三种萝藦科植物中C21甾类成分的研究

　　本实验对三种萝藦科植物通光散(Marsdenia tenacissima)、黑水藤（Biondia insignis Tsiang）和徐长卿（Cynanchum Paniculatum）中的C21甾体化合物进行了研究。从通光散藤茎乙酸乙酯提取物的水解产物中，分离得到两类八个C21甾类甙元。经光谱鉴定，它们的结构分别为11α-O-(2-甲基)-丁酰基-12β-O-顺芷酰通光藤甙元乙（1），11α-O-乙酰基-12β-O-乙酰基通光藤甙元乙（2），11α-O-（2-甲基）-丁酰基-12β-O-乙酰基通光藤甙元乙(3), 11α-O-苯甲酰基-12β-O-乙酰基通光藤甙元乙(4)， 11α-O-顺芷酰基-12β-O-乙酰基通光藤甙元乙（5）11α-O-顺芷酰基-12β-O-顺芷酰基通光藤甙元乙（6），12β-O-乙酰基通光藤甙元甲（7）和12β-O-顺芷酰基通光藤甙元甲（8）。其中，化合物2和8为新化合物。从黑水藤乙醇提取物的水解产物中，分离得到了两个C21甾体化合物。经1D、2D NMR技术鉴定，分别为（3β，14β，15β，17β）-3, 14-二羟基-15，16-裂-孕甾-5-烯-15-醛-16-半缩醛-20-酮（1）和白前甙元C（2）。其中1为15，16-裂环的新骨架类型的C21甾体化合物，命名为黑水藤甙元甲。从采购于昆明、浙江、湖南三地药材市场的徐长卿的根及根茎中，分离得到了18个化合物（其中昆明徐长卿6个，浙江徐长卿9个，湖南徐长卿3个）。经光谱数据分析，这些化合物被鉴定为：cynapanoside-C (1), cynapanoside-A (2), 白前甙元B 3-O-β-D-磁嘛吡喃糖甙(3)，白前甙元C 3-O-β-D-葡萄吡喃糖基-(1 → 4)-α-L-2-脱氧洋地黄吡喃糖基-（1 → 4）-β-D-洋地黄吡喃糖基-（1 → 4）-β-D-夹竹吡喃糖甙（4），白前甙元C3-O-β-D-葡萄吡喃糖基-(1 → 4)-α-D-夹竹桃吡喃糖基-(1 → 4)-β-D-洋地黄吡喃糖基-(1 → 4)-β-D-夹竹桃吡喃糖甙（5），白前甙元C 3-O-β-D-葡萄吡喃糖基-（1 → 4）-β-D-葡萄吡喃糖基-（1 → 4）-α-D-夹竹桃吡喃糖基-（1 → 4）-β-D-洋地黄吡喃糖基-（1 → 4）-β-D-夹竹桃吡喃糖甙（6），cynatratoside-B (7)，cynatratoside-C (8);glaucoside A(9)，新白薇甙元B 3-O-β-L-磁嘛吡喃糖基-（1 → 4）-β-D-洋黄吡喃糖基-（1 → 4）-β-D-夹竹桃吡喃糖甙（10），白前甙元 A 3-O-α-L-磁嘛吡喃糖基-（1 → 4）-β-D-洋地黄吡喃糖基-（1 → 4）-β-D-磁嘛吡喃糖甙（11），白前甙元 B 3-O-α-L-磁糖基吡喃糖基-(1 → 4)-β-D-磁嘛吡喃糖基-(1 → 4)-β-D-磁嘛吡喃糖甙(12)，白前甙元D 3-O-α-L-磁嘛吡喃糖基（1 → 4）-β-D-洋地黄吡喃糖基-（1 → 4）-β-D-磁嘛吡喃糖甙（13），白前甙元 C 3-O-β-D-葡萄吡喃糖基-（1 → 4）-β-D-葡萄吡喃糖基-（1 → 4）-α-L-2-脱氧洋地黄吡喃糖基-（1 → 4）-β-D-磁嘛吡喃糖基-（1 → 4）-β-D-夹竹桃吡喃糖甙（14），白前甙元 C 3-O-β-D-葡萄吡喃糖基-（1 → 4）-β-D-葡萄吡喃糖基-（1 → 4）-α-L-磁嘛吡喃糖基-（1 → 4）-β-D-洋地黄吡喃糖基-（1 → 4）-β-D-夹竹桃吡喃糖甙（15），白前甙元 B 3-O-α-D-夹竹桃吡喃糖基-(1 → 4)-β-D-洋地黄吡喃糖基-（1 → 4）-β-D-磁嘛吡喃糖甙（16），白前甙元 D 3-O-α-D-夹竹桃吡喃糖基-(1 → 4)-β-D-洋地黄吡喃糖基-(1 → 4)-β-D-磁嘛吡喃糖甙(17)，白前甙元月B 3-O-α-L-磁嘛吡喃糖基-(1 → 4)-β-D-洋地黄吡喃糖基-(1 → 4)-β-D-磁嘛吡喃糖甙（18）。其中，化合物3-6，10-18为新化合物，10的甙元为一个新甙元，命名为新白薇甙无B。

四种药用植物的化学成分及芍药苷的微生物转化研究

本学位论文共有5章。第一章报道白芍的化学成分及芍药苷的微生物转化研究成果；第二章报道天山雪莲的化学成分研究；第三章报道两面针的化学成分研究；第四章报道通关藤的化学成分研究成果；第五章概述了花椒属植物中最近十年报道的新化合物及药理研究情况。 在第1章的第一部分报道了白芍（Paeonia lactiflora Pall.）的化学成分。我们采用正、反相硅胶柱层析等各种分离方法，从白芍的干燥根中共分离出14个化合物，其中1个为新化合物，其结构通过波谱分析证实为没食子酰白芍苷，另外还有2个为首次从该植物中分离得到。第二部分报道了芍药苷的微生物转化生产芍药苷代谢素-I的研究，从15株厌氧菌中筛选出10株有转化活性的菌株，其中短乳杆菌Lactobacillus brevis AS1.12的转化活性最好，对其转化条件进行了初步的筛选，确定了相对合理的转化工艺。 在第2章报道了天山雪莲（Saussurea involucrate Kar.et Kir.）全草乙醇提取物化学成分的分离纯化和结构鉴定。通过正、反相硅胶柱层析等分离纯化和MS、NMR等波谱解析，共分离鉴定了28个化合物，结构类型分属于黄酮、倍半萜和木脂素等，其中2个新倍半萜化合物的结构分别表征为6α-羟基云木香酸6-β-D-吡喃葡萄糖苷和11βH-11,13-二氢去氢云木香内酯8α-O-(6′-乙酰)-β-D-吡喃葡萄糖苷。 第3章报道了两面针（Zanthoxylum nitidum （Roxb.）DC.）干燥根的乙醇提取物化学成分的分离纯化和结构鉴定。通过正、反相硅胶柱层析等分离纯化和MS、NMR等波谱解析以及X-射线单晶衍射，共分离鉴定了16个生物碱，结构类型分属于苯并啡啶类、喹啉类和阿朴啡类等，其中2个新苯并啡啶类生物碱的结构分别表征为二聚双氢两面针碱和丙酮基双氢崖定椒碱。 第4章报道了通关藤（Marsdenia tenacissima (Roxb.) Wight et Arn.）水提取物化学成分的分离纯化和结构鉴定。通过正、反相硅胶柱层析等分离纯化和MS、NMR等波谱解析以及X-射线单晶衍射，共分离鉴定了14个化合物，结构类型均属于C21多羟基甾醇，其中4个新化合物tenacigenoside A, tenacigenoside B, tenacigenoside C和tenacigenoside D的结构分别表征为3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-17β-tenacigenin B (62), 3-O-2,6- dideoxy-4-O-methyl-D-lyxo-hexopyranosly-11α-O- methylbutyryl-12β-O-acetyl-tenacigenin B (63), 3-O-6-deoxy-3-O-methyl-β-D- allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α-O-tigloyl-tenacigenin C (64)和3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α-O-2- methylbutyryl-tenacigenin C (65)。 第5章概述了花椒属植物的化学成分及药理活性研究进展。 This dissertation consists of 5 chapters. The first chapter elaborate the phytochemical investigation of Paeonia lactiflora Pall., and microbial transformation of paeoniforin. The second, third and four chapters elaborate the phytochemical investigation of Saussurea involucrate Kar.et Kir., Zanthoxylum nitidum (Roxb.) DC. and Marsdenia tenacissima (Roxb.) Wight et Arn., respectively. Chapter 5 is a review on chemical constituents and bioactivities of Zanthoxylum species. The part one of chapter 1 focus on the isolation and identification of chemical constituents from P. lactiflora. Fourteen compounds were isolated from the roots of P. lactiflora by repeat column chromatography over normal and reversed phase silica gel. Among them, one is a new compound and the structure was suggested as galloyl-albiflorin by spectral evidence. In addition, two compounds were firstly reported in this plant. The part 2 is about microbial transformation of paeoniforin. Chapters 2, 3 and 4 were isolations and identifications of chemical constituents from S. involucrate, Z. nitidum and M. tenacissima, respectively. From the aerial parts of S. involucrate, 28 compounds including 7 flavonoids and 13 sesquiterpenoids were isolated and identified. Among them, 2 new compounds were characterized as 6α-hydroxycostic acid 6-β-D-glucoside and 11βH-11,13-dihydrodehydro- costuslactone 8α-O-(6'-acetyl)-β-D-glucoside, respectively, by means of spectroscopic analysis. Otherwise, 11 ones were firstly reported from this plant. The third chapter is about the phytochemical investigation of Z. nitidum. Sixteen compounds were isolated and identified. Among them, 2 new benzophenanthridine alkaloids were characterized as 8-acetonyldihydrofagaridine and 1,3-bis(8-dihydronitidinyl)-acetone by spectroscopic analysis. The fourth chapter is about the phytochemical investigation of M. tenacissima. Fourteen compounds were isolated and identified. Among them, 4 new compounds, tenacigenosides A~D, were characterized as 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-17β- tenacigenin B, 3-O-2,6-dideoxy-4-O-methyl-D-lyxo-hexopyranosly-11α-O-methyl butyryl-12β-O-acetyl-tenacigenin B, 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl- (1→4)-β-D-oleandropyranosyl-11α-O-tigloyl-tenacigenin C, and 3-O-6-deoxy-3-O- methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α-O-2-methylbutyryl- tenacigenin C. Chapter 5 is a review on recent progress in bioactive constituents from plants of Zanthoxylum species.

First record of Aecidium sp. on Marsdenia sp. in Australia

Aecidium sp. (Pucciniaceae) on Marsdenia sp. is reported for the first time from Australia.

Contribution to the knowledge of the Stipa-tenacissima Iberian grasslands (All. Stipion tenacissimae)

A syntaxonomic revision of the Iberian "espartales" -dry grasslands dominated by Stipa tenacissima L.- is reoprted. Nomenclatural types as well as floristic, ecologic, dynamic and biographic data  (Stipion tenacissimae, Lygeo-Stipetalia, Lygeo Stipetea) is described for the thermophilous Alcoian espartales, and the name Heteropogono conforti-Stipeton tenacissimae is also validated for the thermophilous Valencian-Tarraconensian ones.

Avaliação das atividades antioxidante, anticoagulante e antiproliferativa de extratos aquosos de marsdenia megalantha

The species of the genus Marsdenia, Apocynaceae, are widely used in folk medicine of several countries. In Brazil is found several species belonging to this genus. The in vitro antioxidant, anticoagulant and antiproliferative activities were evaluated to aqueous extracts of stalk, leaf and root of Marsdenia megalantha. In the total antioxidant capacity assay (expressed as ascorbic acid equivalents) the stalk extract showed 76.0 mg/g, while leaf and root extracts 141.3 mg/g and 57.0 mg/g, respectively. The stalk and leaf extracts showed chelating activity around 40% at 1.5 mg/mL, while root extract, at the same concentration showed, 17%. Only the leaf extract showed a significant ability in superoxide scavenging (80% at 0.8 mg/mL). Any extract was able in scavenge hydroxyl, as well anticoagulant activity. The antiproliferative activity of the extracts was evaluated against HeLa tumor cell line. The extracts inhibited in a dose-dependent manner the cell growth. However, the leaf extract showed 80% of inhibition at 1.0 mg/mL, while stalk and root extracts inhibited 63% and 30%, respectively. To assess the mechanism of cell death caused by the leaf extract in HeLa, was performed flow cytometry and western blot. The results show that leaf extract induces cell death by apoptosis through an activation caspase-independent pathway. These data indicate that stalk and leaf extracts obtained have potential to be used as antioxidants and anticancer drugs

Assessment of species diversity and state of "Stipa tenacissima" steppes

North African steppes are subjected to extreme degradation resulting in the reduction of their surface, genetic erosion of resources, and decrease in biodiversity. "Stipa tenacissima" steppes, which constitute one of the most representative vegetation types in the driest areas of the Mediterranean basin, are continuously degrading. With the aim of contributing to a better knowledge of the floristic composition and diagnosing the state of degradation of these steppes, we conducted a phytoecological analysis of 10 "S. tenacissima" sites in Tunisia. Floristic inventory compiled a systematic list of 46 vascular plant species belonging to 43 genera and 26 families. Species richness ranged from 4 to 18 species per 900 m2. Total vegetation cover was moderate and fluctuated between 22.8% and 49.9%. Our results revealed also a decreasing trend in species richness with increasing elevation (ρ = –0.585). Indeed, species richness was negatively correlated with slope (ρ = –0.19) and positively correlated with sand content (ρ = 0.262). Biological types were dominated by chamaephytes; this chamaephytization is due to the phenomenon of aridization and overgrazing. Moreover, the low species cover and the appearance of nonpalatable species highlighted the vulnerability of these steppes to degradation.

Contribution a l’identification des principaux facteurs de degradation des steppes a Stipa tenacissima du sud-ouest Algerien

La steppe algérienne est confrontée depuis plusieurs décennies à un problème de dégradation induit par l’effet combiné de facteurs anthropiques et naturels. Les résultats obtenus dans la lutte contre ce phénomène, malgré les moyens mobilisés, restent très mitigés et révèlent l’inefficacité des approches et méthodes adoptées. Toute action de préservation et de réhabilitation de la steppe doit reposer dans un premier temps sur un diagnostic permettant d’identifier et d’évaluer le poids de chaque facteur dégradant. Parmi les facteurs souvent soulignés on note le climat, le parcours, le défrichement et la pratique d’une agriculture pluviale sans une justification ni estimation de cet impact. Le diagnostic ciblant et classant les principaux facteurs de régression des formations steppique de Stipa tenacissima est l’objectif assigné à cette publication. La démarche retenue traitera de l’état des formations steppiques face aux pressions pour une identification des facteurs causaux de cette situation et une évaluation de leur impact futur.

Avaliação das atividades antioxidante, anticoagulante e antiproliferativa de extratos aquosos de marsdenia megalantha

The species of the genus Marsdenia, Apocynaceae, are widely used in folk medicine of several countries. In Brazil is found several species belonging to this genus. The in vitro antioxidant, anticoagulant and antiproliferative activities were evaluated to aqueous extracts of stalk, leaf and root of Marsdenia megalantha. In the total antioxidant capacity assay (expressed as ascorbic acid equivalents) the stalk extract showed 76.0 mg/g, while leaf and root extracts 141.3 mg/g and 57.0 mg/g, respectively. The stalk and leaf extracts showed chelating activity around 40% at 1.5 mg/mL, while root extract, at the same concentration showed, 17%. Only the leaf extract showed a significant ability in superoxide scavenging (80% at 0.8 mg/mL). Any extract was able in scavenge hydroxyl, as well anticoagulant activity. The antiproliferative activity of the extracts was evaluated against HeLa tumor cell line. The extracts inhibited in a dose-dependent manner the cell growth. However, the leaf extract showed 80% of inhibition at 1.0 mg/mL, while stalk and root extracts inhibited 63% and 30%, respectively. To assess the mechanism of cell death caused by the leaf extract in HeLa, was performed flow cytometry and western blot. The results show that leaf extract induces cell death by apoptosis through an activation caspase-independent pathway. These data indicate that stalk and leaf extracts obtained have potential to be used as antioxidants and anticancer drugs

Vascular Plants and Biocrusts Modulate How Abiotic Factors Affect Wetting and Drying Events in Drylands

Understanding how organisms control soil water dynamics is a major research goal in dryland ecology. Although previous studies have mostly focused on the role of vascular plants on the hydrological cycle of drylands, recent studies highlight the importance of biological soil crusts formed by lichens, mosses, and cyanobacteria (biocrusts) as a major player in this cycle. We used data from a 6.5-year study to evaluate how multiple abiotic (rainfall characteristics, temperature, and initial soil moisture) and biotic (vascular plants and biocrusts) factors interact to determine wetting and drying processes in a semi-arid grassland from Central Spain. We found that the shrub Retama sphaerocarpa and biocrusts with medium cover (25–75%) enhanced water gain and slowed drying compared with bare ground areas (BSCl). Well-developed biocrusts (>75% cover) gained more water, but lost it faster than BSCl microsites. The grass Stipa tenacissima reduced water gain due to rainfall interception, but increased soil moisture retention compared to BSCl microsites. Biotic modulation of water dynamics was the result of different mechanisms acting in tandem and often in opposite directions. For instance, biocrusts promoted an exponential behavior during the first stage of the drying curve, but reduced the importance of soil characteristics that accentuate drying rates. Biocrust-dominated microsites gained a similar amount of water than vascular plants, although they lost it faster than vascular plants during dry periods. Our results emphasize the importance of biocrusts for water dynamics in drylands, and illustrate the potential mechanisms behind their effects. They will help to further advance theoretical and modeling efforts on the hydrology of drylands and their response to ongoing climate change.

Aridity modulates N availability in arid and semiarid Mediterranean grasslands

While much is known about the factors that control each component of the terrestrial nitrogen (N) cycle, it is less clear how these factors affect total N availability, the sum of organic and inorganic forms potentially available to microorganisms and plants. This is particularly true for N-poor ecosystems such as drylands, which are highly sensitive to climate change and desertification processes that can lead to the loss of soil nutrients such as N. We evaluated how different climatic, abiotic, plant and nutrient related factors correlate with N availability in semiarid Stipa tenacissima grasslands along a broad aridity gradient from Spain to Tunisia. Aridity had the strongest relationship with N availability, suggesting the importance of abiotic controls on the N cycle in drylands. Aridity appeared to modulate the effects of pH, plant cover and organic C (OC) on N availability. Our results suggest that N transformation rates, which are largely driven by variations in soil moisture, are not the direct drivers of N availability in the studied grasslands. Rather, the strong relationship between aridity and N availability could be driven by indirect effects that operate over long time scales (decades to millennia), including both biotic (e.g. plant cover) and abiotic (e.g. soil OC and pH). If these factors are in fact more important than short-term effects of precipitation on N transformation rates, then we might expect to observe a lagged decrease in N availability in response to increasing aridity. Nevertheless, our results suggest that the increase in aridity predicted with ongoing climate change will reduce N availability in the Mediterranean basin, impacting plant nutrient uptake and net primary production in semiarid grasslands throughout this region.

Community attributes determine facilitation potential in a semi-arid steppe

Studies on positive plant–plant relations have traditionally focused on pair-wise interactions. Conversely, the interaction with other co-occurring species has scarcely been addressed, despite the fact that the entire community may affect plant performance. We used woody vegetation patches as models to evaluate community facilitation in semi-arid steppes. We characterized biotic and physical attributes of 53 woody patches (patch size, litter accumulation, canopy density, vegetation cover, species number and identity, and phylogenetic distance), and soil fertility (organic C and total N), and evaluated their relative importance for the performance of seedlings of Pistacia lentiscus, a keystone woody species in western Mediterranean steppes. Seedlings were planted underneath the patches, and on their northern and southern edges. Woody patches positively affected seedling survival but not seedling growth. Soil fertility was higher underneath the patches than elsewhere. Physical and biotic attributes of woody patches affected seedling survival, but these effects depended on microsite conditions. The composition of the community of small shrubs and perennial grasses growing underneath the patches controlled seedling performance. An increase in Stipa tenacissima and a decrease in Brachypodium retusum increased the probability of survival. The cover of these species and other small shrubs, litter depth and community phylogenetic distance, were also related to seedling survival. Seedlings planted on the northern edge of the patches were mostly affected by attributes of the biotic community. These traits were of lesser importance in seedlings planted underneath and in the southern edge of patches, suggesting that constraints to seedling establishment differed within the patches. Our study highlights the importance of taking into consideration community attributes over pair-wise interactions when evaluating the outcome of ecological interactions in multi-specific communities, as they have profound implications in the composition, function and management of semi-arid steppes.

Drought and grazing combined: Contrasting shifts in plant interactions at species pair and community level

The combined effects of drought stress and grazing pressure on shaping plant–plant interactions are still poorly understood, while this combination is common in arid ecosystems. In this study we assessed the relative effect of grazing pressure and slope aspect (drought stress) on vegetation cover and soil functioning in semi-arid Mediterranean grassland–shrublands in southeastern Spain. Moreover, we linked these two stress factors to plant co-occurrence patterns at species-pair and community levels, by performing C-score analyses. Vegetation cover and soil functioning decreased with higher grazing pressure and more south-facing (drier) slopes. At the community level, plants at south-facing slopes were negatively associated at no grazing but positively associated at low grazing pressure and randomly associated at high grazing pressure. At north-facing slopes, grazing did not result in a shift in the direction of the association. In contrast, analysis of pairwise species co-occurrence patterns showed that the dominant species Stipa tenacissima and Anthyllis cytisoides shifted from excluding each other to co-occurring with increasing grazing pressure at north-facing slopes. Our findings highlight that for improved understanding of plant interactions along stress gradients, interactions between species pairs and interactions at the community level should be assessed, as these may reveal contrasting results.