A New Penta β-D-Glucopyranosyl Diterpene from Stevia rebaudiana

A new steviol diterpene glycoside, 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-{2-O-[6-O-β-D-glucopyranosyl)-βD-glucopyranosyl]-β-D-glucopyranosyl} ester (1) has been isolated from the commercial extract of the leaves of Stevia rebaudiana Bertoni. Structure of the new compound has been established on the basis of extensive NMR spectroscopy (H &C, TOCSY, HMQC, and HMBC) and High Resolution (HR) mass spectroscopic data as well as enzymatic and acid hydrolysis studies.


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 regions, particularly in China, Japan, Taiwan, Korea, India, Thailand and Indonesia.Extracts of the leaves of S. rebaudiana have been used for decades to sweeten food and beverages in Japan, South America and China [1].The major constituents in the leaves of S. rebaudiana are the potently sweet glycosides namely stevioside, and rebaudiosides A; which are glycosides of the diterpene steviol, ent-13-hydroxykaur-16-en-19-oic acid [2] [3].As a part of our ongoing research in discovering new natural sweeteners and their potential usage into food and beverage industry [4] [5] [6]; we have isolated a new diterpene glycoside from the commercial extract of the leaves of S. rebaudiana from China.The structure of new compound has been characterized as 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oicacid-{2-O-[6-O-β-D-glucopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranosyl} ester (1) based on extensive spectroscopic (NMR and MS) and hydrolysis studies (Figure 1), and in comparison with the spectral data of the known steviol glycoside rebaudioside E (2) reported earlier from S. rebaudiana.

General Instrumentation
An Agilent (Wilmington, DE) 1100 HPLC System, including a quaternary pump, a temperature controlled column compartment with an additional 6 port switching valve, an auto sampler and VWD absorbance detector was used for analysis.The detector was set-up at UV 210 nm and the data acquisition was done using a Chemastation A 10.02 software.The column used for HPLC analysis was a reversed-phase C18 ( 2

Plant Material
The commercial sample of stevia extract from the leaves of S. rebaudiana which is a mixture of diterpene glycosides was obtained from Sinochem Qingdao Co Ltd, China with Lot No.: 20161201.The authenticity of the commercial extract was confirmed by performing its retention time (t R ) comparison with the internal standard compounds of known JECFA steviol glycosides isolated from S. rebaudiana using the preparative HPLC method as reported earlier [7].A voucher specimen is deposited at Wisdom Natural Brands.

Enzymatic Hydrolysis of 1
Compound 1 (500 μg) was dissolved in 5.0 mL of 0.1 M sodium acetate buffer (pH 4.5) and crude pectinase from Aspergillus niger (250 µL, Sigma-Aldrich, P2736) was added.The mixture was stirred at 50˚C for 96 hr.The product precipitated out during the reaction was filtered and then purified using reversedphase preparative TLC using water: MeOH (70:30) yielded a pure compound, which was identified as steviol (3) by comparison with co-TLC and 1 H-NMR of an authentic sample as well as from the spectral data from the literature [11].

Results and Discussion
The molecular formula of compound 1 has been deduced as C 50 H 80 O 28 on the basis of its positive high resolution (HR) mass spectrum which showed an ion corresponding to [M+ Na] + at m/z 1151.4713 and this composition was supported by the 13 C NMR spectral data.The 1 H NMR spectral data of 1 showed the presence of two methyl singlets at δ 1.15 and 1.39, two olefinic protons as singlets at δ 5.04 and 5.68 of an exocyclic double bond, nine sp3 methylene and two sp3 methine protons between δ 0.76-2.65,very similar to the ent-kaurane diterpenoids isolated earlier from S. rebaudiana [12] [13] [14].The 1 H NMR spectrum of 1 also showed the presence of five anomeric protons resonating at δ  (China).The new compound was identified and characterized based on the basis of NMR and HR mass spectral data as well as hydrolysis studies.This is the first report of the isolation of this new diterpene glycoside from S. rebaudiana in nature, which is an important addition in expanding our understanding of the diversity of the β-D-glucopyranosyl units attached at multiple positions on diterpenoid glycosides present in the S. rebaudiana and their structure-activity relationship.
) 100 A Phenomenex (Torrance CA) (250 × 4.6 mm, 5 µm); pH was measured using meter Metler Toledo seven compact pH/ion S220 (Switzerland); Branson Ultrasonic Cleaner Model 2510 (Maplewood, NJ) was used for degassing HPLC solvents.NMR spectra were acquired on Bruker Avance DRX 500 MHz or Varian INOVA 600 MHz instrument instruments using standard pulse sequences.High Resolution Mass Spectral (HRMS) data were generated with a LTQ Orbitrap Discovery instrument with its resolution set to 30k.The needle voltage was set to 4 kV; the other source conditions were sheath gas = 25, aux gas = 0, sweep gas = 5 (all gas flows in arbitrary units), capillary voltage = 30V, capillary temperature = 300˚C, and tube lens voltage = 75.Sample was diluted with 2:2:1 CH 3 CN:MeOH:water (same as infusion eluent) and injected 50 microliters.TLC was performed on Baker Si-C 18 F plates with mobile phase H 2 O-MeOH (80:20).Identification of the spots on the TLC plate was carried out by spraying 10% H 2 SO 4 in EtOH and heating the plate at about 80˚C.
The mixture was refluxed for 16 hours and then neutralized with saturated sodium carbonate after cooling to room temperature.The aqueous phase was extracted with ethyl acetate (EtOAc, 2 × 15 ml) to separate an EtOAc fraction containing the aglycone part.The aqueous layer was concentrated and compared with standard sugars using the TLC system EtOAc/n-butanol/water (2:7:1) and CH 2 Cl 2 /MeOH/ water (10:6:1) [8] [9][10]; the sugars were identified as D-glucose.
a assignments made on the basis of TOCSY, HMQC and HMBC correlations; b Chemical shift values are in δ (ppm); c Coupling constants are in Hz.