Clerodane Furanoditerpenoids from Tinospora bakis (A.Rich.) Miers (Menispermaceae)

Tinospora bakis (A.Rich.) Miers (Menispermaceae) has traditionally been used to alleviate headaches, rheumatism, mycetoma, and diabetes, among others. Despite its extensive use, the active components of the plant have never been investigated. In this work, a series of furanoditerpenoids (1–18) and five compounds from other classes (19–23) were isolated from T. bakis. Notably, two new compounds were discovered and named: tinobakisin (1) and tinobakiside (10). Their molecular structures were elucidated with NMR, MS, UV, IR, and ECD spectra. Additionally, known compounds (2–9 and 11–23) were corroboratively identified through spectral comparisons with previously reported data, while highlighting and addressing some inaccuracies in the prior literature. Remarkably, compounds 6, 7, 13, and 17 exhibited a superior anti-glycation effect, outperforming established agents like rutin and quercetin in a lab model of protein glycation with glucose. The overall findings suggest that furanoditerpenoids play a crucial role in the antidiabetic properties of T. bakis. This research marks the first comprehensive phytochemical investigation of T. bakis, opening the door for further investigation into furanoditerpenoids and their biological mechanisms.


Introduction
Diabetes has been a severe health and economic burden globally, with increasing prevalence year by year.It is worth mentioning that around 422 million adults were living with diabetes in 2014 globally, in comparison to 108 million in 1980, and the prevalence is expected to approach 629 million worldwide in 2045 [1,2].Therefore, treating diabetes and its associated comorbidities has been a hot field, attracting the attention and effort of scientists [3].Natural products have been indispensable sources for new drug discovery.Among the approved drugs from 1981 to 2014, natural products or their derivative accounted for 26% [4].Folk herbal medicines have been used worldwide for controlling blood glucose levels in patients with diabetes since ancient times [5].Many investigations have been conducted to explore the active components for advanced antidiabetic drug discovery [6].
Tinospora bakis (A.Rich.)Miers is a deciduous climber, wildly growing in Africa.Its whole plant, root, or leaves have been used to treat headaches, rheumatism, mycetoma, diabetes, etc., in Africa [8,17,18].Despite its therapeutic importance, the phytochemicals of T. bakis have never been investigated, except for the main component, columbin [17].Although the extracts from the roots or whole plant of T. bakis have been studied through in vivo pharmacological approaches, demonstrating antipyretic, antidiabetic, and immunomodulatory effects, most of the active components in T. bakis remain unknown [18][19][20].
In this work, a systematic phytochemical investigation was conducted on T. bakis for the first time.A total of 23 compounds were isolated from the EtOAc fraction of T. bakis.The structures of all compounds were elucidated based on multiple pieces of spectroscopic evidence, such as NMR, MS, UV, IR, and ECD.Among them, 18 compounds (1-18) were furanoditerpenoids, as shown in Figure 1.Moreover, an in vitro anti-glycation essay on the obtained components was performed, resulting in several potential antidiabetic leads, which shed light on the molecular basis of the antidiabetic property of T. bakis.Further investigation of the in vivo evidence of the active components and related mechanisms might be required [21][22][23].

Results and Discussion
Compound 1 was obtained as white needle-like crystals.The molecular formula was deduced as C 21 H 26 O 7 according to the molecular ion peak [M] + at m/z 390.1687 (calcd.for 390.1679) in HR-EI-MS, with nine unsaturation degrees.The 1 H-NMR data (Table 1) displayed the characteristic signals for a furan ring at δ H 7.54 (br s), 7.39 (t, J = 1.2 Hz), and 6.73 (d, J = 1.2 Hz).An olefinic proton at δ H 6.57, two oxymethine signals at δ H 5.15 (dd, J = 11.2, 4.8 Hz) and 4.46 (m), two methyls at δ H 1.44 (s) and 1.05 (m), and a methoxy at δ H 3.73 were also observed.The 13 C-NMR (Table 1) and DEPT spectra revealed the existence of 21 carbons, including two carbonyl signals at δ C 178.3 and 169.5; 4 carbons for the furan group at δ C 144.1, 142.0, 127.6, and 110.9; 2 olefinic carbons at δ C 141.1 and 140.4; 3 oxygen-substituted alkyl carbons at δ C 90.4, 74.3, and 65.1; two methyls at δ C 30.6 and 22.2; and a methoxy at δ C 52.1, along with another 2 quaternary carbons, one methine, and four methylenes in the upfield.All the data agreed with the aglycon of tinospinosides B (2) [11].The structure was further confirmed by correlations in the 1 H-1 H COSY, HSQC, and HMBC spectra, which are assigned in Figure 2. Correlations of H-2 (δ H 4.46)/CH 3 -20 (δ H 1.05), H-10 (δ H 1.92)/CH 3 -19 (δ H 1.44), and H-10 (δ H 1.92)/H-12 (δ H 5.15) in the NOESY spectrum indicated the configurations of chiral carbons.Since enantiomers of clerodane diterpenoids commonly exist naturally [24], the relative configuration of 1 was also further proved by curve fitting the experimental and calculated ECD.As shown in Figure 3, the calculated ECD of the proposed structure showed an excellent agreement with the experimental ECD of 1.Therefore, the structure of compound 1 was confirmed and named tinobakisin.a 1 H-NMR data (δ) were measured in CD 3 OD at 400 MHz. 13 C-NMR data (δ) were measured in CD 3 OD at 125 MHz.b 1 H-NMR data (δ) were measured in CD 3 OD at 500 MHz. 13C-NMR data (δ) were measured in CD 3 OD at 125 MHz.
Compound 10 was isolated as an amorphous, colorless semisolid.The molecular formula was deduced as C 25 H 32 O 6 , through the molecular ion peak [M + H] + at m/z 493.2061 (calcd.for 493.2074,C 25 H 33 O 6 ) in positive HR-FAB-MS, suggesting 10 degrees of unsaturation.The 1 H-NMR data (Table 1) displayed the characteristic signals for a furan moiety at δ H 7.59 (br s), 7.49 (t, J = 2.0 Hz), and 6.54 (d, J = 2.0 Hz); an olefinic proton at δ H 6.30 (dd, J = 6.5, 2.5 Hz); an oxymethine signal at δ H 5.57 (dd, J = 12.5, 4.0 Hz); and two methyls at δ H 1.28 (s) and 0.97 (s). 13C-NMR (Table 1) revealed the presence of two carbonyl carbons at δ C 200.5 and 174.8; aromatic carbons at δ C 149.2, 145.0, 141.4,126.8, 124.3, and 109.6; and two angular methyls at δ C 29.0 and 26.6.All these data of 1 were similar to those of sagitone, a novel 18-norclerodane furanoditerpenoid isolated from the roots of Tinospora sagittata var.yunnanensis [14].However, additional signals belonging to a glucopyranoside at δ C 102.3, 74.6, 78.3, 71.3, 77.6, and 62.5, corresponding to δ H 4.62, 3.84, 3.65, 3.39, 3.38, 3.34, and 3.32 in the HSQC spectrum, were also observed [28].After acid hydrolysis, the sugar moiety was determined as D-glucose, based on the optical rotation detected by an optical detector through HPLC.Also, the large coupling constant exhibited by the anomeric proton at δ H 4.62 (1H, d, J = 7.0 Hz) suggested the relative configuration as β-oriented (Figure 4).In addition, the experimental ECD of 10 was close to 1 (Figure 5), indicating a similar relative configuration.Thus, the structure of 10 was established and named tinobakiside.Compound 11 was elucidated as palmatoside G [29].Compounds 12 and 13 were determined to be jateorin and isojateorin, respectively, based on the NMR data [30,31].The X-ray structure of jateorin was reported in 1986 as a major component of Tinospora cordifolia [32].Tinosporide was previously wrongly identified from Tinospora cordifolia and then later confirmed by X-ray to be identical to jateorin [33].Chasmanthin and palmarin from Jateorrhiza palmate, Jateorhiza palmate, and Fibraurea chloroleuca were reported as the 12-epimer of jateorin and isojateorin, respectively [30,31,34], and also found in Tinospora cordifolia [35], which proved the common existence of these compounds in the Menispermaceae family.The structure of 14 was initially deduced, as reported by Hanuman et al. [36], because the NMR values were identical.However, after careful elucidation by 2D NMR, it was determined as the columbin, which was also isolated from several other species of the Tinospora genus [14,16,37,38] and found to be the major component of T. bakis [17].This literature might wrongly determine the structure [36] in which 2D NMR was not performed.In addition, this is the only report of this structure.Compound 14 had β-H at C-8. Compound 15 had α-H (δ 2.98) at C-8 as its isomer [30].Compounds 16 and 17, palmatoside C and D, the glucosides of 14 and 15, were isolated together from Jateorhiza palmata Miers in 1987 [29].Later, palmatoside C was also found in the Tinospora genus [14,37].Its configuration can be confirmed by the correlations with CH 3 -20 (δ 1.26) and H-10 (δ 1.84) in the NOESY spectrum.Compound 18 was elucidated as 8-hydroxycolumbin [39].

General Experiment Procedures
The Bruker AMS-400 and AMX-500 (Bruker, Billerica, MA, USA) were used to record NMR spectra.For LR-FAB-MS and LR-EI-MS, JEOL MS Route JMS 600H (JEOL Ltd., Akishima, Japan) mass spectrometer was used, and, for HR-FAB-MS, JEOL JMS-HX110 mass spectrometer was used.A UV/visible spectrophotometer was used to accomplish the UV/visible method.The optical rotations were completed on the p-2000 Polarimeter, and the infrared (IR) spectra were recorded on the Attenuated Total Reflectance Infrared Spectrophotometer (ATRIR) FTIR iS50 (Fourier-Transform Infrared Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA) in the KBr disc.Electronic Circular Dichorism (ECD) measurements were performed on Jaso-J-8-10 Circular Dichorism Spectro-Polarimeter.Normal silica gel (E.Merck, 70-230 Mesh) was utilized for fractionation by column chromatography.C18 (Wakogel, 38-63 Mesh), Sephadex LH-20 (GE healthcare, Chicago, IL, USA), and normal and reverse-phase HPLC were employed for purification.The purity of the sample was confirmed using normal and reverse-phase precoated TLC.UV light at 254 nm was used to evaluate TLC plates.Dragendroff, ceric sulfate, and vanillin were employed to visualize the spots on TLC plates.

Plant Material
The whole plant of T. bakis was collected from the Nuba Mountains in western Sudan from December 2018 to January 2019 and taxonomically identified by Yahya Sulieman Mohamed at Herbarium of Medicinal and Aromatic Plants and Traditional Medicine Research Institute, National Center for Research, Khartoum, Sudan.A voucher specimen (M-95-17-MAPTRI-H) was preserved in the Herbarium of Medicinal and Aromatic Plants and Traditional Medicine Research Institute, National Center for Research, Khartoum, Sudan, for further reference.

Acid Hydrolysis
First, 5 mL 1 M HCl was used to dissolve compound 10 (2 mg), and then the mixture was stirred at 80 • C.After 8 h, the reaction mixture was extracted by CH 2 Cl 2 .Then, the obtained aqueous layer was evaporated under a vacuum and diluted multiple times to provide a neutral residue.The residue was subjected to analytical HPLC (Jasco LC-4000, Tokyo, Japan) with a Jasco OR-4090 optical rotation detector.The hydrolyzed sugar moiety indicated a positive rotation [45,46].

ECD Calculation
The ECD calculation of 1 was performed as reported, and the details are provided in Supplementary Data [10,47].Conformational search with systematic algorithm was performed in Yinfo Cloud Platform (https://cloud.yinfotek.com/,accessed on 26 September 2023) using Confab at MMFF94 force field.Conformers were filtered by an RMSD threshold of 0.5 Å and an energy window of 7 kcal/mol.The energies and populations of dominative conformers are provided in Table S1.Structures for ECD calculation are shown in Table S2.All structures were confirmed by vibration frequency analysis, and no imaginary frequencies were found.Table S3 indicates the standard orientations of all configurations for ECD calculation at B3LYP/6-311G(d,p) level in methanol.

Anti-Glycation Activity
The anti-glycation effect was performed by the BSA (bovine serum albumin)-glucose glycation model in vitro, as previously described [48].Glucose (0.5 M), BSA (10 mg/mL), and sodium azide (NaN 3 ) (0.1 mM) were dissolved in sodium phosphate buffer (pH 7.4) as the reaction reagent to perform with or without the tested sample.The test compounds and standard references (rutin and quercetin) were dissolved in 10% DMSO.BSA without a glycating agent was used as a negative control.All samples were initially screened at 1 mM concentration.The assay was performed in triplicates, and a final reaction volume of 200 µL was maintained in each well of a 96-well plate.The reaction mixture was incubated at 37 • C for 7 days in a dark, sterile condition.The fluorescence (excitation 340 nm and emission 440 nm) was measured using a Varioskan Lux microtitre plate reader (Thermo Fisher Scientific, Waltham, MA, USA).
The compounds that exhibited more than 50% inhibition were processed for IC 50 .The two-fold dilution was made, and multiple concentrations (1, 0.5, 0.25, 0.125, 0.06, and 0.03 mM) of active compounds and standards (rutin and quercetin) were incubated with BSA-glucose at 37 • C for 7 days.The fluorescence was measured, and then the IC 50 (µM) was calculated using the EZ-FIT Enzyme Kinetics Program (Perrella Scientific Inc., Amherst, MA, USA).

Conclusions
In summary, our phytochemical investigation of Tinospora bakis uncovered a plethora of clerodane furanoditerpenoids, including the discovery of two new compounds, tinobakisin (1) and tinobakiside (10), and provided clarifications on previously reported compounds.The observed superior anti-glycation effects of compounds 6, 7, 13, and 17 underscore the potential therapeutic relevance of T. bakis in managing diabetic complications.This research offers the first in-depth chemical profile of T. bakis.It lays the foundation for future studies to understand the mechanistic details of these compounds' biological activities and their potential therapeutic applications, thus paving the way for new avenues in treating chronic diseases.More in vivo pharmacological and mechanism investigation might be required to further understand the active components' antidiabetic properties, leading to the discovery of potential lead compounds for drug discovery.
Figure S24.NOESY spectrum of compound 10.S1.Detailed methodology of ECD calculation of compound 1.Table

Author
Contributions: Conceptualization, A.S.K. and Y.W.; methodology, A.S.K., M.A.S., N.N.S. and M.A.; formal analysis, Y.W., M.A.S. and M.A.; investigation, A.S.K., M.A.S., M.A., M.K., A.F. and N.N.S.; resources, A.S.K.; writing-original draft preparation, M.A.S. and Y.W.; writing-review and editing, Y.W. and F.-A.K.; visualization, Y.W.; supervision, Y.W., F.-A.K. and H.J.; project administration, Y.W. and F.-A.K.; funding acquisition, A.S.K., F.-A.K. and Y.W.All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by the National Administration of Traditional Chinese Medicine (no.GZYYGJ2020023) and the Higher Education Commission of Pakistan (NRPU 2021 #17603), Sindh Higher Education Commission (SHEC/SRSP/MS-1/7/2020-21).We acknowledge The World Academy of Science (TWAS) for the 2019 ICCBS-TWAS Sandwich Postgraduate Fellowship awarded to Ahmed Saeed Kabbashi with FR number 3240305623.The authors appreciate the support from the Medicinal and Aromatic Plants and Traditional Medicine Research Institute (MAPTMRI) and the Faculty of Pure and Applied Sciences, International University of Africa, Khartoum, Sudan.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.