基于UPLC-Q-Orbitrap MS技术的沙棘黄酮类成分分析

利用UPLC-Q-Orbitrap MS技术对沙棘(Hippophae rhamnoides L.)果实中黄酮类物质进行系统剖析,并对不同产地沙棘果实中黄酮类化合物进行比较分析,为中药沙棘的化学物质基础和资源利用研究奠定基础。根据保留时间比对,精确分子量匹配和MS/MS碎片信息分析,共鉴定出58个化合物,包括34个黄酮醇、4个黄酮、14个花色素、1个二氢黄烷酮、2个二氢黄烷醇、2个黄烷醇和1个二氢查尔酮,其中14个花色素类化合物为首次在沙棘果实中鉴定。最终筛选得到38种差异积累的黄酮类化合物,其中大部分在青海和西藏产地的沙棘果中呈显著积累。研究丰富了沙棘果中黄酮类化合物化学物质基础研究,揭示了不同产地沙棘果中黄酮类化合物的组成及差异。

中国沙棘(Hippophae rhamnoides L．ssp．sinensis Rousi)的开花特性及风媒传粉距离的检测

连续两年对雌雄异株的中国沙棘（Hippophae rhamnoides L．ssp．sinensis Rousi）的花期物候、开花式样进行了观察，检测了柱头可授性、花粉活力和花粉一胚珠比，并进行厂人工授粉、套袋实验，检测结实率。结果显示中国沙棘风媒传粉发生在4月下旬或5月上旬，单花花期约7d；传粉盛期在第3天到第5大，柱头的可授性从第2天到第4天或第5天。中国沙棘花先叶开放、花小、无花冠、花药成熟时萼片从两个侧向的纵缝中裂开，形成有利于化粉散布的对流风洞。花粉活力和花粉．胚珠比都很高，具有典型的适应风媒传粉的花部特征和性状。中国沙棘花粉的传播距离受外界环境影响较大，通过重力玻片法检测可以看出，在风速小于3m／s时，主要集中在15-25m之间，当风速大于3m／s时，顺风向可超过85m以外。传粉效率对结实率的影响较大。

Effects of Total Flavonoids ofHippophae RhamnoidesL.on Intracellular Free Calciumin Cultured Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats and Wistar-Kyoto Rats

To explore the effects of total flavonoids of Hippophae rhamnoides L. （TFH） quercetin （Que） and isorhamnetin （Isor） on the intracellular free calcium （[Ca^2＋]） in vascular smooth muscle cells （VSMC） of spontaneously hypertensive rats （SHR） and Wistar-Kyoto rats （WKY）. Metheds： Fluo 3-acetoxymethylester（Fluo-3/AM） was used to observe the effects of TFH （100mg/L） and its essential monomers, namely Que （10^-4mol/L） and Isor （10^-4mol/L） on changes of [Ca^2＋]1 in cultured SHR and WKY VSMC （abbr. to Ca-SHR ＆ Ca-WKY） following exposure to high K^＋, norepinephrine （NE） and angiotensin Ⅱ （AngⅡ）, and to compare with the effects of verapamil （Ver）. Results： （1） TFH, Que and Isor had inhibitory effects on resting Ca-SHR （P〈0.05）, but had no significant effects on Ca-WKY （P〉0.05）. （2） High K^＋ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）; TFH, Que and Isor could inhibit the elevation of [Ca^2＋]1 induced by high K^＋ -depolarization, with the effects similar to that of Ver, and the effect on Ca-SHR was more significant than that on Ca-WKY （P〈0.05）. （3） NE and Ang Ⅱ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）, TFH, Que and Isor had remarkably inhibitory effect on the elevation of Ca-SHR and Ca-WKY induced by NE or Ang Ⅱ. （4） In the absence of extracellular Ca^2＋ , TFH, Que and Isor also had certain inhibitory effect on Ca-SHR and Ca-WKY induced by NE, and the effect on the former was more significant than that on the latter（P〈0.05）. Ceaclusiea： TFH, Que and Isor might decrease the levels of [Ca^2＋], in VSMCs by blocking both voltage-dependent calcium channels （VDC） and receptoroperated calcium channels （ROC） in physiological or pathological state, which may be one of the important mechanisms of their hypotensive and protective effects on target organs in patients with hypertension.

Molecular identification of sex in Hippophae rhamnoides L. using isozyme and RAPD markers

In many dioecious plants, gender affects economic value, breeding schemes and opportunities for commercial harvests. Hippophae rhamnoides L. is a dioecious plant species in which female genotypes are commercially preferred over male genotypes. Its berries have rich medicinal, nutritional and pharmaceutical properties because of their large amounts of vitamins, essential oils, proteins, fatty acids, free amino acids and flavanoids. Primary limitation for breeding H. rhamnoides L. is its dioecious nature, since gender cannot be identified by traditional methods. Therefore, some reliable and quick methods need to be developed. This commu- nication deals with the development of isozyme and RAPD markers for early sex identification in this dioecious tree. The isozyme analysis was conducted with four enzyme systems, viz. peroxidase, esterase, malate dehydrogenase and catalase. The peroxidase enzyme system produced a female specific sex marker, which successfully differentiated between the staminate and pistillate geno- types ofH. rhamnoides L. Thirty five random decamer primers were used in our study and one male sex linked marker was identified. OPD-20 （5＇-ACTTCGCCAC-3＇） displayed a band at 911 bp that expressed polymorphism between male and female genotypes. The staminate and pistillate genotypes could be distinguished using RAPD marker OPD-209n. These results revealed the immense poten- tial of peroxidase isozyme patterns and RAPD as genetic markers for sex identification in H. rhamnoides L.

Analysis of Effective Composition of Hippophae Rhamnoides L.

The vitamin E, proteins, amino acids, sugar, trace elements and fatty acids in the oil of Hippophae Rhamnoides L. seeds were quantitatively analyzed. Several methods for analyzing vitamin E were compared with each other and the content of gossypol in the oil was first determined.

Short-term responses to salinity of seabuckthorn (Hippophae rhamnoides L.) seedlings in the extremely cold and saline Qinghai region of China

The investigation of the mechanisms of plant adaptation to stressor action is one of the leading directions of current biological studies. To understand the mechanism of salt tolerance of seabuckthom （Hippophae rhamnoides L.） and identify its ability to cope with the salinity effect in the saline and extremely cold region of Qinghai, China, a test was conducted with two-year-old seedlings subjected to 0, 200, 400 and 600 mmol·L^-1 NaClsolutions for 30 d. The results show that with an increase in salinity, the biomass of H. rhamnoides seedlings clearly decreased. Leaf water potential （ψw） and relative water content （RWC） were significantly reduced under salinity, with severe water shortages appearing in leaves. At the same time, the total chlorophyll content declined markedly. When salinity increased and stress time prolonged, the net CO2 assimilation rate （A） significantly declined. Intercellular CO2 concentration （Ci） declined at first and was then followed by an increase over the stress time. We conclude that H. rhamnoides grown in the extremely cold and saline region of Qinghai has a certain resistance to salt, which can be planted at appropriate salinity levels.

Comparative Analysis of Antioxidants in Hippophae rhamnoides L. Leaves by UPLC

[Objectives]The research aimed to compare antioxidation abilities of different extraction phases of Hippophae rhamnoides L. leaves extract and find its active marker. [Methods] H. rhamnoides L. leaves were extracted firstly by 75% of ethanol,and then were extracted by petroleum ether,chloroform,ethyl acetate and n-butanol in turn after decompression concentration. Via DPPH and ABTS^+ radical scavenging experiments and Fe^(3+) reduction experiment,antioxidation activity of each phase was evaluated. And chemical compositions related to its antioxidation activity were determined by using UPLC for qualitative identification. [Results] Antioxidation activity sequence of each phase was ethyl acetate phase≈n-butanol phase > alcohol extract of raw material > water phase > chloroform phase > petroleum ether phase. 33 kinds of chemical compositions were isolated from alcohol extract and extracts by using UPLC. [Conclusions] UPLC and antioxidation activity test were used simultaneously to analyze alcohol extract and extracts of H. rhamnoides L. leaves,which could fast and accurately determine extraction phase with the strongest antioxidation ability. The research is significant to study active ingredients and mechanism of antioxidation from H. rhamnoides L. leaves.

Direct somatic embryogenesis from leaves,cotyledons and hypocotyls of Hippophae rhamnoides

Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in Hippophae rhamnoides L. was achieved. The influence of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants was studied. The highest frequency of somatic embryo production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg/L kinetin (KIN) and 0.2, 0.5 mg/L indole-3-butyric acid (IAA). Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55%. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.

Protective Effects of Polyphenol Extracts from Sea Buckthorn (Hippophaë rhamnoides L.) on Rat Hearts

The objective of present study was to investigate the antioxidative and cardioprotective effects of polyphenol extracts from sea buckthorn (PESB) against myocardial ischemia reperfusion injury (MIRI). PESB was analyzed using HPLC-DAD and their antioxidant activities were evaluated by measuring the intrinsic free radicals cavenging capacity and inhibiting the formation of hydroxyl radical capacity. Experiments on the isolated rat hearts were carried out according to the improved Langendorff retrograde perfusion technology. Coronary effluent of lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB) activities were analyzed to determine the extent of myocardial injury. Protein expression levels of Beclin-1, microtubule-associated protein 1 light chain 3 (LC3) I and II were measured to evaluate the degree of autophagy. In conclusion, coupled results from both in vivo and in vitro experiments have confirmed that PESB has potential health implications in both prevention and amelioration of MIRI, which may be attributed to the inhibition of autophagy.

The Effects of Total Flavones of Hippophae Rhamnoides L. on Cardiac function and Hemodynamics of Normal Subjects

Healthy volunteers were randomly divided into TFH group(22 subjects) and placebo control group (20 subjects). We used double-blind method. The cardiac performance and hemodynamics of the subjects before and after administration of TFH or placebo were determined by Admittance and STI methods, Oral TFH in a dose of 10 mg could significantly decrease the PEP/LVET, ICT/LVET and ICT of the mormal subjects(P<0.05). Oral TFH could increase SV(P<0.05). CO, CI, SWI, CWI and LI also tended to increase. Oral TFH also markedly reduced TPR (P<0.05) and increasedc (P<0.05). After administration of TFH, HR and HP did not significantly change(P>0.05). After administration of placebo, the parameters observed did not significantly change(P>0.05). The above results show that TFH can strengthen myocardial contractility and pump function of the heart, reduce total peripheral vascular resistance and increase vascular clasticity in normal subjects.

FL-1多功能吸附树脂对沙棘叶总黄酮的分离纯化

目的 考察 FL- 1多功能吸附树脂对沙棘叶中总黄酮的分离纯化方法。方法 根据沙棘叶黄酮的结构特征 ,考察了 FL - 1树脂的吸附性能 ,并采用高效液相色谱法对沙棘叶总黄酮进行了定量分析。结果 FL - 1树脂对沙棘叶总黄酮具有较高的吸附选择性 ,70 %乙醇作为脱附剂 ,产品中总黄酮含量为 4 0 .2 %。结论 FL- 1树脂用于沙棘叶总黄酮的分离纯化简便有效。

柱前衍生气相色谱法测定沙棘中脂肪酸的含量

本研究旨在建立柱前衍生-气相色谱法测定沙棘中脂肪酸的含量。采用索氏提取法提取沙棘中的脂肪油,甲酯化后,采用气相色谱法测定沙棘中脂肪酸成分。结果表明,柱前衍生测定的肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸、α-亚麻酸线性范围良好,r均大于0.999 8;平均回收率均大于93.1%,RSD均小于2.8%;通过Spass软件分析沙棘果不同部位的种子、果皮、果肉分别被聚为一类表明3种药用部位的脂肪酸类成分可以分别作为鉴别沙棘不同部位的重要指标成分。其中种子中的亚油酸含量约为果肉、果皮的2倍,α-亚麻酸含量种子中高于果肉、果皮15倍以上通过棕榈酸与棕榈油酸的比值判断,随着贮藏时间的增长棕榈酸与棕榈油酸的比值越大;沙棘果在通过蒸制20min后干燥处理,脂肪酸类成分含量最佳。通过柱前衍生-气相色谱法测定沙棘中脂肪酸的含量,可用于沙棘的质量评价。

沙棘果硫酸化酯多糖HR_3SL的制备与分析

从沙棘果皮分离提取了相对分子质量较均一的杂多糖ＨＲ３ ．用氯碘酸吡啶法对ＨＲ３ 进行硫酸化修饰后，相对分子质量明显增大，ＨＲ３ＳＬ的紫外光谱与红外光谱分析表明，在２０８ ｎｍ ，２６８ ｎｍ ，２８６ ｎｍ 和１ ２４０ ｃｍ －１ ，６２０ ｃｍ －１ 有硫酸酯键的特征吸收峰．紫外光谱法与硫酸钡质量法测定ＨＲ３ＳＬ中ＳＯ２－４ 含量约为２１ ％ ．

HPLC法测定沙棘不同部位槲皮素和异鼠李素的含量

目的:建立沙棘茎中槲皮素、异鼠李素含量测定方法,并测定沙棘不同部位中两种苷元的含量。方法:以甲醇-0.4%磷酸(58:42)为流动相,370nm为检测波长,对甲醇提取酸水解后的样品进行含量测定。结果:沙棘茎、叶、果实中槲皮素的含量分别为:0.276、2.11、0.574mg/g,异鼠李素的含量为:0.463、1.60、0.729mg/g。结论:该方法简便、快速、结果可靠。

沙棘NaCl胁迫下差异表达基因的转录组分析

通过测定盐胁迫下沙棘生理指标的变化,利用生物信息学方法分析沙棘在盐胁迫下转录组数据,挖掘沙棘盐胁迫下差异表达基因,为沙棘在土壤盐渍化区域的开发及其利用提供理论依据。生理指标测定结果表明,在300mmol/L的盐胁迫下,丙二醛含量呈平稳上升的趋势。分析300mmol/L NaCl胁迫14d的沙棘叶片与对照总RNA,发现差异表达基因富集在光合作用、光合生物固碳、丙酮酸代谢、苯丙醇合成、乙醛酸盐代谢及丝氨酸、甘氨酸和苏氨酸代谢通路这6大通路中。其中,与光合作用通路相关的差异表达基因达到了27个,是所有代理通路中差异表达基因最多的。在gene ontology(GO)功能数据库中,差异表达基因主要富集在催化活性、氧化还原过程和氧化还原酶活性三大功能中。由于光合作用的影响,造成丙酮酸、苯丙醇、乙醛酸和氨基酸代谢的差异,有可能是沙棘抗盐胁迫的重要原因。

新疆不同产地沙棘营养成分对比

对新疆不同产地沙棘果中总糖、总酸、磷、微量营养元素、4种重金属污染物含量以及沙棘籽油脂肪酸组成成分进行对比分析。结果表明:不同产地、不同品种的沙棘果中,青河县沙棘(状元黄和深秋红)总糖含量(4.24%~5.59%)高于温宿县沙棘(沙棘1#、沙棘2#、沙棘3#和沙棘4#)总糖含量(2.45%~3.31%);同一产地的沙棘,不同品种的总糖含量有明显差异,青河县的状元黄含量平均值为5.53%,深秋红含量平均值为4.28%;不同产地、不同品种的沙棘总酸含量有明显差异,青河县状元黄平均值为1.91%,青河县深秋红平均值为2.87%,温宿县4个样品总酸平均值为2.43%;不同产地沙棘微量营养元素钙、钾含量有较明显差异;沙棘籽油中脂肪酸以不饱和脂肪酸为主,不同品种、产地的沙棘籽油饱和脂肪酸含量差异不明显,不饱和脂肪酸含量有一定差异;所有样品中砷、镉、铬和铅4种重金属含量均未超标。

辽西地区‘朝阳’沙棘扦插育苗及建园关键技术

‘朝阳’沙棘具有适应性强、生长强壮、高产稳产等特性,适宜在辽西北地区栽培推广。2020年国有阜新蒙古族自治县建设林场建立高标准示范林6.3 hm^(2),2023年硬枝、嫩枝扦插育苗分别为6.7万株、3.5万株,成活率分别为87.8%、92.5%,优质苗率90%以上,每公顷鲜果产量3124.5 kg,经济效益显著。为进一步促进‘朝阳’沙棘在辽西地区的推广栽培,本文结合当地生产实践,对‘朝阳’沙棘扦插育苗及建园关键技术进行总结,旨在提升标准化生产管理水平。

山西省沙棘品种AFLP的初步分析

沙棘体内酚类物质和蛋白质含量较高,影响了基因组DNA提取的产量。采用改良CTAB法对观光木基因组DNA进行提取,经过紫外吸收、琼脂糖凝胶电泳、双酶切和AFLP标记检测,结果表明:改良CTAB法提取的基因组DNA吸光值OD260/OD280为1.7～1.9,DNA无降解现象和杂质污染;基因组DNA双酶切彻底,并且APLP扩增的条带较多而且清晰。从而建立了适合沙棘AFLP分子标记的反应体系,为利用AFLP标记对沙棘进行分子生物学研究及分子育种奠定了基础。

沙棘生防内生真菌的筛选、鉴定及产纤维素酶活性分析

为从沙棘中筛选生防内生真菌,从健康沙棘叶片和果实中采用组织匀浆法分离内生真菌,从中筛选具有拮抗活性的菌株,对其进行形态学和分子生物学鉴定,并对活性菌株进行产纤维素酶活性分析.从97株内生真菌中筛选得到16株对8种植物病原真菌具有较好抑菌作用的菌株,其中菌株SJ-83和SJ-116对8种病菌的抑制作用最佳.对16株活性内生真菌进行鉴定,发现菌株SJ-93产生具有横纵隔膜、呈褐色的分生孢子,为链格孢属真菌;菌株SJ-83菌丝棉絮状,浅绿褐色,产生褐色具横隔的分生孢子,为平脐蠕孢属真菌.采用透明圈法在刚果红培养基上进行产纤维素酶活性测定发现,菌株SJ-116产纤维素酶的活性最高,透明圈半径达1.6 cm.研究结果为后续开发沙棘生防内生真菌制剂提供基础材料.

柱前衍生化-HPLC法同时测定注射用沙棘籽油中脂肪酸的含量

目的建立沙棘籽油中脂肪酸的HPLC测定法。方法以2，4＇-二溴苯乙酮为衍生化试剂，18-冠醚-6为相转移催化剂，采用Kromasil C8反相色谱柱（250mm×4．6mm，5μm），检测波长254nm，以甲醇-乙腈-（体积比60：30：10）为流动相等度洗脱，柱温为室温，流速为1．0mL·min%^-1.七烷酸为内标，一次基线分离5种脂肪酸，建立注射用沙棘籽油的脂肪酸含量测定的方法。结果α-亚麻酸的线性范围为2．02～20．22ng，相关系数为0．9991；亚油酸的线性范围为2．90～20．90ng，相关系数为0．9991；棕榈酸的线性范围为1．27-12．67ng，相关系数为0．9990；油酸的线性范围为1．92—19．16ng，相关系数为0．9990；硬脂酸的线性范围为0．30—3．04ng，相关系数为0．9991。α-亚麻酸、亚油酸、棕榈酸、油酸、硬脂酸的平均回收率分别为106．07％、104．13％、100．81％、99．43％、95．62％，RSD分别为2．64％、2．42％、3．14％、1．18％、2．24％（n=9）。结论本方法重现性好，定量准确，可作为注射用沙棘籽油中脂肪酸测定的定量方法，用于沙棘籽油的质量控制。