Triterpenoid Saponins from the Cultivar “Green Elf” of Pittosporum tenuifolium

Four oleanane-type glycosides were isolated from a horticultural cultivar “Green Elf” of the endemic Pittosporum tenuifolium (Pittosporaceae) from New Zealand: three acylated barringtogenol C glycosides from the leaves, with two previously undescribed 3-O-β-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, 3-O-β-d-galactopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, and the known 3-O-β-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C (Eryngioside L). From the roots, the known 3-O-β-d-glucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyloleanolic acid (Sandrosaponin X) was identified. Their structures were elucidated by spectroscopic methods including 1D- and 2D-NMR experiments and mass spectrometry (ESI-MS). According to their structural similarities with gymnemic acids, the inhibitory activities on the sweet taste TAS1R2/TAS1R3 receptor of an aqueous ethanolic extract of the leaves and roots, a crude saponin mixture, 3-O-β-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, and Eryngioside L were evaluated.


Introduction
The Pittosporaceae family, belonging to the Apiales order according to APGIII classification, is distributed from tropical Africa to the Pacific islands. The genus Pittosporum, which comprises about 200 species, is well known for its horticultural uses. From a phytochemical point of view, these species are rich in triterpene-type glycosides with biological interests, such as antimicrobial, antioxidant, cytotoxic, and antiproliferative activities [1][2][3]. Among this species, P. tenuifolium Banks & Sol. ex Gaertn., native to New Zealand, is a small tree named Kohuhu in Maori. The fresh gum resin is traditionally used for its scent, and it can be mixed with thickened juice of Puha (Sonchus genus) and chewed as a masticatory [4]. Moreover, several cultivars of this species are sold in greenhouses for garden use. Thus, as a continuation of our study on the glycosylated derivatives from the Pittosporum genus [5][6][7], we investigated the phytochemical interest of the "Green Elf" cultivar of P. tenuifolium. In the present paper, we report the isolation and structural elucidation of two undescribed triterpene saponins (Figure 1), together with a known one from the leaves and a known one from the roots. Their structures were elucidated by spectroscopic methods, including 600 MHz 1D and 2D experiments ( 1 H, 13 C, HSQC, HMBC, COSY, TOCSY, and ROESY) in from the leaves and a known one from the roots. Their structures were elucidated troscopic methods, including 600 MHz 1D and 2D experiments ( 1 H, 13 C, HSQC, COSY, TOCSY, and ROESY) in combination with mass spectrometry (ESI-MS), th comparison of their physical and spectral data with literature values.
When we compare the structure of the isolated triterpene-type glycosid gymnemic acids, some similarities appeared. Accordingly, we tested the inhibito ity on the sweet taste TAS1R2/TAS1R3 receptor of an aqueous ethanolic extrac leaves and roots, a crude saponin mixture, the pure compound 1, and Eryngiosid

Results and Discussion
The aqueous ethanolic extract of the leaves of P. tenuifolium was fractionated uum liquid chromatography (VLC) and purified by several medium-pressure liqu matography (MLPC) runs on normal-and reverse-phase silica gel, as well as semi ative HPLC, yielding compounds 12 and the known Eryngioside L [8]. The kno drosaponin X [9] was also isolated from an aqueous ethanolic extract of the roo the same protocol. All compounds were obtained as amorphous powders. Their st were established mainly by spectroscopic methods including 600 MHz NMR expe and mass spectrometry, and the structural analysis of the newly identified compo detailed below.

Results and Discussion
The aqueous ethanolic extract of the leaves of P. tenuifolium was fractionated by vacuum liquid chromatography (VLC) and purified by several medium-pressure liquid chromatography (MLPC) runs on normal-and reverse-phase silica gel, as well as semipreparative HPLC, yielding compounds 1-2 and the known Eryngioside L [8]. The known Sandrosaponin X [9] was also isolated from an aqueous ethanolic extract of the roots using the same protocol. All compounds were obtained as amorphous powders. Their structures were established mainly by spectroscopic methods including 600 MHz NMR experiments and mass spectrometry, and the structural analysis of the newly identified compounds is detailed below.
The presence of three sugar moieties in 1 was evidenced by the 1 H-NMR sp which displayed signals of three anomeric protons at δH 4.85 (d, J = 7.3 Hz), 5.38 (d The deshielded chemical shift of CH-21 at δ C /δ H 81.0/6.40 ppm and CH 2 -28 at δ C /δ H 66.2/4.26 ppm suggested an acylation at these positions. This was confirmed by the HMBC cross-peaks at δ H /δ C 6.40 (H-21)/168.8 (Ang-1) and δ H /δ C 4.26 (H 2 -28)/171.0 (Ac-1). At the C-21 position, the substituent was composed of two vinylic methyl groups at 2.01 (s) and 2.07 (d, J = 6.7 Hz) ppm, which correlated in the HMBC spectrum with one ethylenic quaternary carbon at 129.0 and an ethylenic methine carbon at 136.2 ppm. These data revealed an angeloyl group acylating the C-21 position (Table 1) [5]. The NMR signals for the acylating group at C-28 were in accordance with an acetyl function. Thus, the structure of the acylated aglycone was elucidated as 21-O-angeloyl-28-O-acetylbarringtogenol C.
The presence of three sugar moieties in 1 was evidenced by the 1 H-NMR spectrum which displayed signals of three anomeric protons at δ H 4.85 (d, J = 7.3 Hz), 5.38 (d, J = 7.6 Hz), and 5.65 (d, J = 7.0 Hz), giving correlations in the HSQC spectrum with three anomeric carbons at δ C 104.6, 104.2, and 103.0, respectively (Table 2). Complete assignments of each sugar were achieved by extensive 1D-and 2D-NMR analyses, allowing the identification of one GlcA, one Glc, and one Ara unit. In the HMBC spectrum, correlations at δ H /δ C 4.85 (GlcA-1)/90.  The HR-ESI-MS of compound 2 was the same as compound 1, with a molecular formula of C 54 H 84 O 22 . Extensive 2D-NMR analysis (Table 1)  Natural similar compounds of 1, 2 with acylated 3-O-glucuronopyranosylbarringtogenol C derivatives have already been isolated from a "variegatum" cultivar of P. tenuifolium [5].
Some saponins are known for their sweet taste such as glycyrrhizin from licorice, as well as for their sweet inhibitor activity such as gymnemic acids (GS), which correspond to a saponin mixture from Gymnema sylvestre (Apocynaceae) [11,12]. The sweet taste is mediated by the TAS1R2/TAS1R3 receptor found in the oral cavity and in various extraoral tissues such as the pancreas, brain, and bones [13]. The aglycone of GS named gymnemagenin is a polyhydroxylated oleanane-type derivative (3β,16β,21β,22α)3,16,21,22,23,28-hexol-olean-12-ene. This aglycone differs from barringtogenol C (3β,16α,21β,22α)-3,16,21,22,28-pentololean-12-ene by only one hydroxylation at the C-23 position. Moreover, GS structures possess acylation at the 21 and 22 positions, in addition to a 3-O-heterosidic linkage with a glucuronopyranosyl moiety. The interaction between this glucuronopyranosyl residue and the transmembrane domain of hTAS1R3 has already been described [14]. According to these similarities between the isolated compounds from Pittosporum tenuifolium "Green Elf" and GS, an aqueous ethanolic extract of the leaves (PTGE L) and roots (PTGE R), a crude saponin mixture (CSM), the pure compounds 1, and Eryngioside L (EL) were tested as TAS1R2/TAS1R3 inhibitors. Human embryonic kidney HEK293T-Gα16gust44 cells were transiently transfected with two plasmids coding for hTAS1R2 and hTAS1R3 subunits, respectively. Then, the cellular responses of cells to sucralose were measured by calcium mobilization assay after application of increasing concentrations of GS or plant extracts. Firstly, PTGE L, PTGE R, CSM, 1, and EL were evaluated for stimulation of the sweet taste receptor, with sucralose as a positive control (EC 50 = 52 ± 7 µM) (Supplementary Figure S11). None of them showed activation of the TAS1R2/TAS1R3. Then, the same compounds and GS (isolated from a commercial product; see Section 3) were tested for inhibition of the sucralose response (Figure 4). At higher concentrations, between 3 and 10 µg/mL, a decrease in sucralose response was observed, but this may be linked to a toxic effect. Actually, this toxicity could be observed for the control cells. On the contrary, GS showed an inhibitory effect at IC 50 = 2.97 ± 0.64 µg/mL (Supplementary Figure S12), according to results previously published [15]. From a structure/activity relationship point of view, the lack of toxic effects of GS compared with the toxicity of molecules isolated from Pittosporum tenuifolium "Green Elf" could be related to the presence of a secondary alcoholic function at the C-23 position of gymnemagenin. This conclusion needs to be proven by further tests with saponins possessing gymnemagenin-type aglycones.
(3β,16β,21β,22α)3,16,21,22,23,28-hexol-olean-12-ene. This aglycone differs fro ringtogenol C (3β,16α,21β,22α)-3,16,21,22,28-pentol-olean-12-ene by only one hyd tion at the C-23 position. Moreover, GS structures possess acylation at the 21 and tions, in addition to a 3-O-heterosidic linkage with a glucuronopyranosyl moiety teraction between this glucuronopyranosyl residue and the transmembrane do hTAS1R3 has already been described [14]. According to these similarities between lated compounds from Pittosporum tenuifolium "Green Elf" and GS, an aqueous e extract of the leaves (PTGE L) and roots (PTGE R), a crude saponin mixture (CS pure compounds 1, and Eryngioside L (EL) were tested as TAS1R2/TAS1R3 in Human embryonic kidney HEK293T-Gα16gust44 cells were transiently transfect two plasmids coding for hTAS1R2 and hTAS1R3 subunits, respectively. Then, the responses of cells to sucralose were measured by calcium mobilization assay afte cation of increasing concentrations of GS or plant extracts. Firstly, PTGE L, PTGE 1, and EL were evaluated for stimulation of the sweet taste receptor, with sucral positive control (EC50 = 52 ± 7 μM) (Supplementary Figure S11). None of them activation of the TAS1R2/TAS1R3. Then, the same compounds and GS (isolated commercial product; see Section 3) were tested for inhibition of the sucralose r (Figure 4). At higher concentrations, between 3 and 10 μg/mL, a decrease in su response was observed, but this may be linked to a toxic effect. Actually, this toxici be observed for the control cells. On the contrary, GS showed an inhibitory effect 2.97 ± 0.64 μg/mL (Supplementary Figure S12), according to results previously pu [15]. From a structure/activity relationship point of view, the lack of toxic effec compared with the toxicity of molecules isolated from Pittosporum tenuifolium "Gr could be related to the presence of a secondary alcoholic function at the C-23 po gymnemagenin. This conclusion needs to be proven by further tests with sapon sessing gymnemagenin-type aglycones.

Extraction and Isolation
Dried powdered leaves of P. tenuifolium "Green Elf" (29 g) were submitted to ultrasonicassisted extraction at room temperature, five times, 30 min each, with a mixture of EtOH-

Acid Hydrolysis and Absolute Configuration Determination
An aliquot (180 mg) of a rich saponin fraction was hydrolyzed with 2 N aqueous CF 3 COOH (25 mL) for 3 h at 95 • C. After extraction with CH 2 Cl 2 (3 × 15 mL), the aqueous layer was evaporated to dryness with H 2 O until neutral to give the sugar residue (64 mg). Glucuronic acid, glucose, galactose, and arabinose were identified by comparison with authentic samples by TLC using CH 3

Bioactivity Assay
For functional experiments on the sweet taste receptor, HEK293T cells stably expressing the chimeric G-protein subunit Gα16gust44 were seeded into 96-well plates as previously described [16]. Then, 24 h after seeding, cells were transiently transfected with hTAS1R2 and hTAS1R3 cDNAs, cloned into pcDNA6 and pcDNA4 vectors (Life Technologies, Carlsbad, CA, USA), respectively, with plasmid pCMV-GCaMP5G (Addgene) used as a genetically encoded calcium indicator. HEK293T-Gα16gust44 cells transfected with an empty vector served as a negative control. Then, after 24 h, cells were washed two times with C1 solution (130 mM NaCl, 5 mM KCl, 10 mM Hepes, 2 mM CaCl 2 , 5 mM sodium pyruvate, pH 7.4). Test substances were initially solubilized in DMSO, followed by in C1 solution for further dilution, before being subjected to 10 min stimulation on cells. After washing with C1 buffer, cells were stimulated by automatic injection of 300 µM sucralose, and changes in intracellular calcium levels were measured at 510 nm in a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices) after excitation at 488 nm. For data analysis of dose-response curves, signals of wells receiving the same treatment were averaged, the fluorescent signal of mock cells was subtracted from receptor-transfected cells, and the net signal was normalized to background (∆F/F 0 , F 0 fluorescence light before stimulus application). For calculation of the half-maximal effective concentration EC 50 and halfmaximal inhibitory concentration IC 50 values, ∆F/F 0 was plotted against concentration of the test substance using a four-parameter logistic equation [f(x) = min + (max − min)/(1 + (x/EC 50 ) nH )] with curves fitting of Sigma Plot software.