched β-D-glucobiosyl unit at C-13 with an additional β-D-glucosyl at C-19 position. Based on the above spectral and chemical results, 2 was assigned as 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid b-D-glucopyranosyl ester (stevioside). CONCLUSION The complete 1H and 13C NMR spectral assignments for steviolbioside (1) and stevioside (2) were made on the basis of extensive 1D and 2D NMR and MS spectral data as well as the acid and enzymatic hydrolysis studies. ACKNOWLEDGEMENT We wish to thank Pure Circle (Kuala Lumpur, Malaysia) for providing the stevia extract SG-95 and AMRI, Bothell, WA, USA for obtaining some selected spectral data. REFERENCES 1. Mosettig E, Nes WR. Stevioside. II. The structure of the aglucon.

J.Org.Chem.1955;20: 884-899. 2. Mosettig E, Beglinger U, Dolder F, Lichiti H, Quitt P, Waters

JA. The absolute configuration of steviol and isosteviol. J. Am. Chem. Soc. 1963; 85: 2305.

3. Brandle JE, Starrratt AN, Gijen M. Stevia rebaudiana: its agricultural, biological and chemical properties. Can. J. Plant Sci. 1998; 78: 527-536.

4. Chaturvedula VSP, Prakash I. A new diterpenoid glycoside from Stevia rebaudiana. Molecules. 2011; 16: 2937-2943.

5. Chaturvedula VSP, Mani U, Prakash I. Diterpene glycosides from Stevia rebaudiana. Molecules. 2011; 16: 3552-3562.

6. Chaturvedula VSP, Prakash I. Structures of the novel diterpene glycosides from Stevia rebaudiana. Carbohydr. Res., 2011; 346: 1057-1060.

7. Chaturvedula VSP, Rhea J, Milanowski D, Mocek U, Prakash I. Two minor diterpene glycosides from the leaves of Stevia rebaudiana. Nat. Prod. Commun. 2011; 6: 175-178.

8. Chaturvedula VSP, Prakash I. Additional minor diterpene glycosides from Stevia rebaudiana. Nat. Prod. Commun. 2011; 6: 1059-1062.

9. Chaturvedula VSP, Clos JF, Rhea J, Milanowski D, Mocek U, DuBois GE, Prakash I. Phytochemistry Lett. 2011 (in press).



10. Chaturvedula VSP, Mani U, Prakash I. Structures of the novel α-glucosyl linked diterpene glycosides from Stevia rebaudiana. Carbohydr. Res. 2011 (in press).

11. Chaturvedula VSP, Prakash I. Stability study of steviol glycosides in mock beverages using fluorescent light exposure under ICH guidelines, Int. J. Pharm. Pharm. Sci. 2011; 3: 316-323.

12. Clos JF, DuBois GE, Prakash I. Photostability of Rebaudioside A and Stevioside in Beverages. J. Agric. Food Chem. 2008; 56:8507-8513.

13. Tanaka T, Nakashim T, Ueda T, Tomii K, Kouno I. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 2007; 55: 899-901.

14. Ohtani K, Aikawa Y, Kasai R, Chou W, Yamasaki K, Tanaka O. Minor diterpene glycosides from sweet leaves of Rubus suavissimus. Phytochemistry 1992; 31: 1553-1559 .

Table 1. RP-HPLC method for the separation of steviolbioside (1) and stevioside (2)

Time (min) % of Mobile Phase A % of Mobile Phase B % of Mobile Phase C 0.0 75 25 0 8.5 75 25 0 10.0 71 29 0 16.5 70 30 0 18.5 0 34 66 24.5 0 34 66 26.5 0 52 48 29.0 0 52 48 31.0 0 70 30 37.0 0 70 30 37.1 0 90 10 40.0 0 90 10 40.1 75 25 0 43.0 75 25 0



Table 2. 1H and 13C NMR chemical shift values for 1–2 recorded in CD3OD a-c.

Position 1 1H 13C

2 1H 13C

1 0.86 (m, 1H), 1.87 (m, 1H)

41.5 0.86 (m, 1H), 1.87 (m, 1H)

41.5

2 1.41 (m, 1H), 1.92 (m, 1H)

19.8 1.41 (m, 1H), 1.92 (m, 1H)

19.8

3 1.05 (m, 1H), 2.21 (m, 1H)

38.9 1.05 (m, 1H), 2.15 (m, 1H)

38.9

4 44.9 44.9 5 1.12 (d, J=11.9, 1H) 58.2 1.14 (d, J=11.9, 1H) 58.2 6 1.85 (m, 1H),

2.04 (m, 1H) 22.8 1.85 (m, 1H),

2.04 (m, 1H) 22.8

7 1.43 (m, 1H), 1.55 (m, 1H)

42.4 1.43 (m, 1H), 1.55 (m, 1H)

42.4

8 41.6 41.6 9 0.98 (m, 1H) 54.9 0.98 (m, 1H) 54.9 10 40.8 40.8 11 1.65 (m, 1H),

1.80 (m, 1H) 21.1 1.65 (m, 1H),

1.80 (m, 1H) 21.1

12 1.52 (m, 1H), 1.97 (m, 1H)

37.8 1.52 (m, 1H), 1.97 (m, 1H)

37.8

13 88.4 87.8 14 1.54 (m, 1H),

2.27 (d, J=11.9, 1H) 45.2 1.51 (m, 1H),

2.26 (d, J=11.1, 1H) 45.2

15 2.04 (d, J=16.7, 1H), 2.14 (d, J=16.7, 1H)

48.4 2.04 (m, 1H), 2.14 (m, 1H)

48.4

16 153.9 153.4 17 4.86 (s, 1H)

5.24 (s, 1H) 105.7 4.84 (s, 1H)

5.21 (s, 1H) 105.3

18 1.19 (s, 3H) 28.5 1.21 (s, 3H) 28.5 19 180.4 178.7 20 1.00 (s, 3H) 16.7 0.98 (s, 3H) 16.0 1′ 4.61 (d, 8.2, 1H) 97.5 5.38 (d, 8.2, 1H) 95.5 2′ 3.46 (m, 1H) 82.3 3.35 (m, 1H) 73.8 3′ 3.54 (m, 1H) 78.5 3.43 (m, 1H) 78.4 4′ 3.32 (m, 1H) 72.1 3.32 (m, 1H) 70.8 5′ 3.26 (m, 1H) 78.7 3.36 (m, 1H) 78.1 6′ 3.64 (m, 1H), 3.86 (m,

1H) 63.4 3.67 (m, 1H), 3.80 (m,

1H) 61.2

1′′ 4.59 (d, 8.2, 1H) 105.2 4.59 (d, 7.8, 1H) 97.4 2′′ 3.25 (m, 1H) 76.4 3.45 (m, 1H) 82.3 3′′ 3.40 (m, 1H) 78.0 3.53 (m, 1H) 78.4 4′′ 3.14 (m, 1H) 71.6 3.34 (m, 1H) 72.3 5′′ 3.38 (m, 1H) 77.9 3.30 (m, 1H) 78.6 6′′ 3.62 (m, 1H), 3.84 (m,

1H) 62.9 3.67 (m, 1H), 3.88 (m,

1H) 63.2

1′′′ 4.57 (d, 7.8, 1H) 105.1 2′′′ 3.27 (m, 1H) 76.2 3′′′ 3.42 (m, 1H) 77.8 4′′′ 3.12 (m, 1H) 71.4 5′′′ 3.36 (m, 1H) 77.8 6′′′ 3.60 (m, 1H), 3.80 (m,

1H) 62.9

a assignments made on the basis of COSY, HMQC and HMBC correlations; b Chemical shift values are in δ (ppm); c Coupling constants are in Hz.



H

CH 3

H 3C O

O H

1

2

45

8

9

10

11 1314

15

1617

18

19

20

O

O

OH O

H OH O

H O

H OR O

sug ar I

sugar II O H

1

O

sugar I

H

CH 3

H 3C O

O

1

2

45

8

9

10

11 1314

15

1617

18

19

20

O

O

OH O

H OH O

H O

H OH O

sugar II

sugar II I O H

2

O

OH O

H OH O

O H

F igure 1. Structures of steviolbioside (1) and stevioside (2)



Source of support: Nil, Conflict of interest: None Declared

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