摘要
山核桃(C.cathayensis)是我国浙、皖两省山区的重要干果。给产区农民带来巨大经济效益的同时,取仁弃之的山核桃外果皮严重污染了当地的农业生态环境。针对山核桃废弃物外果皮的主要无机成分和有机成分及其对农作物的生物活性效应进行了系统研究。主要研究结果如下:
1.采取干、湿两种消化方法分解山核桃外果皮有机物,利用原子吸收分光光度法分析到山核桃外果皮含有钾、钙、铁、锰、锌、镁、铜等灰分元素,其中钾含量高达1.61%,可制备工业钾盐。热处理法制备碳酸钾的研究表明:山核桃外果皮粒度d＜10mm,600℃高温灰化6hr,钾得率约达30%;纯化粗碱经600℃煅烧0.5hr,碳酸钾含量高达98%。化学合成法制备焦磷酸钾的研究表明:山核桃外果皮灰分与磷酸质量比2.24:1～2.30:1,中和反应pH9.0,磷酸氢二钾在500℃聚合1.5 hr,焦磷酸钾含量达97%以上。
2.利用气相色谱-质谱联用仪分析到山核桃外果皮含有酚类、脂肪酸、醇类、酯类、烃类、酮类、甾体、维生素E、喹啉等多种有机成分,其中酚类物质含量较高。对主要酚类黄酮、鞣质和醌化合物的提取分离进行了研究,醌化合物提取分离的最适条件是:70%乙醇、料液比1:10(g/mL)、60℃水浴4hr,醌提取得率达0.84%;硅胶柱分离、液固比3:1(mL/g)、吸附时间30min,醌化合物纯度达86.08%。鞣质化合物提取分离的最适条件是:50%乙醇溶剂、料液比1:12(g/mL)、60℃水浴2hr,鞣质提取得率达26.89%。采用咖啡碱成盐沉淀、甲醇溶解,经氯仿、乙酸乙酯等有机溶剂萃取除去杂质,鞣质纯度达86.42%。黄酮化合物提取分离的最适条件是:70%乙醇、料液比1:15(g/mL)、60℃水浴3hr,黄酮提取得率达0.997%;硅胶柱分离、液固比3:1(mL/g)、吸附时间30min,用20、40、60、70、80、90%乙醇以2mL/min的流速洗脱,黄酮解吸率为89.46%,纯度达84.40%。
3.山核桃外果皮浸提液对作物种子萌发有低浓度促进、高浓度延缓或抑制的生物效应,0.1g/mL浸提液能显著促进小麦、玉米、绿豆、大豆的种子萌发、胚根和胚轴的伸长,小麦与绿豆种子萌发的处理比对照增加了38%和24%,胚根增长幅度分别为21%、26%、29%、32%,差异达显著或极显著;从1.5g/mL开始,小麦、玉米、绿豆种子萌发强烈受阻,发芽率分别比对照降低了100%、43%和49%,处理与对照差异显著;胚根、胚轴伸长受抑效应增强,但对浸提液的敏感性胚轴不如胚根。
4.0.1g/mL浓度的浸提液能提高作物种子a-淀粉酶活性、呼吸速率、降低吲哚乙酸氧化酶活性,增加可溶性糖、氨基酸、吲哚乙酸含量,加快蛋白质和淀粉转化速率;高于0.5g/mL的浓度处理,a-淀粉酶活性下降、吲哚乙酸氧化酶活性增强,种子呼吸速率减弱,细胞内吲哚乙酸减少,淀粉-可溶性糖、蛋白质-氨基酸的转化过程减慢。总体上,四种作物的种子萌发对山核桃外果皮浸提液的敏感顺序是小麦＞绿豆＞玉米＞大豆。
5.四种幼苗根系对矿质元素的吸收大都表现出低浓度促进、高浓度抑制的效应,0.1、0.5g/mL浓度的浸提液能适当降低幼苗新生根系的电导率,提高根系活力和硝酸还原酶的活性;适宜浓度浸提液促进了玉米对N、K、Ca、Mg、Fe、Mn、Cu的吸收,促进小麦对N、K、Ca、Fe、Cu的吸收,促进大豆对N、K、Mg、Fe、Mn、Cu的吸收,促进绿豆对N、K、Ca、Mg、Fe、Mn和Zn的吸收,因作物种类不同适宜浓度各有差异。
6.从作物叶片光合性能变化的角度分析影响作物幼苗生长的机理,0.1、0.5g/mL浓度的浸提液可提高小麦等四种作物幼苗叶片的叶绿素a和叶绿素b、叶绿素总量、光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr),适当增加细胞间隙内部的CO_2浓度(Ci),以0.1g/mL处理效果最明显。四种作物幼苗的鲜重与干重明显高于对照,0.1g/mL浸提液处理的小麦和大豆干物质积累最多,0.5g/mL浸提液处理的玉米和绿豆干物质积累最多。高于0.5g/mL处理,叶绿素a和叶绿素b、叶绿素总量、光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)均有较大幅度的降低,叶绿素a/b比值则有小幅度的上升,细胞间隙内部的CO_2浓度(Ci)快速提高。研究认为,高浓度浸提液造成幼苗叶片光合速率下降,叶肉因素限制是主要原因,可能与浸提液抑制光合作用暗反应的酶活性、使CO_2同化过程受阻以及叶绿素含量下降引起光能吸收和光化学反应速率降低、同化力减少有关。
7.适宜浓度的黄酮、鞣质、醌类三种活性物质及其组合和山核桃外果皮浸提液都能促进四种作物幼苗的株高、新生根系长度,增长幅度一般高于对照6～17.5%:各种活性物质及其组合的最适浓度不同,处理与对照表现的化感效应达到P＜0.01～0.05的差异显著水平;比较了三种活性物质及其组合和山核桃外果皮浸提液对四种作物幼苗细胞膜脂过氧化的影响,分析到适宜浓度的活性物质及其组合能降低幼苗新生根系和新生幼叶细胞膜的相对电导率(降幅为5～14%)、丙二醛(MDA)含量、H_2O_2含量和超氧阴离子自由基(O_2~-·)含量;增加过氧化物酶(POD)、过氧化氢酶(CAT)、超氧物歧化酶(SOD)的活性,提高新生根系和幼叶的呼吸强度和新生根系的活力,对作物幼苗细胞膜脂过氧化的影响顺序是:一种活性物质＜二种活性物质组合＜三种活性物质组合≤山核桃外果皮浸提液,说明山核桃外果皮浸提液对四种作物幼苗生长产生的化感效应是多种活性物质协同作用的结果。适宜浓度的活性物质及其组合通过提高细胞保护酶系统催化的氧化还原反应能力,有效清除细胞内的活性氧有害物质,是增加细胞膜结构稳定性的内在原因。
8.研究了大田栽培牡丹大胡红7、8月份叶片的光合速率日变化动态:7月上旬测定日午间,光合速率(Pn)大幅度下降,Pn日变化呈双峰型,出现明显的光合午休:8月上旬光合午休比7月份加重,Pn日变化呈“一降不起型”。两个月份测定日Pn下降时,气孔导度(Gs)降幅很小,细胞间隙内部CO_2浓度(Ci)不降反升,叶肉因素限制是光合午休的主要原因。叶绿素荧光测定显示,午间PSⅡ最大光化学效率(Fv/Fm)、PSⅡ实际光化学效率(ΦPSⅡ)明显下降,光化学猝灭系数(qP)分别下降34.4%、37.9%,非光化学猝灭(NPQ)分别增加44.4%和49%,叶绿体PSⅡ的电子传递活性大幅度下降,吸收光能用于非光化学反应的比例大幅度增加,牡丹叶片发生了严重的光合作用光抑制。
9.利用0.06mg/mL的山核桃胡桃醌溶液喷施牡丹植株,处理叶片的光合速率比对照提高10.5%～12.8%,8月份的Pn最高值比对照推迟1h出现;对照叶片的Fv/Fm两个月分别降低51%和89%,处理叶片分别降低20%和26%;对照叶片ΦPSI分别下降49.5%和67%,处理叶片下降18.1%和19.9%,下午7、8月份对照叶片ΦPSⅡ恢复较差或接近早晨水平,处理叶片则完全恢复:7月份对照和处理的qP分别下降39%和10%,8月分别下降43%和11.6%;7月份NPQ分别上升49.3%和17.3%,8月份上升49%和14.3%。研究认为,适宜浓度的胡桃醌能缓解牡丹叶片光合作用的光抑制、减轻光合午休,并能较好地修复强光损伤的叶绿体,提高牡丹叶片对光能的利用率。
C.cathayensis,a Juglandaceae Carya Nutt plant,is an important nut in Zhejiang and Anhui mountain areas.While C.cathayensis brings great economic benefits to peasants,the discarded C.cathayensis exocarp severely pollute local agricultural ecological environment.This research aims to study the main inorganic and organic constituents in C.cathayensis exocarp and their bioactive influence on plants.
1.Adopting wet digestion and dried digestion method respectively to decompose C.cathayensis exocarp,as well as the atomic absorption spectrometry to analyze the inorganic constituents in C.cathayensis exocarp,the result shows that there are ash elements—K,Ca,Fe, Mn,Zn,Mg,Cu,etc.—in C.cathayensis exocarp,among which,the content of K is about 1.61%. The research of adopting thermal treatment to prepare potassium carbonic acid shows that with material diameter＜10mm,ashing temperature 600℃,ashing time 6hr,the yield ratio of K is about 30%;with purified alkali calcination temperature 600℃,calcination time 0.5 hr,the content of potassium carbonic acid can be up to 98%.Adopting chemical synthesis to prepare pyrophosphate potassium,the result shows that the quality ratio of ash to phosphoric acid 2.24: 1～2.30:1,pH in neutralization reaction 9.0,dipotassium hydrogen phosphate polymerization temperature 500 and time 1.5hr,the content ofpyrophosphate potassium can be up to 97%.
2.Adopting GC-MS,the result shows that there are many organic components in C.cathayensis exocarp—phenol,fatty acid,mellow,ester,hydrocarbon,ketone,sterides, vitamin E,quinoline etc,of which the content of phenol is relatively higher.This research extracts and separates flavone,tannin,quinones,and gets the optimal extraction condition of quinones:with 70%ethanol,solid-liquid ratio 1:10,temperature 60,and reflux time 4 hours, the extraction ratio of quinones is 0.84%;separating silica gel,liquid-solid ratio 3:1,absorption time 30 min,the purity of quinones compound is 86.08%.The optimal extraction condition of tannin is:using 50%ethanol as solvent,solid-liquid ratio 1:12(g/mL),lixiviation temperature 60,and lixiviation time 2 hours,the extraction ratio of tannin is 26.89%;using theine to produce salt precipitation,methanol to dissolve,removing impurity through fractional extraction by different polar organic solvent of aether,chloroform and ethyl acetate,the tannin purity is 86.42%.The optimal extraction condition of flavones is:70%ethanol,solid-liquid ratio 1:15 (g/mL),extraction time 3 hours,extraction temperature 60℃,the maximum extraction ratio of flavone compound is 0.997%;separating silica gel,liquid-solid ratio 3:1(mL/g),absorption time 30 min,gradiently eluted by 20,40,60,70,80,90%ethanol at the speed of 2mL/min,the recovery of flavone is 89.46%,and its purity is 83.76%.
3.The study showed that after treatment with the lixiviation drench of C.cathayensis exocarp,the seed germination is accelerated at low concentration and delayed or inhibited at high concentration.The 0.1g/ml lixiviation drench obviously promotes seed germination and radicles elongation of wheat,corn,soybean and mung bean.The wheat and mung bean seeds germination rates are increased by 38%and 24%;the elongation of their radicles is increased by 21%、26%、29%、32%respectively,and the difference is significant.From 1.5g/ml on,the seed germination of wheat,corn and mung bean are greatly inhibited,with germination rate decreased by 100%,43%and 49%respectively.Meanwhile,the lixiviation drench exerts more inhibition on the elongation of radicles and hypocotyls.In addition,hypocotyls are less sensitive to the lixiviation drench than radicles.
4.The study indicates that the 0.1 g/ml lixiviation drench stimulates the activity ofα-amylase and inhibits that of IAA oxidase.Consequently the respiratory rate of the seeds,the content of soluble sugar,amino acid,and indoleacetic acid(IAA) increase;the content of starch and protein decreases,and their conversion rates accelerate.In addition,the lixiviation drench with its concentration higher than 0.5g/ml reduces the activity ofα-amylase and stimulates that of IAA oxidase.Hence the respiratory rate of the seeds decreases,cellular IAA decreases,and the conversion process from starch to soluble sugar and that from protein to amino acid decelerate. Generally the sensitivity of seed germination of the above four crops to the lixiviation drench goes in a descending order as follows:wheat＞mung bean＞corn＞soybean.
5.The lixiviation drench with low concentration promotes the absorption of the mineral elements of the seedling root systems,while the lixiviation drench with high concentration inhibits the absorption.The 0.1g/ml lixiviation drench and the 0.5g/ml lixiviation drench decrease the electric conductivity of the seedling root system,increase the activity of root system and nitrate reductase.With optimal concentration the lixiviation drench improves the absorption of nitrogen(N),potassium(K),calcium(Ca),magnesium(Mg),ferrum(Fe),manganese(Mn), and copper(Cu) of corn,the absorption of N,K,Ca,Fe,and Cu of wheat,the absorption of N,K, Mg,Fe,Mn,and Cu of soybean,and the absorption of N,K,Ca,Mg,Fe,Mn,and zinc(Zn) of mung bean.The optimal concentration of lixiviation drench varies according to different crops.
6.The research analyzes the way in which photosynthetic performance of the leaves influences the seedling growth.The 0.1g/ml lixiviation drench and the 0.5g/ml lixiviation drench have increased the content of chlorophyll a and chlorophyll b and the total chlorophyll content, photosynthetic rate(Pn),stomatal conductance(Gs),and transpiration rate(Tr) in the seedling leaves of the above four crops,and adequately increased the intercellular CO_2 concentration(Ci), with the 0.1g/ml lixiviation drench producing the most remarkable effect.The fresh weight and dry weight of their seedlings are obviously heavier than the control.The wheat and soybean treated with 0.1g/ml lixiviation drench accumulate the most dry substances,while the corn and mung bean treated with 0.5g/ml lixiviation drench accumulate the most dry substances. Treatment with the concentration higher than 0.5g/ml results in great decrease in the content of chlorophyll a and chlorophyll b and the total chlorophyll content,photosynthetic rate(Pn), stomatal conductance(Gs),and transpiration rate(Tr),and results in slight increase in the ratio of chlorophyll a:chlorophyll b,and rapid increase in the intercellular CO_2 concentration(Ci).The research believes that the lixiviation drench with high concentration,particularly the mesophyll limitation to photosynthesis,causes the decrease of photosynthetic rate of the seedling leaves. The reason for this may be that the lixiviation drench restrains the enzymatic activity in the dark reaction so as to hinder the assimilation of CO_2 or that the decrease of chlorophyll reduces the absorption of light energy,lowers the photochemical reaction rate,and decreases the assimilatory power.
7.The study indicates that optimal concentration of flavone,tannin,quinones and their combinations as well as the lixiviation drench of C.cathayensis exocarp,can enhance the height of wheat,corn,soybean,and mung bean seedlings and the length of their nascent root systems, 6%to17.5%higher than the control in general.The allelopathy between the experiment and control has significant difference with P＜0.01～0.05 in terms of various active substances and their combinations.The research compares influences of three active substances and their combinations as well as lixiviation drench of C.eathayensis exocarp on the peroxidation of cellular membrane lipids of these four seedlings,and finds that active substances and their combinations with optimal concentration can decrease relative electric conductivity(the range of decreasing is 5%to 14%),and the content of MDA,hydrogen peroxide,and superoxide anion radical(O_2~-) nascent root systems and spires of seedlings,promote the activity of peroxidase (POD),catalase(CAT),and superoxide dismutase(SOD),increase the respiratory intensity of both nascent root systems and spires,and reinforce the vigor of nascent root systems.The order of influence on peroxidation of cellular membrane lipids of these four seedlings is as follows: unieus active substance＜two-active-substance combination＜three-active-substance combination≤lixiviation drench of C.cathayensis exocarp,which indicates that allelopathy of lixiviation drench of C.cathayensis exocarp on the above four seedlings is the result of many active substances working together.Active substances and their combinations with optimal concentration can strengthen the stability of cellular membrane structures by improving the ability of oxidation-reduction reaction activated protectase system in cells and effectively clearing harmful active substances in cells.
8.The research studies the photosynthesis diurnalvariation dynamics of the Da Hu Hong leaf—a type of Da Tian peony—under natural sunlight of July and August:during the noon in early July,the photosynthetic rate(Pn) substantially declines,and the Pn diurnalvariation shows two peaks and there also appears a clear_photosynthetic noon break.On the measure day of early August the photosynthetic noon break is more severe than in July and the Pn diumalvariation of the peony leaves shows a trend of "declining straightly".During the phase when Pn declines at noon on the measure days of the two months,the declination of stomatal conductance is in a small scale,the Ci of CO_2 inside the cell gap does not fall but rises instead and the Mesophyll constraint is the main reason for photosynthetic noon break.Chlorophyll fluorescence test indicates that during noon time of the measure days,Fv/Fm and PSⅡphotochemical efficiency(ΦPSⅡ) decline obviously,the photochemical quenching factor(qP) of the two months decreases by 34.4%and 37.9%respectively;meanwhile,the non-photochemical quenching factor(NPQ) increases by 44.4%and 49%,the electronic transferring activity of chloroplast declines significantly,the absorbed light energy used for non-photochemical reaction substantially increases,and severe photosynthetic photoinhibition happens in peony leaves.
9.Spraying the peony leaves with 0.06mg/mL juglone solution,the result indicates that Pn is more obvious than the control,the maximum Pn of the two months enhances 10.5%and 12.8% respectively than the control,and the maximum Pn in August appears one hour later than the control;Fv/Fm of the two months drops by 51%and 89%respectively and the processed leaf reduces by 20%and 26%separately,the contrastΦPSⅡof the two months decreases by 49.5% and 67%respectively,while the processed leaves only decreases by 18.1%and 19.9%;in the afternoon of July and August,ΦPSⅡof the leaf in control restores poorly or almost to the morning level,while the processed leaf can recover completely;in July,the compared qP and processed qP drop by 39%and 10%respectively and in August,they drop by 43%and 11.6% respectively;NPQ in July rises by 49.3%and 17.3%respectively and in August,it rises by 49% and 14.3%respectively.The research believes that juglone with optimal concentration can relieve peony leave photosynthetic photoinhibition,alleviate photosynthetic noon break,restore damage to the chloroplast and enhance the peony leaves' utilizing ratio of light.
引文
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