Hydroxyoleoside-type seco-iridoids from Symplocos cochinchinensis and their insulin mimetic activity

As part of an ongoing study of new insulin mimetic agents from medicinal plants, the 70% EtOH extract of Symplocos cochinchinensis was found to have a stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. The intensive targeted isolation of this active extract resulted in ten new hydroxyoleoside-type compounds conjugated with a phenolic acid and monoterpene (1–6 and 8–11), as well as four known compounds (7 and 12–14). The chemical structures of the new compounds were determined based on spectroscopic data analysis (1H and 13C NMR, HSQC, HMBC, NOESY and MS). The absolute configurations of the isolated compounds were determined by electronic circular dichroism (ECD) analysis of derivatives obtained after a series of reactions, such as those with dirhodium (ІІ) tetrakis (trifluoroacetate) and dimolybdenum (ІІ) tetraacetate. In vitro, compounds 3, 7 and 8 moderately increased the 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) uptake level in differentiated 3T3-L1 adipocytes. For further studies, we evaluated their effects on the expression of glucose transporter-4 (GLUT4), its translocation, protein tyrosine phosphatase 1B (PTP1B) inhibition and expression of phosphorylated Akt. Our results strongly suggest that the traditional uses of this plant can be described as active constituents by hydroxyoleoside-type compounds.

Globally, the number of people with diabetes mellitus is growing rapidly, and the incidence rate of diabetes is also accelerating, especially as the elderly and obese population increases 1 . The number of patients with diabetes is expected to increase from 171 million in 2000 to 366 million globally by 2030. According to the American Diabetes Association, the incidence of diabetes is approximately 25.2% of the total elderly population in the United States and 12.0 million seniors suffered from diabetes in 2015. As the increase in diabetic patients is associated with a dramatic increase in the cost of diabetes-related complications, direct healthcare costs and productivity losses in the US alone are estimated to be $ 176 billion and $ 69 billion in 2012, respectively 2 .
Diabetes is a chronic disease that occurs when the pancreas does not produce enough insulin (type 1 diabetes) or the insulin produced does not function effectively at the site of action (type 2 diabetes, T2D). When insulin does not function properly, the increased blood sugar in the body can cause serious damage to the heart, blood vessels, kidneys, eyes and nerves. Since T2D associated with insulin resistance is the most prevalent 3 , it is urgently necessary to develop new anti-diabetic agents, especially those of targeting type 2 diabetes. While many diabetes medications lower blood glucose levels in the short term, they often cause weight gain as a side effect and prolonged use worsens the insulin resistance of diabetic patients 3 . Insulin mimetics used as oral diabetic agents, which act similar to insulin but do not synthesize fats, have been suggested as a good solution for the treatment for diabetes. Interestingly, food intake and body weight decrease when insulin is selectively delivered to the brain, but not when it is delivered to the whole body 4 . These results suggest that insulin mimetics that separate glucose-lowering action from the weight gain are a very good pharmacological solution for overcoming insulin resistance as the side effects of diabetes therapies.
Symplocos cochinchinensis (Lour.) S. Moore (www.theplantlist.org) is an evergreen tree that grows up to 35 meters in height and belongs to the Symplocaceae family. This plant is distributed in East Asia, including China, Japan, India, Vietnam and Malaysia 5 . Ethnobotanical uses of this plant include treatment of diabetes mellitus in traditional "Ayurvedic" Indian medicine 6  antidiabetic 7,8 , antilipidemic and antioxidant activity 9 , but there have been few studies on the chemical constituents of S. cochinchinensis. The genus Symplocos contains a large amount of seco-iridoid and phenolic compounds 10 . Recent reports on the antidiabetic activity of oleuropein, which is abundant in olive tree leaves 11 , led us to isolate active compounds by a special dereplication method aimed at seco-iridoids.
In the search for new insulin mimetics from S. cochinchinensis, the 70% EtOH extract of the plant showed a moderate increase in glucose uptake in differentiated adipocyte cells. Bioassay-guided fractionation resulted in the isolation of ten new hydroxyoleoside-type compounds, including eight phenolic hydroxyoleosides, symplocochinside A-H (1-6, 8 and 9), and two monoterpene-derivatized hydroxyoleosides, symplocochinside I-J (10)(11), along with four known compounds, including a megastigmane and triterpene glycosides (Fig. 1). The absolute configurations of the monoterpene attached to hydroxyoleoside (10) and megastigmane (12) were assigned by chemical methods coupled with spectroscopic analysis. All isolates were evaluated for glucose uptake level, GLUT-4 translocation, PTP1B activity and Akt phosphorylation. In this paper, we report the isolation, structural elucidation, determination of absolute configuration and anti-diabetic properties of these isolates.
Symplocochinside B (2) was purified as a brownish gum, and its molecular formula was established as C 26 (Table 1) were similar to those of 1 except for the configuration of the ferulic acid double bond. The J values of H-7′ (δ H 6.86, d, J = 13.0 Hz) and H-8′ (δ H 5.78, d, J = 13.0 Hz) of compound 1 are indicative of its cis-configuration. Whether the cis form of compound 2 is a plant-derived compound was determined by the retention time and abundance when the partial extract of S. cochinchinensis was co-injected with 2 on LC/MS. Hence, the structure of 2 was characterized as 10-O-cis-feruloyl-10-hydroxyoleoside. Symplocochinside C (3) (Fig. 1) was isolated as a brownish gum, and its molecular formula was established as C 25 H 28 O 14 by HRESI mass spectrum, which showed a ion peak at m/z 575.1371 [M + Na] + (calcd for C 25 H 28 NaO 14 , 575.1371). The distinct UV patterns of 3, which indicate the presence of a cinnamic acid moiety, and the characteristic proton peaks of H-1 (δ H 5.90, s) and H-3 (δ H 7.46, br s) showed the common features of this seco-iridoid. The 1 H and 13 C NMR spectra of 3 were similar to those of 1 except for the absence of the methoxy group. Since the trans-cinnamic acid derivative can be converted to the cis-isomer through photoisomerization 18 , the structure of compound 3 was determined after conversion to the trans form by the reaction with iodine 19 . Thus, the structure of 3 was determined as 10-O-trans-p-coumaroyl-10-hydroxyoleoside.
Symplocochinside D (4) was obtained as a brownish gum, and its molecular formula was established as C 25 H 28 O 14 from the HRESI mass spectrum with a peak at m/z 575.1381 [M + Na] + (calcd for C 25 H 28 NaO 14 , 575.1371). The 1D NMR of 4 (Tables 1 and 2) showed almost same patterns as those of 3 except for the coupling constants of H-7′ (δ H 6.87, d, J = 11.2 Hz) and H-8′ (δ H 5.77, d, J = 12.8 Hz), indicating that compound 4 is the cis-isomer of compound 3. The pure form of cis-configured compound 4 could be obtained. Hence, the structure of 4 was identified as 10-O-cis-p-coumaroyl-10-hydroxyoleoside.
Symplocochinside E (5) (Fig. 1) was isolated as a brownish gum, and its molecular formula was established as C 27 H 32 O 15 from the HRESI mass spectrum, which showed a sodium adduct ion peak at m/z 619.1673 [M + Na] + (calcd for C 27 H 32 NaO 15 , 619.1633). The NMR spectra of 5 (Tables 1 and 2 Fig. S26) of H-10 with C-7′ at δ C 167.8 showed that the benzoic acid group is connected to C-10. Thus, the chemical structure of 8 was assigned as 10-O-benzoyl-10-hydroxyoleoside.
Symplocochinside H (9) (Fig. 1) was purified as a brownish gum, and its molecular formula was established as C 18  Symplocochinside I (10), a yellowish gum, had the molecular formula C 26   , which are three sp 3 primary carbons, three sp 3 methylene carbons and one sp 3 quaternary carbon with a deshielded chemical shift (δ C 70.1) implying the presences of one oxygenated quaternary carbon, one carbonyl group, and two olefinic carbons. The HMBC correlations of H-10 with C-1′, H-9′ with C-2′/C-4′, of H-6′ with C-4′/C-5′, of H-5′ with C-3′ and of H-4′ with C-2′/C-5′ suggested the connectivity of the 10-hydroxyoleoside skeleton with 3-hydroxydimethyloctenoic acid 20,21 , which is a monoterpene also known as 3-hydroxycitronellic acid. The isolation of 3-hydroxycitronellic acid from 10 by selective hydrolysis was not successful due to racemization (data not shown). Therefore, after the reaction with dirhodium (ІІ) tetrakis (trifluoroacetate), the empirical ECD method was employed for the determination of the absolute configuration at C-3′ 22 . The acetate form of compound 10 was purified after the peracetylation reaction (Supplementary Table S1) and was subjected to complexation with [Rh 2 (OCOCF 3 ) 4 ] in CDCl 3 . According to the bulkiness rule, the negative Cotton effect at 350 nm (band E) was observed (Fig. 3B), which means that the absolute configuration of C-3′ is 3′R. Thus, compound 10 was elucidated as (3′R)-10-O-3′-hydroxycitronellyl-10-hydroxyoleoside. Symplocochinside J (11) was obtained as a yellowish gum, and its molecular formula was established as C 26 Fig. S38) of H-9′ with C-2′/C-4′ along with the similar pattern of HMBC correlations with 10 signified that 11 is an analogue of 10-hydroxyoleoside substituted with a different monoterpene, namely, geranic acid, which is supported  by the comparison with previously reported data 23 . Thus, the structure of compound 11 was elucidated as 10-O-geranyl-10-hydroxyoleoside. Compound 12 (Fig. 1) was obtained as a brownish gum, and its molecular formula was established as C 13 H 20 O 3 from the HRESI mass spectrum with a peak at m/z 225.1484 [M + H] + (calcd for C 13 H 21 O 3 , 225.1485). The comparison with previously reported NMR data 24 showed the compound has the same planar megastigmane structure (Supplementary Table S1). However, the NOESY spectrum ( Supplementary Fig. S43) indicated the possibility of different configurations at C-8 and C-9 from those of the known compound 8,9-dihydromegastigmane-4,6-diene-3-one. In the NOESY spectrum, the correlations between H-7 at δ H 6.06 (d, J = 9.2 Hz)/H-13 at δ H (2.15, d, J = 0.8 Hz) and H-8 at δ H (4.68, dd, J = 9.4, 5.3 Hz)/H-9 at δ H (3.75, m) indicated that the olefinic carbon of C-7 is in the E configuration and that the relative configuration of C-8 and C-9 is [8R*, 9S*]. The specific rotation of the known compound is +54.0 (c 1.52 MeOH), whereas that of 12 was −88.9 (c 0.2 MeOH). Since the planar structure of the known compound was reported without the absolute configuration, the absolute configuration of compound 12 was determined by the helicity rule using the ECD measurement after derivatization with dimolybdenum tetraacetate 25,26 . It is possible to apply this method because compound 12 is erythro-1,2-diol and there is a bulkiness difference between the two substituents around the hydroxyl groups. The CD measurement after complexation with [Mo 2 (OAc) 4 ] showed a negative CE in the band II region (403 nm, −0.15 mdeg) used as the diagnostic band (Fig. 3C). Accordingly, the structure of 12 was determined as (8R,9S)-8,9-dihydromegastigmane-4,6-diene-3-one.

Measurements of insulin mimetic activity with 2-NBDG on differentiated 3T3-L1 adipocytes.
Unlike the insulin in the brain, insulin at the periphery acts as an anabolic factor and causes weight gain as a side effect if the energy usage of the patient is not increased. Insulin mimetics have similar characteristics to insulin in terms of the reduction in food intake and body weight in rats when administered intracerebroventricularly. Considering the abovementioned difference, insulin memetics appear to be more advantageous than insulin because of their potential to pass through the blood-brain barrier (BBB), which allows us to find insulin mimetics from natural resources with fewer side effects. To measure for insulin mimetics, the 2-NBDG assay, which is used a fluorescent-tagged glucose analogue for monitoring the glucose uptake in cells, was introduced. All isolates (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) were evaluated for 2-NBDG uptake in differentiated 3T3-L1 adipocytes at a concentration of 40 μM (Fig. 4A and Supplementary Fig. S49). Most of the phenolic acid-derivatized seco-iridoid showed activity, whereas compound 9 with an acetyl group and compound 10 with a monoterpene showed weak activity. Comparison of the activities of the compounds with the trans and cis forms showed that the trans isomers had stronger activity than cis. Among these compounds, compounds 3, 7 and 8 showed stronger activities compared to others. Thus, fluorescent signals measurement was performed using a fluorescence microscopy for assessing the transport efficacy of 2-NBDG into cells. Increased signal intensities after treatment of the compounds were more strongly observed in cells treated with 3, 7 and 8 at 40 μM compared to those in cells of the control group (DMSO, Fig. 4B). It was also observed that selected compounds 3, 7 and 8 increased the 2-NBDG uptake in a dose-dependent manner (Fig. 5A,B). Taken together, these results suggest that derivatization of seco-iridoids with trans-configured phenolic acids is relevant to activity. These results are consistent with the ethnopharmacological history of this plant as a diabetes remedy in Ayurvedic formulations.  Figs S61 and S62). These results provide insight into how these molecules stimulate glucose uptake through up-regulation of the Akt pathway. The present data strongly indicate that compound 3 promotes glucose uptake by GLUT4 translocation associated with increased level of GLUT4 expression. Although the mechanism of 3 is still unclear and further studies are in need near future, we propose this compound could regulate the glucose metabolism and insulin sensitivity.
Protein tyrosine phosphatase 1B (PTP1B) inhibitory activity of isolated compounds. Upon insulin binding, glucose uptake is increased by a series of signals including phosphorylation of the insulin receptor (IR), transformation of phosphatidylinositol (4,5)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3) by phosphatidylinositol 3-kinase (PI3K), activation of Akt, and translocation of GLUT4. PTP1B exerts negative regulation of insulin and leptin receptor signalling by dephosphorylation of activated IR and Janus kinase 2 31 . Since some seco-iridoids increased glucose uptake through 2-NBDG and GLUT4 translocation, the PTP1B inhibitory activity, which is important in the relevant signal transduction process, was also assessed 32 . When all isolated compounds were subjected to the PTP1B assay, compound 3 was found to be a good candidate for use as a PTP1B inhibitor, whereas 7 and 8 showed moderate activity at 50 μM ( Supplementary  Fig. S53). Among these compounds, the IC 50 of 3 is 19.54 ± 0.76 μM, showing moderate activity compared to the positive control tested (Supplementary Fig. S54). Compound 3 was further examined in kinetic experiments and was shown to inhibit PTP1B in a non-competitive manner at different concentrations (10, 20 and 40 μM, Fig. 5D). Molecular docking analysis of compound 3 into the active site of PTP1B (PDB ID code 1Q6T) was performed according to the CDOCKER protocol in the CHARMm-based docking algorithm 33 . As shown in Fig. 5E, the C-7 carboxylic acid group forms a conventional hydrogen bond and a carbon-hydrogen bond with Asp548 and GLY759, respectively. Moreover, the benzene ring shows affinity for Phe682 via a π-π interaction ( Supplementary Fig. S55). These key residues have been proposed as active sites A and B of PTP1B. Additionally, the CDOCKER interaction energy was calculated to be −61.18 kcal/mol. Overall, the observed effects appear to contribute to the inhibitory activity of compound 3 against PTP1B.

Conclusions
In this paper, eight new analogues of phenolic acid-conjugated 10-hydroxyoleoside type (1-6 and 8-9) and two new monoterpene-conjugated compounds (10 and 11), along with one known seco-iridoid (7), one megastigmane (12) and two triterpenoids, (13)(14) were isolated from S. cochinchinensis. The absolute configurations of these compounds were determined by ECD analysis, dirhodium (ІІ) tetrakis (trifluoroacetate) reaction or dimolybdenum (ІІ) tetraacetate reaction. Compounds 3, 7 and 8 exhibited 2-NBDG uptake increasing activity in differentiated 3T3-L1 adipocytes by GLUT4 translocation which was evident in Western blot analysis. Compound 3 increased GLUT4 expression level and direct GLUT4 translocation through the PI3K/Akt pathway via a PTP1B inhibition. These results also imply the structure activity relationship to some degree such that derivatization with a phenolic acid and trans configuration contribute to the activity. In conclusion, investigation of new seco-iridoids as ant-diabetic compounds enriches the chemical profile of S. cochinchinensis and provides evidence for the traditional ethnopharmacological uses of this plant.

Methods
General experimental procedures. Optical rotations were recorded on a JASCO P-2000 polarimeter (JASCO International Co. Ltd., Tokyo, Japan). ECD spectra were measured using Chirascan plus (Applied photophysics Ltd., Surrey, United Kingdom). IR data were obtained using a Nicolet 6700 FT-IR spectrometer (Thermo Electron Corp., Waltham, MA, USA). The 1D and 2D NMR spectra were obtained in deuterated solvents using an AVANCE 800 MHz spectrometer (Bruker, Germany). HRESIMS values were obtained using an Agilent Technologies 6530 Q-TOF MS spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA). Regular column chromatography (CC) was carried out with silica gel (particle size: 63-200 μm, Zeochem, Lake Zurich, Switzerland), RP-C 18 (particle size: 75 μm, nacalai tesque, Kyoto, Japan), and Sephadex LH-20 (GE Healthcare, Little Chalfont, UK). Silica gel 60 F 254 and RP-18 F 254 S TLC plates were obtained from Merck (Darmstadt, Germany). A Gilson HPLC purification system was used at a flow of 2 mL/min and UV detection at 205, 254, and 300 nm using an Optima Pak C 18 column (10 × 250 mm, 5 μm particle size; RS Tech, Seoul, Korea) and a COSMOSIL 5C 18 -MS-II column (10 × 250 mm, 5 μm particle size; Nacalai Tesque, Kyoto, Japan). Analyticalgrade solvents were used for extraction and isolation. Extraction and isolation. The stems and leaves of S. cochinchinensis (4 kg) were extracted with 70% EtOH (4 × 11 L, for 4 h each) at 60 °C. The combined extract was concentrated by an evaporator to yield a dried residue (752.6 g). Dried extract was suspended in H 2 O and then partitioned with n-hexane, EtOAc and n-BuOH successively. The EtOAc portion (57.3 g) was subjected to silica gel CC (8 × 50 cm) and eluted with gradient system of n-hexane/acetone from 5:1 to 0:1 to yield five fractions (F.1-F.5). F.5 (12 g) was subjected to reversed-phase chromatography (  Determination of absolute configuration of sugars. Compound 9 (1.0 mg) was hydrolysed by 0.5 M HCl (1.0 mL) at 90 °C for 1 hour 35 . The solvent was neutralized with Na 2 CO 3 and concentrated in vacuo. L-Cysteine methyl ester hydrochloride in anhydrous pyridine (0.5 mg) was added to the resulting residue, followed by heating for 1 hour at 60 °C. Phenyl isothiocyanate (0.1 mL) was added and heated at 60 °C for 1 hour. The solution was then analyzed by reversed-phase HPLC under the following conditions: an INNO C 18 column (120 Å, 4.6 × 250 mm, 5 μm); MeCN/H 2 O mobile phase (27:73, v/v); a diode array detector; a detection wavelength of 254 nm; and a flow rate of 0.6 mL/min. Comparisons of the retention time of the derivative of compound 9 with that of the derivative of an authentic sample of D-glucose (retention time: 23.18 min) proved the D-configuration of the glucose moiety in compound 9.
Absolute configuration of the tertiary alcohol moiety in 10. Compound 10 (13.2 mg) was kept in pyridine/Ac 2 O 1:1 (4 mL) at room temperature for 19 hours to give peracetylated compound 10a. The mixture was neutralized with NaHCO 3 and dried under vacuum. A colorless gum (10.6 mg, 80.3%) was obtained by extraction with EtOAc and the product was further purified to over 99% using prep-HPLC. Compound 10a (0.5 mg) was then dissolved in a dry solution of [Rh 2 (OCOCF 3 ) 4 ] (1.0 mg) in CDCl 3 (600 μL). The resulting mixture was used for CD measurements and the obtained CD spectrum was compared with that of compound 10a for clarity. The Cotton effect at 350 nm (E band) was correlated with the absolute configuration of the tertiary alcohol 36 . preparation of Mo 2 -complexes of compound 12. The CD spectra were measured at room temperature in DMSO with 1.0 nm/step scans using a 2 mm cell over the range of 250-650 nm according to the Snatzke's method 25 . To form complexes, compound 12 (0.18 mg, 1.33 mM/L) was dissolved in a solution of [Mo 2 (OAc) 4 ] (0.34 mg, 1.33 mM/L) in DMSO at a 1/1 ratio of the molybdenum complex to the diol. Measurement of glucose uptake using 2-NBDG in differentiated 3T3-L1 adipocytes. To determine the level of glucose uptake into 3T3-L1 adipocytes, a fluorescent derivative of glucose (2-NBDG) (Invitrogen, OR, USA) was used as previously described with slight modifications 34,37 . First, 3T3-L1 preadipocytes were differentiated by Dulbecco's Modified Eagle's Medium (DMEM) (HyClone, IL, USA) containing 10% fetal bovine serum (FBS) (Gibco, NY, USA), 1 μM dexamethasone (Sigma, MO, USA), 520 μM 3-isobutyl-1-methyl-xanthine