Functional analysis of flavanone 3-hydroxylase(F3H)from Dendrobium officinale,which confers abiotic stress tolerance

Flavonoids are important bioactive components in Dendrobium officinale,a medicinal orchid.They are involved in many biological activities,including protecting plants against biotic and abiotic stresses.Research on the key genes related to flavonoid biosynthesis in D.officinale is limited.In this study,one of the key flavonoid biosynthesis genes,flavanone 3-hydroxylase(F3H),was characterized from D.officinale.The open reading frame of DoF3H was 1134 bp long and it encoded a 377-amino acid protein.The DoF3H protein showed considerably high homology with F3H proteins from other plant species and shared a common evolutionary ancestor with other F3Hs.DoF3H transcripts were detected in different organs of adult plants and mainly accumulated in flowers,followed by roots,stems and leaves,a pattern that was similar to the content of flavonoids.Recombinant DoF3H protein,which was localized in the cytosol,could convert naringenin to dihydrokaempferol.The mRNA levels of DoF3H were significantly induced by salt and cold stresses.Furthermore,the heterologous expression of DoF3H in Escherichia coli conferred it higher tolerance to salt and cold stresses.These results provide insight into the molecular function of DoF3H in the biosynthesis of flavonoids,and provide a new application for improvement of abiotic tolerance in D.officinale.

Study on Mechanism of Dendrobium officinale Kimura et Migo in Preventing and Treating Exercise-induced Muscle Damage(EIMD)in Rats

[Objectives]This study was conducted to observe the mechanism of Dendrobium officinale Kimura et Migo on gastrocnemius muscle in rats with exercise-induced muscle damage(EIMD).[Methods]The micro-injury model of skeletal muscle was established by treadmill training.Forty two SD rats were randomly divided into a control group,1,12 and 24 h exercise groups,D.officinale 2 ml+1 h exercise group,D.officinale 2 ml+12 h exercise group,and D.officinale 2 ml+24 h exercise group,with 6 rats in each group.Various D.officinale groups were given the drug once in the morning and once in the evening at a dose of 2 ml/time,a week in advance.Except for the quiet group,the samples were collected from the 1,12 and 24 h exercise groups after anesthesia following 1,12 and 24 h of exercise for the last time,respectively,and the D.officinale 2 ml+1 h exercise group,D.officinale 2 ml+12 h exercise group and D.officinale 2 ml+24 h exercise group were also sampled after anesthesia following 1,12 and 24 h of exercise for the last time,respectively.The contents of ATP,CK-MM and CK in rat serum were determined by enzyme-linked immunosorbent assay(ELISA).The histopathological changes of gastrocnemius muscle were observed by HE staining.PCR and Western-blot detection were carried out to analyze the effects of D.officinale on IGF-1 mRNA and protein expression in gastrocnemius muscle.[Results]Compared with the quiet group,the ATP contents in the serum of rats in the 1,12 and 24 h exercise groups significantly decreased(P<0.01),while the CK and CK-MM contents significantly increased(P<0.01).The expression of IGF-1 mRNA and protein in the gastrocnemius muscle tissue significantly increased(P<0.01).Compared with the 1 h exercise group,the ATP content and IGF-1 protein expression in the gastrocnemius muscle tissue of the D.officinale liquid+1 h exercise group significantly increased(P<0.05),while the CK and CK-MM contents significantly decreased(P<0.01).Compared with the 12 h exercise group,the D.officinale liquid+12 h exercise group showed a significant increase in ATP content(P<0.01),significant increases in IGF-1 mRNA and protein expression in the gastrocnemius muscle tissue(P<0.01),and significant decreases in CK and CK-MM contents(P<0.01).Compared with the 24 h exercise group,the ATP content and IGF-1 mRNA and protein expression in the gastrocnemius muscle tissue of the D.officinale liquid+24 h exercise group significantly increased(P<0.01),while the CK and CK-MM contents significantly decreased(P<0.01).From the pathological tissue morphology of the gastrocnemius muscle in rats with EIMD treated with D.officinale,it could be concluded that the gastrocnemius muscle of each exercise group was significantly damaged,and the damage was significantly alleviated after administration of D.officinale liquid.[Conclusions]The effects and mechanism of D.officinale on prevention and treatment of EIMD in rats might be related to the promotion of IGF-1 mRNA and protein expression in injured tissues by reducing ATP energy consumption,CK-MM and CK activity.

药蒲公英(Taraxacum officinale Weber)耐1．5％NaCl变异体的筛选及特性分析

为提高药蒲公英的耐盐性,用20～30 d大小的药蒲公英叶片诱导愈伤,获得的愈伤以NaCl作为选择因子,用直接筛选的方法,每3周进行一次继代培养,经3个月继代筛选获得了耐1.5%NaCl的药蒲公英愈伤组织,将耐1.5%NaCl的药蒲公英愈伤组织接种在分化培养基上分化出芽,之后将再生芽转接到生根培养基中进行生根培养,经4个月得到了12株耐1.5%NaCl的药蒲公英再生植株。与野生型相比,耐盐植株叶片宽大、叶柄粗短、叶表面覆盖白色细毛,根粗壮较短,花茎中部具2 cm左右的苞叶。RAPD(Random amplified polymorphic DNA,随机扩增的多态性DNA)和SDS-PAGE检测表明,耐盐植株与对照植株在DNA及蛋白水平上均存在明显差异。1.5%NaCl处理后,与普通再生植株相比,耐盐株系的抗氧化酶活性明显提高,脯氨酸含量上升幅度更为显著,而丙二醛(MDA)含量降低,其主要药用成分黄酮的含量显著增加。这些结果说明耐盐植株的抗氧化防御能力明显增强。以上结果表明耐1.5%NaCl的药蒲公英再生植株为耐1.5%NaCl药蒲公英变异体,这些耐盐变异体有望成为抗盐耐海水蔬菜家族的新成员。同时,这些耐盐变异体植株比普通植株具有更高的医用商业价值。耐1.5%NaCl的药蒲公英再生变异体遗传稳定性的研究正在进行中。

耐盐药蒲公英(Taraxacum officinale Weber)愈伤组织筛选及生理生化特性分析

为获得耐1.5%NaCl的药蒲公英(Taraxacum officinale Weber)愈伤组织,以药蒲公英叶片外植体为材料诱导愈伤组织。以NaCl为选择因子,从愈伤组织直接筛选。在选择培养基上,大部分愈伤组织褐化死亡,个别褐化死亡的愈伤组织周围有少量新的细胞团长出,将其转接到新鲜的选择培养基上,每3周继代一次,经3个月继代筛选获得了耐1.5%NaCl的药蒲公英细胞团。以普通愈伤组织为对照,发现随着NaCl浓度升高,耐盐愈伤组织的相对生长率下降但显著高于对照;且随着盐胁迫处理时间延长持续升高,而普通愈伤组织对照几乎停止生长,说明耐盐愈伤组织具有相对稳定的耐盐性。在蛋白水平上,耐盐愈伤组织与对照愈伤组织差异明显,SDS-PAGE分析显示:耐盐愈伤组织比对照多出一条34kD大小的蛋白带,且30kD、18kD左右的蛋白带明显上调。相同处理条件下耐盐愈伤组织脯氨酸的增加幅度高于对照。盐胁迫条件下,耐盐愈伤组织的超氧化物歧化酶(Super oxidase dimutase,SOD)、过氧化物酶(Peroxidase,POD)和过氧化氢酶(Catalase,CAT)活性明显高于对照,且随着处理时间的延长和盐浓度的增加呈现升高的趋势,而对照则呈现先升高后下降的趋势。结果说明耐盐愈伤组织一方面通过小分子有机溶质如脯氨酸的方式调节其渗透平衡,另一方面还可通过提高抗氧化能力降低盐分造成的次级伤害。积累蛋白也可能是耐盐愈伤组织调节渗透平衡的一种方式。通过生理生化分析确定我们获得的耐盐愈伤组织为耐盐变异体。

Antibacterial effect of Allium sativum cloves and Zingiber officinale rhizomes against multiple-drug resistant clinical pathogens

Objective:To evaluate the antibacterial properties ot Allium sativum(garlic) cloves and Zingiber officinale(ginger) rhizomes against multi-drug resistant clinical pathogens causing nosocomial infection.Methods:The cloves of garlic and rhizomes of ginger were extracted with 95%(v/v) ethanol.The ethanolic extracts were subjected to antibacterial sensitivity test against clinical pathogens.Results:Anti-bacterial potentials of the extracts of two crude garlic cloves and ginger rhizomes were tested against five gram negative and two gram positive multi-drug resistant bacteria isolates.All the bacterial isolates were susceptible to crude extracts of both plants extracts.Except Enterobacter sp.and Klebsiella sp.,all other isolates were susceptible when subjected to ethanolic extracts of garlic and ginger.The highest inhibition zone was observed with garlic(19.4S mm) against Pseudomonas aeruginosa(P.aeruginosa).The minimal inhibitory concentration was as low as 67.00 μg/mL against P.aeruginosa.Conclusions:Natural spices of garlic and ginger possess effective anti-bacterial activity against multi-drug clinical pathogens and can be used for prevention of drug resistant microbial diseases and further evaluation is necessary.

Research Progress of Medicinal Secondary Metabolites and Gene Cloning of Dendrobium officinale

Dendrobium officinale is one of the most precious medicinal plants in China. Its main medicinal ingredients are its secondary metabolites. However,it has the characteristics of limited sources,low active ingredient content and high cost,limiting the use of D. officinale.Studying the network structure and rate-limiting steps of secondary metabolites of medicinal components of D. officinale,analyzing the secondary metabolic synthesis process,mastering the production rules of its medicinal components and carrying out gene cloning or biosynthesis,etc.are of great significance for the rational development and utilization of D. officinale resources. This paper briefly reviews the progress of the research on the secondary metabolites of D. officinale,including the detection and identification of metabolites and the identification and cloning of key metabolic enzymes.

Evaluation of androgenic activity of Zingiber officinale and Pentadiplandra brazzeana in male rats

Aim: Aqueous extracts of Zingiber officinale and Pentadiplandra brazzeana were tested for their possible androgenic activity in male Wistar rats. Methods: The aqueous extracts of the two plants were gavaged separately to 2 groups of rats at a similar dose of 600 mg.kg^1.day-1 for 8 days. At the end of the treatment, the animals were killed and the blood, testis, epididymis, seminal vesicles and prostate were collected for biochemical analysis. Results: The aqueous extract of Z. officinale significantly increased in the relative weight of the testis, the serum testosterone level, testicular cholesterol level and epididymal α-glucosidase activity. The aqueous extract of P. brazzeana significantly increased the weights of the testis, seminal vesicles and prostate. It also significantly increased the serum and testicular testosterone level. The fructose, α-glucosidase and cholesterol levels in P. brazzeanatreated rats were increased by 28%, 35% and 114%, respectively. Conclusion: The aqueous extracts of both P. brazzeana and Z. officinale have an androgenic activity, which seems to be more potent with P. brazzeana than with Z. officinale. (Asian J Androl 2002 Dec; 4: 299-301)

Two new compounds from Zingiber officinale

A new cyclic diarylheptanoid, 1,5-epoxy-3-hydroxy-1-（3-methoxy-4,5-dihydroxyphenyl）-7-（4-hydroxyphenyl）-heptane （1）, as well as a new monoterpene, 10-O-β-n-glucopyranosyl-hydroxy cineole （2） were isolated from the rhizomes of Zingiber officinale. The structures of compounds 1 and 2 were established based on their spectral data. In addition, the antioxidant activities of these compounds were also measured.

Densitometric HPTLC analysis of 8-gingerol in Zingiber officinale extract and ginger-containing dietary supplements,teas and commercial creams

Objective:To develop and validate a simple,accurate HPTLC method for the analysis of 8-gingerol and to determine the quantity of 8-gingerol inZingiber officinaleextract and gingercontaining dietary supplements,teas and commercial creams.Methods:The analysis was performed on 10×20 cm aluminium-backed plates coated with 0.2 mm layers of silica gel 60 F254(E-Merck,Germany)with n-hexane:ethyl acetate 60:40(v/v)as mobile phase.Camag TLC Scanner III was used for the UV densitometric scanning at 569.Results:This system was found to give a compact spot of 8-gingerol at retention factor(Rf) value of(0.39依0.04)and linearity was found in the ranges 50-500 ng/spot(r2=0.9987).Limit of detection(12.76 ng/spot),limit of quantification(26.32 ng/spot),accuracy(less than 2%)and recovery(ranging from 98.22-99.20)were found satisfactory.Conclusions:The HPTLC method developed for quantification of 8-gingerol was found to be simple,accurate,reproducible,sensitive and is applicable to the analysis of 8-gingerol in Zingiber officinaleextract and ginger-containing dietary supplements,teas and commercial creams.

Two New Diarylheptanoids from the Rhizomes of Zingiber officinale

Two new diaryheptanoids, (5S)-5-acetoxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3- heptanone (1) and (3S,5S)-3,5-diacetoxy-1,7-bis(4-hydroxy-3-methoxyphenyl)heptane (2) were isolated from the rhizomes of Zingiber officinale. Their structures were elucidated by spectral methods.

Investigation on the Morphological Characteristics of Dendrobium officinale Plantlets Propagated from Different Explants

[ Objective] This study aimed to investigate the differences in morphological characteristics of Dendrobium officinale plantlets propagated from different explants. [ Method] Randomly 1 000 D. offtcinale plantlets propagated via different regeneration pathways were selected for morphological investigation and classification. [ Result] D. officinale plantlets propagated from stem segment explants exhibited highly consistent morphological characteristics, while those propagated from seed explants exhibited a variety of morphological characteristics. [ Conclusion] Therefore, using seed explants for regeneration can effectively broaden the germplasms resources of D. officinale.

Evaluation of Heavy Metals and Pesticide Residues in Dendrobium officinale Produced in Guangxi

[Objectives]To establish a method for the determination of heavy metals and pesticide residues in Dendrobium officinale produced in Guangxi and its culture substrate.[Methods]The contents of five heavy metals(Pb,Cd,As,Hg and Cu)were determined by the atomic absorption spectrophotometric(AAS)method;residues of 9 organochlorine pesticides(α-BHC,β-BHC,γ-BHC,δ-BHC,p,p′-DDT,p,p′-DDE,p,p′-DDD,o,p′-DDT and pentachloronitrobenzene)were determined by gas chromatography(GC).[Results]The contents of five heavy metals in the medicinal materials of D.officinale produced in Guangxi were 0.96,0.21,0.021 mg/kg,and not detected,and 0.35 mg/kg.The contents of five heavy metals in the culture substrate were 0.03,0.19,0.21,0.004 and 1.80 mg/kg,respectively;the content of 9 organochlorine pesticide residues in the sample was not detected.[Conclusions]The detection results of heavy metal content and organochlorine pesticide residues in D.officinale produced in Guangxi and its substrate are consistent with the limit requirements of the Chinese Pharmacopoeia.This experiment is expected to provide reliable data reference for ensuring the safety of the drug resources of D.officinale in Guangxi.

Optimization of Rooting Medium for Tissue Culture Seedlings of Dendrobium officinale by Response Surface Methodology

[Objectives] To increase the survival rate of tissue culture seedlings of Dendrobium officinale,and optimize the conditions of rooting medium by the response surface methodology( RSM). [Methods]The effects of 6-BA concentration,NAA concentration,potato amount and the amount of mashed banana on the growth of seedlings were determined by single factor experiment and were analyzed by Box-Behnken design and response surface methodology. [Results]The optimal culture conditions: rooting medium 1/2 MS + 6-BA 0. 24 mg/L + mashed banana 87. 63 g/L + potato 89. 30 g/L + NAA 0. 52 mg/L + sucrose 20. 0 g/L + activated carbon 4. 0 g/L + agar 7. 0 g/L,p H 5. 8,and light intensity 2 000 Lx. [Conclusions]The model established by response surface methodology has a good predictability and could be used to optimize the conditions of tissue culture and rooting medium of D. officinale.

The bibenzyl derivatives of Dendrobium officinale prevent UV-B irradiation induced photoaging via SIRT3

Dendrobium officinale is a valuable medicinal herb that is widely used in traditional Chinese medicine.The chemical constituents of D.officinale have attracted much attention and a large number of compounds have been reported including many bibenzyl derivatives.13 bibenzyl derivatives from D.officinale were sent for molecular docking,sur-face plasmon resonance(SPR)assay and after detection of Mn-SOD and SIRT3 activities in or not in HaCaT cells,it was concluded that bibenzyl derivatives did not directly activate Mn-SOD but promoted SIRT3 proteins.In addition,HaCaT cells were irradiated with UV-B to induce an oxidative stress model in vitro to further verify the effect of bibenzyl derivatives.The results show that bibenzyl derivatives could directly bind to SIRT3,enhance the deacetylation and then activate Mn-SOD,so as to protect UV-B induced skin photoaging.

Comparative Study on Effects of Rheum officinale and Wine Prepared Rheum palmatum Dachengqi Decoction on Laxative Effect of Mice

[Objectives] To study the differences in the effects of Rheum officinale and wine prepared Rheum palmatum Dachengqi Decoction(DCQ) on laxative effect of mice.[Methods]First,72 mice were randomly divided into positive control group,blank control group,6 and10 g/kg dose R.officinale DCQ groups,6 and 10 g/kg dose wine prepared R.palmatum DCQ groups.After administration at the dose of20 m L/kg,metabolic cage method and carbon powder propulsion were used to conduct Na+-K+-ATPasc activity experiment,and the comparison was made for the differences in the normal laxative effect of R.officinale and wine prepared R.palmatum on DCQ,the carbon powder propulsion rate,and Na+-K+-ATPasc activit.[Results]There are differences in the laxative effect of R.officinale and wine prepared R.palmatum on DCQ,but their differences in changes are,to some extent,connected with R.officinale and wine prepared R.palmatum.From comparison of R.officinale DCQ group,wine prepared R.palmatum DCQ group,blank control group,and positive control group,it is found that there are differences in the normal laxative effect of normal mice,carbon powder propulsion,and inhibition of Na+-K+-ATPase activity.In addition,the comparison of drug administration groups indicates that the differences in the effects of R.officinale DCQ group are most significant,and there are significant differences in the 6 and 10 g/kg groups.[Conclusions]There are certain differences in the influence of R.officinale and wine prepared R.palmatum Dachengqi Decoction on normal laxative effect of mice,which may be connected with the fact that changes in main chemical components of king drugs R.officinale and wine prepared R.palmatum lead to changes in the dissolution amount of anthraquinones in the compound Dachengqi Decoction.

Antimicrobial Activity of Essential Oil of <i>Zingiber officinale</i>Roscoe (Zingiberaceae)

Zingiber officinale, commonly known as ginger, is a species native of tropical Asia. It is grown and used in tropical countries of America, including Mexico, where it is used for the treatment of throat infections. The aim of this study was to determine the chemical composition of essential oil of Z. officinale rhizome and evaluate their antimicrobial activity. The essential oil was obtained by hydrodistillation, and the chemical composition of the oil was determined by GC-MS. To determine the antimicrobial activity diffusion, agar dilution and radial growth inhibition methods were employed. Microbial growth curves were performed on the strains most susceptible. It was identified 28 compounds in the essential oil, the most abundant were eudesmol (8.19%), γ-terpinene (7.88 %), a-curcumene (7.28%), alloaromadendrene (6.56%), zingiberene (6.06 %), α-pinene (5.76 %), δ-cadinene (3.84%), elemol (3.39%), farnesal (3.45%), E-β-farnesene (3.57%), neril acetate (2.8%) and β-myrcene (2.94%). In antimicrobial activity, the essential oil inhibited the growth of 15 bacterial strains, 3 of yeast fungi and 4 of mycelia fungi. The most susceptible strains were S. aureus FES-I (MIC = 0.25 mg/mL) S. epidemidis FES-C (MIC = 0.5 mg/mL), E. faecalis ATCC 14506 (MIC = 1.0 mg/mL), C. tropicalis (CMI = 0.125 mg/mL) and T. mentagrophytes (CF50 = 0.08 mg/mL). In the microbial growth curves the essential oil showed bactericidal effect on S. aureus FES-I and S. marcescens ATCC 14756 from the first hour of exposure of the strains to oil, eliminating 99.9% of CFU in concentrations of 0.5 and 0.75 mg/mL respectively. The results validate the medicinal use of Z. officinale in the treatment of diseases of possible infectious origin.

Correction:Haplotype-resolved genome of diploid ginger (Zingiber officinale)and its unique gingerol biosynthetic pathway

After online publication of the article 1,the authors noticed the affiliation“College of Landscape Architecture and Life Science/Institute of Special Plants,Chongqing University of Arts and Sciences,Yongchuan,Chongqing,China”for author Yiqing Liu was missing.

Physiological Responses of Dendrobium officinale under Exposure to Cold Stress with Two Cultivars

This study aimed to explore the cold tolerance of two cultivars of Dendrobium officinale(MG1,MG2)grown in different regions of China.Under-2℃ incubation,cultivar MG1 remained active after 3 d,and continued to grow after returning to room temperature.However,MG2 could only maintain its activity after 2 d treatment at−2℃,and the seedlings died with the low temperature treatment time.Investigation of the characteristics of the plants grown in the south(Hangzhou)or north(Zhengzhou)of China indicated that the leaves of MG1 also had reduced stomatal density,the highest thickness,and a compact microstructure.The contents of proline and soluble sugars were higher in MG1 than those in MG2.The cultivar MG1 had higher SOD enzyme activity than MG2,while CAT and POD activities in samples from Zhengzhou were higher than those from Hangzhou.The contents of polysaccharides and alkaloids in stems of in MG1 were higher than those in MG2,while the content of flavonoids in the Zhengzhou samples was higher than that in the Hangzhou samples.In addition,plant heights,stem diameters,and chlorophyll content were higher in MG1.Overall,MG1 had better cold resistance than MG2.MG1 is a cold tolerant cultivar with thick leaves and reduced stomatal density,higher contents of soluble sugars,proline,CAT,POD,polysaccharides,flavonoids and alkaloids,which together make it more adaptable to low temperatures.Thus,the cultivar MG1,with its demonstrated cold tolerance,can accordingly be grown on a large scale in cold regions,thereby expanding the available planting area for this important traditional medicinal plant to meet the increasing commercial demand for it.

Evaluation of Various Crude Extracts of <i>Zingiber officinale</i>Rhizome for Potential Antibacterial Activity: A Study <i>in Vitro</i>

In vitro antibacterial activity of crude aqueous and organic extracts of rhizome of Zingiber officinaleRoscoe (ginger) was studied against both Gram-negative (Escherichia coli and Salmonella typhi) and Gram-positive (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus and Streptococcus pyogenes) bacterial strains. The present study reveals that the pattern of inhibition varied with the solvent used for extraction and the organism tested. Plant extracts prepared in organic solvents provided more consistent antibacterial activity as compared to aqueous extracts. Methanol extract was the most active against maximum number of bacterial species tested. Gram-positive bacteria were found the most sensitive as compared to Gram-negative bacteria. Staphylococcus aureus was significantly inhibited by almost all the extracts even in very low MIC followed by other Gram-positives. Escherichia coli (a Gram-negative bacterium) was showing the least inhibition with highest MIC values, while Salmonella typhi was found completely resistant. Methanol extract yielded the presence of terpenoids, flavonoids, alkaloids and tannins in phytochemical screening. Results of the present study sign the interesting assurance of designing a potentially active antibacterial agent from Zingiber officinale.

密蒙花(Buddlija officinalis)基因转化受体系统的建立

以密蒙花(BuddlijaofficinalisMaxim)芽、叶、茎段为外植体,在B5,MS,White培养基上获得愈伤组织.结果表明:芽、幼叶脱分化能力差异不大;不同时期的叶片差异较大,以幼叶诱导为佳;作者筛选出的愈伤组织最佳培养基为B5+6 BA0.5mg/L+2,4 D0.5mg/L.在继代培养阶段,B5比MS和White更适合细胞的快速生长和繁殖;并且以叶片的愈伤组织生长较快,芽、茎段次之.最佳继代培养基为B5+6 BA0.5mg/L+NAA0.5mg/L.继代的愈伤组织可在B5+6 BA1mg/L+NAA0.1mg/L+GA0.1mg/L培养基中分化出幼苗,在生根培养基1/2MS+NAA0.2mg/L或1/2MS+NAA0.6mg/L中分化出根且获得再生植株.将长4～6cm的再生小苗练苗3d后移植,幼苗成活率达100%,并于当年开花.