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.     

优化喜树果中喜树碱和10-羟基喜树碱的匀浆提取工艺

采用匀浆提取的方法从喜树果中提取喜树碱和10-羟基喜树碱，考察乙醇体积分数、匀浆时间和液料比等因素对提取率和纯度的影响，并通过响应面法进行工艺优化，分别得到以喜树碱、10-羟基喜树碱的提取率和纯度为响应值的回归方程，进而获得最佳工艺条件为乙醇体积分数52．41％；匀浆时间2．93min；液料比11．11（mL：g）。最佳条件下的验证实验表明：喜树碱提取率为0．0923％，浸膏中喜树碱的纯度为1．0633％，与模型值相对偏差分别为0．75％和0．88％；10-羟基喜树碱提取率为0．0094％，浸膏中10-羟基喜树碱的纯度为0．1078％，与模型值相对偏差分别为0．53％和0．78％，说明回归方程可以很好地预测实验结果。     

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 ofC. 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 100 plants·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 fromC. acuminata, the optimal planting density ofC. acuminata seedlings should be designed as 25 plants·m⁻². Foundation item: This paper was supported by the National Natural Science Foundation of China (No. 3970086) and Heilongjiang Province Foundation for Distinguished Youth Scholars (JC-02-11) Biography: Wei Huan-yong (1978-), male, Graduate in College of Life Sciences, Northeast Forestry University, Harbin 150040, P. R. China. Responsible editor: Zhu Hong     

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.     

喜树种子中喜树碱和10-羟基喜树碱高效液相色谱法分析

建立了一种简便、快速、准确的测定喜树(Camptotheca acuminata Decne.)种子中喜树碱和10-羟基喜树碱含量的高效液相色谱分析方法;色谱条件为:采用日本KYA HIQ sil C18柱(250 mm×4.6 mm,5 μm),流速为1 mL/min,梯度洗脱程序为:在前 15 min流动相乙腈 -水的体积比由10 %线性增加至40%,在随后的3 min乙腈 -水的体积比线性降至10 %并保持恒定3 min,在21 min时停止该程序.检测器为二极管阵列检测器,喜树碱定量分析波长254 nm,10-羟基喜树碱定量分析波长266 nm,进样量10 μL.样品制备以60 %的乙醇作溶剂,在60 ℃下超声波提取喜树种子50 min.利用以上方法分别测定了喜树种子胚乳、胚轴、子叶和种皮中的喜树碱和10-羟基喜树碱含量,喜树碱的含量是胚乳＞胚轴,子叶＞种皮,10-羟基喜树碱的含量是胚乳＞种皮＞胚轴＞子叶.     

Optimization of ultrasound-assisted extraction of camptothecin from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> 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.     

超声波辅助提取脱脂红松仁中水溶性多糖的研究

采用超声波辅助提取法对脱脂红松仁中水溶性粗多糖的提取工艺进行了研究.通过正交试验,分别考察了提取温度、液料比、提取时间及醇沉浓度4个因素对红松仁多糖提取率的影响,得出优化的工艺条件：提取温度为70℃,液料比为20：1,提取时间为40 min,醇沉浓度为80%,此条件下多糖的提取率为3.65%,平均含量为45.38%.结果表明,超声波辅助提取的效率和含量均优于传统热水浸提法,且具有提取温度低、时间短及效率高的优势.     

高效液相色谱法测定羟基喜树碱含量方法的研究

The determination method of 10-hydroxycamptothecin inCamptotheca acuminatu 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 acetonitrile-water (3:7, V:V), flow rate 1 mL·min⁻¹ and UV detective wavelength 266 nm. Extracting 10-hydroxycamptothecin by ultrasonic method from fruits ofC. acuminata to prepare samples for analysis was systematically discussed. The optimal extraction condition was carried out by 60% alcohol solution at 60°C for 50 minutes. Foundation Item: This paper was supported by National Natural Science Foundation of China (No. 30070086). Biography: MA Mei-fang (1976-), female, Graduate Student of Northeast Forest University. Responsible editor: Song Funan     

印度黄檀叶多酚提取工艺优化及抗氧化活性研究

目的]印度黄檀叶含有多酚及类黄酮物质,研究印度黄檀叶多酚及其抗氧化活性,可为其利用提供依据。[方法]以印度黄檀叶为原料,乙醇为提取液,经单因素实验与正交试验设计,检测在不同乙醇浓度、提取时间、提取温度及超声功率120 w时3个因素进行响应面优化试验,确定印度黄檀多酚的提取工艺;同时,鉴定印度黄檀叶乙醇-水提取液对DPPH-自由基的清除能力。[结果]低浓度印度黄檀叶多酚能发挥更强的抗氧化能力,其提取液对清除DPPH自由基的半数抑制质量浓度(IC_(50))约为3.2 mg·L~(-1),略大于Vc的2.5 mg·L~(-1);不过,其还原能力略低于Vc。[结论]印度黄檀叶内富含多酚类物质,具有很强的体外抗氧化活性,可作为天然抗氧化植物资源开发利用。     

Contributions of salmon-derived nitrogen to riparian vegetation in the northwest Pacific region

We examined the relationship between the annual escapement of salmon and the δ ¹⁵N of willow (Salix spp.) leaves to evaluate the contribution of marine-derived nutrients (MDN) to riparian vegetation around the Pacific Northwest and Northeast regions. Foliar δ ¹⁵N values ranged from −3.42‰ to 4.65‰. The value increased with increasing density of carcasses up to 500 fish/km and 1500 fish/km. δ ¹⁵N values were variable at carcass densities below 500 fish/km. Possible factors affecting the fluctuation of δ ¹⁵N at reference sites are: (1) denitrification; (2) the presence of N₂-fixing trees, such as alder; and (3) agricultural runoff. δ ¹⁵N values at the sites with carcass densities over 500 fish/km were consistently high, while a value of δ ¹⁵N below zero was observed at only one site (Rusha River; δ ¹⁵N = −1.87‰). At this site, most adult pink salmon returned to limited locations near the estuary because steeper channel gradients acted as a migration barrier, resulting in the negative δ ¹⁵N value. Nevertheless, we concluded that our results showed evidence of the feedback of MDN to terrestrial vegetation, although the use of the δ ¹⁵N value as a terrestrial end member at spawning sites is limited. If the relationship between the enrichment index, which is expressed as the values using a mixing model, and salmon abundance was estimated, the availability of MDN in riparian ecosystems could possibly be evaluated and will lead to the establishment of escapement goals. An erratum to this article is available at .     

正交试验法优化元宝枫叶中黄酮类提取工艺

以元宝枫叶为原料,优化槲皮素、山奈酚的提取工艺。运用正交试验法考察提取条件对提取率的影响。结果表明,元宝枫叶提取槲皮素、山奈酚最佳工艺为:以20倍样品质量的水煎煮提取2次,每次3 h。干膏得率1.02%,山奈酚质量分数为50.4 mg/g,相对标准偏差(RSD)为2.2%;槲皮素质量分数为48.2 mg/g,RSD为1.6%。该工艺可使样品中2种活性成分有效提取分离,且经济、环保。     

Investigation of anti-salt stress on tetraploid Robinia pseudoacacia

Tetraploid Robinia pseudoacacia was used as a main test material and diploid R. pseudoacacia was used as the control. The indices of shape, physiology and biochemistry, photosynthesis and anatomic structure of the young plants were investigated under salt stress (NaCl and Na₂SO₄). The treatment time was 30 d with an interval time of 7 d. Before and after treatment, the indices were measured. Results show that: 1) the growth of diploid R. pseudoacacia inhibited an evident symptom of salt damage and the leaf moisture content was lower under salt stress than that of control. But the tetraploid R. pseudoacacia was contrary. 2) The relative electric conductivity and proline (Pro) of tetraploid R. pseudoacacia increased slightly and had no significant difference compared with its control, which was contrary to diploid R. pseudoacacia. At the same time, three protective enzymes including perocidase (POD), superoxide (SOD) and catalase (CAT) kept higher activities at a post stage of salt stress to tetraploid R. pseudoacacia, which enhanced its anti-salt characteristics. Diploid R. pseudoacacia was sensitive to salt and had contrary information. 3) Salt stress had little influence to photosynthesis of tetraploid R. pseudoacacia. The net photosynthetic rate (P _n) and intercellular CO₂ concentration (C_i) had no significant changes, but those of diploid R. pseudoacacia decreased singificantly. 4) After salt stress, the anatomic structure of tetraploid R. pseudoacacia had a positive reaction, including the palisade parenchyma of diachyma, was prolonged and arranged more tightly. The spongy parenchyma was shrunk and was arranged tightly, which was contrary with diploid R. pseudoacacia. These data demonstrate that tetraploid R. pseudoacacia had superior anti-salt performance. __________ Translated from Journal of Plant Ecology, 2008, 32(3): 654–663 [译自: 植物生态学报]     

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.     

Ultrasound-associated extraction of seed oil of Korean pine

Experiment on ultrasound- associated extraction of seed oil of Korean pine (Pinus koraiensis) was conducted in Northeast Forestry University, Harbin, China. The factors affecting extraction yield, such as ultrasonic frequency, extracting temperature, extracting time and the ratio of material to liquid (ratio of Korean pine seed to absolute alcohol), were analyzed under specific condition and the optimal extracting parameters were obtained as the ultrasonic frequency 32 000 Hz, the extracting temperature 80℃, the extracting time 50 rain, and the ratio of material to liquid 1: 30. The study demonstrates that ultrasound is a reliable and great efficiency tool for the fast extraction of Korean pine seed oil。     

Antimicrobial activity against <Emphasis Type="Italic">Streptococcus sobrinus</Emphasis> and glucosyltransferase inhibitory activity of taxifolin and some flavanonol rhamnosides from kempas (<Emphasis Type="Italic">Koompassia malaccensis</Emphasis>) extracts

Twenty plant materials collected from the islands of Java and Kalimantan in Indonesia were extracted with 50% aqueous ethanol (crude extract). The crude extracts were assayed for antimicrobial activities against Streptococcus sobrinus and for glucosyltransferase (GTase) inhibition. Fourteen extracts inhibited the growth of S. sobrinus by more than 50% and six extracts inhibited GTase activity by more than 50% at a concentration of 100 μg/ml. Koompassia malaccensis (kempas) extracts showed 90% depression of S. sobrinus growth and 80% inhibition of GTase activity at a concentration of 100 μg/ml. Kempas crude extracts were subjected to column chromatography using Sephadex LH-20 and then preparative high-performance liquid chromatography to isolate four compounds A, B, C, and D. These compounds were identified as taxifolin and the flavanonol rhamnoside isomers neoastilbin, astilbin, and isoastilbin, respectively, from ¹H and ¹³C nuclear magnetic resonance (NMR) spectra and other two-dimensional NMR techniques (COSY, HMBC, and HMQC). Each compound depressed the growth of S. sobrinus over a concentration range of 9.3242.7 μg/ml and showed GTase inhibitory activity with IC₅₀ values in the range 27.4–57.3 μg/ml. Taxifolin and flavanonol rhamnoside isomers isolated for the first time from kempas could be potent compounds for preventing dental caries. Part of this report was presented at the 57th Annual Meeting of the Japan Wood Research Society Conference, Hiroshima, 2007     

Box-Behnken法优化大叶榉树黄酮类超声辅助提取方法及初步鉴定

为了探讨大叶榉树Zelkova schneideriana叶片黄酮类成分,以1年生大叶榉树叶片为材料,在超声波辅助下,通过单因素和Box-Behnken试验,探究并优化了大叶榉叶片黄酮类乙醇提取方法;采用常规显色法,对大叶榉树叶片中黄酮类物质种类进行初步鉴定。结果表明:(1)优化后的大叶榉树黄酮类提取方法为:乙醇浓度63%、液料比31:1mL·g^-1、提取时间1.7 h,大叶榉树黄酮类提取量为(31.067±0.11)mg·g^-1;(2)初步鉴定发现:大叶榉树叶片中含有黄酮、具有邻二酚羟基结构的黄酮类化合物、具有邻二苯酚羟基或兼有3-OH,4=O或5-OH,4=O的化合物;可能有黄酮醇、二氢黄酮、二氢黄酮醇等;不含查尔酮和橙酮。     

Preparation technology of ultra-fine powders ofAuricularia auricular

Conventional mechanical method and mechanical method combined with vacuum freeze-drying technology were used to make the ultra-fine powders of edible fungus (Auricularia auricular). The content of basic nutrients, amino acid, micro structure and their properties of raw edible fungus and the edible fungus powders obtained with the two methods were analyzed and compared. The granularity size and micro-structure of the pulverized samples were analyzed by SEM and TEM technology. The average granularity size of the edible fungus powder obtained with mechanical method was 1–5 μm, while that obtained with mechanical method combined with vacuum freeze-drying process was 0.5–1 μm. The ultra-fine powders of edible fungus obtained with the two methods had better water recovery capability and quality, and their preserving time was longer than that of raw edible fungus. All the properties of the ultra-fine powders of edible fungus obtained with the vacuum freeze-drying technology were evidently superior to that of the conventional mechanical method. Biography: YANG Chun-yu (1975), female, Ph. D. in Engineering Technology Center of Forestry and Wood Workine, Machinery, Northeast Forestry University, Harbin 150040, P. R. China. Responsible editor: Zhu Hong     

提取骨碎补中柚皮苷的研究

采用超声波对骨碎补中的柚皮苷进行辅助提取,在单因素试验的基础上,应用Box-Behnken响应面优化设计,得到最适的超声提取工艺条件:按1∶24的料液比加入55%(V/V)乙醇溶液,在500W功率、70℃的温度下超声处理32min,在此条件下骨碎补中的柚皮苷的提取量为15.07mg/g。     

微波技术提取刺三加叶中绿原酸的研究

采用L9（34）正交实脸设计对影响微波法提取刺三加叶片中绿原酸的溶剂浓度、微波功率、提取时间和提取次数四个因素进行了研究,结果表明,提取刺三加叶中绿原酸的最佳条件是A2B2C3D3,即乙醇浓度60%,功率300W,提取时间3min,提取次数3次。影响因子的主次顺序为乙醇浓度〉提取次数〉提取时间〉微波功率。     

Rapid changes of induced volatile organic compounds in Pinus massoniana

Using the thermal-desorption cold trap gas chromatography/mass spectrometer (TCT-GC-MS) technique, the composition and relative contents of volatile compounds were analyzed in undamaged (control), insect-damaged (ID) and artificially-damaged (AD) leaves of Pinus massoniana in field at different times and levels of damage. Results showed that although volatile substances were highly released earlier in AD leaves plants, they were significantly less abundant in AD than in ID leaves treatments. Also, the damage level considerably influenced the changes of induced volatile products from leaves. Compared with the control, the emission rate of camphene, β-pinene, phellandrene, caryophyllene and (E)-farnesene was high after 1 h in 25%–40% ID-affected leaves, whereas that of tricyclene, myrcene, camphene, β-Pinene, phellandrene and caryophyllene reached its maximum after 24 h in 60%–75% ID-affected leaves. In the same manner, some volatile compounds in the AD leaves treatment displayed their peaks just after 1 h, but others after 24 h. The AD and ID leaves at the damage level of 25%–40% did not exhibit an obvious regularity with time; however, in 60%–75% AD leaves, peaks of volatile substances were attained after 1 or 2 h. Our results also showed that the relative content of β-pinene increased and was higher in damaged than control plants. β-pinene plays an important role in inducing the insect resistance of P. massoniana trees. __________ Translated from Scientia Silvae Sinicae, 2006, 42(4): 65–70 [译自: 林业科学]