Novel azulene-based derivatives as potent multi-receptor tyrosine kinase inhibitors.

A series of azulene-based derivatives were synthesized as potent inhibitors for receptor tyrosine kinases such as FMS-like tyrosine kinase 3 (FLT-3). Systematic side chain modification of prototype 1a was carried out through SAR studies. Analogue 22 was identified from this series and found to be one of the most potent FLT-3 inhibitors, with good pharmaceutical properties, superior efficacy, and tolerability in a tumor xenograft model.

Publisher Correction: Non-Toxic Gold Nanoclusters for Solution-Processed White Light-Emitting Diodes.

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[An experimental study on percutaneous autologous bone marrow mesenchymal stem cell grafting for bone defect].

OBJECTIVES: To investigate in vive osteogenic potential in size-critical bone defect after percutaneous autologous grafting of culture-expanded rabbit autologous BMSCs, osteo-induced BMSCs and combination of both. METHODS: BMSCs were cultured and then induced with osteogenic supplement (OS) medium. BMSCs with and without OS induction were collected and percutaneously autologously injected respectively into the 15 mm bone defect of 20 experimental rabbit model. The grafts were BMSCs, osteo-induced BMSCs, BMSCs and osteo-induced BMSCs, BMP combined with fibrin sealant, and 0.9% NaCl solution. Osteogenesis at the defect areas were observed by regular radiography, histology and biomechanics. RESULTS: The group transplanted with BMSCs + osteo-induced BMSCs achieved complete bone healing with medullary cavity united, which showed the largest quantity of new bone measured by X-ray analysis, and also their maximal load were better than those in other groups. CONCLUSION: The bone-forming ability of rabbit osteo-induced BMSCs combined with BMSCs in bone defect is superior to those of BMSCs and osteo-induced BMSCs.

Non-Toxic Gold Nanoclusters for Solution-Processed White Light-Emitting Diodes.

Solution-processed optoelectronic devices are attractive because of the potential low-cost fabrication and the compatibility with flexible substrate. However, the utilization of toxic elements such as lead and cadmium in current optoelectronic devices on the basis of colloidal quantum dots raises environmental concerns. Here we demonstrate that white-light-emitting diodes can be achieved by utilizing non-toxic and environment-friendly gold nanoclusters. Yellow-light-emitting gold nanoclusters were synthesized and capped with trioctylphosphine. These gold nanoclusters were then blended with the blue-light-emitting organic host materials to form the emissive layer. A current efficiency of 0.13 cd/A was achieved. The Commission Internationale de l'Eclairage chromaticity coordinates of (0.27, 0.33) were obtained from our experimental analysis, which is quite close to the ideal pure white emission coordinates (0.33, 0.33). Potential applications include innovative lighting devices and monitor backlight.

Alterations of scapular kinematics and associated muscle activation specific to symptomatic dyskinesis type after conscious control.

BACKGROUND: Scapular orientation and movements can affect the function of the shoulder. However, evidence is limited on whether symptomatic subjects can actively maintain the scapula in a neutral position through conscious control. OBJECTIVE: To investigate whether symptomatic subjects with scapular dyskinesis can achieve optimal scapular movements and associated muscle activities through conscious control. DESIGN: A cross-sectional study. METHODS: Sixty subjects with scapular dyskinesis (16 inferior angle pattern I, 16 medial border pattern II, and 28 mixed pattern) performed 3 selected exercises (arm elevation, side-lying elevation, and side-lying external rotation) with and without conscious control. Three-dimensional electromagnetic motion and electromyography were used to record the scapular kinematics and muscle activation during the exercises. RESULTS: For scapular kinematics, significant increases in scapular external rotation (4.6 ± 3.2°, p < 0.0125) were found with conscious control during arm elevation and side-lying elevation in three groups. Significant increases in activation of the middle and lower trapezius (MT: 4.9 ± 2.4% MVIC; LT: 10.2 ± 6.8% MVIC, p < 0.0 25) were found with conscious control in 3 exercises among the 3 dyskinesis groups. Increased serratus anterior activation (SA: 11.2 ± 4.8% MVIC, p < 0.0 25) was found in the concentric phase of side-lying external rotation in the pattern I and I + II groups. CONCLUSION: Conscious control of the scapula can alter scapular orientation and MT, LT, and SA activation during 3 selected exercises in subjects with symptomatic dyskinesis. Specifically, conscious control during side-lying external rotation can be applied to increase SA activity in pattern I and I + II dyskinesis.

[Case-control study on bone-setting manipulation for the treatment of isolated systolic hypertension combined with cervical spondylosis].

OBJECTIVE: To investigate clinical effect and safety of bone-setting manipulation in treating isolated systolic hypertension combined with cervical spondylosis. METHODS: From January 2012 to January 2015, 320 patients suffered from isolated systolic hypertension combined with cervical spondylosis were randomly divided into treatment group and control group. In treatment group, there were 160 patients including 84 males and 76 females with an average age of (39.82 ± 10.33) years old, average blood pressure was (149.61 ± 10.75)/(81.01± 8.25) mmHg, NPQ score was 24.61 ± 8.14; treated with flexion top spin and lock bone-setting manipulation of cervical spine, once every two days for 20 days. While in control group, there were 160 patients including 90 males and 70 females with an average age of(41.37 ± 9.42) years old, average blood pressure was (151.48 ± 11.32)/ (79.65 ± 9.32) mmHg, NPQ score was 25.78 ± 9.53; treated with manipulation of reposition cervical spine by rotation, once every two days for 20 days. Blood pressure and NPQ score were tested and compared for evaluating clinical effects. RESULTS: Before and after a period treatment, systolic pressure in treatment group was (149.61 ± 10.75) mmHg and (129.67 ± 12.26) mmHg; (151.48 ± 11.32) mmHg and (132.02 ± 11.73) mmHg in control group. After treatment, systolic pressure in both two groups was obviously decreased, and treatment group was better than control group. Before and after a period treatment, diastolic pressure in treatment group was (80.01 ± 8.25) mmHg and (78.15 ± 10.34) mmHg, (79.65 ± 9.32) mmHg and (76.89 ± 9.79) mmHg in control group, and there was no significant difference between two groups. NPQ score in treatment group was 24.61 ± 8.14 before treatment, 12.46 ± 7.94 after treatment, while in control group was 25.78 ± 9.53, 14.17 ± 8.86; NPQ score of the two groups after treatment was better than before treatment, while there was no obviously significance between two groups after treatment. The whole clinical effect in treatment group was better than control group. CONCLUSION: Bone-setting manipulation for isolated systolic hypertension combined with cervical spondylosis at early stage could receive good clinical result, and flexion top spin and lock bone-setting manipulation of cervical spine was better and safety than manipulation of reposition cervical spine by rotation.

[Extracellular splitting pattern of mitochondria and the depressant effects of CsA on the process].

OBJECTIVE: To investigate extracellular splitting pattern of mitochondria and the depressant effects of CsA on the process and explore the mechanism of post-traumatic SIRS and its therapeutic strategy. METHODS: Ten male SD rats with 60 to 70 days age and 240 to 280 g weight were used for mitochondrial isolation. Freshly isolated mitochondria were randomly divided into two groups, which were cultured in blood plasma with or without CsA respectively for 8 h. COX and MDH were assayed by ELISA every 30 min. Meanwhile, Rat macrophage cell line NR8383 were treated as follows, control (group A): cultivation with normal medium; NR8383+CsA co-culture group (group B): culture medium was supplemented with CsA of 10 mmol/L; NR8383+intact mitochondria co-culture group (group C): culture medium was supplemented with intact mitochondria (mtDNA=5 g/ml); NR8383+intact mitochondria+CsA co-culture group (group D): culture medium was supplemented with intact mitochondria (mtDNA=5 µg/ml)and CsA of 10 mmol/L; NR8383+disrupted mitochondria co-culture group (group E): culture medium was supplemented with disrupted mitochondria (mtDNA=5 µg/ml); NR8383+disrupted mitochondria+CsA co-culture group (group F): culture medium was supplemented with disrupted mitochondria (mtDNA=5 µg/ml)and CsA of 10 mmol/L. TNF-α and IL-6 concentrations in supernatant were assessed at 1, 3, 5 h after culture. RESULTS: In the mitochondria plasma cultures, MDH and COX levels were increased with the time and peaked at about 3 h and 3.5 h; CsA can delay the appearance of peak to 4.5 h. Among different treated groups,there was no significant difference in TNF-α and IL-6 between group A and group B; there was significant difference in TNF-α and IL-6 other groups. After 1 h culture, compared with group C, no significant difference of TNF-α and IL-6 was observed in group D, while TNF-α and IL-6 were significant higher in group E; after 3 h culture, compared with group C, TNF-α and IL-6 were significantly lower in group D, while TNF-α and IL-6 were significantly higher in group E; after 5 h culture, compared with group C, TNF-α and IL-6 were significantly lower in group D, while no significant difference of TNF-α and IL-6 were observed in group E. At each time point, there was no significant difference in TNF-α and IL-6 between group F and group E. CONCLUSION: Mitochondria can split in serum with time, which will further activate macrophages. CsA has depressant effect to mitochondrial splitting on the process and will therefore inhibit the activation of macrophages.

Differential Expression and Signal Transduction Analysis of MicroRNA Related to Kidney Yang Deficiency in Kidney Tissues of Rats / 中国实验方剂学杂志

Objective: Microarray chip was used to detect the differentially expressed microRNA (miRNA) in kidney tissues of rats with kidney-Yang deficiency induced by adenine,and its significance was analyzed by bioinformatics method. Method: Rats with kidney-Yang deficiency were induced by intragastric administration of 150 mg·kg-1 adenine in model group, while rats in normal group were given the same amount of saline.Kidney tissues were taken for hematoxylin-eosin (HE) staining pathological sections after anesthesia and blood urea nitrogen (BUN), creatinine (SCr) in blood and 24-hour urinary protein (24U-TP) in urine were measured. μParaflo microfluidic chip technology was used to investigate differential expression miRNA in kidney tissues, and microarray results were verified by Real-time PCR. Bioinformatics database was used to analyze the target genes and functions of differential expression miRNAs. Result: Gene chip results showed that there were 50 differentially expressed microRNAs after modeling. Compared with control group, only 9 miRNAs were highly expressed in kidney tissues with significant difference were detected in model group. Compared with the normal group, the expression of rno-miR-21-5p, rno-let-7i-5p, rno-miR-146b-5p and rno-miR-15b-5p in model group increased significantly(PPPPPConclusion: This experiment found 4 miRNAs involved in the regulation of renal interstitial fibrosis (RIF) and 2miRNAs with unknown functions, which provided a new clue for further analysis of the regulatory network of kidney-yang deficiency.

Effect of Zhenwu Tang on regulating of "AVP-V2R-AQP2" pathway in NRK-52E cells / 中国中药杂志

This study was aimed to investigate the effect and mechanism of Zhenwu Tang on AVP-V2R-AQP2 pathway in NRK-52E cells . Forty eight male SD rats were randomly divided into eight groups with 6 animals in each group. Distilled water or 22.68 g·kg⁻¹·d⁻¹ Zhenwu Tang(calculated by raw drug dosage meter) was given by gavage. Blood samples were collected by cardiac puncture， and the medicated serum was centrifuged from the blood by 3 000 r·min⁻¹. NRK-52E cells were treated with different medicated serum or dDAVP. The condition of cell proliferation was detected by RTCA. The distribution of V2R and AQP2 in cells were detected by immunofluorescence. The expression of V2R， PKA and AQP2 were detected by Western blot and AQP2 mRNA level was detected by real-time PCR. Results showed that the level of AQP2 mRNA(<0.01) and protein expression of V2R， PKA and AQP2(<0.05， <0.01， <0.05) of Z7d group which was treated with Zhenwu Tang medicated serum for 24 h were significantly higher than that of normal rat serum group. And the expression level of V2R， p-AQP2 and AQP2(<0.01， <0.05， <0.01) of Z7d+dDAVP group were significantly increased comparing to normal rat serum group. The results indicate that the applying of Zhenwu Tang medicated serum could increase the expression level of V2R， PKA and AQP2 which exist in AVP-V2R-AQP2 pathway in NRK-52E， and there is synergistic effect between Zhenwu Tang medicated serum and dDAVP. So the pathway of AVP-V2R-AQP2 may be one of the mechanism for which Zhenwu Tang regulate balance of water transportation.

Protective effect of β-asarone on AD rat model induced by intracerebroventricular injection of Aβ₁₋₄₂ combined 2-VO and its mechanism / 中国中药杂志

This study was aimed to investigate the protective effect and mechanism of β-asarone on the animal model of Alzheimer's disease(AD) which was induced by intracerebroventricular injection of Aβ₁₋₄₂ combined cerebral ischemia. One hundred and five rats were randomly divided into seven groups including sham-operated group, AD model group, β-asarone 10 mg•kg⁻¹ group, β-asarone 20 mg•kg⁻¹ group, β-asarone 30 mg•kg⁻¹ group, donepezil group(0.75 mg•kg⁻¹) and Ginkgo biloba extract group(24 mg•kg⁻¹). Rats' learning and memory abilities, cerebric regional blood flow, pathological changes in hippocampal CA1 region, the expression level of HIF-1α and serum CAT, SOD and MDA level were detected 4 weeks later. The results showed that the application of intracerebroventricular injection of Aβ₁₋₄₂ joint 2-VO could lead to rats' dysfunction of learning and memory, decrease in regional cerebral blood flow. Neurons in CA1 region were arranged in disorder, and amyloid deposition was increased. The number of cerebral cortical cells expressing HIF-1α was increased as well. The level of serum CAT and SOD decreased, while level of serum MDA increased. However these symptoms were improved by 20 mg•kg⁻¹ and 30 mg•kg⁻¹ β-asarone. The results indicated that β-asarone could effectively relieve the symptoms of the AD model induced by intracerebroventricular injection of Aβ₁₋₄₂ combined cerebral ischemia, and the potential mechanism might be that it could attenuate damage of MDA to the body by improving the level of CAT and SOD, meanwhile the level of HIF-1α decreased as the decline of hyperoxide which might attenuate its damage to neuron, so it finally achieved alleviating Alzheimer's disease.

Protective effect of β-asarone on PC12 cells injury induced by Aβ₁₋₄₂ astrocytic activation / 中国中药杂志

This study was aimed to investigate the protective effect and mechanism of β-asarone on PC12 cells injury induced byAβ₁₋₄₂ activated astrocytes, and provide experimental basis for β-asarone application in the prevention and control of Alzheimer's disease (AD). Firstly, RA-h and PC12 cells were co-cultured in the special transwell chamber, and the Real time cell analysis (RTCA) system was used to real-time observe its effect on PC12 cells survival rate in the co-culture system after astrocytes injury induced by Aβ₁₋₄₂. The best intervention time of β-asarone was selected according to the survival curve and parameters generated automatically. β-asarone with different concentrations was used for intervention on astrocytes, then the changes of PC12 cells survival rate in the co-culture system were observed. Secondly, MTT assay was used to detect the effect of Aβ₁₋₄₂ on PC12 cells survival rate as well as the intervention effect of β-asarone, and verify the testing results of RTCA. The levels of IL-1β, TNF-α and BDNF in culture media of the lower chamber were detected by ELISA. The NF-κB activity and phosphorylation levels of ERK, p38 and JNK were detected by Western blot. Results showed that β-asarone (55.5 mg•L⁻¹) could significantly slowdown the decline of PC12 cells survival rate caused by Aβ₁₋₄₂-induced RA-h activation (P<0.01), significantly reduce the levels of IL-1β, TNF-α and the phosphorylation levels of ERK, p38 and JNK in culture media of the lower chamber (P<0.01). β-asarone(166.7 mg•L⁻¹) could promote the release of BDNF in culture media of the lower chamber(P<0.05). These results indicated that Aβ₁₋₄₂ could induce RA-h activation and its release of IL-1β, TNF-α and other inflammatory factors to aggravate the PC12 cells injury; β-asarone could reduce the levels of IL-1β, TNF-α, promote the release of BDNF, and inhibit the NF-κB activity as well as phosphorylation levels of ERK, p38 and JNK protein in PC12 cells.

Ischemic stroke in Williams-Beuren syndrome: a case report.

A 15-year-old girl was admitted because of an acute onset of facial palsy and right hemiparesis. The patient had a history of moderate mental retardation and developmental delay. On admission, her vital signs were stable, except for high blood pressure. Magnetic resonance imaging demonstrated an infarct involving the left internal capsule and putamen. Because of the patient's young age, an extensive stroke survey was performed. Williams-Beuren syndrome was finally confirmed by fluorescent in situ hybridization. Compared with the previously reported cases, no evidence of cerebral arterial stenosis or cardiac abnormalities was found by noninvasive imaging techniques. Because Williams-Beuren syndrome is a complex, multiple congenital anomaly syndrome with prominent cardiovascular features, regular assessment and antihypertensive treatment are necessary to minimize the lifelong cardiovascular risk in patients with this syndrome.

Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibits LPS-induced apoptosis in hippocampus-derived neural stem cell.

Fluoxetine is a widely used antidepressant compound which inhibits the reuptake of serotonin in the central nervous system. Recent studies have shown that fluoxetine can promote neurogenesis and improve the survival rate of neurons. However, whether fluoxetine modulates the proliferation or neuroprotection effects of neural stem cells (NSCs) needs to be elucidated. In this study, we demonstrated that 20 microM fluoxetine can increase the cell proliferation of NSCs derived from the hippocampus of adult rats by MTT test. The up-regulated expression of Bcl-2, Bcl-xL and the cellular FLICE-inhibitory protein (c-FLIP) in fluoxetine-treated NSCs was detected by real-time RT-PCR. Our results further showed that fluoxetine protects the lipopolysaccharide-induced apoptosis in NSCs, in part, by activating the expression of c-FLIP. Moreover, c-FLIP induction by fluoxetine requires the activation of the c-FLIP promoter region spanning nucleotides -414 to -133, including CREB and SP1 sites. This effect appeared to involve the phosphatidylinositol-3-kinase-dependent pathway. Furthermore, fluoxetine treatment significantly inhibited the induction of proinflammatory factor IL-1beta, IL-6, and TNF-alpha in the culture medium of LPS-treated NSCs (p<0.01). The results of high performance liquid chromatography coupled to electrochemical detection further confirmed that fluoxentine increased the functional production of serotonin in NSCs. Together, these data demonstrate the specific activation of c-FLIP by fluoxetine and indicate the novel role of fluoxetine for neuroprotection in the treatment of depression.

Identification of the active material of anti-hepatic fibrosis from Amydae Carapax / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To identify the active material of anti-hepatic fibrosis from Amydae Carapax.</p><p><b>METHODS</b>Membrane separation technology was adopted to screen active fraction in Amydae Carapax, and the active components were isolated from the active fraction using gel chromatography and high performance liquid chromatography. The purified active components in Amydae Carapax were further analyzed using 4700 series time-of-flight mass spectrometer.</p><p><b>RESULTS</b>Proteins and peptides of Amydae Carapax with molecular weight less than 6000 were proved to have biological activity. 8 components (Bj1-Bj8) were isolated from the active fraction. Bj4, Bj6 and Bj7 were screened as active components. Bj7 was further purified, resulting in 7 components (Bj701-Bj707). Bj704 and Bj707 showed significant biological activity. Mass spectrometry showed three molecular ion peaks with highest abundance, i.e. m/e 526, 542 and 572, i.e. m/e 526, 542 and 572, in Bj707 -A The amino acid sequences of above three peptide compounds were NDDY (Asn-Asp-Asp-Tyr), NPNPT (Asn-Pro-Asn-Pro-Thr), and HGRFG (His-Gly-Arg-Phe-Gly), respectively. And M572 was the most abandunt components.</p><p><b>CONCLUSION</b>Three active peptide compounds of anti-hepatic fibrosis of Amydae Carapax were identified.</p>

Comparison of the distribution of breviscapine in the brain by different administration routes / 药学学报

By comparing the drug distribution of breviscapine administered intranasally, orally and intrgvenous injected in rats' brain. After 0.4 mg x kg(-1) breviscapine was given by tail vein, intranasal and gastric perfusion administration to SD rats, cerebrospinal fluid was obtained by erebellomedllery cisternal puncture at different times. 125I labeling was used to determine the drug content of cerebrospinal fluid, cerebrum, cerebellum, medulla oblongata, olfactory region, olfactory bulb and blood in rats. AUCs were calculated by trapezoidal rule. The results showed that AUCs(0-240 min) (microg x min x g(-1)) of brain tissues were 11.686 +/- 1.919, 5.676 +/- 1.025, 7.989 +/- 0.925, 7.956 +/- 1.159, 17.465 +/- 2.136, 24.2 +/- 2.906 and 78.51 +/- 12.05, respectively, in the intranasal administration group; while those in the tail vein administration groups were 6.79 +/- 0.661, 6.251 +/- 0.40, 10.805 +/- 1.161, 9.146 +/- 1.04, 9.892 +/- 1.532, 7.871 +/- 0.842 and 173.91 +/- 10.02; and oral administration group were 0.868 +/- 0.167, 1.708 +/- 0.266, 2.867 +/- 0.725, 2.067 +/- 0.313, 1.361 +/- 0.308, 1.206 +/- 0.255 and 45.2 +/- 7.52, respectively. AUCs(0-240 min) of the brain tissues after oral, tail vein and intranasal administration were 22.29%, 29.18%, 95.49% of that of blood, respectively, it means that the absorption rate and drug distribution in the brain tissues after intranasal administration were higher than those of oral and tail vein administration. It is worth to investigate further the pharmacodynamic relationship.

Adverse drug reactions induced by Mailuoning injection: literature review on 162 cases / 中国中药杂志

To understand the adverse drug reaction (ADR) induced by Mailuoning injection. 162 ADRs due to the drug were retrieved from national medical journals of 1988-2007 for statistics. It was shown that there was no relationship between ADR and dosage, but ADR appeared mostly in middle-aged and old groups, and more in male than in female. The occurrence of ADR was commonly within 30 min after injection. It involved injuries of various systems and organs. As for the 123 cases of allergy, 38 were anaphylactic shock (accounting for 23.46%), two people died. ADR characterized by immediate type on initial use and tachy type. It should be paid more attention to the Mailuoning injection for the immediate hypersensitivity reaction (such as anaphylactic shock).

The changes of three endogenous hormones during flower bud differentiation of Schisandga chinensis / 中国中药杂志

<p><b>OBJECTIVE</b>To reveal the relation between endogenous hormones and the flower bud differentiation in Schisandga chinensis.</p><p><b>METHOD</b>Top buds of extremely short branch and axillary buds of long branch in the same plant of S. chinensis were used as material and the contents of endogenous hormones were measured during different periods of the flower bud differentiation with HPLC.</p><p><b>RESULT</b>The result showed that flower bud differentiation and the formation of female flower were inhibited by high concentration of GA3 and were promoted by high concentration of ABA or ZT. Low ratio of GA3/ABA has the same result.</p><p><b>CONCLUSION</b>There was a correlation between endogenous hormones and the flower bud differentiation of S. chinensis.</p>

Experimental study of traditional Chinese medicine in the treatment of vascular dementia / 中国中药杂志

To explore the new progress of the experimental study of traditional Chinese medicine in the treatment of vascular. Consulted the relevant papers and integrated the elementary theory, we summarized the virtue of Chinese medicine in the treatment of vascular dementia and insufficient and future development of the experimental study of it. It was confirmed that the traditional Chinese medicine has a lot of target spots in the brain and less ill-effect, it could ameliorate several phenotypes of pathophysiology of vascular dementia and improve the ability of learning and memory of animals with vascular dementia.

Antioxidative effect of total axtract of astragalosides / 中国临床药理学与治疗学

Aim The relationship between the therapeutic effect of TEA (total extract of astragalosides) on adjuvant arthritic (AA) rats and its antioxidative effects were studied . Methods The volume of non-injected hind paw of AA rats and malondialdehyde(MAD) content of arthritic synoviocytes from AA rats were measured and the proliferative responses of fibroblast, the level of superoxide anion() and the hydroxyl radical (?OH) generated in vitro were detected . Results The anti-inflammatory effect of TEA might be related to its antioxidative activity.In vitro low-level oxidative stress promoted the proliferative responses of fibroblast in rats synovium, which was marked by inhibited by TEA in a concentration-dependant manner. Further study showed that TEA could inhibit NBT reduction induced by both xanthine-xanthine oxidase and non-enzyme generated , but the inhibitory effect of this compound on activity of xanthine oxidase was obtained only at high concentration (more than 80 ?g?ml-1). It was also found that TEA could dose-dependantly inhibit the hydroxylation of benzoic acid induced by Fenton reaction generated ?OH. Conclusion TEA has significant therapeutic effects on AA rats, which might be related to its antioxidative effects

Induction of hairy roots of Panax ginseng and studies on suitable culture condition of ginseng hairy roots / 生物工程学报

Ginseng is a valuable medicinal plant with ginsenosides as its mian effective components. Because ginseng is a perennial plant and has a very strict demand for soil conditions, the way of cultivating ginseng by cutting woods is still used in China at present and thus forest resources has been extremely destroyed. Increasing attention has been paid to the hairy roots induced by the infection of Agrobacterium rhizogenes in the production of plant secondary metabolic products for the hairy roots are characterized by rapid growth and stable hereditary and biochemical traits. That has opened a new way for the industrial production of ginseosides. However, there is little report for such studies from China. In this paper, hairy roots of ginseng were induced from the root explants of two-year-old ginseng by Agrobacterium rhizogenes A4 with directly inoculating. The transformed hairy roots could grow rapidly on MS medium and 1/2 MS medium without hormones. The cultured clones of the hairy roots were established on a solid 1/2 MS medium. After 4 - 5 subcultures the hairy roots still maintained a vigorous growth. A pair of primers were designed and synthesized according to the analytical results of RiA4TL-DNA sequence by Slightom et al . 0.8kb rolC was obtained by PCR using the genome DNA of hairy root of ginseng. Transformation was confirmed by PCR amplification of rolC genes from the hairy roots of P. ginseng. Growth rate of hairy roots on liquid medium increased by 2 times then that of the solid medium. The growth of the hairy roots can be divided into three stages: high speed in the first two weeks, middle speed in the 3 - 4 weeks and low speed hereafter. Changing the culture solution at 2 weeks regular intervals is conductive to maintaining the rapid growth of the hairy roots. By means of determination for specific growth rate and ginsenosides content, the high-yield hairy root clone R9923 was selected. The content of monomer gisenoside of Rg1, Re, Rf, Rbl, Rc, Rb2 and Rd in hairy root clone R9923 was determined by the HPLC. The total ginsenosides content in the hairy toot clone R9923 came up to 15.2 mg/g. The suitable culture conditions for ginseng hairy roots growing were 1/2 MS liquid medium (30 g/L glucose), in a shaker at 110 r/min, changing the culture solution at 2 weeks and subculture time 4 weeks. In the liquid fermented culture of 2L medium, the yield of the hairy roots could amount to 270.10 g in 4 weeks. The industrial production of ginsenosides has been preliminarily realized. Effect factors on biomass and ginsenosides content such as culture volume, inoculation, in steps cultural technology at the scale-up process of hairy roots culture were also explorated. Our results have laid a foundation for defining optimum culture manner for large-scale cultivation and large-scale production of ginsenosides.