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Objective: To select the appropriate reference genes for calibrating the quantitative real-time PCR detection of gene expression in different tissues and leaves with different treatments of Morinda officinalis. Methods: With different groups and different processing leaves of M. officinalis as materials, 10 internal genes, including GAPDH, CYP, TUA, Actin and so on, were selected as candidate genes according to the M. officinalis transcriptome data. The expression stability of internal reference genes was analyzed by using real-time fluorescence quantification technique combined with software such as geNorm, NormFinder and BestKeeper, so as to select stable reference genes in different tissues and leaves of M. officinalis with different treatments. Finally, appropriate internal reference genes were selected to analyze the relative expression levels of DXS and DXR genes in different tissues and leaves with different treatments. Results: Internal reference genes GAPDH and UBQ were the most stable in different tissues of M. officinalis, the double internal reference combination of GAPDH + UBQ can more accurately analyze the relative expression levels of target genes in different tissues of M. officinalis, while the most stable reference genes in leaves with different treatments were GAPDH and Actin; The selection of the double reference combination of GAPDH + Actin can ensure the reliability of the target gene expression results. In different tissues of M. officinalis, the relative expression of DXS target gene was in sequence of root < stem < leaf, while the relative expression of DXR was stem < root < leaf. The relative expression levels of DXS and DXR genes in leaves with different treatments were increased compared with those untreated leaves (CK). Conclusion: The selected stable internal reference genes lay a foundation for the subsequent study on the expression of related genes of M. officinalis. Using the combination of two stable internal references to homogenize the target genes is conducive to improving the accuracy of the analysis of the expression of target genes.
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Objective To observe the clinical effect of Morinda officinalis How oligosaccharide capsule (MOs) combined with psychological therapy in the treatment of postpartum depression .Methods 60 patients with postpartum depression were randomly divided into two groups ,30 cases in each group.The control group was adopted the psychological therapy,and the observation group received combined treatment of MOs and psychological therapy . The HAMD-17 and Chinese medicine scale for the quality of kidney deficiency syndrome (CMSKDS) were used to assess the treatment effect after 2,4,6 weeks treatment.The TESS was adopted to evaluate the side effect of treatment . Results There were no statistically significant differences between the two groups on HAMD and CMSKDS before treatment(all P >0.05).After treatment,the effective rates of HAMD and CMSKDS (reducing score ≥25%) of the two groups were 67.62%(control group) and 70.00%(observation group),the defferences between the two groups were statistically significant (all P <0.01).There was no statistically significant difference in TESS result ( P >0.05).Conclusion The clinical effect of combined MOs and psychological therapy in the treatment of postnatal depression is remarkable,with little side effect,which can be strongly recommended .
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Objective To develop a method for determination of iridoid glycosides in Morinda officinalis How.and optimize the extraction methods for iridoid glycosides in Morinda officinalis How.Methods The iridoid glycosides, including monotropein, deacetyl asperulosidic acid,asperulosidic acid and asperuloside as standards, HPLC method was developed to determine the content of iridoid glycosides in Morinda officinalis How.The separation was performed on Venusil MP C18 (250 mm×4.6 mm, 5 μm) column.The mobile phase was acetonitrile (A)-0.2% phosphoric acid and 0.01 disodium hydrogen phosphate buffer salt (B) with gradient elution (0-12 min, 1%-2% A;12-30 min, 2%-25% A).The detection wavelength was 235 nm.The flow rate was set at 1.0 ml/min and the column temperature at 25 ℃.The injection volume was 20 μl.Single factor analysis and orthogonal test were used to optimize extraction method of iridoid glycosides in Morinda officinalis How.Results Monotropein, deacetyl asperulosidic acid, asperulosidic acid and asperuloside showed good linearity (r>0.999 5) in the ranges of 0.375-12 μg, 0.13-4.16 μg, 0.016-0.516 μg and 0.012-0.384 μg, respectively.This validated method has good repeatability, precision, recovery and stability.It was conformed to meet the requirements and regulation.The optimal extraction method included soaking the raw materials with 16 times of 10% ethanol for 9 h, and then extraction by percolation with the flow rate of 0.8 BV/h.Conclusion The HPLC method sensitively and precisely determined the content of iridoid glycosides in Morinda officinalis How.The optimized extraction method extracted these constituents effectively.
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OBJECTIVE: To investigate the contents of oligosaccharides in 37 batches of Morinda officinalis How samples from different habitats and germplasm resources at various ages. METHODS: HPLC-ELSD method was used to determine the contents of four oligosaccharides, i.e. sucrose, 1-kestose, nystose and 1F-fructofuranosylnystose in Morinda officinalis How at different ages from different habitats and germplasm resources. The relationships among the several factors were analyzed. RESULTS: The samples from Guangdong Province had larger amounts of sucrose, 1-kestose and 1F-fructofuranosylnystose than those from Fujian, Guangxi and Hainan Provinces. The content of nystose in the samples from Guangdong Province was similar with those from Fujian Province. The contents of sucrose and 1-kestose were the highest in the samples of 2.5 years old, while the contents of nystose and 1F-fructofuranosylnystose were the highest in the samples of 4 years old. The germplasm resources of small leaf had higher content of oligosaccharides than the large leaf germplasm in Guangdong Province, and different germplasm resources of Morinda officinalis How also had different morphological characteristics. CONCLUSION: The contents of four Morinda officinalis How oligosaccharides vary with habitat, germplasm and age. This research may provide references for the quality control of Morinda officinalis How.
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OBJECTIVE:To optimize the extraction technology of oligosaccharides of Morinda officinalis. METHODS:The liq-uid/solid ratio,extraction time and temperature were chosen as factors,and the yield of oligosaccharides was estimated as index. On the basis of single-factor experiments,using a 3-factor,3-level Box-behnken central composite experimental design,two-poly-nomial regression equation of extraction rate of oligosaccharide was established,and it was analyzed by response surface methodolo-gy to obtain the optimum extraction conditions,and the verification test was conducted. RESULTS:The optimum extraction condi-tions were as follows as material/liquid ratio of 23∶1(ml/g),extraction time of 1.7 h,and extraction temperature of 93℃,extract-ing for twice. Under these conditions, the estimated and observed average values of extraction rate of oligosaccharides were 10.37% and 10.29%(RSD=0.20%,n=3),respectively. The deviation value was 0.06%. CONCLUSIONS:The response surface methodology can be used to optimize the extraction process of oligosaccharides of M. officinalis.
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OBJECTIVE: To determine the contents of six oligosaccharides, i. e. glucose, fructose, sucrose, 1-kestose, nystose, 1-fructofuranosylnystose, in the roots of Morinda officinalis in different growing periods by HPAEC-PAD method. METHODS: The separation was performed on Hamilton RCX-10 column(4.1 mm × 250 mm, 7 μm) with gradient elution using mobile phase composed of 100 mmol · L-1 NaOH(A) and mixture of 100 mmol · L-1 NaOH and 500 mmol · L-1 NaOAc (B) at a flow rate of 0.8 mL · min-1 and the temperature was kept at 35℃. The injection volumn was 25 μX. The gradient elution program was as follows: 0-5 min, 1%B; 5-15 min, 1%-9% B; 15-20 min, 9%-12% B; 20-25 min, 12%-16% B; 25-40 min, 16%-50% B; 40-43 min, 50% B; 43-46 min, 50%-1% B. RESULTS: With the increase of growing periods, the content of monosaccharide progressively reduced while that of sucrose gradually increased. The contents of oligosaccharides with polymerization degree of 3 or greater, including 1-kestose, nystose, and 1-fructofuranosylnystose, slowly increased during 1-5 years, then slowed down. CONCLUSION: The contents of six oligosaccharides in the roots of Morinda officinalis vary greatly in different growing periods, indicating that the biosynthesis of oligosacchride in Morinda officinalis is related to growing periods. The result provides a reference for determination of appropriate collection time.
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Aim To investigate the angiogenic promoting effect of Morinda officinalis How oligosaccharides(MOO) in the ischemic myocardium of rats after acute myocardial infarction(AMI).Methods 40 male Wistar rats were established into AMI model successfully and were randomly divided into 5 groups equally, i. e. the low, medium and high doses of MOO groups, the Shexiangbaoxin group and the model group. They were treated with different doses of the water fraction of the ethanolic extract of Radix morinda officinalis (0.7, 1.4, 2.8 mg·kg~(-1) ·d~(-1)), suspension liquid of Shexiangbaoxin Pill(30 mg·kg~(-1) ·d~(-1)) and distilled water with the same volume respectively.Besides, a sham operated group with 10 rats was set up for control. All rats were sacrificed after 6-week-treatment.The Ⅷ coagulation factor, vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF) protein in ischemic myocardium of rats in each group were detected by immunohistochemistry assay.The microvessel density(MVD) was calculated. Gray values of protein expression of VEGF and bFGF in ischemic myocardium were calculated and analyzed by image analysis system.Results The MVD, the gray values of VGF and bFGF were higher in the medium and high doses of MOO groups than those in the model group(P <0.05), but still lower than those in the Shexiangbaoxin group(P <0.05). The MVD and the gray values of VEGF among 3 doses of MOO groups showed significant differences (P <0.05).Significant differences of gray value of bFGF were observed between small and middle doses of MOO groups, also between small and large doses of groups(P <0.05).Conclusion MOO can obviously promote angiogenesis in the ischemic myocardium of the rats after AMI.And up-regulating expressions of VEGF and bFGF protein in the ischemic myocardium may act as one of its angiogenic promoting mechanisms.
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Introduction: To date, there has not been any systematical and experimental research aimed at indicating the effects of Morinda officinalis How on the genital organs of male rats. \r\n', u"Objectives: To determine the effectiveness of Morinda officinalis How (MOl-I) on the genital organs of male rats, to describe the changes in histology of testicles, and evaluate the testosterone concentration in mature male rats' bloods when dosed with MOH.\r\n", u'Subjects and method: Mature and immature rats were divided into 3 groups: mature rats, castrated and non - castrated immature rats. The rats have a daily dosage of MOH: 20g/kg. The above groups of rats were euthanized after 10 days; their testicles, seminal vesicles, prostates and Cowper land were weighed; specimens of histology of testicles were made; testosterone concentration in the bloods of the mature rats was measured. Results were compared amongst experimental groups. \r\n', u'Results and conclusion: Compared with the control group, MOH (dosage 20g/kg) increased the weight of testicles, seminal vesicles, prostates, and Cowper lands (p < 0.05). In mature rats, MOH increased the testosterone concentration in the blood in comparison with the control group (p < 0.05). MOH did not change the histology of testicles, diameter of seminal ducts, but it did change the rate of seminal ducts having sperm compared with the control group and the testosterone injected group (p < 0.05). \r\n', u'
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GenitaliaABSTRACT
The herbal remedy Morinda officinalis How (Rubiaceae) has been used to invigorate the kidney-yang, strengthen muscles and bones, and in impotence. In this study, a micropropagation protocol for the plant was proposed. Aseptic shoot cultures were established from single-node stem cuttings taken from 3-year old, field growing plants, on Murashige and Skoog (MS) medium, supplemented with 0.5 mg/l Kin. 100% of the shoot apices and single-node stem cuttings (1-1.5 cm long) from these aseptic in vitro shoots produced multiple shoots in MS medium containing 3 mg/l BAP. Shoots arose both directly from axillary buds and indirectly from the periphery of the basal cells of the explants. Maximum rates of multiplication averaging 18.808.99 and 23.529.26 were obtained for shoot apices and stem cuttings, respectively. The basal callus portions gave healthy shoots, and therefore were used for subcultures. Rooting of the single shoots was inhibited by both NAA and IBA at the concentrations 0.1-0.5 mg/l. 50-60% in vitro rooting was obtained in MS medium without any growth regulators in about 30 days, and plantlets were successfully transferred to the nurseries.
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Plants , Medicine, Traditional , Biochemistry , RubiaceaeABSTRACT
Objective To study the effect of different treatment methods on seed germination of wild Morinda officinalis How(MOH),which will provide evidence for breeding new cultivars of MOH.Methods We observed the morphological feature,thousand-seed weight and hygroscopicity of wild MOH,and studied the effect of different treatment methods on the germination rate,germination vigor,and germination index of the seeds.Results The thousand-seed weight was 38.03 g and the seed coat had good hygroscopicity.Stripping seed coat,acid etching with sulfuric acid,and the combined treatment of stripping seed coat with exogenous hormones of gibberellin(GA) and 6-benzyladenine(6-BA) had an effect on increasing germination rate,and the germination rate arrived 88.89% after the combined treatment of stripping seed coat with 4 mg/L 6-BA.Conclusion The germination inhibitors containing in the seed coat mainly contribute to the difficulty of the germination of MOH,and the germination inhibitors containing in the seed also have some influences on the germination.
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Objective To investigate the genetic diversity of five populations of cultivated Morinda officinalis in Guangdong Province using random amplified polymorphic DNA(RAPD).Methods Sixty-four individuals in five populations of cultivated M.officinalis were analyzed by RAPD markers to determine the genetic variations among the populations.The data of genetic diversity were analyzed with Popgen32 software.Results The levels of genetic variation and patterns of population structure in M.officinalis were investigated using RAPD markers.Of the 100 primers screened,15 primers produced highly reproducible RAPD bands,using these primers,224 discernible DNA fragments were generated with 112(50.00%) polymorphic fragments,indicating considerable genetic variation at the species level.In contrast,there were relatively high levels of polymorphism at the population level with the percentage of polymorphic bands ranging from 37.05% to 53.13%,and the mean percentage of polymorphic loic(P=45.86%).Genetic variation among populations was detected based on Nei's genetic diversity analysis(0.175 6),Shannon's diversity index(0.287 6) for every population with Shannon's index 0.103 3—0.236 2 and Nei's indexes 0.074 5—0.154 0.Conclusion There is a little genetic differentiation among populations of cultivated M.officinalis and result in different cultivated types,the genetic diversity with M.officinalis(POP2) is higher than that of other populations.It may be the main reason of the difference of cultivated M.officinalis quality.
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Objective To compare the rDNA-ITS differentiation and its regulation between Morinda (officinalis) and its counterfeit species, and provide DNA molecular markers for the fingerprint identification of them. Methods The rDNA-ITS regions of M. officinalis and its counterfeit species were amplified and sequenced, then analyzed by means of CLUSTRAL X and MEGA softwares. Results The internal transcribed spacers (ITS) including ITS1, 5.8S, ITS2, and partial 18S and 26S were determined. In DNA DIST analysis, the range of diversity among M. officinalis and M. shughuaeusis, M. umbellata was (2.9%-)5.8% and 2.9%-4.2% based on ITS1 and ITS2; the range of diversity between M. officinalis and Damnacanthus indicus was 21.2% and 18.9% based on ITS1 and ITS2. Phylogenetic tree based on ITS and 5.8S sequence data indicated the M. umbellata and M. shuanghuaensis were closely related then with M. officinalis, while D. indicus was monophyletic group. Conclusion The rDNA-ITS sequence is a better molecular marker for idertification of M. officinalis and its counterfeit species.