Structure–activity relationships of eight <Emphasis Type="Italic">ent</Emphasis>-kaurene diterpenoids from three <Emphasis Type="Italic">Isodon</Emphasis> plants

Eight ent-kaurene diterpenoids were isolated from three Isodon plants, Isodon excisoides (Sun ex C. H. Hu) C. Y. Wu et H. W. Li, Isodon weisiensis C. Y. Wu, and Isodon racemosa (Hemsl.) Hara. Their cytotoxicities were tested by SRB assay against four human tumor cell lines, HepG2, Tb, HO-8910 and SGC-7901. The DNA damage degrees on their Hep G2 cells were evaluated by Comet assay. The results showed that ent-kaurene diterpenoids were selectively toxic against these four tumor cell lines. Weisiensic C had the lowest cytotoxicity against four cell lines and degree of DNA damage on the Hep G2 cell line. Leukamenin E and Glaucocalyxin A had similar IC₅₀ values and also induced a similar degree of DNA damage. The cytotoxicity and degree of DNA damage of eight ent-kaurene diterpenoids on Hep G2 was in the order Leukamenin E > Glaucocalyxin A > Wangzaozin A > Kamebanin > Macrocalyxin D > Weisiensin B > Excisanin K > Weisiensic C. The structure–activity relationships indicated that there was some correlation between the substituents and their structures of ent-kaurene diterpenoids and their activities on Hep G2 cells.     

Determination of Diterpenoids and Flavonoids in Isodon rubescens by LC-ESI-MS-MS

The diterpenoids and flavonoids in Isodon rubescens were analyzed simultaneously by high-performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS-MS) for the first time. The fragmentation pathway of rubescensin A (oridonin) and rubescensin B (ponicidin) in an electrospray ion trap mass spectrometer was also investigated. A total of 10 compounds, including five diterpenoids and five flavonoids, were identified or preliminarily characterized based on their mass spectra. Six of them were reported from Isodon species for the first time.     

Isolation and purification of diterpenoids from the aerial parts of Isodon excisoides target-guided by UPLC-LTQ-Orbitrap-MS

Cytotoxic diterpenoids were enriched and orientation prepared from the aerial parts of Isodon excisoides target-guided by UPLC-LTQ-Orbitrap-MS. Four diterpenoids were obtained, including a novel compound: 1α-acetoxy-7α, 14β, 20α-trihydroxy-ent-kaur-16-en-15-one (1); together with three known compounds kamebakaurin (2), lasiokaurin (3), enmenol-1-β-glucoside (4). Their structures were elucidated on the basis of spectroscopic methods in conjunction with published data for their analogues. All compounds were tested for their cytotoxic effects against five human cancer cell lines HCT-116, HepG2, A2780, NCI-H1650 and BGC-823, respectively. Compounds 1 and 2 showed obviously cytotoxic activity against the five cancer cell lines with IC₅₀ ranging from 1.06 to 3.60 μM. Compounds 3 and 4 showed selective cytotoxic activity.     

ent‐Kaurane Diterpenoids from Isodon phyllostachys

Phytochemical investigation of the medicinal plant Isodon phyllostachys led to the isolation of four new ent‐kaurane diterpenoids, phyllostacins F–I ( 1 – 4 , resp.), together with 11 known compounds, rosthorin A ( 5 ), rabdoternin C ( 6 ), enmenol ( 7 ), oridonin ( 8 ), lasiocarpanin ( 9 ), xerophilusin B ( 10 ), ponicidin ( 11 ), macrocalin B ( 12 ), phyllostachysin A ( 13 ), sculponeatin C ( 14 ), and macrocalyxoformin E ( 15 ). The structures of the new compounds were established by spectroscopic methods, including extensive 1D‐ and 2D‐NMR analyses. Compounds 1, 2, 7, 10 , and 13 were evaluated for their inhibitory activity against K562 and HepG2 cell lines.     

Protection against DNA damage and inhibition of lipid peroxidation by flavones from Eremosparton songoricum (Litv) Vass

Seven flavonoids were isolated from Eremosparton songoricum (Litv) Vass and their antioxidation actions were examined. To investigate protective ability against DNA damage induced by H₂O₂, human peripheral blood mononuclear cells (PBMCs) were incubated with 5-hydroxy-7,4′-dimethoxyflavone,5,7-dihydroxy-3′,4′-dimethoxyflavone, acacetin, chrysoeriol, pedalitin, flavogadorinin and 5,7,4′-trihydroxy-3′-methoxyflavone-4′-O-D-glucoside separately and then treated by H₂O₂. The percentage of damaged cells were determined by single-cell gel electrophoresis. To examine their inhibition of lipid peroxidation, the compounds were incubated with rat microsomes and the reaction was initiated by Fe²⁺/Vc. The lipid peroxidation product thiobarbituric acid reactive substances (TBARS) was determined. The result showed most of flavonoids had concentration-dependent antioxdative activities. The relationship of antioxidation and the structure is discussed.     

ent‐Kaurane Diterpenoids from Isodon japonicus

Two new 6,7‐seco‐ent‐kaurane diterpenoids, isojaponins A ( 1 ) and B ( 2 ), together with 18 known ent‐kaurane diterpenoids were isolated from the aerial parts of Isodon japonicus. The structures of the two new compounds were elucidated by extensive 1D‐ and 2D‐NMR spectroscopic methods in combination with MS experiments.     

Studies on constituents of Isodon japonicus hara : The structures and absolute stereochemistry of isodonal,trichodonin and epinodosin

Seven diterpenoids have been isolated from Isodon japonicus Hara. Isodonal, trichodonin and epinodosin are formulated by chemical and spectroscopic evidence as the structures 2, 4 and 5 Antimicrobial activity has been tested.     

ent‐Clerodanoids from Isodon scoparius

Three new ent‐clerodane diterpenoids, isoscoparin A, B, and C ( 1 – 3 ), were isolated from Isodon scoparius. Their structures were determined by NMR‐spectra analysis. The biogenetic implication of the diterpenes from Isodon genus is discussed.     

Cytotoxic ent-kauranoid derivatives from Isodon rubescens

An extensive study of the diterpenoids produced by the species of Isodon rubescens, has led to the isolation of 12 new ent-kaurane diterpenoids, hebeirubescensins A-L (1-12), and 19 known analogues. Their structures were determined on the basis of spectroscopic analysis. Selected compounds were assayed for their inhibitory ability against human A549, HT-29, and K562 cells. Among them, hebeirubescensins B and C exhibited significant cytotoxicity with IC₅₀ values of <2.0 μM. The structure-activity relationships were discussed.     

New Phenolic Tricyclic Diterpenoids from Rhizomes of Isodon hispida (Benth.) Hara

Three new phenolic tricyclic diterpenoids, including two sempervirane diterpenoids, hispidanols A and B ( 1 and 2 , resp.), and one totarane diterpenoid, totara‐8,11,13‐triene‐13→16‐hemiacetal ( 3 ), along with 14 known compounds, 4 – 17 , were isolated from the AcOEt‐soluble fraction of the 70%‐acetone extract of rhizomes of Isodon hispida. Their structures were elucidated based on the analyses of extensive spectroscopic data and physicochemical properties.     

Ent-kaurane diterpenoids from Isodon rubescens var. lushanensis

Four new ent-kaurane diterpenoids lushanrubescensins F-I (1-4), together with 11 known ones, lasiodonin (5), oridonin (6), ponicidin (7), isodonoiol (8), isodonal (9), rabdosin B (10), rabdoternins A and B (11 and 12), enmenol (13), epinodosin (14), and inflexusin (15), were isolated from Isodon rubescens var. lushanensis, and the structures were elucidated by spectroscopic analysis. The inhibitory effect against the K562, Bcap37, BGC823, BIU87, CA, CNE, and Hela cell lines of compounds 3 and 5-10 were evaluated.     

An immunomodulator from Tinospora cordifolia with antioxidant activity in cell-free systems

Several plant products are known to exhibit immense medicinal value against human diseases. Our earlier studies showed that dry stem crude extract (DSCE) ofTinospora cordifolia contained a polyclonal B cell mitogen, G1-4A. DSCE as well as G1-4A also enhanced immune response in mice. In order to explore the possibility of using G1-4A/PPI (partially purified immunomodulator) to modulate radiation induced immunosuppression, the antioxidant effect of PPI from this plant was examined against reactive oxygen and nitrogen species (ROS/RNS), generated by photosensitization/peroxynitrite. Levels of lipid peroxidation products, superoxide dismutase (SOD) and catalase in liver/spleen homogenate from mouse were monitored. Photosensitization induced significant increase in thiobarbituric acid reactive substances (TBARS) in liver. The activities of SOD and catalase were reduced considerably. PPI, present during photosensitisation, prevented lipid peroxidation and restored the activities of both the enzymes. Likewise, oxidative damage induced by peroxynitrite was inhibited by PPI. The degradation of proteins due to photosensitization as assessed by SDS-PAGE was effectively reduced by simultaneous treatment with PPI during photosensitization. Selective inhibitors of ROS like mannitol, SOD, sodium azide and antioxidants, GSH and vitamin C brought about significant inhibition of formation of TBARS suggesting possible involvement of O₂ ^•,^•OH and¹O₂. Photosensitization in deuterated buffer enhanced formation of TBARS thus indicating generation of¹O₂. Thus, the action of PPI may be against oxidative damage through Type I and II photosensitization mechanisms. Therefore, the immunomodulator fromTinospora cordifolia may also be beneficial as an antioxidant.     

Diterpenoids from Isodon sculponeatus

Further investigation on the leaves of Isodon sculponeatus afforded two new ent-kaurane diterpenoids,sculponeatins J and K .Their sturctures were elucidated on the basis of their spectral properties,as well as X-ray crystallographic analysis ,The cytotoxicities of these two new compounds against human tumor cells K562 and T24 were also tested ,and sculponeation J showed significant inhibitory effects with IC50 values less than 1.0 μg/mL。     

New ent‐Abietanoids from Isodon rubescens

Six new ent‐abietane diterpenoids, rubescensins I–M ( 1 – 5 ) and P ( 6 ), along with two related acetonide derivatives ( 7 and 8 ), were isolated from Isodon rubescens. Their structures were elucidated by detailed spectroscopic analysis. Compound 5 is the first N‐containing diterpenoid from the genus Isodon, exhibiting notable cytotoxicity against human tumor K562 cells.     

A sensitive analysis method for 7 diterpenoids in rat plasma by liquid chromatography-electrospray ionization mass spectrometry and its application to pharmacokinetic study of Isodon serra extract

A simple and sensitive LC-MS/MS method has been developed and validated for the identification and quantification of epinodosin, epinodosinol, nodosin, oridonin, lasiokariurinol, lasiokaurin and rabdoternin A in rat plasma using sulfamethoxazole as the internal standard. The plasma sample pre-treatment consisted of a liquid-liquid extraction. Chromatographic separation was achieved on a C18 column with linear gradient elution using water and methanol, which were both acidified with 0.1% formic acid, at a flow rate of 0.7 mL/min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source. A novel multi-determination-periods program was executed to achieve a higher sensitivity by setting five scanning periods. The method presented here utilizes a novel determination strategy, enabling the application of positive and negative ESI-MS in a single run. The optimized mass transition ion-pairs (m/z) for quantitation were 361.2/287.1 for epinodosin, 382.3/347.3 for epinodosinol, 363.3/281.2 for nodosin, 365.3/347.3 for oridonin, 407.3/329.1 for lasiokariurinol, 405.2/59.0 for lasiokaurin, 363.2/283.1 for rabdoternin A and 254.1/156.0 for IS. The total run time was 20.50 min (including 5 min equilibration time) between injections. The specificity, linearity, accuracy, precision, recovery, matrix effect and several validation results demonstrate that this method is sensitive, specific and reliable. The proposed method was further applied to investigate the pharmacokinetics of all analytes after a single oral administration of Isodon serra extract to rats.     

Novel ent ‐Kaurane Diterpenoids from Isodon xerophilus

Further investigation on the leaves of Isodon xerophilus afforded three novel ent‐kaurane diterpenoids, xerophilusins D‐F (1–3), together with seven known compounds, phyllostachysin A (4), oleanolic acid, caffeic acid, rosmarinic acid, rutin, quercetin‐3‐O‐β‐D‐glucopyranoside, and quercetin. Structures of 1–3 were elucidated on the basis of their spectral properties and X‐ray crystallographic analysis. Compound 1 showed broad spectra inhibiting human tumor cells and significant cytotoxicity.     

Immunosuppressive ent‐Kaurene Diterpenoids from Isodon serra

Three new enmein‐type ent‐kaurenoids, i.e., the two pairs 1 and 2 of 20‐epimers and the (20R)‐isomer 3 , besides the seven known diterpenoids 4 – 10 , were isolated from the aerial parts of Isodon serra. Their structures were elucidated by spectroscopic techniques and X‐ray diffraction. The immunosuppressive effect for T‐lymphocytes proliferation induced by Con A in BALB/c mouse was evaluated for the isolates 1 – 10 . They all displayed a remarkable inhibitory effect, with multi‐glycosides of Tripterygium wilfordii as positive reference substance (Table 3).     

Symmetric and asymmetric ent-kaurane dimers isolated from Isodon japonicus

Bisjaponins A (1) and B (2), two new dimeric ent-kaurane diterpenoids connected with a rare four-membered carbon ring, which was formed by [2+2] reaction, were isolated from the aerial parts of Isodon japonicus. Their structures were elucidated by the analysis of spectroscopic evidence including extensive 2D NMR and MS data. Both the compounds were inactive for their cytotoxicity against human tumor cell lines, K562 and HepG2.     

Diterpenoids from Isodon Eriocalyx

Two new diterpenoids, Maoecrystal U and epi‐maoecrystal P , together with nine known diterpenoids and eight other known compounds were isolated from Isodon eriocalyx ( ). The structures of two new diterpenoids were elucidated as 6β‐acetoxy‐15β‐hydroxy‐3α,20‐epoxy‐ent‐kaur‐16‐ene‐1,7‐dione and 16(S)‐methoxymethy‐6β,7β‐dihydroxy‐7α,20‐epoxy‐ent‐kaur‐2,3‐ethenylene‐1,15‐dione on the basis of spectroscopic evidence, especially by 2D NMR techniques.