Low Specificity but Dissimilar Mycorrhizal Communities Associating with Roots May Contribute to the Spatial Pattern of Four Co-Occurring Habenaria (Orchidaceae) Species.

Fungi with orchid roots have been increasingly proven to play important roles in orchid growth, spatial distribution, and coexistence of natural communities. Here, we used 454 amplicon pyrosequencing with two different primer combinations to investigate the spatial variations in the community of OMF and endophytic fungi associates within the roots of four co-occurring Habenaria species. The results showed that all investigated Habenaria species were generalists and the different fungi communities may contribute to the spatial separation of the four Habenaria species. Firstly, the fungal OTUs identified in the roots of the four species overlapped but their presence differed amongst species and numerous distinct OMF families were unique to each species. Second, NMDS clustering showed samples clustered together based on associated species and PERMANOVA analyses indicated that fungi communities in the roots differed significantly between the Habenaria species, both for all endophytic fungi communities and for OMF communities. Third, the network structure of epiphytic fungi was highly specialized and modular but demonstrated lowly connected and anti-nested properties. However, it calls for more soil nutrition and soil fungal communities' studies to elucidate the contribution of habitat-specific adaptations in general and mycorrhizal divergence.

Preparation, In Vitro and In Vivo Evaluation of Nanoemulsion In Situ Gel for Transnasal Delivery of Traditional Chinese Medicine Volatile Oil from Ligusticum sinense Oliv.cv. Chaxiong.

Ischemic stroke is a difficult-to-treat brain disease that may be attributed to a limited therapeutic time window and lack of effective clinical drugs. Nasal-brain administration is characterized by low systemic toxicity and is a direct and non-invasive brain targeting route. Preliminary studies have shown that the volatile oil of Chaxiong (VOC) has an obvious anti-ischemic stroke effect. In this work, we designed a nanoemulsion thermosensitive in situ gel (VOC-NE-ISG) loaded with volatile oil of Chaxiong for ischemia via intranasal delivery to rat brain treatment of cerebral ischemic stroke. The developed VOC-NE-ISG formulation has a suitable particle size of 21.02 ± 0.25 nm and a zeta potential of -20.4 ± 1.47 mV, with good gelling ability and prolonged release of the five components of VOC. The results of in vivo pharmacokinetic studies and brain targeting studies showed that intranasal administration of VOC-NE-ISG could significantly improve the bioavailability and had excellent brain-targeting efficacy of nasal-to-brain delivery. In addition, the results of pharmacodynamics experiments showed that both VOC-NE and VOC-NE-ISG could reduce the neurological deficit score of model rats, reducing the size of cerebral infarction, with a significant effect on improving ischemic stroke. Overall, VOC-NE-ISG may be a promising intranasal nanomedicine for the effective treatment of ischemic stroke.

A comparative study on the reproductive success of two rewarding Habenaria species (Orchidaceae) occurring in roadside verge habitats.

BACKGROUND: Most orchid species have been shown to be severely pollination limited, and the factors affecting reproductive success have been widely studied. However, the factors determining the reproductive success vary from species to species. Habenaria species typically produce nectar but exhibit variable fruit set and reproductive success among species. Here, we investigated the influence of the flowering plant density, inflorescence size, breeding system, and pollinator behaviour on the reproductive success of two rewarding Habenaria species. RESULTS: Our observations indicated that Habenaria limprichtii and H. petelotii co-occur in roadside verge habitats and present overlapping flowering periods. Both species were pollination limited, although H. limprichtii produced more fruits than H. petelotii under natural conditions during the 3-year investigation. H. petelotii individuals formed distinct patches along roadsides, while nearly all H. limprichtii individuals clustered together. The bigger floral display and higher nectar sugar concentration in H. limprichtii resulted in increased attraction and visits from pollinators. Three species of effective moths pollinated for H. limprichtii, while Thinopteryx delectans (Geometridae) was the exclusive pollinator of H. petelotii. The percentage of viable seeds was significantly lower for hand geitonogamy than for hand cross-pollination in both species. However, H. limprichtii may often be geitonogamously pollinated based on the behaviours of the pollinators and viable embryo assessment. CONCLUSIONS: In anthropogenic interference habitats, the behaviours and abundance of pollinators influence the fruit set of the two studied species. The different pollinator assemblages in H. limprichtii can alleviate pollinator specificity and ensure reproductive success, whereas the more viable embryos of natural fruit seeds in H. petelotii suggested reducing geitonogamy by pollinators in the field. Our results indicate that a quantity-quality trade-off must occur between species with different breeding strategies so that they can fully exploit the existing given resources.

Are fungi from adult orchid roots the best symbionts at germination? A case study.

We studied mycobionts from advanced seedlings and adult mycorrhizal roots of the terrestrial orchid Arundina graminifolia. Fungi were isolated, identified by ITS sequencing, and tested for their impact on seed germination, protocorm formation, and development of advanced seedlings (emergence of first leaf) in vitro. Among the six fungal species isolated, four were not standard orchid mycorrhizal fungi (Fusarium solani, Cylindrocarpon sp., Acremonium sp., and Phlebiopsis flavidoalba) and did not support germination beyond imbibition and greening of the seeds during a span of 35 days. Over the same time, one Tulasnella species isolated from adult mycorrhiza allowed protocorm formation but not further development. However, another Tulasnella species isolated from advanced seedlings facilitated development to the advanced seedling stage. Our results support (i) the inability of occasional orchid root colonizers to support late seed germination, and (ii) the growing literature showing that fungal associates can change over orchid development. Functionally, we show that mycorrhizal taxa isolated from advanced seedlings can be more efficient than those from adults in supporting germination in some species, leading to recommendations for ex situ orchid conservation.

Preparation, in vitro and in vivo Evaluation of Thermosensitive in situ Gel Loaded with Ibuprofen-Solid Lipid Nanoparticles for Rectal Delivery.

Background: Ibuprofen (IBU), a nonsteroidal anti-inflammatory drug, shows poor gastrointestinal absorption due to its low solubility, which limits its clinical application. Objective: In the present study, we aimed to develop thermosensitive gel-mediated ibuprofen-solid lipid nanoparticles (IBU-SLN-ISG) to improve the dissolution and bioavailability of IBU after rectal delivery. Methods: IBU-loaded SLNs (IBU-SLNs) were developed and optimized applying Box-Behnken design. The optimized IBU-SLNs were characterized by physicochemical parameters and morphology. Then, the optimized IBU-SLNs was incorporated into the gel and characterized for gel properties and rheology and investigated its release in vitro, pharmacokinetics in vivo, rectal irritation and rectal retention time. Results: The optimized SLNs had an EE of 90.74 ± 1.40%, DL of 11.36 ± 1.20%, MPS of 166.77 ± 2.26 nm, PDI of 0.27 ± 0.08, and ZP of -21.00 ± 0.59 mV. The FTIR spectra confirmed successful encapsulation of the drug inside the nanoparticle as only peaks responsible for the lipid could be identified. This corroborated well with XRD spectra, which showed a completely amorphous state of the IBU-SLNs as compared to the crystalline nature of the pure drug. The gelation temperature of the prepared IBU-SLN-ISG was 33.30 ± 0.78°C, the gelation time was 14.67 ± 2.52 s, the gel strength was 54.00 ± 1.41 s, and the mucoadhesion was (11.54±0.37) × 102dyne/cm2. The in vitro results of IBU-SLNs and IBU-SLN-ISG showed a biphasic release pattern with initial burst release followed by sustained release. More importantly, IBU-SLN-ISG produced much better absorption of IBU and improved bioavailability in rats. In addition, IBU-SLN-ISG caused no irritation or damage to rectal tissues, and could be retained in the rectum for a long time. Conclusion: Thermosensitive in situ gel loaded with IBU-solid lipid nanoparticles might be further developed as a more convenient and effective rectal dosage form.

Pharmacokinetics of five phthalides in volatile oil of Ligusticum sinense Oliv.cv. Chaxiong, and comparison study on physicochemistry and pharmacokinetics after being formulated into solid dispersion and inclusion compound.

BACKGROUNDS: The dried rhizome of Ligusticum sinense Oliv.cv. Chaxiong has been used to treat cardiovascular and cerebrovascular diseases, atherosclerosis, anemia and stroke. A high purity extract from chaxiong (VOC, brownish yellow oil) was extracted and separated. Its main components were senkyunolide A (SA, 33.81%), N-butylphthalide (NBP, 1.38%), Neocnidilide (NOL, 16.53%), Z-ligustilide (ZL, 38.36%), and butenyl phthalide (BP, 2.48%), respectively. Little is known about the pharmacokinetics of these phthalides in Chaxiong, and different preparations to improve the physicochemistry and pharmacokinetics of VOC have not been investigated. METHODS: At different predetermined time points after oral administration or intravenous administration, the concentrations of SA, NBP, NOL, ZL and BP in the rat plasma were determined using LC-MS/MS, and the main PK parameters were investigated. VOC-P188 solid dispersion and VOC-ß-CD inclusion compound were prepared by melting solvent method and grinding method, respectively. Moreover, the physicochemical properties, dissolution and pharmacokinetics of VOC-P188 solid dispersion and VOC-ß-CD inclusion compound in rats were assessed in comparison to VOC. RESULTS: The absorptions of SA, NBP, NOL, ZL and BP in VOC were rapid after oral administration, and the absolute bioavailability was less than 25%. After the two preparations were prepared, dissolution rate was improved at pH 5.8 phosphate buffer solution. Comparing VOC and physical mixture with the solid dispersion and inclusion compound, it was observed differences occurred in the chemical composition, thermal stability, and morphology. Both VOC-P188 solid dispersion and VOC-ß-CD inclusion compound had a significantly higher AUC and longer MRT in comparison with VOC. CONCLUSION: SA, NBP, NOL, ZL and BP in VOC from chaxiong possessed poor absolute oral bioavailability. Both VOC-P188 solid dispersion and VOC-ß-CD inclusion compound could be prospective means for improving oral bioavailability of SA, NBP, NOL, ZL and BP in VOC.

Multiple factors contribute to reproductive isolation between two co-existing Habenaria species (Orchidaceae).

Reproductive isolation is a key feature that forms barriers to gene flow between distinct plants. In orchids, prezygotic reproductive isolation has been considered to be strong, because their associations with highly specific pollinators. In this study, the reproductive ecology and reproductive isolation of two sympatric Habenaria species, H. davidii and H. fordii, was investigated by floral phenology and morphology, hand-pollination experiments and visitor observation in southwest China. The two species were dependent on insects for pollination and completely self-compatible. A number of factors have been identified to limit gene flow between the two species and achieved full reproductive isolation. Ecogeographic isolation was a weak barrier. H. fordii and H. davidii had completely overlapped flowering periods, and floral morphology plays an important role in floral isolation. The two species shared the same hawkmoth pollinator, Cechenena lineosa, but the pollinaria of the two orchids were attached on different body parts of pollinators. Prezygotic isolation was not complete, but the interspecific pollination treatments of each species resulted in no seed sets, indicating that unlike many other orchid species, in which the postzygotic reproductive isolation is very weak or complete absence, the post-zygotic isolation strongly acted in the stage of seed production between two species. The results illustrate the reproductive isolation between two species involves multiple plant life-history stages and a variety of reproductive barriers can contribute to overall isolation.