Minor Hydroxylated Triterpenoids Produced in Engineered Yeast by the Enzymes OSC and CYP716s from the Plant Enkianthus chinensis and Their Anti-Inflammatory and Hepatoprotective Activities.

Triterpenoids are a type of specialized metabolites that exhibit a wide range of biological activities. However, the availability of some minor triterpenoids in nature is limited, which has hindered our understanding of their pharmacological potential. To overcome this limitation, heterologous biosynthesis of triterpenoids in yeast has emerged as a promising and time-efficient production platform for obtaining these minor compounds. In this study, we analyzed the transcriptomic data of Enkianthus chinensis to identify one oxidosqualene cyclase (EcOSC) gene and four CYP716s. Through heterologous expression of these genes in yeast, nine natural pentacyclic triterpenoids, including three skeleton products (1-3) produced by one multifunctional OSC and six minor oxidation products (4-9) catalyzed by CYP716s, were obtained. Of note, we discovered that CYP716E60 could oxidize ursane-type and oleanane-type triterpenoids to produce 6ß-OH derivatives, marking the first confirmed C-6ß hydroxylation in an ursuane-type triterpenoid. Compound 9 showed moderate inhibitory activity against NO production and dose-dependently reduced IL-1ß and IL-6 production at the transcriptional and protein levels. Compounds 1, 2, 8, and 9 exhibited moderate hepatoprotective activity with the survival rates of HepG2 cells from 61% to 68% at 10 µM.

Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch.

Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (-)-cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (-)-cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo-electron microscopy-derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure-guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311-S419-C458]/[M311-V419-A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons.

Cadinane sesquiterpenes from the stems and branches of Illicium ternstroemioides.

Three new cadinane sesquiterpenes (1-3) and three known sesquiterpenes were isolated from the stems and branches of Illicium ternstroemioides A. C. Smith. The structures of the new compounds were elucidated by extensive analysis of spectroscopic and HRESIMS data. The structures of illiternins A-C (1-3) were confirmed by single crystal X-ray diffraction, allowing for the determination of their absolute configurations. Compounds 3 and 6 exhibited antiviral activity against Coxsackievirus B3 with IC50 values of 33.3 and 57.7 µM, respectively.

Prenylated C6-C3 derivatives from the root of Illicium ternstroemioides with antiviral activity.

Six previously undescribed prenylated C6-C3 derivatives (1-6) were isolated from the root of Illicium ternstroemioides A. C. Smith. Their structures were elucidated based on extensive spectroscopic analyses (UV, IR, 1D and 2D NMR, and HRESIMS). The absolute configurations of 1-3 were determined using electronic circular dichroism (ECD), and Mo2(OAc)4 induced circular dichroism (ICD). Compound 3 exhibited weak activity against Coxsackievirus B3 with an IC50 value of 33.3 µM, and compound 5 exhibited more potent activity against Coxsackievirus B3 with an IC50 value of 6.4 µM.

Diverse prenylated C6-C3 derivatives from the stems and branches of Illicium ternstroemioides A. C. Smith with anti-inflammatory and antiviral activities.

Fifteen unreported prenylated C6-C3 derivatives (1-15) were isolated from the stems and branches of Illicium ternstroemioides A. C. Smith, including one bis-prenylated C6-C3 derivative (1), three prenylated C6-C3 derivative-shikimic acid ester hybrids (2-4) and 11 prenylated C6-C3 monomers (5-15). The structures of compounds 1-15 were elucidated by spectroscopic analysis (UV, IR, 1D and 2D NMR, and HRESIMS). The absolute configurations of the compounds were determined using electronic circular dichroism (ECD), induced circular dichroism (ICD), and the modified Mosher's method. Among the isolates, compounds 11, 12, and 15 exhibited significant anti-inflammatory activities by inhibiting the nitric oxide with IC50 values ranging from 1.89 to 24.83 µM in lipopolysaccharide-stimulated murine RAW 264.7 macrophages and murine BV2 microglial cells; compounds 2, 3, and 7 exhibited antiviral activitives against Coxsackievirus B3 with an IC50 value of 33.3, 25.9, and 27.8 µM, respectively.

Bergamotane-type sesquiterpenes from the leaves and twigs of Illicium oligandrum and their anti-inflammatory activities.

Eight undescribed ß-bergamotene-type sesquiterpene oliganins A-H (1-8) and one known α-bergamotene-type sesquiterpene (9) were isolated from the leaves and twigs of Illicium oligandrum Merr. & Chun. The structures of compounds 1-8 were elucidated by extensive spectroscopic data, and the absolute configurations were determined by using a modified Mosher's method and electronic circular dichroism calculations. The isolates were further evaluated in terms of their anti-inflammatory potential on nitric oxide (NO) generation in lipopolysaccharide-stimulated RAW264.7 and BV2 cells. Compounds 2 and 8 exhibited potent inhibitory effects on the production of NO with IC50 values ranging from 21.65 to 49.28 µM, which were greater than or comparable to those of dexamethasone (positive control).

[Chemical constituents from leaves of Craibiodendron yunnanense].

The present study investigated the chemical constituents from the leaves of Craibiodendron yunnanense. The compounds were isolated and purified from the leaves of C. yunnanense by a combination of various chromatographic techniques including column chromatography over polyamide, silica gel, Sephadex LH-20, and reversed-phase HPLC. Their structures were identified by extensive spectroscopic analyses including MS and NMR data. As a result, 10 compounds, including melionoside F(1), meliosmaionol D(2), naringenin(3), quercetin-3-O-α-L-arabinopyranoside(4), epicatechin(5), quercetin-3'-glucoside(6), corbulain Ib(7), loliolide(8), asiatic acid(9), and ursolic acid(10), were isolated. Compounds 1 and 2 were two new compounds, and compound 7 was isolated from this genus for the first time. All compounds showed no significant cytotoxic activity by MTT assay.

Minor terpenoids from the leaves of Craibiodendron yunnanense.

One new taraxastane-type triterpenoid, three new grayanane-type diterpenoids (2 - 4), and 12 known compounds (5 - 16) were isolated from the leaves of Craiobiodendron yunnanens W. W. Smith. The structures of these compounds were elucidated on the basis of their spectroscopic data and chemical evidence. Compounds 1 and 8 exhibited partly anti-inflammatory activity based on the inhibition of NF-κB activity in SW480 cells at 10 µM with inhibition ratios of 60.53 and 59.20%, respectively. Compounds 10 and 13 showed excellent cytotoxicity against human leukemia cell (MV4-11) at 10 µM with inhibition ratios of 43.02 and 49.11%, respectively.

seco-Sesquiterpenes and acorane-type sesquiterpenes with antiviral activity from the twigs and leaves of Illicium henryi Diels.

Chemical investigation of an alcohol extract from the twigs and leaves of Illicium henryi Diels resulted in the isolation of two new acorane-related seco-sesquiterpenes (1 and 3), two new acorane-related seco-norsesquiterpenes (2 and 4), one new 2-epi-cedrane sesquiterpene (5), eight new acorane-type sesquiterpenes (6-13), and a known major constituent of acorenone B (14). Their structures were established by interpreting extensive spectroscopic data, including HRESIMS, NMR (1H and 13C NMR, 1H-1H COSY, HSQC, and HMBC), and NOE difference spectra analysis. The absolute configurations of 1, 2, 4-7, 9, 10, and 14 were determined by X-ray crystallography, while chemical transformation methods were performed with compound 14 as the starting material to elegantly solve the absolute configuration issue of compounds 8 and 11-13. Notably, 1 and 2 are seco-sesquiterpenes that are related to acorane and possess an unusual ketal-linked hemiacetal in a 6,8-dioxabicyclo[3.2.1]octan-7-ol scaffold ring system. Plausible biosynthetic pathways for compounds 1-14, which were derived from the acorane skeleton, were proposed. All the isolated compounds (1-14) were evaluated for their antiviral and cytotoxic activities.

Indole alkaloids from the bark of Acacia confusa and their potential antinociceptive and anti-inflammatory activities.

A pair of novel trimeric indole alkaloid enantiomers [(±)-8], five new bisindole alkaloids [4-7 and (±)-9], and three pairs of new monomeric indole alkaloid enantiomers [(±)-1-(±)-3], together with seven known alkaloids (10-16), were isolated from the bark of Acacia confusa. Their structures were determined on the basis of spectroscopic methods, especially by NMR data analyses combined with single-crystal X-ray diffraction and electronic circular dichroism analyses. Compounds 4 and 11-16 exhibited significant antinociceptive activities in an acetic acid-induced writhing test. Compounds (+)-9 and (-)-9 displayed anti-inflammatory activities through the inhibition of the NF-κB pathway, with inhibitory rates of 68.9% and 59.5%, respectively, at a concentration of 10 µM.

Monoterpenoid indole alkaloids isolated from the stems and twigs of Strychnos cathayensis.

Seven undescribed monoterpenoid indole alkaloids, two N(4)-chloromethylation artifacts, and 10 known alkaloids were isolated from the stems and twigs of Strychnos cathayensis. The corresponding structures were elucidated via spectroscopic data interpretation and electronic circular dichroism. The absolute configuration of (17S)-12-hydroxy-11-methoxydiaboline, the major anomer of 12-hydroxy-11-methoxydiaboline, was characterized by X-ray diffraction analysis for the first time. At an intraperitoneal dose of 30 mg/kg, 12-hydroxy-11-methoxy-N(4)-chloromethyldiaboline and (-)-macusine A exhibited potential analgesic effects with prolongation rates of 99% and 47% for the latency time of hind-paw licking, respectively, compared to the blank control. 12-Hydroxy-11-methoxydiaboline, 12-hydroxy-11-methoxydiaboline N(4)-oxide, retuline N-oxide, and (-)-vincosamide exhibited antiviral activity against Coxsackie virus B3 (CVB3) with IC50 values of 33.33 µM.

Chemical constituents from the fruits of Illicium simonsii and their antiviral activity and neuroprotective effect.

One undescribed diterpenoid illisimonone A, four undescribed sesquiterpenes named (±)-simonones A, simonterpenoids A and B, and two undescribed lignans, illisimonins A and B, along with five known compounds were isolated from the fruits of Illicium simonsii. Their structures were elucidated by extensive spectroscopic data. The absolute configuration of illisimonone A was determined by single-crystal X-ray diffraction analysis. Illisimonone A showed potential antiviral activity against the Coxsackie B3 virus, with an IC50 value of 3.70 µM. Illisimonin B and henrylactone A showed potential neuroprotective effects against oxygen-glucose deprivation induced cell injury in SK-N-SH cells, with survival rates of 57.6%, 58.0%, respectively.

Minor terpenoids from the stems and twigs of Rhododendron ovatum.

Four minor undescribed terpenoids, including a monoterpenoid (1) and three triterpenoids (3, 6 and 7), together with 26 known terpenoids were isolated from the stems and twigs of Rhododendron Ovatum. Their structures were identified by extensive spectroscopic analyses and electronic circular dichroism (ECD) techniques. Compound 10 showed excellent cytotoxicity against human colon cancer cell (HCT-116) with IC50 value of 2.56 µM. Compounds 9 and 19 exhibited partly inhibitory effects on nitric oxide production stimulated by lipopolysaccharide-induced neuroinflammation in microglia cells at 10 µM with inhibition ratios of 39.70% and 28.08%, respectively.

New lignans and diterpenoid glycosides from the fruits of Xanthium italicum Moretti.

A pair of new lignans [(+)- 1 and (-)- 1] and three new compounds (2-4), together with a known compound 5, were isolated from the fruits of Xanthium italicum Moretti. The structures of these compounds were determined on the basis of spectroscopic analysis, particularly HR-ESI-MS and 1 D and 2 D NMR. Compounds 2 and 3 showed antinociceptive effects in an acetic acid-induced writhing test in mice with the writhe inhibition rates of 80.50% and 67.89% at the dose of 20 mg/kg, respectively.

[A new seco-prezizaane-type sesquiterpene lactone from stems and branches of Illicium ternstroemioides].

Three seco-prezizaane-type sesquiterpene lactones, one phenylpropanoid, and two lignans were isolated from the 95% ethanol extract of stems and branches of Illicium ternstroemioides with silica gel column chromatography, ODS column chromatography, and preparative HPLC. Based on the spectral data, they were identified as burmanicumolide D(1), veranisatin A(2), veranisatin B(3), dihydroconiferylalcohol(4), pinoresinol(5),(-)-matairesinol(6), respectively. Among them, compound 1 was a new seco-prezizaane-type sesquiterpene lactone, and 2-6 were obtained from this plant for the first time. None of these compounds display antiviral or cytotoxic activities.

Prenylated C6-C3 derivatives from the stems and branches of Illicium ternstroemioides A. C. Smith with antiviral activity.

Thirteen compounds were isolated from the lipid-soluble extracts of Illicium ternstroemioides A. C. Smith, including eleven previously undescribed prenylated C6-C3 compounds, a previously undescribed prenylated C6-C3 derivative-abscisic acid ester hybrid, and a known compound (4S)-illicinone I. Their structures and configurations were mainly elucidated by spectroscopic analyses, CD experiments and X-ray crystallography. (2S,4R,11S)-4-O-methyl-12-chloroillifunone C, (2S,4R,11R)-2,3-dihydro-4-O-methyl illioliganfunone D, and illiternfunol A were found to exhibit weak activity against Coxsackievirus B3, with IC50 values ranging from 27.8 to 33.3 µM. Illiternone B exhibited more potent activities against Coxsackievirus B3 and influenza virus A than did its geometric isomer illiternone A, with IC50 values of 7.7 µM and 2.5 µM, respectively. None of these compounds displayed cytotoxic activities.

Library construction of stereochemically diverse isomers of spirooliganin: their total synthesis and antiviral activity.

The construction of libraries of stereoisomers of natural products serves as an important approach to investigating the correlation between the stereostructure and biological activity. However, the total synthesis and isomerzation of polycyclic scaffolds with multiple chrial centers are rare. Spirooliganin (1), a new skeleton natural product isolated from the plant Illicium oligandrum, was structurally characterized by comprehensive analysis of NMR spectroscopic data and ECD which revealed an unprecedented 5-6-6-6-7 polycyclic framework with six chiral centers. Here we report a 17-step total synthesis to prepare a library of stereochemically diverse isomers of spirooliganin, including 16 diastereoisomers and 16 regioisomers. In addition to a regioselective hetero-Diels-Alder cycloaddition, the synthetic strategy involves a photo-induced stereoselective Diels-Alder reaction, which gives only the abnormal trans-fused product as rationalized by density functional theory calculations. Preliminary biological evaluation showed that spirooliganin and regioisomers 39 exhibited potent inhibition of Coxsackievirus B3. It also revealed the pharmacophore effect of the D-ring (16R,18R,24R, and 26R) for their antiviral activities.

Humulane-type and germacrane-type sesquiterpenoids from the fruits of Xanthium spinosum Linn.

Eight undescribed humulane-type sesquiterpenoids (xanthspinol A-E, I, J and N), three undescribed germacrane-type sesquiterpenoids (xanthspinol F, G and O) and twelve known compounds were isolated from the fruits of Xanthium spinosum. The structures of the undescribed compounds were elucidated by analyses of spectroscopic data, electronic circular dichroism (ECD) calculations, dimolybdenum tetraacetate [Mo2(OAc)4]-induced circular dichroism (ICD) spectra, a CD exciton chirality method and the modified Mosher's method. Xanthspinol A and B featured a humulane skeleton containing a 2,5-dihydrofuran fragment. Putative biosynthetic pathways for the undescribed compounds are proposed. Xanthspinol N, 8-epi-isoxanthanol and deacetyl-4-epixanthanol showed moderate activity against Coxsackie virus B3 (CVB3) with IC50 values of 8.70, 3.70 and 3.70 µM, respectively.

Oxygenated pentacyclic triterpenoids from the stems and branches of Enkianthus chinensis.

Thirty new pentacyclic triterpenoids, including five oleanane-type (1-5), twenty-three ursane-type (9-23, 26-33) and two taraxerane-type (24 and 25), along with fourteen known triterpenoids, were isolated from the stems and branches of Enkianthus chinensis. Their structures were elucidated by extensive spectroscopic analyses, X-ray crystallographic data and electronic circular dichroism (ECD) techniques. Sixteen compounds (1-5, 9-13, 20, 22, 32, 34-36) bearing a gem-hydroxymethyl group at C-4 represent rare examples of pentacyclic triterpenoids. In the in vitro biological activity evaluation, compounds 8, 9, 12-14, 17, 24, and 44 exhibited potent hepatoprotective effects at 10 µM. Moreover, compound 25 showed latent activity against HSV-1 with an IC50 value of 6.4 µM.