Safety Evaluation of Natural Drugs in Chronic Skeletal Disorders: A Literature Review of Clinical Trials in the Past 20 years

Chronic skeletal disorders (CSDs), including degenerative diseases such as osteoporosis (OP) and autoimmune disorders, have become a leading cause of disability in an ageing society, with natural drugs being indispensable therapeutic options. The clinical safety evaluation (CSE) of natural drugs in CSDs has been given priority and has been intensively studied. To provide fundamental evidence for the clinical application of natural drugs in the elderly population, clinical studies of natural drugs in CSDs included in this review were selected from CNKI, Web of Science, PubMed, Science Direct and Google Scholar since 2001. Seventeen randomized controlled trials (RCTs) met our inclusion criteria: four articles were on OP, seven on osteoarthritis (OA), four on rheumatoid arthritis (RA) and two on gout. Common natural drugs used for the treatment of OP include Epimedium brevicornu Maxim [Berberidaceae], Dipsacus asper Wall ex DC [Caprifoliaceae] root, and Phalaenopsis cornu-cervi (Breda) Blume & Rchb. f[ Orchidaceae], which have been linked to several mild adverse reactions, such as skin rash, gastric dysfunction, abnormal urine, constipation and irritability. The safety of Hedera helix L [Araliaceae] extract, Boswellia serrata Roxb [Burseraceae] extract and extract from perna canaliculus was evaluated in OA and upper abdominal pain, and unstable movements were obsrerved as major side effects. Adverse events, including pneumonia, vomiting, diarrhoea and upper respiratory tract infection, were reported when RA was treated with Tripterygium wilfordii, Hook. F [Celastraceae][TwHF] polyglycosides and quercetin (Capsella bursa-pastoris (L.) Medik [Brassicaceae]). The present review aimed to summarize the CSE results of natural drugs in CSDs and could provide evidence-based information for clinicians.

A new method to localise and quantify oxidative stress in live juvenile mussels

Stress and survival of juvenile New Zealand green-lipped mussel, Perna canaliculus, is a poorly-understood bottleneck in the ecological and economic performance of a significant aquaculture crop. This species was therefore selected as a model organism for the development of a new method to quantify oxidative stress in whole individuals. An in vivo ROS-activated stain (CellROX™) was administered to anaesthetised, translucent juveniles that were subsequently formaldehyde-fixed and visualised using confocal microscopy. Subsequent application of image analysis to quantifying ROS-positive tissue areas was successfully used to detect stress differences in juvenile mussels exposed to varying levels of emersion. This integrated method can be used to localise and quantify ROS production in individual translucent bivalve life stages (larval and juvenile), while relative stability following fixation greatly expands potential practical field applications.

Does Seston Quality Limit the Presence of Mussels on Wellington’s South Coast? FlowCAM investigation of seston particle type

<p>Temperate rocky shores around the world are characterised by so-called ‘universal’ zonation. An interesting exception to this rule can be seen along the shores of Wellington’s South Coast where there is a virtual absence of mussels and poorly developed rocky shore intertidal community. Yet just kilometres away in Wellington Harbour there is a fully developed intertidal community, including extensive multi-species mussel beds. This thesis aims to determine if the quality of seston is limiting the presence of mussels on Wellington’s South Coast. We now have the technology to see what types of particles the mussels are selecting in low and high quality seston conditions using a FlowCAM that allows identification of particle types and their physical properties. This study compared environmental data for Wellington Harbour (seston-rich) and the South Coast (seston-poor). These data included chlorophyll a, turbidity and suspended sediment concentrations and were collected by CTD and satellite remote sensing. Mussel feeding experiments took place under a high and low quality diet during summer, autumn and winter using Perna canaliculus and Mytilus galloprovincialis. The physiological responses recorded were clearance rate, absorption efficiency and net energy balance for individual mussels. Environmental variables recorded were total particulate matter, particulate organic matter and percent organic matter. Water samples were collected during the feeding experiments and processed using the FlowCAM. By comparing the control chambers to chambers that had mussels feeding in them it was possible to see what particles the mussels were selecting. The environmental variables revealed that Wellington Harbour had a much higher quality seston whereas in Cook Strait the seston quality was too low for mussels to be able to inhabit, as the chlorophyll a concentrations did not reach the required levels for mussel growth. Perna canaliculus and Mytilus galloprovincialis both showed physiological responses that would allow them to grow in Cook Strait waters, both species had positive absorption efficiencies and net energy balances. These responses were greater in the high quality diet in the enriched pond water in Nelson during summer and winter. The FlowCAM analysis revealed an inter-specific difference in preferential particle selection, which varied as a function of site and season. With more particles being preferentially selected in the high quality diet compared to when the mussels were feeding on Cook Strait seawater. This new information of particle selection helps to determine why mussels are absent from Wellington’s South Coast and contributes to the extensive information on mussel feeding.</p>

Does Seston Quality Limit the Presence of Mussels on Wellington’s South Coast? FlowCAM investigation of seston particle type

<p>Temperate rocky shores around the world are characterised by so-called ‘universal’ zonation. An interesting exception to this rule can be seen along the shores of Wellington’s South Coast where there is a virtual absence of mussels and poorly developed rocky shore intertidal community. Yet just kilometres away in Wellington Harbour there is a fully developed intertidal community, including extensive multi-species mussel beds. This thesis aims to determine if the quality of seston is limiting the presence of mussels on Wellington’s South Coast. We now have the technology to see what types of particles the mussels are selecting in low and high quality seston conditions using a FlowCAM that allows identification of particle types and their physical properties. This study compared environmental data for Wellington Harbour (seston-rich) and the South Coast (seston-poor). These data included chlorophyll a, turbidity and suspended sediment concentrations and were collected by CTD and satellite remote sensing. Mussel feeding experiments took place under a high and low quality diet during summer, autumn and winter using Perna canaliculus and Mytilus galloprovincialis. The physiological responses recorded were clearance rate, absorption efficiency and net energy balance for individual mussels. Environmental variables recorded were total particulate matter, particulate organic matter and percent organic matter. Water samples were collected during the feeding experiments and processed using the FlowCAM. By comparing the control chambers to chambers that had mussels feeding in them it was possible to see what particles the mussels were selecting. The environmental variables revealed that Wellington Harbour had a much higher quality seston whereas in Cook Strait the seston quality was too low for mussels to be able to inhabit, as the chlorophyll a concentrations did not reach the required levels for mussel growth. Perna canaliculus and Mytilus galloprovincialis both showed physiological responses that would allow them to grow in Cook Strait waters, both species had positive absorption efficiencies and net energy balances. These responses were greater in the high quality diet in the enriched pond water in Nelson during summer and winter. The FlowCAM analysis revealed an inter-specific difference in preferential particle selection, which varied as a function of site and season. With more particles being preferentially selected in the high quality diet compared to when the mussels were feeding on Cook Strait seawater. This new information of particle selection helps to determine why mussels are absent from Wellington’s South Coast and contributes to the extensive information on mussel feeding.</p>

Ecology of Biofouling and Impacts on Mussel Aquaculture: A Case Study with Didemnum Vexillum

<p>Over the past decade, several species of non-indigenous ascidian have had adverse effects in the marine environment and on associated industries. The colonial ascidian Didemnum vexillum is a recent successful invader in temperate marine communities worldwide, proving problematic to mussel aquaculture in New Zealand. At the inception of this thesis, control strategies to manage the threat from Didemnum to mussel aquaculture were implemented in the absence of information on the biological processes underpinning the species’ invasion success. Background information on Didemnum presented in Chapter 2 recognises this paucity of information on several key biological attributes as well as negative impacts of this species. The ability to obtain larvae and culture colonies in the laboratory was a crucial first step. Thus, Chapter 3 presents laboratory experiments that describe the first successful methods to induce spawning in adult Didemnum colonies, as well as techniques for the successful settlement and metamorphosis of the larvae produced, and for laboratory culture of juveniles. Chapters 4 to 6 address key aspects of the biological characteristics of Didemnum that relate to its invasiveness and spread. The recruitment and reproductive development of Didemnum were assessed over a 20-month period at two locations in central New Zealand. Results indicated that the reproductive season for Didemnum in New Zealand (at least 9 months) is considerably longer than previously believed, with recruitment patterns strongly correlated with seasonal water temperature fluctuations at each location. Secondly, the natural dispersal ability of Didemnum was assessed using a weight-of-evidence approach that combined laboratory and field studies. Larval competency trials revealed that > 70 % of larvae were able to settle and undergo metamorphosis following an artificial settlement delay of 2 hours. Larval viability decreased with increasing delay duration; however 10 % of larvae remained viable following a 36 hour delay. These findings were supported by a field-based study documenting larval recruitment at distances up to 250 m from source populations. Exponential decay models indicated that, given favourable conditions, larval dispersal distances greater than 1 km were theoretically possible, which is a much greater distance than previously assumed. Lastly, the impacts of Didemnum on cultured New Zealand green-lipped mussels (Perna canaliculus) were investigated. At the level of invasiveness experienced in a field experiment, only small mussel size classes were vulnerable to direct Didemnum impacts, with negative effects restricted to fouling-related displacement of mussels as opposed to reduced size or condition. However, at the greater levels of invasiveness evident at other places and times, Didemnum impacts have the potential to be considerably larger. As such, the ability to predict invasiveness, and hence impacts, is critical for stakeholders. However, for reasons discussed in the thesis, making reliable specific predictions of invasiveness is difficult. Despite such limitations, it is clear that an understanding of a species’ basic biological attributes can still greatly assist with management decisions, as highlighted throughout the chapters in this thesis. My research findings have led to a better awareness of commercial impacts and potential spread of this species. Simultaneously, my research also highlights the limitations inherent in inferring invasiveness from other situations (e.g. places, times, and related species); there is a need to specifically evaluate a species’ biological attributes and invasive behaviour when introduced into a novel environment.</p>

Ecology of Biofouling and Impacts on Mussel Aquaculture: A Case Study with Didemnum Vexillum

<p>Over the past decade, several species of non-indigenous ascidian have had adverse effects in the marine environment and on associated industries. The colonial ascidian Didemnum vexillum is a recent successful invader in temperate marine communities worldwide, proving problematic to mussel aquaculture in New Zealand. At the inception of this thesis, control strategies to manage the threat from Didemnum to mussel aquaculture were implemented in the absence of information on the biological processes underpinning the species’ invasion success. Background information on Didemnum presented in Chapter 2 recognises this paucity of information on several key biological attributes as well as negative impacts of this species. The ability to obtain larvae and culture colonies in the laboratory was a crucial first step. Thus, Chapter 3 presents laboratory experiments that describe the first successful methods to induce spawning in adult Didemnum colonies, as well as techniques for the successful settlement and metamorphosis of the larvae produced, and for laboratory culture of juveniles. Chapters 4 to 6 address key aspects of the biological characteristics of Didemnum that relate to its invasiveness and spread. The recruitment and reproductive development of Didemnum were assessed over a 20-month period at two locations in central New Zealand. Results indicated that the reproductive season for Didemnum in New Zealand (at least 9 months) is considerably longer than previously believed, with recruitment patterns strongly correlated with seasonal water temperature fluctuations at each location. Secondly, the natural dispersal ability of Didemnum was assessed using a weight-of-evidence approach that combined laboratory and field studies. Larval competency trials revealed that > 70 % of larvae were able to settle and undergo metamorphosis following an artificial settlement delay of 2 hours. Larval viability decreased with increasing delay duration; however 10 % of larvae remained viable following a 36 hour delay. These findings were supported by a field-based study documenting larval recruitment at distances up to 250 m from source populations. Exponential decay models indicated that, given favourable conditions, larval dispersal distances greater than 1 km were theoretically possible, which is a much greater distance than previously assumed. Lastly, the impacts of Didemnum on cultured New Zealand green-lipped mussels (Perna canaliculus) were investigated. At the level of invasiveness experienced in a field experiment, only small mussel size classes were vulnerable to direct Didemnum impacts, with negative effects restricted to fouling-related displacement of mussels as opposed to reduced size or condition. However, at the greater levels of invasiveness evident at other places and times, Didemnum impacts have the potential to be considerably larger. As such, the ability to predict invasiveness, and hence impacts, is critical for stakeholders. However, for reasons discussed in the thesis, making reliable specific predictions of invasiveness is difficult. Despite such limitations, it is clear that an understanding of a species’ basic biological attributes can still greatly assist with management decisions, as highlighted throughout the chapters in this thesis. My research findings have led to a better awareness of commercial impacts and potential spread of this species. Simultaneously, my research also highlights the limitations inherent in inferring invasiveness from other situations (e.g. places, times, and related species); there is a need to specifically evaluate a species’ biological attributes and invasive behaviour when introduced into a novel environment.</p>

Protein Identification Strategies for the Greenshell Mussel Perna canaliculus

<p>The greenshell mussel Perna canaliculus is considered to be a suitable biomonitor for heavy metal pollution. This is due to their ability to accumulate and tolerate heavy metals in their tissues. These characteristics make them useful for identifying protein biomarkers of heavy metal pollution, as well as proteins associated with heavy metal detoxification and homeostasis. However, the identification of such proteins is restricted by the greenshell mussel being poorly represented in sequence databases. Several strategies have previously been used to identify proteins in unsequenced species, but only one of these strategies has been applied to the greenshell mussel. The objective of this thesis was to examine different protein identification strategies using a combined two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry approach. The protein identification strategies used include a Mascot database search, as well as de novo sequencing approaches using PEAKS DB and SPIDER homology searches. In total, 155 protein spots were excised and a total of 68 identified. Fifty-six proteins were identified using a Mascot search against the Mollusca, NCBInr and Invertebrate EST database, with seven single-peptide identifications. De novo sequencing strategies identified additional proteins, with two from a PEAKS DB search and 10 from an error-tolerant SPIDER homology search. The most noticeable protein groups identified were cytoskeletal proteins, stress response proteins and those involved in protein biosynthesis. Actin and tubulin made up the bulk of the identifications, accounting for 39% of all proteins identified. This multifaceted approach was shown to be useful for identifying proteins in the greenshell mussel Perna canaliculus. Mascot and PEAKS DB performed equally well, while the error-tolerant functionality of SPIDER was useful for identifying additional proteins. A subsequent search against the Invertebrate EST database was also found to be useful for identifying additional proteins. Despite this, more than half of all proteins remained unidentified. Most of these proteins either failed to produce good quality MS spectra or did not find a match to a sequence in the database. Future research should first focus on obtaining quality MS spectra for all proteins concerned and then examine other strategies that may be more suitable for identifying proteins for species with poor representation in sequence databases.</p>

Protein Identification Strategies for the Greenshell Mussel Perna canaliculus

<p>The greenshell mussel Perna canaliculus is considered to be a suitable biomonitor for heavy metal pollution. This is due to their ability to accumulate and tolerate heavy metals in their tissues. These characteristics make them useful for identifying protein biomarkers of heavy metal pollution, as well as proteins associated with heavy metal detoxification and homeostasis. However, the identification of such proteins is restricted by the greenshell mussel being poorly represented in sequence databases. Several strategies have previously been used to identify proteins in unsequenced species, but only one of these strategies has been applied to the greenshell mussel. The objective of this thesis was to examine different protein identification strategies using a combined two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry approach. The protein identification strategies used include a Mascot database search, as well as de novo sequencing approaches using PEAKS DB and SPIDER homology searches. In total, 155 protein spots were excised and a total of 68 identified. Fifty-six proteins were identified using a Mascot search against the Mollusca, NCBInr and Invertebrate EST database, with seven single-peptide identifications. De novo sequencing strategies identified additional proteins, with two from a PEAKS DB search and 10 from an error-tolerant SPIDER homology search. The most noticeable protein groups identified were cytoskeletal proteins, stress response proteins and those involved in protein biosynthesis. Actin and tubulin made up the bulk of the identifications, accounting for 39% of all proteins identified. This multifaceted approach was shown to be useful for identifying proteins in the greenshell mussel Perna canaliculus. Mascot and PEAKS DB performed equally well, while the error-tolerant functionality of SPIDER was useful for identifying additional proteins. A subsequent search against the Invertebrate EST database was also found to be useful for identifying additional proteins. Despite this, more than half of all proteins remained unidentified. Most of these proteins either failed to produce good quality MS spectra or did not find a match to a sequence in the database. Future research should first focus on obtaining quality MS spectra for all proteins concerned and then examine other strategies that may be more suitable for identifying proteins for species with poor representation in sequence databases.</p>

The Effect of the Decorator Crab Notomithrax Minor on Cultivated Greenshell Mussel Spat (Perna Canaliculus) Survival, Growth, and Byssal Characteristics

<p>The decorator crab Notomithrax minor is common on Greenshell mussel (Perna canaliculus) farms in the Marlborough Sounds, New Zealand. Individuals in the Greenshell mussel industry have suggested that the presence of N. minor, found on mussel lines, is related to substantial losses of Greenshell mussel spat. Laboratory and field investigations were used to assess the effect of N. minor presence on the retention and productivity of Greenshell musselTM spat. Specific consideration was given to predation pressure and induced anti-predator defenses, both of which can cause financial losses to mussel farmers. High (12 crabs/cage-1) and low (3 crabs/cage-1) densities of large (males: >20mm, females: >15mm TCW) and medium (males: 15-20mm, females: 10-15mm TCW) decorator crabs were placed in cages on commercial Greenshell mussel farm droppers at two sites in the Pelorus Sound. The byssal characteristics, spat retention rate and spat shell length were assessed at 8 and 11 weeks after trial initiation. Greenshell mussel density on the experimental droppers decreased significantly when medium and high densities of the decorator crabs (N. minor) were introduced. N. minor presence induced the remaining Greenshell mussel spat to produce more and thicker byssus threads which consequently lead to increased mussel attachment. The decrease in retention rate and the increase in mussel attachment strength were more pronounced in small recently seeded spat. Laboratory experiments to assess the consumption rate of small (≤5mm) Greenshell mussel spat by decorator crabs showed that mussel consumption by N. minor peaked at 56.43 (± 13.02 (95% C.I.)) crab-1 hr-1, however the rate of consumption decreased significantly over the duration of the three day trial. N. minor prey size preference was also assessed using Laboratory trials; crabs were offered 4 size classes of mussels (small (<5 mm), small-medium (5-10 mm), medium-large (10-15 mm), large (>15 mm) simultaneously. Female crabs consumed more mussels in the <5 mm and 5-10 mm size classes than in the two larger mussel size classes (10-15 mm and >15 mm), whereas the male crabs showed a numerical preference for mussel spat in the small-medium and medium-large size classes. This study provides preliminary evidence that the decorator crab N. minor is a previously overlooked and under-estimated threat to the Greenshell Mussel industry in the Marlborough Sounds that deserves closer scrutiny and experimental testing.</p>