Formononetin recovered injured nerve functions by enhancing synaptic plasticity in ischemic stroke rats.

BACKGROUND AND OBJECTIVES: Formononetin has protective effect against ischemic stroke. It's unclear whether it can restore the nerve functions after stroke. METHODS: SD rats were subjected with middle cerebral artery occlusion (MCAO), and divided into sham, model and formononetin (30 mg/kg) groups. Neurobehavioral tests (modified Neurological Severity Score [mNSS] and rotarod) were performed before and at 1, 3, 7 and 14 days after MCAO. Then, the rats were sacrificed and the brain sections were processed for neuronal differentiation and synaptic plasticity. RESULTS: Compared with the sham group, the scores of mNSS were significantly increased, and the residence time on the rotating drum was significantly decreased in the MCAO rats. Compared with the model group, the scores of mNSS were significantly decreased, and the residence time on the rotating drum was increased in the formononetin (30 mg/kg) group. Formononetin significantly increased the number of neuronal dendritic spines and the expression of ß III-tubulin, GAP-43, NGF, BDNF, p-Trk A, p-Trk B, p-AKT and p-ERK 1/2. CONCLUSIONS: Formononetin recovered injured nerve functions after ischemic stroke. PI3K/AKT/ERK pathway might involve in the beneficial effect of formononetin on the neuronal differentiation and synaptic plasticity.

A new bilobalide isomer and two cis-coumaroylated flavonol glycosides from Ginkgo biloba leaves.

A new bilobalide isomer (1), together with two flavonol glycosides (2, 3), have been isolated and elucidated from the extract of Ginkgo biloba leaves. Significantly, 1 was a new sesquiterpene lactone with two lactone ring groups, both 2 and 3 were two flavonol glycosides with a same cis-coumaroylated fragment. Their chemical structures were elucidated by NMR and MS spectroscopic date and the absolute configuration of 1 was specific established by Cu-Kα X-ray crystallographic analyses. However, 1-3 showed no obvious anti-platelet aggregation activity.

Monitoring Antiplatelet Aggregation In Vivo and In Vitro by Microtiter Plate Method.

BACKGROUND: The current light transmission aggregation method is a recognized conventional method for platelet function evaluation, but it is time-consuming and poor in parallelism and cannot simultaneously monitor multiple inducers at multiple levels. The microtiter plate method has been established because of the high-throughput characteristic, but it needs more practical applications. OBJECTIVES: To evaluate the microtiter plate method by using aspirin and clopidogrel in vivo and in vitro. METHODS: In vitro, the platelet aggregations inhibited by aspirin (0.3, 1, 3, 10, 30, 90 µM) and clopidogrel (1, 3, 10, 30, 100, 300 µM) were evaluated with the presence of arachidonic acid (AA) and adenosine diphosphate (ADP) agonists. Using the combination index (CI), the effect of the combination of aspirin and clopidogrel on platelet aggregation was evaluated. In vivo, New Zealand rabbits (n = 18) were randomly divided into 3 groups, aspirin group (5 mg/kg, intragastrical gavage [i.g.]), clopidogrel group (14 mg/kg at the first day, followed by 4 mg/kg, i.g.), and the combination of these two drugs, administered (i.g.) continuously for 7 days. Then, the blood was collected to measure platelet aggregation. RESULTS: Different concentrations of AA (12.5, 25, 50, 100 µM) and ADP (1.25, 2.5, 5, 10 µM) could promote platelet aggregation in concentration-dependent manner, and the most stable induction concentrations of AA and ADP were 50 and 5 µM. In vitro, with the above optimized detection system, aspirin and clopidogrel alone or in combination had concentration-dependent antiplatelet aggregation. The combination of aspirin and clopidogrel also showed synergistic inhibition effect within the concentration range studied. In vivo, aspirin and clopidogrel alone or in combination inhibited platelet aggregation induced by multiple concentrations of AA and ADP agonists, and the combined inhibition was more significant during the administration than aspirin or clopidogrel alone. CONCLUSIONS: The improved microtiter plate method combining the use of multiple levels of multiple agonists avoids the variation of the effective inducer concentrations due to individual different response of platelets to agonists. It may be a potential approach in the detection of platelet aggregation.

Simvastatin ameliorates graft-vs-host disease by regulating angiopoietin-1 and angiopoietin-2 in a murine model.

Angiopoietins play an important role in vascular endothelial function. Endothelial damage is an important pathogenesis relating with acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), protecting endothelial cells (ECs) from damage may be a potent prophylaxis and therapeutic strategy of acute GVHD (aGVHD). In this study, we explored changes in Angiopoietin-1 (Ang-1) and Ang-2 expression in a aGVHD mouse model and determined whether simvastatin prevents GVHD through regulating Ang-1 and Ang-2 expression. In vitro simvastatin administration increased Ang-1 production and release but conversely inhibited Ang-2 release from EA.hy926 ECs. Simvastatin improved the survival of aGVHD mice, attenuated the histopathological GVHD grades and plasma levels of Ang-2, and elevated the plasma levels of Ang-1 as well as the aortic endothelial levels of Ang-1 and Ang-2. In summary, simvastatin represents a novel approach to combat GVHD by increasing Ang-1 production while suppressing Ang-2 release to stabilize endothelial cells.

Endothelial microparticles carrying hedgehog-interacting protein induce continuous endothelial damage in the pathogenesis of acute graft-versus-host disease.

Accumulating evidence suggests that endothelial microparticles (EMPs), a marker of endothelial damage, are elevated in acute graft-versus-host disease (aGVHD), and that endothelial damage is implicated in the pathogenesis of aGVHD, but the mechanisms remain elusive. In this study, we detected the plasma EMP levels and endothelial damage in patients and mice with aGVHD in vivo and then examined the effects of EMPs derived from injured endothelial cells (ECs) on endothelial damage and the role of hedgehog-interacting protein (HHIP) carried by EMPs in these effects in vitro. Our results showed that EMPs were persistently increased in the early posttransplantation phase in patients and mice with aGVHD. Meanwhile, endothelial damage was continuous in aGVHD mice, but was temporary in non-aGVHD mice after transplantation. In vitro, EMPs induced endothelial damage, including increased EC apoptosis, enhanced reactive oxygen species, decreased nitric oxide production and impaired angiogenic activity. Enhanced expression of HHIP, an antagonist for the Sonic hedgehog (SHH) signaling pathway, was observed in patients and mice with aGVHD and EMPs from injured ECs. The endothelial damage induced by EMPs was reversed when the HHIP incorporated into EMPs was silenced with an HHIP small interfering RNA or inhibited with the SHH pathway agonist, Smoothened agonist. This work supports a feasible vicious cycle in which EMPs generated during endothelial injury, in turn, aggravate endothelial damage by carrying HHIP into target ECs, contributing to the continuously deteriorating endothelial damage in the development of aGVHD. EMPs harboring HHIP would represent a potential therapeutic target for aGVHD.

Angiogenic factors are associated with development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-ß in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-ß promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.

Umbilical cord blood-derived mesenchymal stem cells ameliorate graft-versus-host disease following allogeneic hematopoietic stem cell transplantation through multiple immunoregulations.

Although mesenchymal stem cells (MSCs) are increasingly used to treat graft-versus-host disease (GVHD), their immune regulatory mechanism in the process is elusive. The present study aimed to investigate the curative effect of third-party umbilical cord blood-derived human MSCs (UCB-hMSCs) on GVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their immune regulatory mechanism. Twenty-four refractory GVHD patients after allo-HSCT were treated with UCB-hMSCs. Immune cells including T lymphocyte subsets, NK cells, Treg cells and dendritic cells (DCs) and cytokines including interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were monitored before and after MSCs transfusion. The results showed that the symptoms of GVHD were alleviated significantly without increased relapse of primary disease and transplant-related complications after MSCs transfusion. The number of CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells decreased significantly, and that of NK cells remained unchanged, whereas the number of CD4(+) and CD8(+) Tregs increased and reached a peak at 4 weeks; the number of mature DCs, and the levels of TNF-α and IL-17 decreased and reached a trough at 2 weeks. It was concluded that MSCs ameliorate GVHD and spare GVL effect via immunoregulations.

Rearrangement of the nad1 gene in Pristaulacus compressus (Spinola) (Hymenoptera: Evanioidea: Aulacidae) mitochondrial genome.

The mitochondrial genome of the Pristaulacus compressus (Spinola, 1808) (Hymenoptera: Aulacidae) (GenBank accession No. KF500406) is reported in this study. This is the first sequenced mitochondrial genome from the family Aulacidae of the order Hymenoptera. The length of this mitochondrial genome is 15,563 bp with an A + T content of 84%, including 13 protein-coding, 2 rRNA and 22 tRNA gene, and an A + T-rich region (Table 1). Three tRNA and one protein-coding genes were rearranged in the P. compressus mitochondrial genome, in which, the trnY was inverted, while the trnQ was shuffled to the downstream of tRNA cluster trnI-trnQ-trnM. The trnS1 was translocated to the downstream of the A + T-rich region together with the protein-coding gene nad1. The gene arrangement pattern of this mitochondrial genome is new to the Hymenoptera. All protein-coding genes start with ATN start codon. Ten protein-coding genes stop with termination codon TAA, whereas one protein-coding gene uses incomplete stop codon TA and two use T. The A + T-region is located between rrnS and trnS1 with a length of 780 bp.

Sequencing and characterization of the Monocellicampa pruni (Hymenoptera: Tenthredinidae) mitochondrial genome.

The mitochondrial genome of the Monocellicampa pruni (Hymenoptera: Tenthredinidae) (GenBank accession No. JX566509) has been reported in this study. This is the first sequenced mitochondrial genome from the family Tenthredinidae of the order Hymenoptera. The sequenced region of this mitochondrial genome is 15,169 bp with an A + T content of 77.21%, including 13 protein-coding, 2 rRNA and 19 tRNA genes, and a partial region of the A + T-rich region. Three tRNA genes, i.e. trnI. trnQ and trnM, between the A + T-rich region and the nad2 gene were failed to sequence because of the present of PolyAT structure. The gene arrangement of the sequenced region was similar to the pupative ancestral arrangement of insects. There are two large non-coding regions located between trnC and trnY. trnF and nad5 with a length of 107 and 177 bp, respectively. All protein-coding genes start with ATN start codon. Eleven protein-coding genes stop with termination codon TAA, whereas one protein-coding gene uses incomplete stop codon TA and one uses T. All of the 22 tRNA genes have a typical cloverleaf structure except for the trnS1, in which, the D-stem pairings in the DHU arm are absent.

Characterization of the complete mitochondrial genome of the black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae).

The complete mitochondrial genome of the black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae) was determined (GenBank accession No. KF163965). The length of this mitochondrial genome is 15,377 bp with an A + T content of 82.5%. There are 37 typical animal mitochondrial genes, that is, 13 protein-coding, 2 rRNA and 22 tRNA gene and an A + T-rich region. The tRNA gene trnM was rearranged to the upstream of the trnI-trnQ-trnM cluster compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN start codon except for the gene cox1, which uses CGA as in other lepidopteran species. Ten protein-coding genes stop with termination codon TAA, whereas three protein-coding gene use incomplete stop codon T. The A + T-region is located between rrnS and trnM with a length of 332 bp and A + T content of 94.88%.

The complete mitochondrial genome of Taeniogonalos taihorina (Bischoff) (Hymenoptera: Trigonalyidae) reveals a novel gene rearrangement pattern in the Hymenoptera.

The family Trigonalyidae is considered to be one of the most basal lineages in the suborder Apocrita of Hymenoptera. Here, we determine the first complete mitochondrial genome of the Trigonalyidae, from the species Taeniogonalos taihorina (Bischoff, 1914). This mitochondrial genome is 15,927bp long, with a high A+T-content of 84.60%. It contains all of the 37 typical animal mitochondrial genes and an A+T-rich region. The orders and directions of all genes are different from those of previously reported hymenopteran mitochondrial genomes. Eight tRNA genes, three protein-coding genes and the A+T-rich region were rearranged, with the dominant gene rearrangement events being translocation and local inversion. The arrangements of three tRNA clusters, trnY-trnM-trnI-trnQ, trnW-trnL2-trnC, and trnH-trnA-trnR-trnN-trnS-trnE-trnF, and the position of the cox1 gene, are novel to the Hymenoptera, even the insects. Six long intergenic spacers are present in the genome. The secondary structures of the RNA genes are normal, except for trnS2, in which the D-stem pairing is absent.

The first complete mitogenome for the superfamily Cossoidea of Lepidoptera: The seabuckthorn carpenter moth Eogystia hippophaecolus.

We determined the first complete mitochondrial genome (mitogenome) for the superfamily Cossoidea of Lepidoptera from the seabuckthorn carpenter moth Eogystia hippophaecolus (GenBank accession No. KC831443). The length of this mitogenome is 15,431 bp with 37 typical animal mitochondrial genes and an A + T-rich region. The tRNA gene trnM was rearranged to the upstream of trnI-trnQ-trnM cluster compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN start codon except for the gene cox1, which uses CGA as in other lepidopteran species. Eight protein-coding genes stop with termination codon TAA. One protein-coding gene uses incomplete stop codon TA and four use T. The A + T region is located between rrnS and trnM with a length of 317 bp and A + T content of 92.74%.

The complete mitogenome of the turnip moth Agrotis segetum (Lepidoptera: Noctuidae).

Abstract The complete mitochondrial genome (mitogenome) of the turnip moth Agrotis segetum (Lepidoptera: Noctuidae) was determined (GenBank accession No. KC894725). This is the first sequenced mitogenome from the subfamily Noctuinae of Noctuidae. The length of this mitogenome is 15,378 bp with a A+T content of 80.7%. There are 37 typical animal mitochondrial genes and a A+T-rich region. The tRNA gene trnM was the only rearranged gene compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN start codon except for the gene cox1, which uses CGA as in other lepidopteran species. Ten protein-coding genes stop with termination codon TAA, whereas three protein-coding gene use incomplete stop codon T. The A+T-region is located between rrnS and trnM with a length of 332 bp and A+T content of 93.5%.

[Enhancement of GA3 and EDTA on Lolium perenne to remediate Pb contaminated soil and its detoxification mechanism].

A pot experiment was conducted to study the effects of plant growth regulator GA3 and metal chelate EDTA on enhancing the remediation of Pb contaminated soil, and the detoxification mechanism of Lolium perenne grown on Pb contaminated soil at 250 and 500 mg · kg(-1). The results showed that cell wall deposition and vacuolar compartmentalization played important roles in the detoxification of Pb in L. perenne shoot. The addition of EDTA alone increased Pb concentration in plants and Pb proportions in soluble fraction and organelles fraction, and enhanced the toxicity of Pb to plant, leading to the significant reduction of the plant biomass (P < 0.05). Foliar spray of lower concentration of GA3 (1 µmol · L(-1) or 10 µmol · L(-1)) alone significantly increased Pb accumulation by L. perenne (P < 0.05), but Pb proportions in soluble and organelles fraction were decreased, which alleviated the adverse effects of Pb on plant, thus improving the growth of plants (P < 0.05), with 1 µmol · L(-1) GA3 being the most effective. In contract, the addition of 100 µmol · L(-1) GA3 decreased Pb concentration in L. perenne, but increased the proportions of Pb in soluble fraction and organelles fraction, resulting in the reduction of plant biomass. Lower concen- tration of GA3 might alleviate the adverse effects of Pb and/or EDTA on plant, since the biomass amounts in the different treatments were in order of GA3 alone of lower concentration > GA3 of lower concentration + EDTA > EDTA alone. The combination application of low concentration of GA3 and EDTA showed a synergistic effect on the Pb accumulation in L. perenne (P < 0.05). Especially, Pb concentration in shoot and Pb extraction efficiency reached 1250.6 mg · kg(-1) and 1.1%, respec- tively, under the treatment of EDTA + 1 µmol L(-1) GA3 on the Pb 500 mg · kg(-1) soil. Therefore, the application of 1 µmol · L(-1) GA3 along with EDTA appeared to be a potential approach for phytoremediation of Pb contaminated soil.

Complete mitogenome of the Argyrogramma agnata (Lepidoptera: Noctuidae).

We determined the complete mitochondrial genome (mitogenome) of the silver looper moth Argyrogramma agnata (Lepidoptera: Noctuidae) (GenBank accession no. KC414791). The length of this mitogenome is 15,261 bp. All the 37 typical animal mitochondrial genes were found. The tRNA gene trnM was rearranged to the upstream of trnI-trnQ-trnM cluster compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN start codon except for the gene cox1, which uses CGA as in other lepidopteran species. Nine protein-coding genes stop with termination codon TAA. One protein-coding gene uses incomplete stop codon TA and three use T. The A+T region is located between rrnS and trnM with a length of 334 bp.

The complete mitochondrial genome of the yellow peach moth Dichocrocis punctiferalis (Lepidoptera: Pyralidae).

The complete mitochondrial genome of Dichocrocis punctiferalis (Lepidoptera: Pyralidae) was determined (GenBank accession number JX448619). The genome is 15,355 bp long with 37 typical animal mitochondrial genes and an A+T-rich region. As in other sequenced mitochondrial genomes of Lepidoptera, trnM was rearranged to the upstream of trnI-trnQ-trnM cluster compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN start codon except for the gene cox1, which uses CGA as in other lepidopteran species. Seven protein-coding genes stop with termination codon TAA. Four protein-coding genes use incomplete stop codons TA and two use T. The A+T-region is located between rrnS and trnM with a length of 338 bp. This is the first completely sequenced mitochondrial genome from the family Pyralidae.

The complete mitochondrial genome of the beet armyworm Spodoptera exigua (Hübner) (Lepodiptera: Noctuidae).

The complete mitochondrial genome of the beet armyworm Spodoptera exigua (Hübner) (Lepodiptera: Noctuidae) was determined (GenBank accession number JX316220). The genome is 15,365 bp long with 37 typical animal mitochondrial genes and an A+T-rich region. As in other sequenced mitochondrial genomes of Lepidoptera, trnM was rearranged to the upstream of trnI-trnQ-trnM cluster compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN start codon except for the gene cox1, which uses CGA as in other lepidopteran species. Eight protein-coding genes stop with termination codon TAA. Five protein-coding genes use incomplete stop codons TA or T. The A+T-rich region is located between rrnS and trnM with a length of 331 bp. This is the third completely sequenced mitochondrial genome from the family Noctuidae of Lepidoptera.

The complete mitochondrial genome of the summer fruit tortrix moth Adoxophyes orana (Lepidoptera: Tortricidae).

The complete mitochondrial genome of the summer fruit tortrix moth Adoxophyes orana (Lepdioptera: Tortricidae) was determined (GenBank accession No. JX872403). The genome is 15,343 bp long with 37 typical animal mitochondrial genes and an A+T-rich region. As in other sequenced mitochondrial genomes of Lepidoptera, trnM was rearranged to the upstream of trnI-trnQ-trnM cluster compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN start codon except for the gene cox1, which uses CGA start codon as in other lepidopteran species. Eight protein-coding genes stop with termination codon TAA. Five protein-coding genes use incomplete stop codons T. The A+T-region is located between rrnS and trnM with a length of 331 bp. This is the fifth completely sequenced mitochondrial genome from the family Tortricidae.

Effect of betulinic acid on the regulation of Hiwi and cyclin B1 in human gastric adenocarcinoma AGS cells.

AIM: To investigate the effect of betulinic acid (BA) on the proliferation, apoptosis and cell cycle of gastric adenocarcinoma cell AGS in vitro and the underlying mechanism. METHODS: The effect of BA on the proliferation of AGS cells was measured by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was analyzed by using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of BA on cell cycle of AGS cells was tested by PI staining. Both FCM and reverse transcription-PCR (RT-PCR) technologies were applied to detect the expression of Hiwi and Cyclin B1. RESULTS: BA exhibited significant cell proliferation inhibition, as well as its potency of inducing apoptosis in AGS cells in vitro in a time- and dose-dependent manner. The IC(50) value for 24 h was 18.25 microg/mL (95% confidence interval: 15.16 to 27.31 microg/mL). Cells treated with BA showed increased cell population in G(2)/M phase, with decreased S phase population. The expression of Hiwi and Cyclin B1 was down-regulated in BA-treated AGS cells in a dose-dependent manner. CONCLUSION: BA exerted potent effect on growth inhibition, G(2)/M cell cycle arrest and induction of apoptosis in AGS cells in vitro, possibly associated with the down-regulation of Hiwi and its downstream target Cyclin B1 expression. The potent antitumor capacity of BA suggested that it could be a promising new experimental anticancer agent in human gastric adenocarcinoma treatment.

[Effect of betulinic acid on inducing apoptosis of human multiple myeloma cell line RPMI-8226].

The aim of this study was to investigate the effect of betulinic acid on inducing apoptosis of human multiple myeloma RPMI-8226 cell line. The inhibitory effect of betulinic acid on proliferation and its inducing apoptosis effect, influence on cell cycle and induced morphological changes of RPMI-8226 were evaluated by MTT, flow cytometry Annexin-V/PI double staining, flow cytometry with PI staining and fluorescence microscopy with Hoechst33258 staining, respectively. The transcription level changes of bcl-xl gene and caspase 3 which are two kinds of apoptosis related protein gene were determined by RT-PCR. The results showed that within a certain range of concentration (0, 5, 10, 15, 20 microg/ml), IC50 of betulinic acid to RPMI-8226 at 24 hours was 10.156+/-0.659 microg/ml, while the IC50 at 48 hours was 5.434+/-0.212 microg/ml, and its inhibiting effect on proliferation of RPMI-8226 showed both time-and dose-dependent manners. Flow cytometry with Annexin-V/PI double staining revealed that apoptotic rate of RPMI-8226 cells increased as betulinic acid concentration increased. Flow cytometry with PI staining showed that the ratio of cells in G0/G1 phase increased, while it in S phase decreased, and ratio of cells at G2/M phase did not present a significant change. Morphological differences were typical and obvious between cells in treated and control groups under fluorescence microscope using Hoechst33258 staining. RT-PCR detection of caspase 3 gene indicated that its transcription level showed an increasing trend as the concentration of betulinic acid increased, while the bcl-xl showed the opposite trend. It is concluded that the betulinic acid can induce apoptosis of RPMI-8226 within a certain range of concentration in a time- and dose-dependent manners. This phenomenon may be related to the transcriptional level increase of caspase 3 gene and decrease of bcl-xl. Betulinic acid also affects G1/S in cell cycle which arrests cells at phase G0/G1.