Probing PXR activation and modulation of CYP3A4 by Tinospora crispa and Tinospora sinensis.

ETHNOPHARMACOLOGICAL RELEVANCE: The two Tinospora species, T. crispa and T. sinensis, native to Southeast Asia, are integral components of various traditional preparations with structure-function claims to treat various disorders, including diabetes and inflammation. AIM OF THE STUDY: To assure the safety of the botanicals finished products, herb-drug interaction potential of T. crispa and T. sinensis was investigated by testing their extracts and compounds for in vitro activation of the pregnane X-receptor (PXR) and the modulation of CYP3A4 isozyme, selectively. MATERIALS AND METHODS: A total of sixteen fully characterized phytochemicals from T. crispa and T. sinensis were evaluated for PXR activation by luciferase reporter gene assay. CYP3A4 inhibition studies were carried out for eleven compounds. In addition, docking studies were performed to elucidate the possible binding modes to the PXR by the compounds using computational methods. RESULTS: Significant activation of PXR (2-fold) was observed for both extracts and non-polar fractions of T. crispa. Among the pure compounds, columbin showed highest activation of PXR (3-fold), which was comparable with the positive control, rifampicin. Vital interactions were predicted with docking simulation of PXR-columbin complex with critical amino acid residues (Trp-299) that are known for the activation of PXR. The methanolic extracts of T. crispa and T. sinensis also showed considerable CYP3A4 inhibition. CONCLUSION: T. crispa and T. sinensis, both demonstrated the potential to mediate herb-drug interaction through PXR activation and inhibition of CYP3A4 isozyme. Moreover, the elucidation of the potential to induce herb-drug interaction, by the phytochemicals of these Tinospora plants, thereby supports the need for further investigation to establish the clinical relevancy of these constituents for possible adverse interactions with pharmaceutical drugs.

Rearranged clerodane diterpenoid from Tinospora crispa.

A new rearranged clerodane diterpenoid, tinocrispide was isolated from the stems of Tinospora crispa along with thirteen known compounds including eight clerodane diterpenoids. Among the known compounds baenzigeride A, (6S, 9 R)-vomifoliol and steponine are being reported for the first time from T. crispa. Their structures were elucidated by 1 D and 2 D NMR and confirmed by HRESIMS. The 13C NMR data of borapetol A has been revised.

Profiling and Quantification of the Key Phytochemicals from the Drumstick Tree (Moringa oleifera) and Dietary Supplements by UHPLC-PDA-MS.

Moringa oleifera is known as a drumstick tree and is cultivated in the subtropics and tropics. It exhibits antihypertensive and antidiabetic effects. An ultra-high-performance liquid chromatography method was developed for the determination of 9 phytochemicals in M. oleifera leaves and marketed products. The efficient separation was achieved within 7 min with a temperature of 45 °C by using a C-18 column as the stationary phase and water/acetonitrile with 0.05% formic acid as the mobile phase. The method was validated for linearity, repeatability, limits of detection, and limits of quantification. The limits of detections of phenolic compounds 1:  - 9: were as low as 0.2 µg/mL. The photodiode array detector at 220 and 255 nm wavelengths was recruited for quantification. The key phytochemicals were detected in the range of 0.42 to 2.57 mg/100 mg sample weight in 13 dietary supplements. This study considers the quantitative analysis for lignans in M. oleifera for the first time. Isoquercitrin (5: ) and quercetin 3-O-(6-O-malonyl)-ß-D-glucopyranoside (6: ) predominates the leaves of M. oleifera with inherent degradable nature detected for compound 6: . Niazirin (2: ) was detected in amounts between 0.010 - 0.049 mg/100 mg while compound 1: was undetectable and potentially an artifact because of the fractionation process. The characterization and confirmation of components were achieved by liquid chromatography-electrospray ionization-mass spectrometry with extractive ion monitoring for the positive and negative ion modes. The developed and validated method is robust and rapid in the conclusive quantification of phytochemicals and authentication of the Moringa samples for quality assurance.

Undescribed phenylpropanoid and a dimeric sesquiterpenoid possessing a rare cyclobutane ring from Tinospora sinensis.

A rare dimeric sesquiterpenoid (tinosposinoside, 1) and a phenylpropanoid (cordifolioside C, 2), two undescribed metabolites, were isolated from the methanolic extract of the stems of Tinospora sinensis together with thirteen known compounds. This is the second example of a sesquiterpene dimer where two monomers linked together through a cyclobutane ring, possibly generated through [2 + 2] cycloaddition. The structures of 1 and 2 were elucidated by NMR and mass techniques.

Evaluation of the hepatotoxic potential of Tinospora crispa and its isolated borapetosides B, C and F in a murine model.

The hepatotoxic potential of the methanolic extract of the stems of Tinospora crispa and of its furano-diterpenoid-borapetosides B, C, and F-was investigated in normal and health-compromised mice. Health-compromised condition was established by a single intraperitoneal injection of LPS (6 mg/kg). Two different sets of experiments were conducted to evaluate the hepatotoxic potential. A 21-day study where the mice were dosed with the extract of T. crispa (1 gm/kg b. wt./day) or standardized combination of borapetosides B, C and F (500 mg/kg b. wt.) with or without a single dose of LPS (1 gm/kg b. wt./day). In the acute toxicity study, mice were dosed with borapetosides B, C, and F (500 mg/kg b. wt.). Results showed that the ALT levels were normal and liver histopathology unaltered. No conclusive hepatotoxicity was observed in the methanolic extract or pure compounds tested under the given experimental conditions.

Comparative Morpho-Anatomical and HPTLC Profiling of Tinospora Species and Dietary Supplements.

Overlapping geographical occurrence, history of traditional use, confusion in species identification, and morphological resemblances among various species are some considerations that necessitate the importance of qualitative analysis for efficient quality control and safer botanical products. This paper provides detailed morpho-anatomies of the leaves and stems of Tinospora cordifolia, Tinospora crispa, and Tinospora sinensis, and stems of Tinospora baenzigeri. Microscopy studies of the selected Tinospora species revealed key diagnostic features that can help distinguish the closely related species of Tinospora as well as to detect any adulteration or substitution in the raw materials. HPTLC profiles of the authenticated plant materials, as well as commercial products claiming to contain Tinospora, were compared to distinguish T. crispa from other closely related species and to establish an efficient method to assess the identity and quality of the products using qualified chemical markers. HPTLC chromatograms of both plant samples and dietary supplements were compared with six reference marker compounds. The analysis revealed that borapetoside B and C were useful to identify T. crispa while tinosineside A was found to be characteristic to authenticate the T. sinensis products.

Bioassay guided isolation of mosquito biting deterrent compounds from Strumpfia maritima.

BACKGROUND: In our natural products screening program against mosquitoes, we screened ethanolic extract of Strumpfia maritima Jacq. which showed high biting deterrent activity in Klun and Debboun (K&D) bioassays against Aedes aegypti L. Through bioguided fractionation we isolated six compounds from the active fractions. The extract and pure compounds were evaluated for larvicidal and biting deterrent activity against Aedes aegypti. RESULTS: Ethanolic extract with proportion not biting (PNB) value of 0.8 ± 0.08 provided biting deterrent activity similar to N,N-dimethyl-m-toluamide (DEET) (PNB = 0.9 ± 0.05) against Ae. aegypti. Five out of eight fractions, prepared through bioassay-guided fractionation, showed biting deterrent activity similar to DEET. Six compounds were isolated from active fractions and characterized as squalene, ursolic acid, betulonic acid, cabraleone, ursolic acid acetate, and 27-p-E-coumaroyloxyursolic acid. The biting deterrent activity of ursolic acid acetate (biting deterrence index, BDI = 0.85), cabraleone (BDI = 0.79), and ursolic acid (BDI = 0.78) was similar to DEET against Ae. aegypti. Strumpfia maritima extract did not show larvicidal activity at the highest screening dose of 125 ppm in the larvicidal bioassay. CONCLUSION: The ethanolic extract and pure compounds showed biting deterrent activity against Ae. aegypti. The high biting deterrent activity of ursolic acid acetate and cabraleone indicated a great potential for these natural compounds to be developed as mosquito repellents. © 2020 Society of Chemical Industry.

Development of a chemical fingerprint as a tool to distinguish closely related Tinospora species and quantitation of marker compounds.

Tinospora species are morphologically similar. Several cases of human toxicity have been reported in association with T. crispa. A chemical fingerprint was developed to differentiate T. crispa from its closely related species and to quantitate its major furanoditerpenes namely as borapetosides B, C and F. The rapid, sensitive and repeatable method was established using ultra-high performance liquid chromatography coupled with photodiode array and single quadrupole electrospray mass spectrometry detectors using a flavonoid, two alkaloids, an amide and six diterpenoids. Qualitative and quantitative determination was performed by UHPLC-UV and confirmed by MS. The intra-day RSD for replicates was between 0.9 and 6.8% and inter-day RSD was between 1.2 and 9.1%. Recovery was 97-103 %. The method is useful to achieve decisiveness in not only identifying but also differentiating T. crispa from T. sinensis and other closely related Tinospora species. Seventeen Tinospora plant samples and seventeen dietary supplements claiming T. crispa, T. sinensis and T. cordifolia were analyzed. The newly developed and validated method successfully resulted in the conclusive identification of two dietary supplements to be mislabeled.

Isolation, synthesis, and drug interaction potential of secondary metabolites derived from the leaves of miracle tree (Moringa oleifera) against CYP3A4 and CYP2D6 isozymes.

BACKGROUND: Moringa oleifera Lam. is known as a drumstick tree that is widely cultivated in various subtropical and tropical provinces. Previous studies indicated that both aqueous and methanolic extracts of M. oleifera leaves have potent inhibitory effects on two major drug metabolizing Cytochrome P450 enzymes, namely, CYP3A4 and CYP2D6. PURPOSE: The current study was aimed to isolate the secondary metabolites from M. oleifera and investigate their cytotoxicity and inhibitory effects on CYP3A4 and CYP2D6 to assess their herb-drug interaction (HDI) potential. METHODS: Chemical structure elucidation was achieved by interpreting the spectroscopic data (UV, IR, 1D, and 2D NMR experiments), confirming by HR-ESI-MS, and comparing with the previously reported data in the literature. All the isolates were evaluated for their cytotoxicity against a panel of cell lines (SK-MEL, KB, BT-549, SK-OV-3, VERO, LLC-PK1, and HepG2) and inhibition of two principal CYP isozymes (CYP3A4 and CYP2D6). RESULTS: Phytochemical investigation of M. oleifera leaves resulted in the isolation and characterization of one new compound, namely omoringone (1), along with twelve known secondary metabolites (2-13) belonging to several chemical classes including flavonoids, terpenoids, lignans, and phenylalkanoids. A plausible biosynthetic pathway for compound 1 was provided. Because of the low isolation yield and limited supply, omoringone (1) and niazirin (12) were successively synthesized. No cytotoxicity was observed on any of the tested cell lines up to 50 µM. The extract exhibited an inhibitory effect on CYP3A4 isoform (IC50 = 52.5 ±â€¯2.5 µg/ml). Among the isolates, 1-4 and 7-9 inhibited CYP3A4 with the IC50 values ranging from 41.5 to 100 µM with no remarkable effect on CYP2D6 isozyme. CONCLUSION: This work aided in ascertaining components of M. oleifera contributing to CYP3A4 inhibition exhibited by the extract using an in vitro assay. Nonetheless, further studies are warranted to determine the bioavailability of the phytochemicals and extrapolate these findings in more physiologically relevant conditions to further establish the clinical relevance of in vitro observations.

Safety Assessment of Phytochemicals Derived from the Globalized South African Rooibos Tea ( Aspalathus linearis) through Interaction with CYP, PXR, and P-gp.

Rooibos tea ( Aspalathus linearis) is a well-known South African herbal tea enjoyed worldwide. Limited reports indicate the potential of rooibos tea to alter the activity of certain cytochrome P450 (CYP450) isozymes. In this study, the phytochemical investigation of MeOH extract of A. linearis (leaves and stems) resulted in the isolation and characterization of 11 phenolic compounds. The MeOH extract exhibited significant inhibition of the major human CYP450 isozymes (CYP3A4, CYP1A2, CYP2D6, CYP2C9, and CYP2C19). The strongest inhibition was observed by the extract for CYP3A4 (IC50 1.7 ± 0.1 µg/mL) followed by CYP2C19 (IC50 4.0 ± 0.3 µg/mL). Among the tested phytochemicals, the most potent inhibitors were isovitexin on CYP3A4 (IC50 3.4 ± 0.2 µM), vitexin on CYP2C9 (IC50 8.0 ± 0.2 µM), and thermopsoside on CYP2C19 (IC50 9.5 ± 0.2 µM). The two major, structurally related compounds aspalathin and nothofagin exhibited a moderate pregnane-X receptor (PXR) activation, which was associated with increased mRNA expression of CYP3A4 and CYP1A2, respectively. These results indicate that a high intake of nutraceuticals containing rooibos extracts may pose a risk of herb-drug interactions when consumed concomitantly with clinical drugs that are substrates of CYP enzymes.