Monoterpene indole alkaloids from the roots of Alstonia rupestris and their anti-inflammatory activity.

Four new monoterpene indole alkaloids (1-4) together with twelve known alkaloids (5-16) were isolated from the roots of Alstonia rupestris. Compound 1 was the first example of C2-symmetric heteroyohimbine-type indole alkaloid homodimer obtained from natural plant resource. Their structures were elucidated on the basis of spectroscopic data. The absolute configuration of 1 was determined by comparison of its calculated and experimental electronic circular dichroism (ECD) spectra. All compounds were evaluated for their anti-inflammatory activities by measuring their NO inhibitory effects in LPS-stimulated RAW 264.7 cells. Compound 2 showed strong NO inhibition with IC50 value of 4.2 ± 1.3 µM. Moreover, compound 2 could decrease the expressions of cyclooxygenase-2 (COX-2) and transforming growth factor beta-1 (TGF-ß1).

[QuEChERS-liquid chromatography-tandem mass spectrometry for determination of 22 triazole pesticide residues in Chinese herbal medicines].

Eight well-known herbals in Zhejiang Province, Zhebawei, are commonly used as traditional Chinese herbal medicines owing to their rich active ingredients. However, the unavoidable use of pesticides during agricultural production has led to pesticide residue problems in these herbs. In this study, a simple, rapid, and accurate method was established to determine 22 triazole pesticide residues in Zhebawei. An improved QuEChERS method was used for sample pretreatment, and Rhizoma Atractylodis Macrocephalae was used as a representative sample. The sample was extracted with acetonitrile to eliminate some polar and nonpolar compounds, pigments, and other impurities, and the purification effects of multiwalled carbon nanotubes (MWCNTs), amino-modified multiwalled carbon nanotubes (MWCNTs-NH2), carboxylated multiwalled carbon nanotubes (MWCNTs-COOH), crosslinked polyvinylpyrrolidone (PVPP), zirconium dioxide (ZrO2), 3-(N,N-diethylamino)-propyltrimethoxysilane (PSA), octadecyl (C18), and graphitized carbon black (GCB) were compared. MWCNTs-COOH and C18 were selected as the purification adsorbents, and their dosages were systematically optimized. The combination of 10 mg of MWCNTs-COOH and 20 mg of C18 was eventually selected as the purification adsorbents. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for analysis, and box graphs were plotted to present the dispersion of each group of recoveries, thus enabling the identification of the data outliers, dispersion distribution, and data symmetry. The established method was systematically verified and showed good linearity over the concentration range of 1-200 µg/L (except for bromuconazole, epoxiconazole, and etaconazole) with correlation coefficients >0.99. The average recoveries of the 22 pesticides at spiked levels of 10, 20, 100, and 200 µg/kg were in the range of 77.0%-115% with relative standard deviations (RSDs) <9.4%. The limits of detection and quantification were 1-2.5 µg/kg and 10-20 µg/kg, respectively. The applicability of the developed method to other herbals was investigated at 100 µg/kg, and the average recoveries of the target pesticides in different matrices ranged from 76.4% to 123% with RSDs <12.2%. Finally, the method established was used to detect triazole pesticide residues in 30 actual Zhebawei samples. The results showed that triazole pesticides were present in Bulbus Fritillariae Thunbergii and Dendranthema Morifolium. Difenoconazole was detected in Bulbus Fritillariae Thunbergii at contents ranging from 41.4 µg/kg to 110 µg/kg, while difenoconazole, myclobutanil, triadimenol and propiconazole were detected in Dendranthema Morifolium at contents ranging from 16.1 µg/kg to 250 µg/kg. The established method can meet the requirements for the accurate quantitative analysis of triazole fungicides in Zhebawei.

Targeting cancer stem cells by nutraceuticals for cancer therapy.

Accumulating evidence has demonstrated that cancer stem cells (CSCs) play an essential role in tumor progression and reoccurrence and drug resistance. Multiple signaling pathways have been revealed to be critically participated in CSC development and maintenance. Emerging evidence indicates that numerous chemopreventive compounds, also known as nutraceuticals, could eliminate CSCs in part via regulating several signaling pathways. Therefore, in this review, we will describe the some natural chemopreventive agents that target CSCs in a variety of human malignancies, including soy isoflavone, curcumin, resveratrol, tea polyphenols, sulforaphane, quercetin, indole-3-carbinol, 3,3'-diindolylmethane, withaferin A, apigenin, etc. Moreover, we discuss that eliminating CSCs by nutraceuticals might be a promising strategy for treating human cancer via overcoming drug resistance and reducing tumor reoccurrence.

Monoterpene indole alkaloids from the roots of Bousigonia mekongensis and their anti-diabetic nephropathy activity.

Four new monoterpene indole alkaloids (1-4) together with six known alkaloids (5-10) were isolated from the roots of Bousigonia mekongensis. Compounds 3 and 4 were the first examples of condylocarpan-adenine type alkaloids obtained from natural plant resource. Their structures were elucidated on the basis of spectroscopic data. All compounds were evaluated for their inhibiting glucose-induced mesanginal cell proliferation and protecting high glucose-evoked podocyte injury activities. (-)-demethoxycarbonyldihydrogambirtannine (5) can significantly antagonize glucose-induced podocyte injury with EC50 value of 6.5 ± 1.2 µM.

A six-herb Chinese medicine composition ointment as a promising candidate for treatment of hypertrophic scars.

Objective: To study the anti-hypertrophic scar effect of the six-herb Chinese medicine composition (SCMC) ointment on the rabbit ear hypertrophic scar models. Methods: The optimal formulation of SCMC ointment matrix was screened by the orthogonal designs and a series of evaluation tests. The SCMC ointment was prepared through emulsifying method. The rabbit ear hypertrophic scar models were established and used to investigate the anti-hypertrophic scar effect of SCMC ointment. Results: Our results demonstrated that all the quality control indications of the SCMC ointment met the requirements. Anti-hypertrophic scar activity results showed that all the rabbit ear scar tissues appeared different degrees of shrink and fading, and took an unobvious but palpable shift from hard to soft texture with the low, middle and high concentration SCMC ointments treatments in vivo. Additionally, on 21st day the scar area and thickness in different concentrations of SCMC ointment groups were significantly reduced than control group, in a concentration-dependent manner. The immunohistochemical results also indicated that the SCMC ointment had good anti-hypertrophic scar properties and could inhibit hypertrophic scar formation. Conclusion: The SCMC ointment could improve the blood circulation condition of hypertrophic scar tissues. Our research has demonstrated the Chinese medicine composition ointment with good anti-hypertrophic scar properties that could be used to treat hypertrophic scars. Meanwhile, it provides a theoretical basis for further clinical application.

Spontaneous resolution of idiopathic intestinal obstruction after pneumonia: A case report.

BACKGROUND: Duodenal obstruction is a common clinical scenario that can either be mechanical or a pseudo-obstruction. Clinical management of intestinal obstruction starts from localization and proceeds to histological examination of the stenotic intestine. Systemic factors and dysfunction of distant organs might contribute to the development of intestinal obstruction. Here, we report a unique case of idiopathic mechanical duodenal obstruction, which resolved spontaneously after 3 mo of conservative treatment, but was followed by intestinal pseudo-obstruction. CASE SUMMARY: An 84-year-old woman presented with worsened postprandial vomiting accompanied by prolonged pneumonia. Thorough noninvasive investigations revealed complete circumferential stenosis in the descending duodenum without known cause. Exploratory surgery was postponed due to septic shock and possible pulmonary fungal infection. Conservative treatment for 3 mo for ileus and control of pulmonary infection resolved the intestinal obstruction completely. Unfortunately, 2 wk later, she had regurgitation and postprandial vomiting again, complicated by deteriorating wheezing and dyspnea. Computed tomography revealed a dilated stomach and proximal duodenum without new intestinal stricture or pulmonary infiltration. The patient fully recovered after combined treatment with antireflux agents, enema, prokinetics, and bronchodilators. CONCLUSION: This complicated case highlights the inter-relationship of local and systemic contributions to ileus and gut dysfunction, which requires multidisciplinary treatment.

Calycosin induces apoptosis in osteosarcoma cell line via ERß­mediated PI3K/Akt signaling pathways.

Previous studies have shown that calycosin, a natural phytoestrogen which is structurally similar to estrogen, inhibits proliferation and induces apoptosis in estrogen­dependent cancer types via the estrogen receptor (ER)ß­induced inhibition of PI3K/Akt. Therefore, the aims of the present study were to investigate the effects of calycosin on human osteosarcoma (OS), and to examine the molecular mechanisms associated with ERß. Human OS MG­63 cells were treated with various concentrations of calycosin, and MTT and flow cytometry assays were used to assess the effects of calycosin on cellular proliferation and apoptosis. In addition, protein expression levels of ERß, phosphorylated (p)­PI3K, p­Akt, cleaved poly (ADP­ribose) polymerase 1 (PARP) and cleaved caspase­3 were evaluated by western blot analysis. The present results suggested that calycosin inhibited proliferation and induced apoptosis in MG­63 cells. Furthermore, increased ERß expression was detected in OS MG­63 cells treated with calycosin, and an ERß inhibitor (PHTPP) reversed calycosin­induced cytotoxicity and apoptosis. Moreover, phosphorylation levels of PI3K and Akt were significantly downregulated after calycosin treatment, whereas PHTPP reversed their phosphorylation. ERß­mediated PI3K/Akt downstream signaling pathways were found to influence the activity of poly (ADP­ribose) polymerase 1 and caspase­3. Thus, the present results indicated that calycosin inhibited proliferation and induced apoptosis in OS MG­63 cells, and that these effects were mediated by ERß­dependent inhibition of the PI3K/Akt pathways.

Chemical constituents of Kopsia officinalis and their antagonizing high glucose-evoked podocyte injury activity.

Four new indole alkaloids (1-4) and twenty known compounds (5-24) were isolated from the leaves and stems, and fruits of Kopsia officinalis. Their structures were confirmed by means of spectroscopic methods. All these isolates were evaluated for their antagonizing high glucose-evoked podocyte injury activity for the first time, and compounds 5-8 showed potent activity with EC50 values of 3.0-12.0 µM.

Four new phenolic constituents from the rhizomes of Gastrodia elata Blume.

Four new gastrodin derivatives containing a trans-cinnamoyl unit (1-4) and nine known compounds (5-13) were isolated from the rhizomes of Gastrodia elata Blume. All these compounds were evaluated for their neuroprotective effects against 6-hydroxydopamine-induced cell death, and compounds 7 and 12 showed potent activities with EC50 values of 10.5 and 10.2 µM, respectively.

Three novel indole alkaloids from Kopsia officinalis.

Three new indole alkaloids, named kopsioffines A-C (1-3), possessing relatively novel ten-membered lactam ring, and one known compound (11,12-demethoxy-16-deoxypauciflorine, 4) were isolated from the leaves and stems of Kopsia officinalis. Their structures were elucidated by means of spectroscopic methods. The absolute configuration of 1 was determined by calculated electronic circular dichroism data. The hypothetical biosynthetic pathway of 1 was postulated. All these isolates were evaluated for their inhibitory effects on α-glucosidase. None of them showed activity with IC50 far beyond 50µM.

Melaxillines A and B, monoterpenoid indole alkaloids from Melodinus axillaris.

Two new monoterpenoid indole alkaloids, melaxillines A (1) and B (2), together with four known alkaloids (3-6), were isolated from the roots of Melodinus axillaris. The structures were determined by extensive spectroscopic methods, including 1D and 2D-NMR spectroscopy, high resolution mass spectrometric data, and by comparison to literature data. New compounds 1-2 might be derived from a common precursor (3) by proposing their biogenetic route, and compound 1 represents the first Melodinus alkaloid featuring an ethyl acetate group skeleton at C-18. The anti-inflammatory effects of all the isolated compounds were evaluated by measuring the inhibitory ratios of ß-glucuronidase release in rat polymorphonuclear leukocytes (PMNs) induced by platelet-activating factor. Compounds 1-4 showed significant anti-inflammatory activities with IC50 values ranging from 1.51 to 21.56µM, respectively.

Pectin enhances rice (Oryza sativa) root phosphorus remobilization.

Plants growing in phosphorus (P)-deficient conditions can either increase their exploration of the environment (hence increasing P uptake) or can solubilize and reutilize P from established tissue sources. However, it is currently unclear if P stored in root cell wall can be reutilized. The present study shows that culture of the rice cultivars 'Nipponbare' (Nip) and 'Kasalath' (Kas) in P-deficient conditions results in progressive reductions in root soluble inorganic phosphate (Pi). However, Nip consistently maintains a higher level of soluble Pi and lower relative cell wall P content than does Kas, indicating that more cell wall P is released in Nip than in Kas. P-deficient Nip has a greater pectin and hemicellulose 1 (HC1) content than does P-deficient Kas, consistent with the significant positive relationship between pectin and root-soluble Pi levels amongst multiple rice cultivars. These observations suggest that increased soluble Pi might result from increased pectin content during P starvation. In vitro experiments showed that pectin releases Pi from insoluble FePO4. Furthermore, an Arabidopsis thaliana mutant with reduced pectin levels (qua1-2), has less root soluble Pi and is more sensitive to P deficiency than the wild type (WT) Col-0, whereas NaCl-treated WT plants exhibit both an increased root pectin content and an elevated soluble Pi content during P-starvation. These observations indicate that pectin can facilitate the remobilization of P deposited in the cell wall. This is a previously unknown mechanism for the reutilization of P in P-starved plants.

Neuroprotective effect of schizandrin A on oxygen and glucose deprivation/reperfusion-induced cell injury in primary culture of rat cortical neurons

Brain ischemia appears to be associated with innate immunity. Recent reports showed that C3a and C5a, as potent targets, might protect against ischemia induced cell death. In traditional Chinese medicine, the fruit of Schizandra chinesis Baill (Fructus schizandrae) has been widely used as a tonic. In the present study, we sought to evaluate the neuroprotective effects of schizandrin A, a composition of S. chinesis Baill, against oxygen and glucose deprivation followed by reperfusion (OGD/R)-induced cell death in primary culture of rat cortical neurons, and to test whether C3a and C5a affected cortical neuron recovery from ischemic injury after schizandrin A treatment. The results showed that schizandrin A significantly reduced cell apoptosis and necrosis, increased cell survival, and decreased intracellular calcium concentration ([Ca2+]i) and lactate dehydrogenase (LDH) release in primary culture of rat cortical neurons after OGD/R. Mechanism studies suggested that the modulation of extracellular-regulated kinase (ERK), c-Jun NH2-terminal kinases (JNK), and p38, as well as caspase-3 activity played an important role on the progress of neuronal apoptosis. C5aR participated in the neuroprotective effect of schizandrin A in primary culture of rat cortical neurons after OGD/R. Our findings suggested that schizandrin A might act as a candidate therapeutic target drug used for brain ischemia and related diseases

Neuroprotective effect of schizandrin A on oxygen and glucose deprivation/reperfusion-induced cell injury in primary culture of rat cortical neurons.

Brain ischemia appears to be associated with innate immunity. Recent reports showed that C3a and C5a, as potent targets, might protect against ischemia induced cell death. In traditional Chinese medicine, the fruit of Schizandra chinesis Baill (Fructus schizandrae) has been widely used as a tonic. In the present study, we sought to evaluate the neuroprotective effects of schizandrin A, a composition of S. chinesis Baill, against oxygen and glucose deprivation followed by reperfusion (OGD/R)-induced cell death in primary culture of rat cortical neurons, and to test whether C3a and C5a affected cortical neuron recovery from ischemic injury after schizandrin A treatment. The results showed that schizandrin A significantly reduced cell apoptosis and necrosis, increased cell survival, and decreased intracellular calcium concentration ([Ca(2+)]i) and lactate dehydrogenase (LDH) release in primary culture of rat cortical neurons after OGD/R. Mechanism studies suggested that the modulation of extracellular-regulated kinase (ERK), c-Jun NH2-terminal kinases (JNK), and p38, as well as caspase-3 activity played an important role on the progress of neuronal apoptosis. C5aR participated in the neuroprotective effect of schizandrin A in primary culture of rat cortical neurons after OGD/R. Our findings suggested that schizandrin A might act as a candidate therapeutic target drug used for brain ischemia and related diseases.

Hyperbaric oxygen intervention on expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in spinal cord injury models in rats.

BACKGROUND: Hyperbaric oxygen (HBO) intervention is a main therapeutic method and the curative effect has been certified for spinal cord injury (SCI), but the mechanisms of the neuroprotective effect of HBO on SCI remain elusive. This study aimed to observe the change in expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) after SCI at different time points and to investigate the neuroprotective mechanism of HBO on SCI in rats. METHODS: A total of 160 adult Sprague-Dawley rats, weighing between 250 and 300 g, were randomly assigned to four experimental groups (n = 40 per group). SCI group: SCI was created with a special NYU impactor of Allen's by a 25 gramcentimeter impacting energy on T10 of the spinal cord. SCI+HBO group: HBO therapy after SCI model was established. Sham operation (SH) group: only laminectomy of T10 and no impact on the spinal cord was done. SH+HBO group: HBO therapy after sham operation. The hindlimb functional recovery was evaluated using Basso, Beattie, and Bresnahan (BBB) score and the expressions of HIF-1α and VEGF were observed with fluorescent quantitation PCR and Western blotting method of six rats picked randomly from each group at different time points of 1, 3, 7, and 14 days after operation. RESULTS: Rats in the SCI group and SCI+HBO group were paralyzed completely after operation with BBB 0-1 score. Rats in the SH group and SH+HBO group could walk after sham operation with BBB 20-21 score. The BBB score of rats in the SCI+HBO group (4.67±1.97 and 10.83±2.23) was higher than that in the SCI group (1.83±0.75 and 6.67±2.16) at 7 and 14 days time points obviously (P < 0.05). The expressions of HIF-1a and VEGF in the SCI group and SCI+HBO group were higher than in the SH group and SH+HBO group at any time point obviously (P < 0.05), while the SCI+HBO group presented the least expression of HIF-1α mRNA and protein (3.82±0.41 and 0.59±0.06; 2.26±0.41 and 0.37±0.05; 1.58±0.26 and 0.29±0.05) than that in the SCI group (6.36±0.58 and 0.76±0.07; 3.55±0.47 and 0.51±0.07; 2.27±0.39 and 0.40±0.06) respectively at 3, 7, and 14 days time points (P < 0.05) with significant difference and more expression of VEGF mRNA and protein (5.83±0.77 and 0.72±0.06; 4.59±0.51 and 0.63±0.06) than that in the SCI group (3.06±0.30 and 0.48±0.07; 2.25±0.24 and 0.39±0.09) respectively at 7 and 14 days time points (P < 0.05) with significant difference. CONCLUSIONS: HBO could improve the hind limb functional recovery after SCI in rats. The elevation and duration of the expression of VEGF and the reduction of expression of HIF-1α by HBO intervention may be inversely related in the repair of damaged spinal cord and neuroprotective effect.

α-Glucosidase inhibitory triterpenoids from the stem barks of Uncaria laevigata.

A phytochemical study on the ethanolic extract of the stem barks of Uncaria laevigata leads to the isolation and characterization of 18 triterpenoid compounds. Ten of these are new, including one novel heptanortriterpenoid (1), four ursane triterpenes (2-4, 10), and five oleanane triterpenes (5-9). Their structures were established by spectroscopic methods, especially by 2D-NMR and MS analyses. All these isolates were evaluated for their inhibitory effects on α-glucosidase: ursolic acid and 3 showed potent activities with IC50 values of 16 ± 2.2 and 49 ± 3.7 µM, respectively.

Developmental changes in the composition of five anthraquinones from Rheum palmatum as quantified by (1) H-NMR.

INTRODUCTION: Rheum palmatum is an important traditional Chinese medicine featuring anthraquinones with several activities. Generally, rhein, emodin, aloe-emodin, physcion and chrysophanol are used as chemical markers for the quality control of rhubarb products. OBJECTIVE: To develop a simple protocol for the quantification of rhein, emodin, aloe-emodin, physcion and chrysophanol in R. palmatum collected at different developmental stages. METHODS: (1) H-NMR spectra were measured on samples dissolved in acetone-d6 , quantification was carried out using the signals of H-4 of rhein (δH 8.36), H-7 of emodin (δH 6.68), CH2 OH of aloe-emodin (δH 4.81), OCH3 of physcion (δH 4.02) and CH3 of chrysophanol (δH 2.50), which were well separated from other signals. Quantitative analysis was based on the relative ratio of the intensity of each compound to the known amount of internal standard maleic acid. RESULTS: The quantitative (1) H-NMR (qHNMR) method developed showed good precision, trueness, linearity, repeatability and stability for the quantification of rhein, emodin, aloe-emodin, physcion and chrysophanol. This method was applied successfully to explore the seasonal variations of the five major anthraquinones in R. palmatum, and provided quantitative results in reasonable agreement with those obtained by the HPLC-UV method. CONCLUSION: Compared with the conventional HPLC-based methods, the qHNMR analysis is rapid, reference-free and convenient with less sample pre-treatment. This technique should be a feasible choice for the quality control of R. palmatum.

Protective effects of Salvia miltiorrhizae on the hearts of rats with severe acute pancreatits or obstructive jaundice.

OBJECTIVE: To investigate the therapeutic effects and mechanisms of Salvia miltiorrhizae (Danshen) in the treatment of severe acute pancreatitis (SAP)- or obstructive jaundice (OJ)-induced heart injury. METHODS: A total of 288 rats were used for SAP- (n=108) and OJ-associated (n=180) experiments. The rats were randomly divided into sham-operated, model control, and Salvia miltiorrhizae-treated groups. According to the difference of time points after operation, SAP rats in each group were subdivided into 3, 6 and 12 h subgroups (n=12), whereas OJ rats were subdivided into 7, 14, 21, and 28 d subgroups (n=15). At the corresponding time points after operation, the mortality rates of the rats, the contents of endotoxin and phospholipase A2 (PLA2) in blood, and pathological changes of the hearts were investigated. RESULTS: The numbers of dead SAP and OJ rats in the treated groups declined as compared with those in the model control group, but not significantly (P>0.05). The contents of endotoxin (at 6 and 12 h in SAP rats and on 7, 14, 21, and 28 d in OJ rats, respectively) and PLA2 (at 6 and 12 h in SAP rats and on 28 d in OJ rats, respectively) in the treated group were significantly lower than those in the model control group (P<0.01 and P<0.001, respectively). Besides, myocardial pathological injuries were mitigated in SAP and OJ rats. CONCLUSION: In this study, we found that Salvia miltiorrhizae improved myocardial pathological changes, reduced the content of PLA2 in blood, and decreased the mortality rates of SAP and OJ rats, exerting protective effects on the hearts of the rats.

[Simultaneous determination of matrine, oxysophocarpin and oxymatrine in rat plasma by HPLC-MS and its application in the pharmacokinetic study].

To establish an HPLC-MS method for simultaneous determination of matrine, oxymatrine and oxysophocarpine in rat plasma after oral administration of herbal preparation, namely Sanwu Huangqin decoction, and the pharmacokinetic porameters were calculated as well. Matrine, oxymatrine, oxysophocarpine, and internal standard pseudoephedrine were extracted from plasma with liquid-liquid extraction, then separated on a Kromasil C18 column by using acetonitrile-0.1% aqueous formic acid (10 : 90) as mobile phase. Electrospray ionization (ESI) source was applied and operated in positive ion mode. The linear calibration curve was obtained in the concentration range of 10 -5 000 ng x mL(-1) for matrine, 2 - 1 000 ng x mL(-1) for oxymatrine, and 2 - 1 000 ng x mL(-1) for oxysophocarpine. The extraction recovery was 89.1% - 93.5%, 83.9% - 91.3%, and 85.4% - 88.0% accordingly. The inter- and intra- day precision (RSD) was below 15.0% calculated from quality control (QC) samples. Matrine, oxymatrine and oxysophocarpine concentration time profile conformed to a two-compartment pharmacokinetic model. The method was shown to be effective, convenient, and suitable for simultaneous pharmacokinetic study of matrine, oxymatrine, and oxysophocarpine in rat.

A new fertility restorer locus linked closely to the Rfo locus for cytoplasmic male sterility in radish.

In this study, we have investigated a new fertility restorer (Rf) locus for cytoplasmic male sterility (CMS) in radish. We have obtained a CMS-Rf system consisting of sterile line '9802A1', maintainer line '9802B1' and restorer line '9802H'. F(1) plants from cross between sterile line '9802A1' and restorer line '9802H' were all male fertile, self pollination of F(1) plants produced an F(2) segregating population consisting of 600 individuals. The segregating population was found to fit a segregation ratio 3:1 for male fertile and sterile types, indicating that male fertility is restored by a single dominant gene (termed Rfo2) in the CMS-Rf system. Based on the DNA sequence of Rfo/Rfk1 (AJ535623), just one full length gene in the sterile line '9802A1', in the restorer line '9802H' and in the male fertile line '2006H', was cloned, respectively. The three sequences correspond to the same gene with two alleles: Rfob in '9802H' and rfob in '9802A1' and '2006H'. These two alleles differ from Rfo/Rfk1 and rfk1 (AJ535624) alleles by two synonymous base substitutions, respectively. Based on the differences between the Rfob and rfob genes, one PCR-based marker was developed, and designated Marker 1, which is identical to the corresponding region of Rfob by sequence analysis. In the F(2) segregating population described above, the Marker 1 was present in 5 sterile plants and in 453 fertile plants, absent in 4 fertile plants and in 138 sterile plants, and was found to fit a segregation ratio 3:1 indicating that Rfob was single copy in '9802H'. Linkage analysis showed that the Rfo2 locus for our CMS-Rf system was distant from the Rfo locus by about 1.6 cM. The sterile line '9802A1' was pollinated by the male fertile line '2006H' and the resulting F(1) plants were all male fertile. These results indicated that the male fertility of radish CMS can be restored by a new Rf locus, which linked tightly to the Rfo locus.