ARHGAP17 enhances 5-Fluorouracil-induced apoptosis in colon cancer cells by suppressing Rac1.

Colon cancer is a common cause of death in the world, and its main cause of therapy failure is chemoresistance. Apoptosis is de-regulated in colon cancer and is one key mechanism of cancer treatment. We recently reported that reduced expression of ARHGAP17, a Rho GTPase activating protein, correlated with a poor prognosis of colon cancer patients. Here we investigated the role of ARHGAP17 in apoptosis induced by 5-fluorouracil (5-FU) in human colon cancer cells and in mouse xenograft tumor model. We observed a decreased protein level of ARHGAP17 in 5-FU resistant colon cancer cells (HCT116/5-FU and HCT8/5-FU). While ARHGAP17 knockdown attenuated apoptosis upon 5-FU treatment in HCT116 and HCT8, and ARHGAP17 overexpression in HCT116/5-FU and HCT8/5-FU cells increased apoptosis induced by 5-FU. We also found that ARHGAP17 knockdown led to a high level of active Rac1 in HCT116 and HCT8, but ARHGAP17 overexpression reduced active Rac1 in HCT116/5-FU and HCT8/5-FU cells. However, Rac1 inhibitor abolished the effect of ARHGAP17 knockdown, and Rac1 overexpression diminished the effect of ARHGAP17 overexpression on apoptosis induced by 5-FU. Apoptosis was also confirmed by cleaved Caspase-3 and cleaved PARP. Further, we observed that overexpression of ARHGAP17 promoted 5-FU-induced apoptosis and attenuated tumor growth in vivo. Collectively, our data indicate that ARHGAP17 sensitizes chemotherapy-resistant colon cancer cells to apoptosis induced by 5-FU, which is in part through suppressing Rac1.

Three new xanthones from the resin of Garcinia hanburyi.

Three new xanthones, 22,23-dihydroxydihydrogambogenic acid (1), 12-hydroxygambogefic acid A (2), and hanburixanthone (3), along with thirteen known compounds were isolated from the resin of Garcinia hanburyi. The structures of the new compounds were determined by detailed analysis of 1D and 2D NMR spectra and by comparison with related model compounds. All compounds were tested for their cytotoxicities against A549, HCT116, and MDA-MB-231, and most of them showed significant effects on the cell lines.

3-Eth-oxy-carbonyl-2-hy-droxy-6-meth-oxy-4-methyl-benzoic acid.

The title compound, C(12)H(14)O(6), a substituted isophthalic acid monoester which was isolated from the lichen Thamnolia vermicularis var. subuliformis, displays intra-molecular carbox-yl-meth-oxy O-H⋯O and hy-droxy-carboxyl O-H⋯O hydrogen-bonding inter-actions. The terminal methyl group of the ethyl ester is disordered over two sets of sites with occupancies of 0.599 (19) and 0.401 (19).

New isoprenylated 2-arylbenzofurans and pancreatic lipase inhibitory constituents from Artocarpus nitidus.

Two new isoprenylated 2-arylbenzofurans, artonitidin A (=(2'R)-2',3'-dihydro-2'-(1-hydroxy-1-methylethyl)-5',7-bis(3-methylbut-2-en-1-yl)-2,4'-bi-1-benzofuran-6,6'-diol; and artonitidin B (=5-[6-hydroxy-7-(3-methylbut-2-en-1-yl)-1-benzofuran-2-yl]-4-(3-methylbut-2-en-1-yl)benzene-1,3-diol; together with 14 known compounds, were isolated from the stems of Artocarpus nitidus Trec. The structures were elucidated by spectroscopic methods. Norartocarpin, cudraflavone C, brosimone I, artotonkin, albanin A, and artopetelin M showed inhibitory effects on pancreatic lipase with IC(50) values ranging from 1.8+/-0.1 to 63.8+/-3.6 microM.

[Studies on the chemical constituents of Coleus forskohlii].

OBJECTIVE: To study the chemical constituents in the aerial parts of Coleus forskohlii. METHODS: The compounds were isolated by various column chromatographic methods, and their structures were identified by spectroscopic methods. RESULTS: Twelve compounds were isolated and identified as chamaecydin (1), 6 alpha-hydroxydemethylcryptojaponol (2), alpha-cedrene (3), oleanolic acid (4), forskolin G (5), forskolin J (6), 1,6-diacetyl-9-deoxyforskolin (7), forskolin A (8), forskolin H (9), 6-acetyl-1-deoxyforskolin (10), betulinic acid (11), beta-sitosterol (12). CONCLUSION: Compounds 1 - 3 are isolated from Coleus genus for the first time, and compound 4 is isolated from C. forskohlii for the first time.

[Phylogeny relationship and molecular identification of ten Huperzia species (Huperziaceae) based on matK gene sequences].

OBJECTIVE: To study the phylogeny relationship and molecular identification of 10 species from Huperzia (Huperziaceae) based on matK gene sequences data. METHOD: Total DNA of nine species from Huperzia was extracted; matK gene sequence was amplified by PCR. PCR product was directly sequenced after purification. RESULT: The chloroplast matK gene nucleotide sequences from 9 species of Huperzia species were sequenced. The matK gene nucleotide sequences length was 1 589 bp. Analysis with Huperzia lucidula matK gene nucleotide sequences (download from GenBank) and taking Lycopodiella cernua as outgroup, Maximum Parsimony, Neighbor-Joining analyses and genetic distances were conducted using MEGA 3.1 software. 35 variable sites and 35 parsimony informative sites have been found. Pairwise genetic distances among 10 species of Huperzia was 1.59% - 0.25%. CONCLUSION: The results were consistent with the taxonomy in morphological of Huperzia. But H. longipetiolata and H. serrata were resolved into in different clade. There are 19 different sites of matK gene sequences between H. longipetiolata and H. serrata, the genetic distances is 0.121%. It is suggested H. longipetiolata should be as an independent species.

[Antidepressant amides from Piper laetispicum C. DC].

AIM: To investigate the antidepressant constituents of Piper laetispicum C. DC. METHODS: The compounds were enriched by column chromatography on silica gel. Structural determination of the pure compounds was based on extensive analyses of modern spectroscopic methods including MS, HR-MS, UV, IR, 1D- and 2D-NMR spectra. The mouse forced swimming test was used for chasing antidepressant activity. RESULTS: Three amides were isolated and identified as N-isobutyl-(3,4-methylendioxyphenyl)-2E, 4E, 9E-undecatrienoamide (I), N-isobutyl-9-phenyl-2E, 4E-nonadienamide (II) and N-isobutyl-7-phenyl-2E, 4E-heptadienamide (III). CONCLUSION: N-Isobutyl-(3,4-methylendioxyphenyl)-2E, 4E, 9E-undecatrienoamide (I) is a new compound named as laetispicine. N-Isobutyl-9-phenyl-2E, 4E-nonadienamide (II) and N-isobutyl-7-phenyl-2E, 4E-heptadienamide (III) were isolated for the first time as natural substance.

[Pharmacological studies on extracts from Piper boehmeriaefolium var. tonkinense].

OBJECTIVE: To study sedative, analgesic, antidepressant and anti-inflammatory effects of the extracts from Piper boehmeriaefolium var. tonkinense. METHOD: EtOAc soluble constituents and EtOH soluble constituents were administered 50, 100 mg x kg(-1) to mice respectively. The sedative effects were determined by autonomic action test. The analgesic effects were observed by the twisting test induced by acetic acid. The antidepressant effects were investigated by the forced swimming test. The anti-inflammatory effects were studied by auricle swelling induced by xylene. RESULT: EtOAc extracts could significantly lower autonomic action times, reduce squirming induced by acetic acid accumulated immobility time in forced swimming and auricle swelling values by xylene. But EtOH extracts could only decrease autonomic action times and accumulated immobility time. CONCLUSION: The active ingredients with sedation, analgesia, antidepressant and anti-inflammation in P. boehmeriaefolium var. tonkinense exist mainly in the EtOAc extracts.

A new lignan and other constituents from Zanthoxylum armatum DC.

A new lignan (1), named armatumin, along with 25 known compounds, was isolated from Zanthoxylum armatum. The structure of compound 1 was elucidated using spectroscopic methods. Compounds 7, 8, 11, 13, 16, 20 and 21 were first isolated from Z. armatum and compounds 14 and 19 were isolated for the first time from Rutaceae.

Cytotoxic alkoxylated xanthones from the resin of Garcinia hanburyi.

Three new xanthones, garcinolic acid (1), 10α-ethoxy-9,10-dihydromorellic acid (2), and 10α-ethoxy-9,10-dihydrogambogenic acid (3), along with six known compounds were isolated from the resin of Garcinia hanburyi. These compounds were tested for their cytotoxicities against A549, HCT116, SK-BR-3 and HepG2, and showed high inhibitory effects on the cell lines.

Simultaneous determination of ten active components in 12 Chinese Piper species by HPLC.

Piper is a genus that is recently valued for the treatment of central nervous system diseases. The major constituents, amides and lignans, are responsible for the antinociceptive and antidepressant activities. This study developed a RP-HPLC-UV method for the simultaneous determination of eight amides and two lignans in twelve different species of Piper. HPLC separation was accomplished on a C18 analytical column (5 µm, 250 mm × 4.6 mm, i.d.) with a gradient mobile phase consisting of acetonitrile and water at a flow rate of 1.0 ml/min. All the calibration curves showed good linear correlation coefficients (r > 0.9997) over the test ranges. The relative standard deviation of the current method was less than 2.90% for intra- and inter-day assays and the average recoveries were between 98.25% and 103.08%. The HPLC method established is appropriate for quality control purposes and allows for the differentiation of Piper species.

Antinociceptive and anti-inflammatory activities of ethyl acetate fraction from Zanthoxylum armatum in mice.

Zanthoxylum armatum DC. is a traditional Chinese medicine that is prescribed to alleviate pain and treat inflammatory disorders. This species is distributed mainly in the southeast and southwest regions of China. In the present study, we found that ethyl acetate fraction of ethanolic extract of Z. armatum could significantly decrease acetic acid-induced writhing numbers, and suppress formalin induced licking times in the first phase at the highest dose and in the second phase at all tested doses. This observation revealed that Z. armatum extract possessed powerful antinociceptive activity. The mechanisms of the antinociceptive effect might be mainly involved in the periphery inflammatory analgesic. In addition, the ethyl acetate fraction also inhibited xylene-induced ear swelling in a dose-dependent manner in mice. Eight lignans [eudesmin, horsfieldin, fargesin, kobusin, sesamin, asarinin, planispine A, and pinoresinol-di-3,3-dimethylallyl] were identified as major components of the ethyl acetate fraction. Considering related studies reporting the anti-inflammatory activity for the identified lignans, lignan might be responsible for its anti-inflammatory activity. Our results confirm that the traditional use of Z. armatum in the treatment of inflammation and pain is warranted.

Studies on antidepressant and antinociceptive effects of ethyl acetate extract from Piper laetispicum and structure-activity relationship of its amide alkaloids.

Piper laetispicum C.DC. (Piperaceae), is an endemic climbing, glabrous plant distributed in the southern part of China. A novel alkaloid amide, Laetispicine, from it has been proven to possess antidepressant activity. In this present study, antidepressant and antinociceptive effects of the ethyl acetate extract (EAE) of P. laetispicum have been studied in forced swimming, open field, acetic acid writhing and formalin tests in KM mice. A significantly antidepressant-like effect was showing at doses of higher than 60 mg/kg, which was not due to an increase in locomotive activity. The EAE also presented an analgesic effect, in our studies. At lower doses (30 mg/kg) the antinociceptive effect was likely mediated via peripheral inflammation and changes in central processing, and at higher doses (120 mg/kg) that was due to both central and peripheral pathways. We also quantitatively analyzed the major components of EAE by HPLC and approached the structure-activity relationship between structure of amide alkaloids and its antidepressant activities. The antidepressant effective components of EAE might be Leatispiamide A and Laetispicine. In their molecular structures, the isolated double bond from benzene ring and conjugated double bond located at 2-3 and 4-5 were necessary for its antidepressant activity.

Molecular analysis of Leonurus species in China based on ITS and matK sequences.

The genus Leonurus has long been recognized as a natural group, but its interspecific relationship has not yet been studied in the light of sequence data. The ITS regions and matK sequences of all subgenera of Leonurus in China were amplified, sequenced and investigated. Phylogenies generated by maximum parsimony and neighbor-joining methods and division of the genus into two major clades. The phylogenetic results indicated that L. chaituroides has the very close phylogenetic relationship with Subg. Cardiochilium and supported the notion that L. macranthus acts as the bridge between Subg. cardiochilium and Subg. Leonurus. According to the analysis of information given by ITS and matK sequences, we suggest that ITS sequences would be more suitable to serve as markers for authentication of Herba Leonuri than matK does.

Pharmacokinetics of laetispicine and its brain distribution in rats.

Laetispicine, which is a novel amide alkaloid isolated from the stem of Piper laetispicum, has been proven to possess antidepressant and antinociceptive effects. This study examined the pharmacokinetic characteristics of laetispicine in plasma and brain distribution in rats by a simple sensitive HPLC-UV method. The separation was performed on a reverse-phase ODS column (250 mm x 4.6 mm, i.d., 5 microm) with a mobile phase composed of acetonitrile-water (75:25, v/v) as the mobile phase at a flow rate of 1.0 ml/min with UV detection at 259 nm. The calibration curve of laetispicine in rat plasma showed excellent linear behavior between 0.005-5.0 microg/ml (r2 = 0.9992), and between 0.02-0.5 microg/ml (r2 = 0.9952) in rat brain samples. The lower limit of quantification (LLOQ) was found to be 0.005 microg/ml in rat plasma and 0.02 microg/ml in rat brain samples. This HPLC assay was a precise and reliable method for the analysis of laetispicine in pharmacokinetic studies. Laetispicine was rapidly and extensively transported across the blood-brain barrier (BBB) and distributed into different brain regions.

Identification of crude drugs from Chinese medicinal plants of the genus Bupleurum using ribosomal DNA ITS sequences.

To determine the variation of rDNA ITS sequences in medicinal plants of the genus Bupleurum and identify corresponding DNA molecular markers associated with medicinally important members, ITS regions were amplified, sequenced, and analyzed by DNAssist version 2.2. We found that the homologous alignment of ITS sequences between members of the genus Bupleurum and associated out-groups was lower than 75%, while within-group alignment was greater than 87%. The conclusion can be drawn that ITS sequences can be used as reliable molecular markers for the identification of Radix Bupleuri.

ITS sequence analysis used for molecular identification of the Bupleurum species from northwestern China.

Radix Bupleuri (Chaihu), a famous Traditional Chinese Medicine, is derived from the dried roots of Bupleurum chinense DC. and B. scorzonerifolium Willd. But nowadays, other nine species and varieties from genus Bupleurum are also used as Radix Bupleuri in northwestern China. The authentic identification of dried roots of B. chinense and B. scorzonerifolium, however, is difficult to just base on the appearance and morphology. A molecular genetic method was developed to help discriminate the original species of Radix Bupleuri. The ITS sequences ( approximately 600 bp) were amplified by the PCR technique from genomic DNA isolated from all the collected samples. According to analyzing the information given by ITS sequences, the conclusion can be made that ITS sequence could serve as markers for authentication of Radix Bupleuri.

[Nuclear ribosomal DNA internal transcribed spacer 1 sequences of 4 Leonurus species].

OBJECTIVE: To analyze the nuclear ribosome DNA (nrDNA) internal transcribed spacer (ITS) sequences of 4 Leonurus species, and the possibility of using them for molecular authentication of the crude drugs from the genus. METHODS: The nrDNA ITS sequence (including ITS1, 5.8S rDNA, ITS2, and partial 18S rDNA and 26S rDNA) of L. japonicus and its 3 adulterant species were amplified and sequenced, and CLUSTRAL X and MEGA software was employed for analysis. RESULTS: The variation of ITS1 and ITS2 between L. japonicus and its adulterant species ranged between 7.2% and 18.8% and between 14.2% and 27%, respectively. The phylogenic tree derived from the dendrograms based on the ITS sequence data contained some discrepancy from the traditional classification. CONCLUSION: The nrDNA ITS sequences can be used potentially as efficient markers for identification of L. japonicus and its adulterants, and further study is needed for studying the phylogeny of Leonurus.

[ITS sequence of 9 Bupleurum species and its application in identification of Chaihu (Radix Buplurei)].

OBJECTIVE: The study the patterns of rDNA ITS sequence variation of 9 medicinal species of genus Bupleurum in China to find the DNA molecular markers for identification of these crude drugs. METHOD: The ITS regions of these species were amplified by PCR and sequenced, and their sequences were analyzed by DNAssist Version 2.2. RESULTS: Homologous alignment indicated that the consanguinity of the ITS sequences between genus Bupleurum and outgroups was lower than 75%, and that within genus Bupleurum was higher than 88%. For the same species, the consanguinity was higher than 99%. CONCLUSION: The ITS sequences may serve as reliable molecular markers for identification of Radix Bupleuri.