Preventive effect and mechanism of ultrafine powder of Dendrobium candidum on photoaging model mice / 中国中药杂志

Chinese herbal medicine ultrafine powder has become a research hotspot for the addition of cosmetic raw materials. Dendrobium candidum is a traditional Chinese herbal medicine. Its extract and stem extract are already cosmetic raw materials and its water extract has the effect of preventing photoaging,but D. candidum ultrafine powder has not been accepted as a raw material for cosmetics,and no relevant research on photoaging prevention has been reported. In this experiment,the ultra-fine powder and fine powder of D. candidum to prevent photoaging were observed and compared,and its mechanism of action was discussed to provide a basis for the prevention of skin photoaging products. Seventy-two female ICR mice were randomly divided into normal group,model group,solvent group,titanium dioxide(Ti O2) group,isooctyl salicylate(2-ES) group,D. candidum ultrafine powder 1(DP1),ultrafine powder 2(DP2) and fine powder(DP3) groups. The photoaging model was established by ultraviolet irradiation for 8 weeks,and the model was intervened while modeling. The skin wrinkle grade,elastic parameters,skin microcirculation blood flow,skin structure and pathological changes(skin thickness,skin collagen fiber,elastic fiber) were observed,the skin transforming growth factor-β1(TGF-β1),Smad3 levels were determined,and the type Ⅰ and type Ⅲ collagen,matrix metalloproteinase-1(MMP-1),activated protein-1(AP-1),VEGF expression were detected. The results showed that ultrafine powder(DP1,DP2) significantly reduced the wrinkle level and skin blood flow of the model mice(P<0. 05,P<0. 01); DP1,DP2 and DP3 could significantly reduce the thickness of the epidermis(P<0. 001),improve collagen fiber,elastic fiber hyperplasia,and distortion and decrease VEGF expression,and DP1 is better than DP2 and DP3; each group could up-regulate type Ⅰ collagen,down-regulate type Ⅲ collagen,AP-1,MMP-1 protein expression,and DP1 improvement optimal. However,it has no obvious effect on TGF-β1 and Smad3. The ultrafine powder and fine powder of D. candidum have certain preventive effect on photoaging,and the effect of ultrafine powder is better than that of fine powder. Ultrafine powder may down-regulate the expression of type Ⅲ collagen,AP-1 and MMP-1 by up-regulating type Ⅰ collagen. Inhibition of collagen degradation plays a role in preventing photoaging.

Protective effect of Dendrobium candidum on isoproterenol induced cardiac hypertrophy in rats / 中国中药杂志

To study the effect and mechanism of Dendrobium candidum on isoproterenol-induced myocardial hypertrophy in rats, 60 healthy SD rats(30 males and 30 females) were randomly divided into 5 groups(12 in each group): normal group, model group, three D. candidum preventive administration groups(0.09, 0.18, 1.1 g·kg⁻¹). Except for the normal group, rats of other groups were injected back subcutaneously with ISO(5 mg·kg⁻¹) for 10 consecutive days. At the same time, preventive administration groups began to give different doses of the sample for 30 days and model group began to give normal saline. Left ventricular systolic pressure(LVSP) was measured in each group by common carotid artery cannulation, and the left ventricle(LW)/tibia length, heart weight index(HWI) and myocardial hydroxyproline(Hydro) content were calculated. Myocardial tissue HE staining and Masson staining were used to observe the myocardial structure and the degree of myocardial fibrosis respectively. Atrial natriuretic peptide(ANP), brain natriuretic peptide(BNP), and cardiac troponin I(cTN-I) concentration were measured by enzyme-linked immunosorbent assay(ELISA). The results showed that as compared with the normal group, the levels of ANP, BNP and cTN-I in plasma were significantly increased in ISO-induced hypertrophic rats; as compared with the model group, D. candidumcan inhibit ISO-induced ventricular pressure and ventricular hypertrophy, reduce myocardial collagen synthesis, improve myocardial fibrosis and ventricular remodeling, and significantly down-regulate ANP, BNP and cTN-I levels in plasma. This study shows that D. candidum has a protective effect on isoproterenol-induced cardiac hypertrophy.

Efficacy of compound Dendrobium on PI3K/AKT/eNOS signaling pathway in hypertensive rats induced by "dietary disorders" / 中国中药杂志

To observe the efficacy of compound Dendrobium on Sprague Dawley rats (SD) hypertension model induced by "dietary disorders" and its relevant mechanism, totally 50 SD rats were fed with high-sugar, high-fat diet and alcohol for four weeks. According to the blood pressure after modeling, the rats were divided into model group, valsartan group (8 mg·kg⁻¹), low, medium and high-dose Dendrobium candidum compound groups (1.65, 3.30, 5.00 g·kg⁻¹), with 10 rats in each group, and the other 10 SD rats were also taken as the normal group. After four weeks of treatment, blood pressure was measured. Orbital blood was collected for the determination of serum cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL), calculation of atherosclerosis index (AI). Nitric acid reductase method was used to detect serum nitric oxide (NO); the levels of serum endothelin-1 (ET-1) and intercellular adhesion molecule-1 (ICAM-1) were measured by ELISA. The rats were put to death after the last administration, and the protein expressions of PI3K/AKT/eNOS in thoracic aorta of rats in each group were detected by Western blot. The aorta was separated and stained with hematoxylin-eosin (HE) to observe the changes in the endothelium and blood vessels in the thoracic aorta. Masson staining was used to observe the formation of aortic collagen. The expressions of nitric oxide synthase (eNOS) and ICAM-1 in aortic endothelial cells were observed by immunohistochemistry. In contrast, the results show D. candidum compound can significantly reduce the blood pressure in hypertensive rats, increase HDL-c, and reduce AI, while increasing serum NO content, decreasing ET-1 and ICAM-1 levels and promoting PI3K/AKT/NOS protein expressions. The lesion degree of the D. candidum compound group was reduced, and the collagen deposition was significantly reduced. Meanwhile, D. candidum compound can significantly increase the expression of eNOS, and reduce the formation of ICAM-1.Therefore, D. candidum compound has an obvious antihypertensive effect on hypertensive rats, which may be related to the increase in PI3K/AKT/eNOS signaling pathways and NO generation, the inhibition of the secretion of ICAM-1 and ET-1, the protection of the vascular endothelium and the improvement of aortic disease.

Protection of polysaccharides of dendrobium candidum on blood-retinal barrier in diabatic rats and its mechanism / 中华实验眼科杂志

Background The pathogenesis of diabetic retinopathy (DR) involves a variety of biological pathways.Recently,inflammation factor has been thought to paly an important role in the pathogenesis of DR.Studies show that the concent of tumor necrosis factor-α (TNF-α) is increased in high-glucose environment,which leads to the abnormality of tight junction protein and follows by blood-retinal barrier (BRB) damage.Polysaccharides of dendrobium candidum (PDC) can inhibit the overexpression of TNF-α,but its effect on TNF-α in early DR procedure has been unelucidated.Objective This study was to investigate the effects of PDC on permeability of BRB and its mechanism in daibetic rats.Methods Fifty clear adult SD rats were divided into normal control group,diabetic model group and low-(100 mg/[kg · d]),moderate-(200 mg/[kg · d]) and high-dose (300 mg/[kg · d]) PDC groups,10 rats for each group.Streptozotocin was intraperitoneally injected to establish diabetic model in 40 rats,expect for normal control group.PDC at the concentrations of 100,200 and 300 mg/(kg · d) was intragastrically administered in the low-,moderate-and high-dose groups respectively at 6 weeks after modeling,and normal saline solution was used at the same way in the normal control group and diabetic model group.Evans blue was perfused via cardic chamber and eyes were obtained.Evants blue leakage was measured to evaluate the BRB permeability.The relative expressions of TNF-α,zonula occludens-1 (ZO-1),occludin and claudin-5 proteins were detected by Western blot;TNF-α contents in the retina and serum of the rats were detected by ELISA.Results The leakage concents of Evans blue in the retinas were (12.68±1.30),(30.45±2.60),(22.12±1.15),(17.99±1.00) and (21.49±1.00) in the normal control group,diabetic model goup and low-,moderate-and high-dose PDC groups,respectively,and the retinal leakage concents in the diabetic model group were significantly higher than those in the normal control group,and the retinal leakage contents in the low-,moderate-and high-dose PDC groups were lower than those in the diabetic model group (all at P ＜ 0.01).Western blot showed that the relative expression level of retinal TNF-α was significantly higher in the diabetic model group compared with the normal control group(1.12±0.10 vs.0.27±0.03),and that in the diabetic model group was significantly higher than that in the normal control group;while the relative expression levels of retinal TNF-α in different doses PDC groups were significantly lower,and the relative expression levels of retinal ZO-1,occludin and claudin-5 were significantly higher than those in the diabetic model group (all at P＜0.05).ELISA showed that the concentrations of retinal and serum TNF-α were higher in the diabetic model group compared with the normal control group,and those in the different doses of PDC groups were lower than those in the diabetic model group (all at P＜0.05).No significant differences were found among various doses of PDC groups (all at P＞0.05).Conclusions PDC can improve the permeability of BRB by down-regulating the expression of TNF-α and up-regulating the expressions of tight junction proteins in the retina of diabetic rats,which is probably related to suppressing the development of early DR.

Effect of different culture conditions on protocorm-like bodies of Dendrobium candidum in bioreactor culture / 中草药

Objective: To explore the effect of different culture conditions on protocorm-like bodies (PLBs) of Dendrobium candidum in bioreactor culture. Methods: Inoculation quantity, ventilation, sucrose concentration, and light intensity were taken to carry out the single factor experiment design. Biomass was determined by drying method, polysaccharide content by phenol-sulfuric acid method, and SPSS16.0 software was used for data analysis. Results: Under the bioreactor culture, the nutrient medium components for the PLBs proliferation of D. candidum and polysaccharide accumulation were 1/2 MS medium supplemented with 1.0 mg/L NAA, 5% coconutmilk, and 3% sucrose, pH value was 6.0. The culture conditions, such as inoculation with 40 g/L, 15 μm pore porous nozzle, aeration volumes by 1.0 and 0.5 L/min used alternately, and photon flux of 2000 lx could improve the proliferation coefficient and polysaccharide contents of PLBs. Conclusion: Different culture conditions have the significant effect on the growth of PLBs and the accumulation of polysaccharide. Suitable culture conditions have the very important practic significance to the biomass of PLBs and main medicinal ingredient production of PLBs.

Primary study of Dendrobium polysaccharides improving immunity activity on mouse / 中国药学杂志

OBJECTIVE: To evaluate the effect on mice immunity activity of Dendrobium devonianum polysaccharides and Dendrobium candidum polysaccharides. METHODS: Cultured splenic lymphocytes of mice in vitro to observe the influence of Dendrobium devonianum polysaccharides and Dendrobium candidum polysaccharides separately cooperated with ConA on proliferation of splenic lymphocyte and content of IFN-γ and IL-2. In animal experiments, respectively offered Dendrobium devonianum polysaccharides and Dendrobium candidum polysaccharides to mouse in different dosage, after 14d, copied the model of toe swelling induced by SRBC to observe the effect of Dendrobium polysaccharides on toe swelling and viscera index. RESULTS: Medium dosage groups of two Dendrobium polysaccharides both can stimulate the proliferation of splenic lymphocyte observably; medium and high dosage groups both can promote the secretion of IFN-γ and IL-2 in splenic lymphocyte: In addition, high dosage groups of two Dendrobium polysaccharides both can increase toe swelling and spleen index of mouse obviously, but every dosage groups have no effect on thymus index of mouse. CONCLUSION: To some extent, Dendrobium devonianum polysaccharides and Dendrobium candidum polysaccharides can improve the immunity activity of mouse.

Biocontrol of Dendrobium candidum damping-off disease with endophytic fungi / 中国药学杂志

OBJECTIVE: To screen out endophytic fungi isolates with good biological control against damping-off in Dendrohium candidum seedlings among the isolates owned by our laboratory, thus to lay a foundation on developing biological control technique and field application. METHODS: Eighteen isolates of endophytic fungi identified by the PDA Petri dish confrontation experiment and athogen of damping-off were used in the biological control experiment. Co-incubation in bottles was conducted to screen out isolates with weak pathogenic and growth promoting effect on Dendrobium candidum seedlings. The chosen isolates were then enrolled in the potted seedlings experiment to determine their biological control effect. RESULTS: Isolates 4829 and 3952 were confirmed to have good biological control effect by statistical analysis. The survival rates of Dendrobium candidum seedlings reached 66.7% and 60.5% respectively. CONCLUSION: Endophytic fungi isolated from Dendrobium sp. shows good biological control against Dendrobium candidum damping-off.

Construction of differential cDNA library on the roots of Dendrobium candidum induced by a symbiotic mycorrhizal fungus / 中国药学杂志

OBJECTIVE: To screen differentially expressed genes on the roots of Dendrobium candidum induced by a symbiotic mycorrhizal fungus, the differential cDNA library was constructed. METHODS: The ds cDNAs were synthesized and enriched by SMARTer PCR cDNA synthesis kit using total RNA of D. candidum roots symbiotic with the fungus as templates. The differential cDNA library was constructed by using suppressive subtraction hybridization (SSH). RESULTS: The differential cDNA library, containing 1975 clones in the storage capacity, was constructed successfully. It was detected that above 90% clones could be amplified effective products. The lengths of the differential cDNA fragments cloned were 150 bp to 1 kb by electrophoresis detection to 20 randomly selected clones. Then, the 20 ESTs similarity analysis based on BLASTx software was carried on and most of the cloned genes were the defensive genes of plant responding to environmental stresses. CONCLUSION: Using this technology system could construct a fine differential cDNA library and be operated easily, especially suitable to the rare species.

Determination of chemical constituents of essential oil from flower of Dendrobium candidum Wall.ex Lind1 / 中华中医药杂志

Objective: To study the chemical constituents of volatile oil from flower of Dendrobium candidum Wall. ex Lind1. Methods: The chemical compositions of volatile oil of the plant which were obtained by steam distillation with hexane were analyzed by GC-MS equipped with a elastic quartz capillary column-HP-5MS5% Phenyl Methyl Siloxane (30 m? 0.25 mm ?0.25 ?m). The constituents were identifi ed by their mass spectra. The relative percentage of the oil constituents was calculated from the GC peak areas. Results: Eighty-nine kinds of chemical constituents in Dendrobium candidum Wall.ex Lind1.flower were separated; of which fifty-nine compounds representing 76.51% of the oil were characterized. Relative contents that were more than 2.0% were determined as Nonanal 9.21%, Eudesma-5,11-dien-8,12-olide 5.55%, (E)-2-Decenal 4.63%, 2,3-Dehydro- 1,8-cineole 4.39%, Pentacosane 4.03%,?-Cedrol 3.69%, Isoalantolactone 3.65%, (E)-2-Heptenal 3.60%, E,E-2,4-Decadienal 2.14%,?-Phorone 2.03%.Conclusion: This paper reports, for the first time, the composition of volatile oils of Dendrobium candidum Wall.ex Lind1.flower by GC/MS.

Histocytological Observation of Somatic Embryogenesis in In-Vitro Cultured Dendrobium candidum wall. Ex Lindl / 广州中医药大学学报

Objective To observe the somatic embryogenesis in in-vitro cultured Dendrobium candidum wall.Ex Lindl.(DCWL),and to supply evidence for its rapid propagation and germplasm preservation.Methods Protocorm-like bodies and callus of DCWL subcultured for 30 days was used as the explants,N6 was used as the basic culture with phytohormone added,and fungal extracts as the elicitor.Protocorm-like bodies and callus of DCWL were used for induction culture.Results Somatic embryogenesis in in-vitro cultured DCWL derived from the epithelial cells or inner cells of callus of DCWL.Conclusion A large amount of buds can be obtained by the induction and culture of protocorm-like bodies and callus of DCWL.

Construction and Analysis of cDNA Expression Library from the Leaves of Dendrobium candidum Wall. ex Lindl / 广州中医药大学学报

[Objective] cDNA expression libraries from the leaves of Dendrobium candidum Wall. ex lindl. were constructed to supply evidence for the clones of the full-length genes involved in the biosynthesis of bioactive compounds. [Methods] Total RNA was isolated and purified from tender leaves of 4-year-growing Dendrobium candidum by using Trizol single-step method. cDNA was synthesized by long distance polymerase chain reaction (LD-PCR) and then was connected to ?TripIEX2. After that, the recombinant bacteriophages were packaged and cDNA library of Dendrobium candidum was constructed. The titer of cDNA library was expressed as the number of phage formation unit (pfu) per milliliter and the inserted fragment was identified by PCR amphfication. [Results] cDNA expression libraries from the leaves of Dendrobium candidum were constructed successfully. The titer of the original library was 4.3?105 pfu/mL and 3.1?105 clones in total, with a recombinant rate of 97.6%. The amplified titer was 6.8?109pfu/mL. PCR amplification suggested that the inserted cDNA fragments ranged from 0.5 to 2.0 kb, mostly from 1.2 to 1.7 kb. [Conclusion] The constructed Dendrobium candidum cDNA library has a higher titer, a higher recombinant percentage and larger inserted fragments.

Construction of Subtractive cDNA Library from the Leaves of Dendrobium candidum Wall, ex Lindl. by Suppression Subtractive Hybridization Technology / 广州中医药大学学报

[Objective] Subtractive cDNA libraries from the leaves of D. candidum were constructed to supply evidence for the clone of dendrobine-biosynthesis-associated genes. [ Methods ] Suppression subtractive hybridization ( SSH) technology was used. With the cDNA of perennial leaf as the tester and that of annual leaf as the driver, forward and reverse hybridization was performed. The two obtained subtracted cDNA fragments were cloned into PointTMXa-1 plasmid vectors, and then the vectors were transformed into E. Coli JM109. The inserted fragments were amplified by PCR and then identified. [Results] The subtractive cDNA libraries related with dendrobine-biosynthesis-associated genes were successfully constructed. The forward and reverse subtractive libraries included 560 and 220 clones respectively. Detected by PCR, the length of the inserted fragments was 550 bp on average. [Conclusion] The constructed subtractive libraries are suitable for further study on the functional genes associated with dendrobine biosynthesis of D. candidum.

Effects of fungal elicitors on growth of Dendrobium candidum protocorms / 中草药

Objective To study the effects of two fungal elicitors on the growth of Dendrobium candidum protocorms cultured on the solid medium.Methods The medium for propagation of protocorms was selected and the growth curve on that medium was obtained.According to the curve,the two fungal elicitors were added at the different growth stages of protocorms.The fresh weight(FW) and dry weight(DW) of protocorms were measured.Results The medium for propagation of protocorms was 1/2MS+20% potato extract+3% sucrose+0.8% agar.Compared with the control MF23 elicitor added at weeks 11 and 13 could significantly increase the FW by 16.4%(P

Fingerprint of dry stem in Dendrobium candidum by HPLC / 中草药

Objective To establish the fingerprint for the dry stem in Dendrobium candidum by RP-HPLC.Methods Kromasil○RKR100-5 C18 column(250 mm?4.6 mm,5 ?m) was used with a mixture of acetonitrile-0.4% phosphorus acid as mobile phase in a gradient mode and the data were analyzed with ″Computer Aided Similarity Evaluation″ software.Results The chemical constituents of D.candidum were optimally separated,among which 15 fingerprint peaks in common were confirmed.Conclusion This method is simple,accurate with good reproducibility,and can be used specifically for the quality control of D.candidum.

RNA Extraction and RT-PCR detection of Dendrobium candidum / 中草药

Objective The high quality RNA from the plantlet of Dendrobium candidum was isolated efficiently.It laid a foundation to study the mechanism of molecular expression on its growth and metabolism under environmental stress.Methods The groups stimulated by sound wave and the control groups were established.Total RNA was prepared by a modified phenol-chloroform method.The content was determined by a spectrophotometer.The integrity of RNA was detected by electrophoresis with 1.0% denatured agarose gel.The total RNA obtained was amplified by RT-PCR.The differential cDNA bands were displayed on 6% denatured polyacrylamide gel by electrophoresis and silver staining.Results The ratios of total RNA A_(260)/A_(230) and A_(260)/A_(280) were 3.16 and 1.96,respectively.The yield was 0.27 ?g/mg fresh weight.The bands were clear on agarose gel and the integrity of RNA was good.The differential cDNA bands were screened with molecular weight of 240—600 bp.Conclusion The method of modified phenol-chloroform can be used to isolate RNA from D.candidium with high quality and high yield.

Characteristics of photosynthesis in three Dendrobium species / 中草药

Object To determine the diumal changes of photosynthesis, its relationships with light intensities and temperatures, and the contents of chloroophyll in leaves of Dendrobium candidum Wall. ex. Lindl., D. wilsonii Rolfe and D. hercoglossum Reichb. f. Methods The net photosynthetic rate (NPR) was measured at intervals of 1.5 h during daytimes 7:30—16:30, under the photosynthetic affective rates 0—140 mmol/(m 2?s), and under the temperatures 29.6—38.2 ℃; contents of chlorophyll a and b in leaves were measured. Results NPR, light saturation point and compensation point were low in each of the three Dendrobium L. species. The NPR peaked during 8:00 and 10:00 am, and thereafter decreased rapidly as the temperature and light intensity increased. Content of chlorophyll b was high in the leaves. Conclusion These results indicated that the Dendrobium L. species, which are sciophyte plants, have great adaptations to their growing habitats.

Protoplast fusion in Dendrobium candidum and Gynostemma pentaphyllum / 中草药

Object To explore the protoplast fusion of Dendrobium candidum Wall ex. Lindl. and Gynostemma pentaphyllum. (Thunb) Makino. Methods The two kinds of protoplasts were fused by PEG method, and then cultured in the modified liquid medium containing 2 mg/L BA and 1 mg/L NAA. Results High yield, viability, and pure mesophyll protoplasts were isolated from D. candidum and G. pentaphyllum. Conclusion The first cell division occurres within three days after fusion. And some of the cells are divided three times.

Studies on industrialization cultivation of Dendrobium candidum / 中草药

Object To optimize the fitting culture media for multiplication of adventitious bud and ra-dication of Dendrobium candidum Wall. ex Lindl. and the length of adventitious bud for succesive transfer multiplication. Methods In the same culture condition, the adventitious bud with different length developed in the various culture media for multiplication and radication. Facing the different growth status, the diversity test and general analysis were carried on for them. Results There were significant difference in different length of adventitious bud, difference between the multiplication and radication of culture media. Conclusion The adventitious buds in (1.2?0.1) cm length are superior for succesive transfer multiplication, MS adding BA 1.0 mg/L, NAA 0.2 mg/L is the best media for the adventitious buds multiplication, 1/2 MS adding NAA 0.2 mg/L is the test media for radication.

Effect of three endophytic fungi on growth of Dendrobium candidum and Anoectochilus roxburghii / 中草药

Object To study the promoting effects of Mycena anoectochila, M dendrobii and M. orchidicola on growth of Dendrobium candidum Wall. ex Ldl. and Anoectochilus roxburghii (Wall ) Ldl Methods The protocorm and plantlet of D candidum and A roxburghii were grown in dual culture with endophytic fungi to observe the proliferation of protocorm and the growth of plantlet. Results The growth of plantlet of D candidium, which was inoculated three endophytic fungi, increased markedly 3 5 times more than those of the control one M dendrobii and M orchidicola also promoted multiplication on protocorm of D candidum (P

Suspension culture of protocorm in Dendrobium candidum / 中草药

Object To study the feasibility of suspension culture of protocorm in Dendrobium candidum Wall ex Lindl and effect of inoculum and medium volume on the growth of protocorm Methods Effect of four basic media MS, 1/2 MS, 67 V, and B 5, inoculum and medium volume on the growth of protocorm were studied by completely random experimental design and orthogonal test design Results The growth of D candidum protocorms in liquid medium was markedly better than that in solid medium (P0 05), B 5 was much better than 1/2 MS (P