Cassane diterpenoids with α-glucosidase inhibitory activity from the fruits of Pterolobium macropterum.

Two new cassane diterpenoids, 14ß-hydroxycassa-11(12),13(15)-dien-12,16-olide (1) and 6'-acetoxypterolobirin B (3), together with a known analogue, identified as 12α,14ß-dihydroxycassa-13(15)-en-12,16-olide (2), were isolated from the fruits of Pterolobium macropterum. Compound 1 is a cassane diterpenoid with a Δ11(12) double bond conjugated with an α,ß-butenolide-type, whereas compound 3 is a dimeric caged cassane diterpenoid with unique 6/6/6/6/6/5/6/6/6 nonacyclic ring system. The structures of 1 and 3 were characterized by extensive spectroscopic analysis combined with computational ECD analyses. The α-glucosidase inhibitory activity of isolated compounds was evaluated, and compounds 1 and 3 showed significant α-glucosidase inhibitory activity with IC50 values of 66 and 44 µM.

Marginaols G-M, anti-inflammatory isopimarane diterpenoids, from the rhizomes of Kaempferia marginata.

Marginaols G-M, a series of undescribed isopimarane diterpenoids, together with four known analogs were isolated from the rhizomes of Kaempferia marginata. The structures of these isolated compounds were characterized using high-resolution mass spectrometry and extensive 1D- and 2D-nuclear magnetic resonance (NMR) analyses. In addition, the absolute configurations of marginaol G and H were determined by X-ray crystallographic analysis and comparison with the literature values. When compared to the standard drug dexamethasone (IC50 4.7 µM), marginaol G, H, and 6ß-acetoxysandaracopimaradien-1α,9α-diol had an intriguing anti-inflammatory effect on NO inhibition in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, with IC50 values ranging from 4.5 to 7.3 µM and being less cytotoxic to the cells. The anti-inflammatory action of these isopimarane diterpenoids from K. marginata supports the use of Thai traditional medicine for inflammation treatment.

The effects of festidinol treatment on the D-galactose and aluminum chloride-induced Alzheimer-like pathology in mouse brain.

BACKGROUND: Festidinol is a flavan-3-ol which has been shown to reduce advanced glycation end products (AGEs) and reactive oxygen species, both of which play a crucial role in the pathology of many neurodegenerative diseases. PURPOSE: This study aimed to investigate the effects of festidinol on oxidative stress, amyloidogenesis, phosphorylated tau (pTau) expression, synaptic function, and cognitive impairment, and the potential mechanisms involved, in a mouse model with an Alzheimer-like pathology. METHODS: D-galactose (150 mg/kg) and aluminum chloride (10 mg/kg) were injected intraperitoneally into 40 mice for 90 days to generate an AD mouse model with cognitive impairment. Festidinol (30 mg/kg) and donepezil (5 mg/kg) were then administered orally for 90 days after which behavior and molecular changes in the brain were measured. RESULTS: The aluminum accumulated and the expression of the cell senescence marker P16 increased after exposure to D-galactose and AlCl3 (2.5 ± 0.5 mg/kg, 149.1 ± 28.1% of control, respectively). Festidinol markedly decreased the escape latency (8.7 ± 4.3 s) and increased the number of platform crossings (8 ± 1.4 time) in the Morris water maze test. Superoxide dismutase activity was significantly elevated after festidinol administration, however there were significant reductions in the levels of 4­hydroxy-2-nonenal, receptor for advanced glycation end products, phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (pNF-κB), and nuclear factor of activated T cells 1 (NFAT1). Festidinol attenuated amyloid beta production by reducing the mRNA of beta-site APP cleaving enzyme 1 (BACE1). Festidinol also significantly decreased the expression of pTau and phosphorylated glycogen synthase kinase 3 (148.6 ± 37.6% of control, 125.3 ± 22.6% of control, respectively). CONCLUSION: Festidinol can ameliorate learning and memory impairments by modulating amyloidogenesis, tau hyperphosphorylation, cholinergic activity, neuroinflammation, and oxidative stress, and by regulating the brain-derived neurotrophic factor signaling pathway.

Biotransformation of 1α,11α-dihydroxyisopimara-8(14),15-diene by Cunninghamella echinulata NRRL 1386 and their neuroprotective activity.

The isopimarane diterpene, 1α,11α-dihydroxyisopimara-8(14),15-diene (1), is the major constituents from the rhizomes of Kaempferia marginata (Zingiberaceae), a Thai medicinal plant. The microbial transformation of parent compound 1 by the fungus Cunninghamella echinulata NRRL 1386 gave five new metabolites, 7α,11α-dihydroxy-1-oxoisopimara-8(14),15-diene (2), 3ß,7α,11α-trihydroxy-1-oxoisopimara-8(14),15-diene (3), 7ß,11α-dihydroxy-1-oxoisopimara-8(14),15-diene (4), 7α-hydroxy-1,11-dioxoisopimara-8(14),15-diene (5) and 1α,7ß,11α-trihydroxyisopimara-8(14),15-diene (6), together with three known metabolites, 7-9. The structures of the new metabolites were elucidated by spectroscopic techniques. The known compounds were identified by comparison of the spectroscopic and physical data with those of reported values. The parent compound 1 and the metabolites have been neuroprotective activities evaluated against Aß25-35-induced damage in human neuroblastoma cells (SK-N-SH). Among them, compounds 1-3, 5 and 7-9 had significant neuroprotective activities at a concentration of 2.5 µM. The results demonstrated that these compounds might be worth for further development into therapeutic agents for the treatment of neurodegenerative diseases.

5,6,7,4'-Tetramethoxyflavanone alleviates neurodegeneration in a dexamethasone-induced neurodegenerative mouse model through promotion of neurogenesis via the Raf/ERK1/2 pathway.

Adult neurogenesis plays an important role in improving cognitive functions. Neurogenesis generates new neurons, a process mediated by neural stem cell proliferation, migration, and differentiation. Long-term exposure to high levels of glucocorticoid results in the suppression of neurogenesis pathways and leads to the onset of cognitive impairment. The induction of neurogenesis by a potent bioactive compound is considered the most promising treatment for neurodegenerative disorders. 5,6,7,4'-Tetramethoxyflavanone (TMF) is a flavonoid compound isolated from Chromolaena odorata (L.) R. M. King & H. Rob. Previous study showed that TMF improved cognitive impairment by attenuating Aß production and pTau expression, thereby increased cell survival and promoted synaptic plasticity. The aim of this study was to investigate the effect of TMF on dexamethasone (DEX)-suppressed neurogenesis in mice. Mice received DEX for 28 days before being treated with TMF for additional 30 days. Mice were randomly divided into four groups: control, TMF, DEX, and DEX + TMF. TMF promoted neurogenesis by increasing BrdU-positive cells, Prox1, doublecortin, and Nestin expression. TMF also upregulated the expression of Raf and extracellular-signal-regulated kinase (ERK)1/2, which are pivotal for neurogenesis signaling. In conclusion, TMF promoted neurogenesis-related protein expression in the proliferation, differentiation, and maturation phases via Raf/ERK1/2 signaling pathway.

Germacrone Reduces Cisplatin-Induced Toxicity of Renal Proximal Tubular Cells via Inhibition of Organic Cation Transporter.

Cisplatin is a widely used chemotherapy for solid tumors; however, its benefits are limited by serious nephrotoxicity, particularly in proximal tubular cells. The present study investigated the renoprotective effect and mechanisms of germacrone, a bioactive terpenoid compound found in Curcuma species on cisplatin-induced toxicity of renal cells. Germacrone (50 and 100 µM) attenuated apoptosis of human renal proximal tubular cells, RPTEC/TERT1 following treatment with 50 µM cisplatin and for 48 h. Co-treating RPTEC/TERT1 cells with cisplatin and germacrone significantly reduced cellular platinum content compared with cisplatin treatment alone. The effect of germacrone on organic cation transporter 2 (OCT2) which is a transporter responsible for cisplatin uptake was determined. Germacrone showed an inhibitory effect on OCT2-mediated methyl-4-phenylpyridinium acetate (3H-MPP+) uptake with IC50 of 15 µM with less effect on OCT1. The germacrone's protective effect on cisplatin-induced cytotoxicity was not observed in cancer cells; cisplatin's anti-cancer activity was preserved. In conclusion, germacrone prevents cisplatin-induced toxicity in renal proximal tubular cells via inhibition OCT2 transport function and reducing cisplatin accumulation. Thus germacrone may be a good candidate agent used for reducing cisplatin-induced nephrotoxicity.

Microbial transformation of isocoronarin D by Cunninghamella echinulata NRRL 1386.

The diterpene isocoronarin D (1) is a bioactive major constituent of labdane diterpene from the aerial parts of Curcuma comosa Roxb. (Zingiberaceae), the Thai medicinal plant. Microbial transformation of 1 was performed by the fungus Cunninghamella echinulata NRRL 1386 to yield three new metabolites, 3ß-hydroxyisocoronarin D (2), 6α-hydroxyisocoronarin D (3) and 3ß,7α-dihydroxyisocoronarin D (4). The structures of the new compounds were elucidated by spectroscopic techniques.

Diterpenoid trigonoreidon B isolated from Trigonostemon reidioides alleviates inflammation in models of LPS-stimulated murine macrophages and inflammatory liver injury in mice.

The roots of Trigonostemon reidioides, Thai medicinal plant, have long been used as an antidote, laxative, and antiasthmatic, and also used as folk remedy for relieving inflammatory symptoms from poisonous insect and snake bites as well as abscesses and sprains. Here, we studied anti-inflammatory effects of a major diterpenoid named trigonoreidon B (TR-B) isolated from T. reidioides roots in lipopolysaccharide (LPS)-activated RAW264.7 macrophages and D-galactosamine (D-GalN)/LPS-induced inflammatory liver injury in mice. RAW264.7 cells were treated with TR-B or other available minor diterpenoids, and cell viability was determined by AlamarBlue. The levels of inflammatory mediators were determined by nitrite assay, ELISA, and luminescence. NF-κB nuclear translocation was investigated by indirect immunofluorescence. Expression levels were determined by real-time PCR and Western blotting. Transaminases and caspase activities were determined by using assay kits. Our results showed that TR-B was able to suppress PI3K/Akt activation and inflammatory induction in LPS-activated macrophages. These events were concomitant with TR-B's ability to hamper activated generation of reactive oxygen species, nitric oxide, prostaglandin E2, and cytokines as well as NF-κB p65 nuclear translocation. In an in vivo model of inflammatory liver injury, an administration of TR-B protected mice from D-GalN/LPS-induced liver injury by suppressing the elevation of serum TNF-α, transaminase activities, and hepatocyte apoptosis as well as an improvement of liver histopathology. During protection against liver damage, TR-B also prevented the loss of Akt phosphorylation. Collectively, the results of this present study suggested that TR-B exerted an anti-inflammatory effect via attenuating macrophage-mediated inflammation and inflammatory liver injury in vivo. TR-B may represent a promising lead compound for anti-inflammatory drug development.

Cyperenoic acid suppresses osteoclast differentiation and delays bone loss in a senile osteoporosis mouse model by inhibiting non-canonical NF-κB pathway.

Cyperenoic acid is a terpenoid isolated from the root of a medicinal plant Croton crassifolius with a wide range of biological activities. In this study, the effects of cyperenoic acid on osteoclast differentiation were investigated both in vitro and in vivo using receptor activator of nuclear factor-κB ligand (RANKL)-induced bone marrow-derived osteoclasts and senescence-accelerated mouse prone 6 (SAMP6). Cyperenoic acid significantly suppressed RANKL-induced osteoclast differentiation at the concentrations with no apparent cytotoxicity. The half maximum inhibitory concentration (IC50) for osteoclast differentiation was 36.69 µM ± 1.02. Cyperenoic acid treatment evidently reduced the expression of two key transcription factors in osteoclast differentiation, NFATc1 and c-Fos. Detailed signaling analysis revealed that cyperenoic acid did not affect MAPK pathways and canonical NF-κB pathway but impaired activation of p100/p52 in the non-canonical NF-κB pathway upon RANKL stimulation. Moreover, the expression of osteoclast-related genes, nfatc1, ctsk, irf8, acp5 and cfos were disrupted by cyperenoic acid treatment. The bone resorption activity by cyperenoic acid-treated osteoclasts were impaired. In a senile osteoporosis mouse model SAMP6, mice fed on diet supplemented with cyperenoic acid showed delay in bone loss, compared to the control. Taken together, plant-derived cyperenoic acid shows great potential as therapeutic agent for osteoporosis.

Determination of the Marker Diarylheptanoid Phytoestrogens in Curcuma comosa Rhizomes and Selected Herbal Medicinal Products by HPLC-DAD.

A method for quantification of diarylheptanoids in Curcuma comosa rhizomes and selected pharmaceutical preparations was established by using HPLC-diode array detector (DAD). The chromatographic separation of three diarylheptanoids [(3S)-1-(3,4-dihydroxy-phenyl)-7-phenyl-(6E)-6-hepten-3-ol (1), (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (2), and (3S)-1,7-diphenyl-(6E)-6-hepten-3-ol (3)] was performed on a Luna C18 analytical column using gradient elution with 0.5% acetic acid in water and acetonitrile with a flow rate of 1 mL/min and a column temperature of 35°C. The calibration curves for the analytes showed good linearity (R2>0.999), high precision (relative standard deviation (RSD) <2%) and acceptable recovery (98.35-103.90%, RSD <2%). The limit of detection (LOD) and limit of quantification (LOQ) were 0.06-0.22 and 0.18-0.69 µg/mL, respectively. The results of all validated parameters were within the limits according to the International Conference on Harmonization (ICH) Guidelines. The established method was successfully applied for qualitative and quantitative determination of the three constituents in different samples of C. comosa and some commercial products in capsules. The simplicity, rapidity, and reliability of the method could be useful for the fingerprint analysis and standardization of diarylheptanoids, which are responsible for the estrogenic activity in raw materials and herbal medicinal products of C. comosa.

LARVICIDAL AND PUPICIDAL ACTIVITIES OF CRUDE AND FRACTIONATED EXTRACTS OF ACACIA PENNATA (L.) WILLD. SUBSP INSUAVIS SHOOT TIPS AGAINST AEDES AEGYPTI (L.) (DIPTERA: CULICIDAE).

Acacia pennata subsp insuavis, or Cha-om in Thai, is a common vegetable found in Thailand. It has been used as a medicinal herb for a long time. From the literature, antinociceptive, anti-inflammatory, antimicrobial, and anti-helminthic activities were reported. In this study, we investigated two new actions of this plant: larvicide and pupicide. The crude ethanolic and fractionated extracts of A. pennata shoot tips were tested against aquatic stages of the dengue virus vector, Aedes aegypti mosquito. The 1st-4th instar larvae and pupae of Ae. aegypti were subjected for bioassays by following the standard protocol of WHO. The larval and pupal mortalities were observed after 24- and 48-hour exposure times. The bioassays demonstrated that stronger efficacy was found from the fractionated extracts than the crude extracts. The LC50 values against the 3rd instar larvae were 39.45-50.75 mg/l (fractionated extracts) and 244.50 mg/l (crude extracts). It also effects the pupae with the LC50 values of 44.10-53.73 mg/l and 87.27 mg/l for the fractionnated and the crude extracts, respectively. The bioassays demonstrated the effective mosquito larvicide and pupicide of A. pennata extracts. It could be an alternative candidate for the development of phytotoxin for controlling mosquito vectors.

Serum lipidomics analysis of ovariectomized rats under Curcuma comosa treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcuma comosa Roxb. (C. comosa) or Wan Chak Motluk, Zingiberaceae family, has been used in Thai traditional medicine for the treatment of gynecological problems and inflammation. AIM OF THE STUDY: This study aimed to investigate the therapeutic potential of C. comosa by determining the changes in the lipid profiles in the ovariectomized rats, as a model of estrogen-deficiency-induced hyperlipidemia, after treatment with different components of C. comosa using an untargeted lipidomics approach. MATERIALS AND METHODS: Lipids were extracted from the serum of adult female rats subjected to a sham operation (SHAM; control), ovariectomy (OVX), or OVX with 12-week daily doses of estrogen (17ß-estradiol; E2), (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (DPHD; a phytoestrogen from C. comosa), powdered C. comosa rhizomes or its crude ethanol extract. They were then analyzed by liquid chromatography-mass spectrometry, characterized, and subjected to the orthogonal projections to latent structures discriminant analysis statistical model to identify tentative biomarkers. RESULTS: Levels of five classes of lipids (ceramide, ceramide-1-phosphate, sphingomyelin, 1-O-alkenyl-lysophosphatidylethanolamine and lysophosphatidylethanolamine) were elevated in the OVX rats compared to those in the SHAM rats, while the monoacylglycerols and triacylglycerols were decreased. The E2 treatment only reversed the levels of ceramides, whereas treatments with DPHD, C. comosa extract or powder returned the levels of all upregulated lipids back to those in the SHAM control rats. CONCLUSIONS: The findings suggest the potential beneficial effects of C. comosa on preventing the increased ceramide levels in OVX rats, a possible cause of metabolic disturbance under estrogen deficiency. Overall, the results demonstrated the power of untargeted lipidomics in discovering disease-relevant biomarkers, as well as evaluating the effectiveness of treatment by C. comosa components (DPHD, extract or powder) as utilized in Thai traditional medicine, and also providing scientific support for its folklore use.

Diarylheptanoids of Curcuma comosa with inhibitory effects on nitric oxide production in macrophage RAW 264.7 cells.

Eight new diarylheptanoids, a 1.2:1 mixture of (3S)- and (3R)-1-(4-hydroxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-ol (1a and 1b), a racemic mixture of (3S)- and (3R)-1-(4-hydroxyphenyl)-3-methoxy-7-phenyl-(4E,6E)-4,6-heptadiene (2a and 2b), a ca. 1:1 mixture of (3S)- and (3R)-1-(4-hydroxy-3- methoxyphenyl)-3-methoxy-7-phenyl)-(4E,6E)-4,6-heptadiene (3a and 3b), 3-acetoxy-1-(3,4-dihydroxyphenyl)-7-phenylheptan-5-ol (4), (3R)-1-(4,5- dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3,2'-epoxide (5), and thirteen known diarylheptanoids, 6-12, a 3:1 mixture of 13a and 13b, and 14-17, were isolated from the rhizomes of Curcuma comosa from Sakon Nakhon, northeastern part of Thailand. The isolated compounds were evaluated for their anti- inflammatory activities on the inhibition of lipopolysaccharide-induced nitric oxide production in macrophage RAW 264.7 cells and the diarylheptanoids 1a and 1b mixture and 14 exhibited potent inhibitory activity.

LARVICIDAL ACTIVITY OF PERESKIA BLEO (KUNTH) DC. (CACTACEAE) FRUIT ENDOCARP CRUDE AND FRACTIONATED EXTRACTS AGAINST AEDES AEGYPTI (L.) (DIPTERA: CULICIDAE).

The use of insecticides can cause adverse effects in vector control, a plant bio-insecticide is an advantageous substitute. Currently, the promising mosquito larvicidal activity from plant extracts has been reported worldwide, including Thailand. In this study, the endocarp of Pereskia bleo (Kunth) DC. fruit was extracted with distilled water and ethanol. Crudes and fractionated groups of the extracts were evaluated for their larvicidal efficacy against the 3rd instar larvae of Aedes aegypti. At 48 hours of exposure, it was found that the activities of the extracts were higher than 24-hour's. The ethanolic extracts showed stronger activities than the aqueous ones, indicating the lower LC50 values of both crude and fractionated group extracts. The most toxic activity was found in a fractionated group of the ethanolic extract, E-Gr3, with significantly lowest LC50 values of 707.94 and 223.12 ppm for 24- and 48-hour detection times, respectively. The bioassay results indicated the larvicidal property against the Ae. aegypti mosquito of the P. bleo plant extracts. A safety for non-target organisms or an action on other mosquito vectors of this plant, should be further investigated.

Long-term effect of phytoestrogens from Curcuma comosa Roxb. on vascular relaxation in ovariectomized rats.

Phytoestrogens have been implicated as promising therapeutic agents to treat the vascular impairment seen in menopausal women. The present study investigated the long-term effects of phytoestrogens from Curcuma comosa Roxb. on vascular relaxation of isolated thoracic aorta from ovariectomized (OVX) rats. Treatment of OVX rats for 12 weeks with C. comosa powder, hexane extract, and a novel phytoestrogen, diarylheptanoid-D3, [(3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol] prevented impairment of the endothelium-dependent relaxation response to acetylcholine in OVX, but not the endothelium-denude aortic ring relaxation in response to sodium nitroprusside. These data suggest that the vascular relaxation effect of C. comosa is mediated via endothelial cells. Treatment with D3 also increased endothelial nitric oxide synthase (eNOS) and estrogen receptor-α (ERα) protein expression in the aorta of OVX rats and suppressed elevated tumor necrosis factor-α (TNF-α) expression in OVX aortic rings. These results indicate that C. comosa treatment prevents impairment of vascular relaxation in estrogen-deficient animals via the ER-eNOS pathway as well as through its ability to promote an anti-inflammatory response.

Bioactive flavonoids of the flowers of Butea monosperma.

One new dihydrochalcone, dihydromonospermoside (7), was isolated from the flowers of Butea monosperma together with three known chalcones, butein (2), monospermoside (4) and isoliquiritigenin (8), one flavone, 7,3',4'-trihydroxyflavone (6), four flavanones, (-)-butin (1a), (-)-butrin (3a), (+)-isomonospermoside (5b) and (-)-liquiritigenin (9a), and three isoflavones, formononetin (10), afrormosin (11) and formononetin-7-O-beta-D-glucopyranoside (12). The structure of the new compound was elucidated by spectroscopic techniques whereas those of the known compounds were identified by comparisons of spectroscopic and some physical data with those of reported compounds. The absolute configurations at the 2-position of the flavanones 1a, 3a, 5b and 9a were established to be 2S, 2S, 2R and 2S, respectively, by circular dichroism spectral measurements and were confirmed by comparison of the optical rotations with those of reported values and by enzymic hydrolysis of the glucosides to the corresponding aglycones. The isolated flavonoids exhibited varying antimycobacterial activity with the chalcone 2 being the most active compound (MIC 12.5 microg/ml).

Diarylheptanoids, new phytoestrogens from the rhizomes of Curcuma comosa: Isolation, chemical modification and estrogenic activity evaluation.

Three new diarylheptanoids, a 1:2 mixture of (3S)- and (3R)-1-(4-methoxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol (13a and 13b) and 1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-one (15), together with two synthetically known diarylheptanoids 1,7-diphenyl-(1E,3E,5E)-1,3,5-triene (9) and 1-(4-hydroxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-one (16), and nine known diarylheptanoids, 2, 8, 10-12, 14, a 3:1 mixture of 17a and 17b, and 18, were isolated from the rhizomes of Curcuma comosa Roxb. The absolute stereochemistry of the isolated compounds has also been determined using the modified Mosher's method. The isolated compounds and the chemically modified analogues were evaluated for their estrogenic-like transcriptional activity using RT-PCR in HeLa cell line. Some of the isolated diarylheptanoids and their modified analogues exhibited estrogenic activity comparable to or higher than that of the phytoestrogen genistein. Based on the transcriptional activation of both estrogenic targets, Bcl-xL and ERbeta gene expression, the structural features for a diarylheptanoid to exhibit high estrogenic activity are the presence of an olefinic function conjugated with the aromatic ring at the 7-position, a keto group at the 3-position, and a phenolic hydroxyl group at the p-position of the aromatic ring attached to the 1-position of the heptyl chain.