Pattara Sawasdee

Anticholinesterase Activity of 7-Methoxyflavones Isolated from Kaempferia parviflora

The rhizome of Kaempferia parviflora or kra‐chai‐dum (in Thai) is used traditionally as a folk medicine. The preliminary cholinesterase inhibitory screening of this plant extract exhibited significant acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Thirteen known methoxyflavones (1–13) were isolated and their structures were completely elucidated based on NMR analysis and compared with literature reports. Minor compounds 12–13 were reported for the first time from this species. The cholinesterase inhibitory test results showed that the highest potential inhibitors toward AChE and BChE were 5,7,4′‐trimethoxyflavone (6) and 5,7‐dimethoxyflavone (7), respectively, with the percentage inhibitory activity varying over 43–85%. The structure‐activity relationship study led to the conclusion that compounds bearing 5,7‐dimethoxy groups and a free substituent at C‐3 had a significant inhibitory effect at a concentration of 0.1 mg/mL, but those bearing a 5‐hydroxyl group reduced the inhibitory potency. On the other hand, flavones bearing a 3′‐ or 5′‐methoxy group did not influence the inhibitory effect. Interestingly, 5,7‐dimethoxyflavone (7) exhibited strong selectivity for BChE over AChE which may be of great interest to modify as a treatment agent for Alzheimers disease. Copyright

Possible mechanisms of vasorelaxation for 5,7-dimethoxyflavone from Kaempferia parviflora in the rat aorta.

The present study investigated the vascular effects of 5,7‐dimethoxyflavone (DMF), isolated from the rhizomes of Kaempferia parviflora (KP), on rat isolated aortic rings and its possible mechanisms. DMF (1–100 μm) caused concentration‐dependent relaxations in aortic rings precontracted with methoxamine. This effect was significantly reduced by removal of the endothelium, and after pretreatment with NG‐nitro‐L‐arginine methyl ester (L‐NAME, 300 μm), indomethacin (10 μm) and 1H‐[1,2,4]oxadiazolo‐[4,3‐a]quinoxalin‐1‐one (ODQ, 10 μm), but not 9‐(tetrahydro‐2‐furanyl)‐9H‐purine‐6‐amine (SQ22536, 100 μm). Relaxant responses to DMF were significantly inhibited by high KCl (60 mm) in both endothelium‐intact and ‐denuded rings. In addition, the relaxations to DMF were significantly reduced by pretreatment with tetraethylammonium (TEA, 5 mm), glibenclamide (10 μm), 4‐aminopyridine (1 mm) or barium chloride (10 μm). Preincubation with DMF (10 and 100 μm) for 30 min significantly inhibited the contractile responses to CaCl2 in a Ca2+‐free, high K+ buffer. The present study demonstrated that DMF causes endothelium‐dependent relaxation that is partly mediated by NO‐cGMP and cyclooxygenase pathways. Interestingly, DMF‐induced responses are mainly due to increasing K+ efflux, and inhibition of Ca2+ influx from the extracellular space. The vasodilator effects of DMF provide experimental support for the potential use of KP as a medical plant in the treatment of cardiovascular diseases. Copyright

Potent and selective butyrylcholinesterase inhibitors from Ficus foveolata

Four stilbenes (1-4), one inseparable mixture of two alkyl diferulates (5a, 5b), one alkyl ferulate (6) and four flavonoids (7-10) were isolated from Ficus foveolata Wall. Except for quercetin (10), the other ten constituents were isolated from F. foveolata for the first time. In addition, one of the two components in mixture 5 is a new compound identified as (1E,22E)-1,22-docosanediol diferulate (5a). All nine isolated compounds, plus the mixture of 5a and 5b known as 5, exhibited a low or no activity against acetylcholinesterase. However, and interestingly, the stilbenes 1-4 showed a high inhibition towards butyrylcholinesterase. Gnetol (4) had the lowest IC(50) value of 1.3μM towards butyrylcholinesterase and showed a reversible and competitive inhibition in the kinetic study.

Acetyl- and butyryl-cholinesterase inhibitory activities of mansorins and mansonones.

Cholinesterase (ChE) inhibitory activities of three coumarins (mansorins A–C) and five naphthoquinones (mansonone C, E, G and H) were evaluated to determine the relationships between the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory effects and the core structures of these compounds. Among the tested compounds, mansonone E exhibited the highest ChE inhibitory activities, with IC50 values in the low micromolar levels. In addition to revealing the ChE inhibitory activities of naphthoquinones for the first time, the results also revealed structure–activity relationship information that could be useful for further modification. Furthermore, the study also supports the hypothesis that mansonones are the active component in Thespesia populnea, a plant that previously has been shown to enhance memory activity in an in vivo study. Copyright

Effects of Kaempferia parviflora Wall. Ex Baker on endothelial dysfunction in streptozotocin-induced diabetic rats

AIM OF THE STUDY The aim of the present study was to investigate an ethanolic extract of Kaempferia parviflora (KPE) reduces oxidative stress and preserves endothelial function in aortae from diabetic rats. MATERIALS AND METHODS Diabetes was induced in Sprague-Dawley rats by streptozotocin (STZ) treatment (55 mg/kg i.v.). Vascular reactivity and superoxide generation were assessed in aortic rings using standard organ bath techniques and lucigenin-enhanced chemiluminescence, respectively. RESULTS Eight weeks after STZ treatment blood glucose was elevated compared to citrate treated control rats and there was an increased aortic generation of superoxide anion. In aortic rings acetylcholine-induced relaxation was impaired whereas endothelium-independent relaxation to sodium nitroprusside was unaffected. When aortic rings were acutely exposed to KPE (1, 10 and 100 μg/ml) there was a significant reduction in the detection of superoxide anion and enhanced relaxation to acetylcholine. Two separate groups of rats (control and diabetic) were orally administered daily with KPE (100 mg/kg body weight) for 4 weeks. KPE treatment reduced superoxide generation and increased the nitrite levels in diabetic aortae, and enhanced acetylcholine-induced relaxation. In the presence of N(G)-nitro-L-arginine (L-NNA), the relaxation to acetylcholine in aortic rings of diabetic rats was only partially inhibited, but was totally abolished in aortic rings from the KPE-treated diabetic rats. Indomethacin did not affect relaxation to acetylcholine in aortic rings of any group. CONCLUSIONS These results suggest that KPE, acutely in vitro or after 4 weeks administration in vivo, reduces oxidant stress, increases NO bioavailability and preserves endothelium-dependent relaxation in aortae from diabetic rats.

Kaempferia parviflora, a plant used in traditional medicine to enhance sexual performance contains large amounts of low affinity PDE5 inhibitors

AIM OF THE STUDY A number of medicinal plants are used in traditional medicine to treat erectile dysfunction. Since cyclic nucleotide PDEs inhibitors underlie several current treatments for this condition, we sought to show whether these plants might contain substantial amounts of PDE5 inhibitors. MATERIALS AND METHODS Forty one plant extracts and eight 7-methoxyflavones from Kaempferia parviflora Wall. ex Baker were screened for PDE5 and PDE6 inhibitory activities using the two-step radioactive assay. The PDE5 and PDE6 were prepared from mice lung and chicken retinas, respectively. All plant extracts were tested at 50 μg/ml whereas the pure compounds were tested at 10 μM. RESULTS From forty one plant extracts tested, four showed the PDE5 inhibitory effect. The chemical constituents isolated from rhizomes of Kaempferia parviflora were further investigated on inhibitory activity against PDE5 and PDE6. The results showed that 7-methoxyflavones from this plant showed inhibition toward both enzymes. The most potent PDE5 inhibitor was 5,7-dimethoxyflavone (IC(50) = 10.64 ± 2.09 μM, selectivity on PDE5 over PDE6 = 3.71). Structure activity relationship showed that the methoxyl group at C-5 position of 7-methoxyflavones was necessary for PDE5 inhibition. CONCLUSIONS Kaempferia parviflora rhizome extract and its 7-methoxyflavone constituents had moderate inhibitory activity against PDE5. This finding provides an explanation for enhancing sexual performance in the traditional use of Kaempferia parviflora. Moreover, 5,7-dimethoxyflavones should make a useful lead compound to further develop clinically efficacious PDE5 inhibitors.

New furoquinoline alkaloids from the leaves of Evodia lepta

Three new furoquinoline alkaloids, leptanoines A-C (1-3) along with three known compounds (4-6) were isolated from the leaves of Evodia lepta. Their structures were identified by interpretation of their spectroscopic data as well as comparison with those reported in the literature. In addition, all isolated compounds were evaluated for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities. Compound 4 showed the highest inhibitory activity towards BChE with an IC50 value of 47.9 μM. On the other hand, Compound 5 showed the highest inhibitory activity towards AChE with an IC50 value of 69.1 μM.

A 5,7-Dimethoxyflavone/Hydroxypropyl-β-Cyclodextrin Inclusion Complex with Anti-Butyrylcholinesterase Activity

This study aimed to improve the water solubility of 5,7-dimethoxyflavone (5,7-DMF) isolated from Kaempferia parviflora by complexation with 2-hydroxypropyl-β-cyclodextrin (HPβ-CD). The phase solubility profile of 5,7-DMF in the presence of HPβ-CD was classified as AL-type and indicated a 1:1 mole ratio. Differential scanning colorimetry, X-ray diffraction, NMR and SEM analyses supported the formation of a 5,7-DMF/HPβ-CD inclusion complex involving the A ring of 5,7-DMF inside the HPβ-CD cavity. This is the first example of CD inclusion with the A ring of non-hydroxyl flavones. The stability and binding constants of the complexes were determined using the phase solubility and UV-vis absorption spectroscopy, respectively. The water solubility of 5,7-DMF was increased 361.8-fold by complexation with HPβ-CD and overcame the precipitation problem observed in aqueous buffers, such as during in vitro anti-butyrylcholinesterase activity assays. The 1:1 mole ratio of the 5,7-DMF/HPβ-CD complex showed a 2.7-fold higher butyrylcholinesterase inhibitory activity (in terms of the IC50 value) compared to the non-complexed compound.

3,5,7-Trimeth­oxy-2-(4-methoxy­phen­yl)-4H-1-benzopyran-4-one

In the title compound, C19H18O6, also known as 3,4′,5,7-tetramethoxyflavone, the dihedral angle between the benzopyran-4-one group and the attached benzene ring is 11.23 (8)°. An intramolecular C—H⋯O hydrogen bond generates an S(6) ring motif. In the crystal, molecules are linked into a two-dimensional network parallel to (01) by intermolecular C—H⋯O hydrogen bonds, which generate R 4 4(20), R 4 4(12) and R 2 2(14) ring motifs. Adjacent networks interact by π–π interactions between the pyran ring and its methoxyphenyl substituent [centroid–centroid distance = 3.5267 (8) Å].

Chemical Constituents of the Stems of Spatholobus parviflorus and Their Cholinesterase Inhibitory Activity

Spatholobus parviflorus (Fabaceae), locally known as “Tao Pun Sai,” is a strong and woody climber. It is widely distributed in many parts of Thailand. An ethnobotanical use of this species by tribesmen in the North is as a medicinal plant to treat rheumatism, anemia, and other diseases. In previous work, the active constituents of the leaves of S. parviflorus were reported [1]. However, to our knowledge the chemical constituents of S. parviflorus stems have not been reported up to now. The stem extract of S. parviflorus was fractionated by a combination of silica gel column chromatography and centrifugal thin-layer chromatography to afford stigmasterol (1) [2], (6aR,11aR)-medicarpin (2) [3], 8-O-methylretusin (3) [4], formononetin (4) [5], biochanin A (5) [6], 7,4 -dihydroxy-8-methoxyisoflavone (6) [7], daidzein (7) [8], trans and cis-4-hydroxymelleins (8 and 9) [9], and coumestrol (10) [10], which were interpreted by spectroscopic analysis, particularly NMR and MS and coincided well with previous reports. All isolated compounds were evaluated for their AChE and BChE activity using a microtiter assay. The AChE and BChE data indicated that compound 10 showed moderate percentage inhibitory activity towards AChE and BChE, with percentage inhibitory activity values of 38.01 and 47.89, respectively. On the other hand, compound 5 also showed moderate percentage inhibitory activity towards BChE only, with a percentage inhibitory activity value of 48.08. Other compounds showed lower inhibitory levels (% inhibition < 20%) at a concentration of 0.1 mg/mL. Plant Material. The stems of S. parviflorus were collected from Mahasarakham Province of Thailand in October 2009 and identified by Ms. Suttira Khumkratok, a botanist at the Walai Rukhavej Botanical Research Institute, Mahasarakham University, where a voucher specimen (Khumkratok No. 2-09) is deposited. Extraction and Isolation. The air-dried and powdered stems (2.0 kg) of S. parviflorus were successively extracted in a Soxhlet apparatus with CH2Cl2. The extract was concentrated under vacuum to yield 52.0 g of crude residue. This material was fractionated by vacuum liquid chromatography (VLC) over silica gel (Merck Art 7730), using successive elutions by hexane, CH2Cl2, EtOAc, and MeOH with increasing polarity. A total of six fractions (A–F) was collected. The VLC fraction B was chromatographed on a silica gel column using a stepwise gradient of hexane–CH2Cl2–EtOAc to furnish 1 (34.9 mg), 2 (14.6 mg), 3 (101.7 mg), and 4 (4.6 mg). Similarly, the VLC fraction C was chromatographed on a silica gel column using a stepwise gradient elution of EtOAc in hexane to afford five fractions (C1–C5). Fraction C3 was separated using a Chromatotron (centrifugal thin-layer chromatograph) eluting with a hexane and EtOAc gradient to obtain 5 (3.3 mg). Similarly, fraction C5 was separated by a Chromatotron eluting with a hexane and EtOAc gradient to yield 6 (2.9 mg) and 7 (2.3 mg). The VLC fraction E was rechromatographed on silica gel using a gradient system of hexane–CH2Cl2–MeOH as eluents to yield five fractions (E1–E5). Fraction E2 was further purified by a Chromatotron using a stepwise gradient of EtOAc in hexane to give 8 (2.1 mg) and 9 (1.3 mg) [9]. Finally, fraction E4 was evaporated and was recrystallized from MeOH–CH2Cl2 (2:9) to obtain 10 (5.3 mg). Stigmasterol (1), colorless needles. ESI-MS m/z 413 [M + H]+. This compound is identical to an authentic sample. (6aR,11aR)-Medicarpin (2), yellowish powder, mp 123–124 C. 1H NMR (400 MHz, CDCl3, , ppm, J/Hz): 3.46–3.54 (1H, m, H-6a), 3.59 (1H, t, J = 10.8, H-6 ), 3.75 (3H, s, 9-OCH3), 4.21 (1H,dd, J = 10.8, 4.8, H-6 ), 5.48 (1H, d,