Camptothecin and 10-Hydroxycamptothecin Accumulate Differentially Under Specific Developmental Control in Camptotheca acuminata

Camptotheca acuminata Decaisne is a tree unique to China, producing terpenoid indole alkaloid (TIA), camptothecin (CPT), and 10-hydroxycamptothecin (HCPT), which possesses strong anti-cancer bioactivity. The dynamics of HCPT and CPT during seed maturation, seed germination, postgerminative development and daily growth were measured by High Performance Liquid Chromatography (HPLC). This article provides some evidence for presenting the theory that each characteristic of HCPT and CPT accumulation is under developmental regulation and then attempt to elucidate the metabolic relationships between them. HCPT accumulates particularly in young tissues and organs and is relatively unstable, while the distribution of CPT is more ubiquitous and stable. Their different distribution properties suggest that HCPT and CPT are differentially regulated and play distinct roles:during development.

Different Responses of Camptothecin and 10-Hydroxycamptothecin to Heat Shock in Camptotheca acuminata Seedlings

Plants interact with their environment by producing a diverse array of secondary metabolites, one of which is alkaloid. In this study, alkaloids, including camptothecin (CPT) and 10-hydroxycamptothecin (HCPT), malondialdehyde (MDA) and chlorophyll contents were measured during heat shock in seedlings of Camptotheca acuminata Decaisne unique to China. Responses of different tissues, including young leaves, old leaves, buds and barks, to heat shock were examined in alkaloid changes. CPT and HCPT concentrations reached their peak values separately at 38 degreesC and 40 degreesC, which were below the lethal heat-shock temperature indicated by MDA and chlorophyll, and their great changes took place in young leaves. These results indicated that CPT and HCPT were involved in the C acuminata resistance against heat shock from its environment. Furthermore, plant rigidly observed the cost-benefit principle and mobilized and allocated limited alkaloid sources to young and reproductive tissues preferentially. In addition, HCPT displayed well-regulated changes during incubation at sublethal temperature, and this indicated that HCPT might play a more positively defensive role in enhancement of plant thermotolerance than CPT does.

Effect of arbuscular mycorrhiza on the growth of Camptotheca acuminata seedlings

Camptotheca acuminata seeds were sown in sterilized sands in the greenhouse in February of 2005. After 90-day growth, seedlings were inoculated with three species of arbuscular mycorrhizal fungi （AMF）, Acaulospora mellea, Glomus diaphanum and Sclerocystis sinuosa.. The height, biomass, and absorptions of nitrogen and phosphorus of C. acuminata seedlings inoculated with AMF were investigated. The results showed that the formation of AM promoted the height growth and biomass accumulation of seedlings significantly and improved the absorption of phosphorus in seedlings. The height and biomass of mycorrhizal seedlings were 1.2 and 1.6 times higher than those of the non-mycorrhizal seedlings. The absorption of nitrogen was less influenced by the formation of AM. The nitrogen content in mycorrhizal seedling was equal to that of non-mycorrhizal seedlings. Compared with non-mycorrhizal seedlings, the nitrogen content of mycorrhizal seedlings inoculated with A. mellea changed considerably in the root, stem and leaves. The difference in nitrogen content was not significant between mycorrhizal seedlings inoculated with G. diaphanum and S. sinuosa. The AM formation stimulated the absorption of phosphorus, especially in roots, and also changed the allocation of nitrogen and phosphorus in different organs of seedlings. Compared with non-mycorrhizal seedlings, the ratio of nitrogen and phosphorus in mycorrhizal roots increased, but reduced in stem and leaves.

Homogenate extraction technology of camptothecine and hydroxycamptothecin from Camptotheca acuminata leaves

Camptothecine （CPT） and hydroxycamptothecin （HCPT）, two kinds of anti-cancer alkaloids, were extracted from Camptotheca acuminata leaves using homogenate extraction technology under different conditions such as the ratio of material to liquid, ethanol concentration, and homogenate time. The optimum technology parameters for homogenate extraction of CPT and HCPT from C acuminata leaves were determined as homogenate time at 8 rain, ethanol concentration at 55% and the ratio of material to liquid at 1：15 （g：mL）. By using the optimized parameters, we obtained 0.639‰ extraction rate for CPT and 0.437‰ for HCPT. The extraction yields of CPT and HCPT extracted by homogenating technology were higher than those by other extractive methods, such as ultrasonic, reflux, shaking in water bath. It is concluded that the homogenate extraction technology was an efficient method for extracting CPT and HCPT from C acuminata leaves, with characteristics of less extraction time and high yield.

Determination of contents of 10-Hydroxycamptothecin in Camptotheca acuminata by high-performance liquid chromatogram

The determination method of 10-hydroxycamptothecin in Camptotheca acuminata fruits by high-performance liquid chromatogram (HPLC) was studied. The HPLC analysis was performed on a HIQ sil C18(4.6×250 mm) column with mobile phase of acetonitrilewater (3:7, V:V), flow rate 1 mLmin-1 and UV detective wavelength 266 nm. Extracting 10-hydroxycamptothecin by ultrasonic method from fruits of C. acuminata to prepare samples for analysis was systematically discussed. The optimal extraction condition was carried out by 60% alcohol solution at 60℃ for 50 minutes.

Nitrate reductase activity and its diurnal variation rhythm for Camptotheca acuminata seedlings

Nitrate reductase activity (NRA) in different plant organs and leaves in different positions of Camptotheca acuminata seedlings was determined by an In vivo assay, the diurnal variation rhythm of NRA in leaves of different positions was observed,and the correlations between leaf NRA, leaf area and lamina mass per unit area (LMA) were also examined. The results showed that NRA in the leaf was significantly highest, compared with that in other organs such as roots, stems and leaves. In this experiment, the 10 leaves were selected from the apex to the base of the seedlings in order. The different NRA occurred obviously in leaves of different positions of C. acuminata seedlings from the apex to the base, and NRA was higher in the 4th-6th leaves.The diurnal change rhythm of leaf NRA showed a one peak curve, and maximum NRA value appeared at about midday (at 12:30 or so). No obvious correlations between NRA and leaf area or lamina mass per unit area were observed. This study offered scientific foundation for the further research on nitrogen metabolism of C. acuminata.

Assessment of factors affecting in vitro shoot regeneration from axillary bud explant of Camptotheca acuminata

Axillary buds from 3-yr.-old seedlings of Camptotheca acuminata in the greenhouse were cultured on the different basal media with different concentrations of growth regulators for shoot regeneration for studying the effects of different basal media, different concen- trations of growth regulators (BA or TDZ), sucrose, agar and pH value on shoot regeneration from axillary bud. The results showed that B5 and WPM media were the optimal basal media and the optimal phyotohormone was BA of 1.0 mg/L or TDZ of 0.1mg/L; The concentrations of sucrose of 30g/L and agar of 6g/L were most suitable for the shoot regeneration; pH value from 5.8 to 6.6 were broadly effective, but the best at pH 5.8.

Effect of planting density on plant growth and camptothecin content of Camptotheca acuminata seedlings

C. acuminata seedlings cultivated in greenhouse were transplanted into the fields with 5 designed planting densities (11, 16, 25,44 and 100 plants·m^-2) in May of 2004 and were harvested in the middle of September of 2004. The seedling growth indexes including plant height and crown width, biomass allocation, camptothecin (CPT) content and CPT yield of different organs (young leaf, old leaf, stem,and root) were studied. For the 5 selected planting densities, the plant biomass, height, crown width, and total leaf area of C. acuminata seedlings all showed highest values at the planting density of 25 plants ·m^-2. CPT content in young leaves was higher than that in other organs of seedlings and presented an obvious change with the variation of planting densities and with the highest value at density of 100plants·m-2, while for other organs no significant variation in CPT content was found with change of planting density. The accumulation of CPT was enhanced significantly at the planting density of 25 plants·m^-2. It is concluded that for the purpose to get raw materials with more CPT from C. acuminata, the optimal planting density of C. acuminata seedlings should be designed as 25 plants·m^-2.

The effects of different nitrogen sources on camptothecin content and related gene expression in Camptotheca acuminata seedlings

Camptotheac acuminata Decne is a unique tree species in China with an important secondary metabolite,camptothecin(CPT),used in the treatment of cancer.Nitrogen(N)is an important element that affects plant growth and the accumulation of CPT.Reports on the effect of N on CPT synthesis from a genetic perspective are scarce.To explore the effects of different N sources and levels on CPT synthesis in C.acuminata,two-year-old seedlings were fertilized with different concentrations of pure ammonium sulphate,source of ammonium N(NH4+-N),and potassium nitrate for nitrate N(NO3--N).Concentrations of 2.5,5,7.5,and10 g pot-1 NH4+-N and NO3--N were used.The results showed that 7.5 g NH4+-N and NO3--N treatments were best for growth and fresh weight of leaves.Compared with the other treatments,the CPT content,tryptophan synthase and tryptophan decarboxylase activities,and expression of the CaTSB and CaTDCl genes under the 2.5 g NH4+-N and NO3--N treatments peaked significantly at 30 days.However,the expression of CaTDC2 surpassed that of the other two genes at 60 days.Therefore,compared with NH4+-N source,the NO3--N source was more beneficial for growth,and NO3--N was better for CPT yield.Consequently,leaves of C.acuminata treated with 2.5 g NO3--N could be harvested after 30 days to obtain maximum CPT content.CaTDC1 is more closely linked to CPT synthesis.The results of this study improved the production of CPT in C.acuminata via fertilization.

Optimization of ultrasound-assisted extraction of camptothecin from Camptotheca acuminata seeds

Naturally occurring camptothecin（CPT） is an important source of chemotherapeutic agents.The extraction from Camptotheca acuminata is still the main approach to obtain CPT compared with total synthesis.In the present study,ultrasound-assisted extractions（UAE） of CPT from C.acuminata seeds with alkaline solutions were investigated and CPT yield were determined by High Performance Liquid Chromatography.The conditions of alkaline species and concentrations,extraction time,extraction temperature and ultrasonic power were optimized.Results show that both Na3PO4 and Na2CO3 solutions gain good extraction yields,whereas Na3PO4 solution has stronger basicity and need higher concentration than Na2CO3 solution does,thus aqueous Na2CO3 is more beneficial for the extraction.The optimal condition was ultrasonically extracted with 0.5% aqueous Na2CO3 at 50°C and ultrasonic power of 400 W for 60 min.Comparing with UAE with ethanol,the extraction with 0.5% Na2CO3 solution achieves higher yield.Moreover,aqueous Na2CO3 as a solvent has various advantages including non-toxicity,inflammable,non-corrosive and low cost,which ensure this UAE method is a superior method with high utilizing prospect.

Camptotheca acuminata Decne Residue after Camptothecin Extract as a Substrate to Produce Mushroom Spawn

Currently in China,no technically and economically viable methods exist to handle large quantities of Camptotheca acuminata Decne residue(CA residue) after camptothecin extract while there is a great demand for low cost alternatives to replace the cottonseed hull-based materials used in commercial mushroom culture.Hence,it is of importance for camptothecin extract factories and mushroom producers to explore the utilization of CA residue in mushroom industry.We conducted a research to study how partially or completely substituting traditional mushroom substrate by CA residue would influence the mycelial growth in mushroom spawn production.5 mushroom strains from 4 species were used in the test,i.e.,oyster mushroom(Pleurotus ostreatu) strains,Zayou No.1 and Xide 33,needle mushroom(Flammulina velutipes) strain Chuanjin No.3,hairy wood ear(Auricularia polytricha) strain Huang Er No.10,and shiitake(Lentinula edodes) strain Wuxiang.The nutrient element composition and heavy metal contents of CA residue were determined to ensure its safety and to determine its appropriate component in the substrate formulation for mushroom spawn production.The four substrate formulations(one control-CK,and three treatments,named,T1,T2,T3,) contained 0%,40%,79%,and 100% CA residue,respectively,to allow comparison of the fungal mycelial growth.The control(CK) was the popularly used formulation in Chinese commercial mushroom production,comprising of 73% cottonseed hulls,10% sawdust,15% wheat bran,1% lime,1% white sugar(percentage by weight).All mushroom spawns of the five strains in the four treatments were incubated under the same conditions.The results showed that mycelia of the five mushroom strains grew significantly faster on the substrates containing CA residue than on the substrate with no CA residue(CK).There were no significant differences in the mycelial growth rate among treatments containing CA residue for the two oyster mushrooms and the needle mushroom,but mycelial growth rate in treatments T2 and T3 was significantly higher than in treatment T1 for hairy wood ear and shiitake.The results suggest that CA residue can be used to culture oyster mushroom,needle mushroom,hairy wood ear,and shiitake spawn,and the medium containing CA residue can stimulate their mycelial growth.The commercial production of mushroom spawn using CA residue not only brings better economical benefits including lower cost to mushroom producers,but also reduces environmental pollution by providing a means to reduce dumping and piling of CA residue.

Effects of medium nutrition on cell growth and isocamptothecin A and B production by suspension cell culture of Camptotheca acuminata

The effects of initial sucrose concentration, nitrate to ammonium ratio, total N concentration and phosphate concentration in medium on cell growth and isocamptothecin A and B synthesis by suspension call culture of Camptotheca acuminata were investigated in 250 mL shake flasks. 30 g L^-1 sucrose concentration was beneficial to secondary metabolites synthesis. The cell growth and metabolites synthesis were also affected by the ratio of NO3^-/NH4^＋ , and nitrate was tavourable for cell growth. The maximum dry weight was achieved when nitrate was used as the sole N souree. The effect of total initial N on the cell cultures was also investigated with NO3^-/NH4^＋ ratio of 1 ： 2. The final dry cell weight was similar throughout culture period and 50 mM initial N was favourable for secondary metabolite synthesis. 50 mM initial phosphate concentration facilitated both cell growth and secondary metabolites synthesis.

Homogenate extraction technology of camptothecine and hydroxycamptothecin from Camptotheca acuminata leaves

Camptothecine(CPT) and hydroxycamptothecin(HCPT), two kinds of anti-cancer alkaloids, were extracted from Camptotheca acuminata leaves using homogenate extraction technology under different conditions such as the ratio of material to liquid, ethanol concentration, and homogenate time.The optimum technology parameters for homogenate extraction of CPT and HCPT from C.acuminata leaves were determined as homogenate time at 8 min, ethanol concentration at 55% and the ratio of material to liquid at 1:15(g:mL).By using the optimized parameters, we obtained 0.639‰ extraction rate for CPT and 0.437‰ for HCPT.The extraction yields of CPT and HCPT extracted by homogenating technology were higher than those by other extractive methods, such as ultrasonic, reflux, shaking in water bath.It is concluded that the homogenate extraction technology was an efficient method for extracting CPT and HCPT from C.acuminata leaves, with characteristics of less extraction time and high yield.

Agrobacterium-mediated genetic transformation of Camptotheca acuminata

UGPase gene related with wood cellulose synthesis was transferred into C. acuminata using the method of Agrobacte- rium-mediated genetic transformation, and an efficient transformation system was developed for C. acuminata on the basis of evaluations of several factors affecting Agrobacterium-mediated DNA transfer rate. The highest transformation rate was achieved when pre-cultttred leaf explants were infected with an Agrobacterium culture corresponding to OD600 （0.5） for 10 min, and cultured on explant regeneration medium for three days. The results of Southern hybridization showed that genomic DNA of the kanamycin-resistant shoots to an UGPase gene probe substantiated the integration of the transgene. Transformation efficiency （6%） was achieved under the optimized transformation procedure, This system should facilitate the introduction of important useful genes into C, acuminata.

Genome-wide identification and analysis of AP2/ERF transcription factors related to camptothecin biosynthesis in Camptotheca acuminata

Camptotheca acuminata produces camptothecin(CPT),a monoterpene indole alkaloid(MIA)that is widely used in the treatment of lung,colorectal,cervical,and ovarian cancers.Its biosynthesis pathway has attracted significant attention,but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor(AP2/ERF)transcription factors(TFs)remains unclear.In this study,a systematic analysis of the AP2/ERF TFs family in C.acuminata was performed,including phylogeny,gene structure,conserved motifs,and gene expression profiles in different tissues and organs(immature bark,cotyledons,young flower,immature fruit,mature fruit,mature leaf,roots,upper stem,and lower stem)of C.acuminata.A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies,including AP2(26 genes),DREB(61 genes),ERF(92 genes),RAV(18 genes),and Soloist(one gene).The combination of gene expression patterns in different C.acuminata tissues and organs,the phylogenetic tree,the co-expression analysis with biosynthetic genes,and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C.acuminata might be involved in CPT synthesis regulation,which exhibit relatively high expression levels in the upper stem or immature bark.Among these,four genes(Cac AP2/ERF123,Cac AP2/ERF125,Cac AP2/ERF126,and Cac AP2/ERF127)belong to the ERF–B2 subgroup;two genes(Cac AP2/ERF149 and Cac AP2/ERF152)belong to the ERF–B3 subgroup;and two more genes(Cac AP2/ERF095 and Cac AP2/ERF096)belong to the DREB–A6 subgroup.These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C.acuminata.

Characteristics of the microbial community in rhizosphere of Camptotheca acuminata cultured with exotic invasive plant Eupatorium adenophorum

The traditional culture-dependent plate counting and culture-independent small-subunit-ribosomal RNA gene-targeted molecular techniques, Single-Strand Conformation Polymorphism (SSCP) and ter-minal Restriction Fragment Length Polymorphism (tRFLP) combined with 16S rDNA clone library were adopted to investigate the impacts of secretion from Camptotheca acuminata (abbreviated to Ca) roots on the quantities and structure of eukaryotic microbes and bacteria in the rhizosphere, and the possi-bility that Ca controls exotic invasive plant Eupatorium adenophorum (Ea). The counting results indi-cated that the number of bacteria increased in turn in rhizospheres of Ea, Ca-Ea mixed culture and Ca, while that of eukaryotic microbes decreased. PCR-SSCP profiles showed eukaryotic microbial bands (corresponding to biodiversity) in rhizosphere of Ea were more complex than those of Ca and CE. Meristolohmannia sp., Termitomyces sp. and Rhodophyllus sp. were the dominant populations in the rhizosphere of Ca. Bacterial terminal restriction fragments (TRFs) profiles showed no difference among three kinds of rhizospheres, and the sequences of the 16S rDNA clone library from Ca rhizospheres were distributed in 10 known phyla, in which phylum Proteobacteria were the absolute dominant group and accounted for 24.71% of the cloned sequences (δ-Proteobacteria accounted for up to 17.65%), and phyla Acidobacteria and Bacteroidetes accounted for 16.47% and 10.59% of the cloned sequences, respectively. In addition, high performance liquid chromatography detected a trace amount of camp-tothecin and hydroxycamptothecin in the rhizospheric soil of Ca and CE, but examined neither camp-tothecin nor hydroxycamptothecin in rhizospheric soil of Ea. Therefore, invasion and diffusion of Ea evidently depended on distinguishing the eukaryotic community structure, but not on that of the bac-terial pattern. Ca was able to alter the eukaryotic community structure of invasive Ea by secreting camptothecin and hydroxycamptothecin into rhizospheres, and may benefit the control of overspread of Ea. This study provided theoretical evidence for rhizospheric microbial aspects on substituting Ca for Ea.

喜树中一株特异产喜树碱内生真菌Aspergillus niger GP3的分离与鉴定

目的:从喜树内生真菌中筛选出具有产喜树碱潜力的菌株,为获得喜树碱提供一条新的药源途径。方法:从喜树根皮分离内生真菌,综合利用高效液相色谱法(HPLC)以及超高效液相色谱-高分辨质谱(UPLC-Orbitrap-MS/MS)法对内生真菌次生代谢产物有效活性成分进行分析,并通过形态学和分子学对其特异性真菌进行鉴定。结果:内生真菌GP3具有产喜树碱的化学性质,其菌落圆形、黑褐色、中心凸起、边缘呈纤毛状,经ITS序列比对,该菌与Aspergillus niger ITS序列相似性为99%。结论:GP3是一株具有产喜树碱潜力的内生真菌菌株,鉴定为黑曲霉菌(Aspergillus niger)。

镉胁迫下γ-氨基丁酸对喜树幼苗光合色素含量和抗氧化酶活性的影响

为提高喜树(Camptotheca acuminata)对镉(Cd)胁迫的抗性,为镉污染地区的喜树栽培提供参考,采用盆栽试验,研究Cd胁迫下γ-氨基丁酸(GABA)对喜树幼苗光合色素含量和抗氧化酶活性的影响。结果表明,Cd处理(1 mg/L)降低了喜树幼苗的叶绿素a、叶绿素b和类胡萝卜素含量及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性。Cd胁迫下,随GABA浓度增加,喜树幼苗的叶绿素a、叶绿素b和类胡萝卜素含量及SOD、CAT和POD活性均呈上升趋势,GABA浓度为2 mmol/L时均最大。相关性分析表明,Cd胁迫下施加GABA,光合色素含量与抗氧化酶活性均呈极显著正相关。因此,GABA能提高Cd胁迫下喜树幼苗的抗性,缓解Cd胁迫对喜树幼苗的毒害。

外源γ-氨基丁酸对喜树镉积累的影响

为了降低喜树对镉的积累,本研究采用盆栽试验,研究了外源γ-氨基丁酸(GABA)对喜树镉积累的影响。结果表明,浓度为1 mg·L^(-1)的镉处理降低了喜树生物量,对喜树产生了胁迫。在镉胁迫条件下,浓度为0.1 mmol·L^(-1)和0.5 mmol·L^(-1)的GABA提高了喜树植株生物量,并提高了根冠比。就镉积累而言,不同浓度0.1、0.5、1.0和2.0 mmol·L^(-1)的GABA均降低了喜树根系和地上部分镉含量、镉积累量和富集系数。浓度为0.1 mmol·L^(-1)的GABA的降镉作用最好,使喜树根系和地上部分镉含量分别较对照降低了31.77%和62.82%。此外,不同浓度的GABA在一定程度上降低了喜树的转运系数。因此,外源GABA能够在一定程度上促进镉胁迫下喜树的生长,并降低喜树对镉的积累,其中,浓度为0.1 mmol·L^(-1)的GABA降镉效果最好。

福建省明溪县极小种群野生植物喜树种群结构与动态特征

喜树(Camptotheca acuminata)为中国特有种,也是中国120种极小种群野生植物之一。在第二次全国重点保护野生植物资源调查中,发现福建省明溪县角溪区域有喜树种群野生分布点。为了探讨该区域喜树种群的生存状况,基于样地调查数据,以径级代替龄级,编制种群静态生命表并引入4个生存分析函数,分析种群数量特征;利用种群动态变化指数和时间序列模型对种群动态和未来发展趋势进行分析和预测。结果表明,(1)喜树种群龄级结构呈倒“J”型,各龄级均有个体分布,其中小树个体数量最多,幼苗、幼树存储量不足;种群动态指数在相邻龄级间存在一定的波动,Vpi和Vpi′的值均大于零,表明该种群当前属于增长型,但种群对外界干扰比较敏感,抗干扰能力较差。(2)静态生命表显示,喜树种群存活数量和个体生命期望值都随着龄级的增加逐渐下降;种群存活曲线趋近于Deevey-Ⅱ型,前期死亡率下降较为迅速,而后期死亡率下降趋于平缓;死亡率和消失率曲线的变化趋势基本一致,均在第Ⅱ、Ⅷ龄级出现峰值。(3)生存分析表明,该种群具有前期迅速下降、中期小幅波动、后期逐渐衰退的变化特点。(4)时间序列分析预测,在经历未来2个龄级时间后,Ⅱ龄级和Ⅴ龄级个体数量均出现不同程度的减少;而经历未来4、6、8个龄级时间后,所有龄级个体数量均呈增长趋势。人为干扰、自然干扰、种子成苗率低是影响该地区喜树种群增长的主要因素。在今后的保护工作中,建议采取就地保护、近地保护和加强宣传等拯救措施,保护和改善生境,促进喜树种群的更新和发展。