Comprehensive Screening and Characterization of α-glucosidase Inhibitory Components in the Edible Medicinal Plant Pholidota cantonensis Rolfe Using UPLC-Q-TOF-MS/MS Analysis and Molecular Docking.

Pholidota cantonensis Rolfe is an edible medicinal plant in the genus Pholidota of the family Orchidaceae. This plant is used to prepare medicated food in China and has been reported to possess anti-α-glucosidase activity. To date, little is known about the active substances responsible for the observed anti-α-glucosidase activity. In the present study, we aimed to screen and characterize the α-glucosidase inhibitory fraction of P. cantonensis using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) analysis and molecular docking. As a result, the 50% ethanol fraction obtained from D101 macroporous adsorption resin column chromatography (D50 fraction) had the highest total phenol content (353.83 ± 6.06 mg GAE/g) and the most prominent α-glucosidase inhibitory activity (IC50 = 30.01 ± 7.30 µg/mL). Forty-five compounds were identified from the D50 fraction by using UPLC-Q-TOF-MS/MS analysis. Molecular docking results showed that six main constituents, namely, crepidatin, 2,7-dihydroxy-4-methoxyl-9,10-dihydrophenylene, 4,4',5,6-tetrahydroxystilbene, 4,7-dihydroxy-2-methoxyl-9,10-dihydrophenylene, (-)-lariciresinol, and thunalbene, in the D50 fraction occupied the catalytic sites of α-glucosidase through strong hydrophobic interactions, hydrogen bonding, and other patterns. The binding energies were between - 29.95 and - 11.41 kJ/mol, indicating good binding between the tested compounds and α-glucosidase. The active ingredients responsible for the α-glucosidase inhibitory activity may include phenanthrenes, stilbenes, dibenzyls, and lignans. The D50 fraction has potential value for developing innovative drugs for the prevention and treatment of diabetes mellitus (DM) and is worthy of in-depth research.

Axially chiral dihydrophenanthrene dimers from Pholidota yunnanensis with anti-neuroinflammatory activities.

Axially chiral compounds are well known in medicinal chemistry of natural products, but their absolute configurations and bioactivities are rarely reported and studied. In this study, eleven undescribed axially chiral dihydrophenanthrene dimers, as well as twenty-five known dihydrophenanthrenes, were isolated from the entire plant of Pholidota yunnanensis. Their structures were elucidated by comprehensive spectroscopic analysis. A method for determining the absolute configurations of enantiomers was developed based on the rotational barriers and calculated ECD spectra. Additionally, the activities of all isolated compounds were assessed in LPS-induced BV-2 microglial cells. Most dihydrophenanthrenes exhibited significant NO inhibitory activities, and compound 7 showed the most potent inhibitory effect with an IC50 value of 1.5 µM, compared to the positive control minocycline. The immunofluorescence and western blot results revealed that compound 7 suppressed the expression of Iba-1, iNOS and COX-2 in LPS-stimulated BV-2 microglial cells.

The gross reproductive morphology of the male Temminck's pangolin Smutsia temminckii (Smuts, 1832).

The Temminck's pangolin (Smutsia temminckii) is one of eight pangolin species worldwide and the only pangolin present in southern Africa. Historically, pangolins have not been able to reproduce successfully in captivity and this may be in part due to the lack of knowledge and understanding with regards to the pangolin reproductive system (anatomy, physiology, biology) in all eight species. This original study describes the gross anatomy of the male Temminck's pangolin from three adult individuals investigated. The male Temminck's pangolin presented a short, conical penis with ascrotal (internal) testes, similar to many other myrmecophagous mammals such as the aardvark (Orycteropus sp.) and anteaters (suborder: Vermilingua). However, the orientation of the penis of the Temminck's pangolin differed in that it was oriented cranioventrally, in contrast to the caudal orientation of the giant anteater. The testes were found to be bilaterally flattened with an elongate oval shape, similar to the aardvark. The specific characteristics of the reproductive tract of the male Temminck's pangolins are thought to be adaptations to their peculiar lifestyle as the male portrays characteristics that indicate adaptation to a lower basal metabolic rate and body temperature as well as to their defensive mechanism of rolling up into a ball. Our study suggests the male Temminck's pangolin reproductive anatomy is most similar and comparable to the Xenarthrans and the aardvark that display the same fossorial activities as pangolins, and the male morphology is not comparable to the phylogenetically closely-related Carnivora.

Ants (Hymenoptera: Formicidae) and termites (Blattodea: Termitoidae) in the diet of wild white-bellied pangolin (Phataginus tricuspis) in forest-savanna habitats of Cameroon.

The white-bellied pangolin Phataginus tricuspis (Rafinesque 1821) is a semiarboreal species occurring in tropical sub-Saharan Africa. It is the world's most trafficked African pangolin species based on volumes recorded in seizures. Reintroduction of confiscated live pangolins and ex-situ rearing are being explored worldwide as a conservation action. However, the husbandry of seized animals is challenging as the diet of the white-bellied pangolin is poorly known and little studied. We analyzed the stomach contents of dead white-bellied pangolins from two forest-savanna protected areas. Stomach content samples from 13 white-bellied pangolin specimens contained ~165,000 Arthropoda, mostly Hymenoptera (60.34%) and Blattodea (39.66%). Overall, we identified 39 termite and 105 ant species consumed as prey by pangolins. Individual pangolins examined had fed on a maximum of 31 ant species and 13 termite species. The termite and ant species richness varied significantly across the pangolins' last consumed meal. We recorded 24 ant genera dominated by Crematogaster (relative importance [RI] = 17.28). Out of 18 termite genera recorded, the genus Pseudacanthotermes (RI = 17.21) was the most important prey. Ten ant species were preferentially eaten by white-bellied pangolin, with Crematogaster acis being the most common prey species. Four species of termite were most frequently eaten with Pseudacanthotermes militaris being the most abundant. The mean abundance of ants and termites varied among pangolin individuals. The season did not influence the mean abundance of termites eaten by pangolin individuals. However, ant abundance in stomach contents was significantly higher in the dry season. An improved understanding of pangolin feeding behavior and prey selection may help inform conservation husbandry efforts. For example, nutritional analysis of the food eaten by wild pangolins can guide the development of nutritional diets for captive pangolins.

Pangolin Genomes Offer Key Insights and Resources for the World's Most Trafficked Wild Mammals.

Pangolins form a group of scaly mammals that are trafficked at record numbers for their meat and purported medicinal properties. Despite their conservation concern, knowledge of their evolution is limited by a paucity of genomic data. We aim to produce exhaustive genomic resources that include 3,238 orthologous genes and whole-genome polymorphisms to assess the evolution of all eight extant pangolin species. Robust orthologous gene-based phylogenies recovered the monophyly of the three genera and highlighted the existence of an undescribed species closely related to Southeast Asian pangolins. Signatures of middle Miocene admixture between an extinct, possibly European, lineage and the ancestor of Southeast Asian pangolins, provide new insights into the early evolutionary history of the group. Demographic trajectories and genome-wide heterozygosity estimates revealed contrasts between continental versus island populations and species lineages, suggesting that conservation planning should consider intraspecific patterns. With the expected loss of genomic diversity from recent, extensive trafficking not yet realized in pangolins, we recommend that populations be genetically surveyed to anticipate any deleterious impact of the illegal trade. Finally, we produce a complete set of genomic resources that will be integral for future conservation management and forensic endeavors for pangolins, including tracing their illegal trade. These comprise the completion of whole-genomes for pangolins through the hybrid assembly of the first reference genome for the giant pangolin (Smutsia gigantea) and new draft genomes (∼43x-77x) for four additional species, as well as a database of orthologous genes with over 3.4 million polymorphic sites.

Adapting camera-trap placement based on animal behavior for rapid detection: A focus on the Endangered, white-bellied pangolin (Phataginus tricuspis).

Pangolin species are notoriously difficult to detect and monitor in the wild and, as a result, commonly used survey techniques fall short in gathering sufficient data to draw confident conclusions on pangolin populations, conservation status, and natural history. The white-bellied pangolin is a semiarboreal species that may be poorly detected in general mammal surveys even with modern techniques such as camera-trapping. As a result, population status information is often derived from hunting, market, and trafficking data. There is therefore a crucial need to improve camera-trap survey methods to reliably detect this species in its natural environment. Here, we test the influence of camera-trap placement strategy on the detectability of the white-bellied pangolin by comparing estimates from targeted ground-viewing camera-trapping and a novel log-viewing placement strategy adapted from local hunters' knowledge. Our results suggest that (1) deploying camera-traps to detect animals walking along logs is an effective strategy for recording several forest species, including the white-bellied pangolin, and (2) that camera-traps targeting logs are more efficient at detecting white-bellied pangolins than camera-traps viewing the ground (>100% increase in detection probability). We also found moderate evidence that there is a relationship between the white-bellied pangolin occurrence at our locality and elevation and weak evidence of an association with distance to the nearest river. Our results suggest an effective new monitoring approach allowing consistent detection of the white-bellied pangolin with moderate survey effort. This highlights the importance of harnessing local knowledge to guide the design of monitoring protocols for cryptic species.

Comparative analyses and phylogenetic relationships of thirteen Pholidota species (Orchidaceae) inferred from complete chloroplast genomes.

BACKGROUND: The orchid genus Pholidota Lindl. ex Hook. is economically important as some species has long been used in traditional medicine. However, the systematic status of the genus and intergeneric relationships inferred from previous molecular studies are unclear due to insufficient sampling and lack of informative sites. So far, only limited genomic information has been available. The taxonomy of Pholidota remains unresolved and somewhat controversial. In this study, the complete chloroplast (cp.) genomes of thirteen Pholidota species were sequenced and analyzed to gain insight into the phylogeny of Pholidota and mutation patterns in their cp. genomes. RESULTS: All examined thirteen Pholidota cp. genomes exhibited typical quadripartite circular structures, with the size ranging from 158,786 to 159,781 bp. The annotation contained a total of 135 genes in each cp. genome, i.e., 89 protein-coding genes, 38 tRNA genes, and eight rRNA genes. The codon usage analysis indicated the preference of A/U-ending codons. Repeat sequence analysis identified 444 tandem repeats, 322 palindromic repeats and 189 dispersed repeats. A total of 525 SSRs, 13,834 SNPs and 8,630 InDels were detected. Six mutational hotspots were identified as potential molecular markers. These molecular markers and highly variable regions are expected to facilitate future genetic and genomic studies. Our phylogenetic analyses confirmed the polyphyletic status of the genus Pholidota, with species grouped into four main clades: Pholidota s.s. was resolved as the sister to a clade containing species of Coelogyne; the other two clades clustered together with species of Bulleyia and Panisea, respectively; species P. ventricosa was placed at the basal position, deviated from all other species. CONCLUSION: This is the first study to comprehensively examine the genetic variations and systematically analyze the phylogeny and evolution of Pholidota based on plastid genomic data. These findings contribute to a better understanding of plastid genome evolution of Pholidota and provide new insights into the phylogeny of Pholidota and its closely related genera within the subtribe Coelogyninae. Our research has laid the foundation for future studies on the evolutionary mechanisms and classification of this economically and medicinally important genus.

Chromosome-length genome assemblies and cytogenomic analyses of pangolins reveal remarkable chromosome counts and plasticity.

We report the first chromosome-length genome assemblies for three species in the mammalian order Pholidota: the white-bellied, Chinese, and Sunda pangolins. Surprisingly, we observe extraordinary karyotypic plasticity within this order and, in female white-bellied pangolins, the largest number of chromosomes reported in a Laurasiatherian mammal: 2n = 114. We perform the first karyotype analysis of an African pangolin and report a Y-autosome fusion in white-bellied pangolins, resulting in 2n = 113 for males. We employ a novel strategy to confirm the fusion and identify the autosome involved by finding the pseudoautosomal region (PAR) in the female genome assembly and analyzing the 3D contact frequency between PAR sequences and the rest of the genome in male and female white-bellied pangolins. Analyses of genetic variability show that white-bellied pangolins have intermediate levels of genome-wide heterozygosity relative to Chinese and Sunda pangolins, consistent with two moderate declines of historical effective population size. Our results reveal a remarkable feature of pangolin genome biology and highlight the need for further studies of these unique and endangered mammals.

Bis(4-glycosyloxybenzyl) 2-isobutyltartrate and dihydrophenanthrene derivatives from the pseudobulbs of Pholidota chinensis and their anti-inflammatory activity.

Six previously undescribed components, bis(4-glycosyloxybenzyl) 2-isobutyltartrate derivatives (pholidotoside A-E) and phenolic glycoside (pholidotosin A), together with twenty known compounds were isolated from the pseudobulbs of Pholidota chinensis. Their structures and absolute configuration were elucidated and established through various spectroscopic and chemical methods. The anti-inflammatory potential of selected compounds was examined using a human neutrophil cell model activated by N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB). Among these, dihydrophenanthrenes exhibited potent inhibitory effect on both superoxide anion generation and elastase release assays with IC50 values ranging from 0.41 ± 0.05 to 7.14 ± 0.30 µM.

The gastrodin biosynthetic pathway in Pholidota chinensis Lindl. revealed by transcriptome and metabolome profiling.

Pholidota chinensis Lindl. is an epiphytic or lithophytic perennial herb of Orchidaceae family used as a garden flower or medicinal plant to treat high blood pressure, dizziness and headache in traditional Chinese medicine. Gastrodin (GAS) is considered as a main bioactive ingredient of this herb but the biosynthetic pathway remains unclear in P. chinensis. To elucidate the GAS biosynthesis and identify the related genes in P. chinensis, a comprehensive analysis of transcriptome and metabolome of roots, rhizomes, pseudobulbs and leaves were performed by using PacBio SMART, Illumina Hiseq and Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS). A total of 1,156 metabolites were identified by UPLC-MS/MS, of which 345 differential metabolites were mainly enriched in phenylpropanoid/phenylalanine, flavone and flavonol biosynthesis. The pseudobulbs make up nearly half of the fresh weight of the whole plant, and the GAS content in the pseudobulbs was also the highest in four tissues. Up to 23,105 Unigenes were obtained and 22,029 transcripts were annotated in the transcriptome analysis. Compared to roots, 7,787, 8,376 and 9,146 differentially expressed genes (DEGs) were identified in rhizomes, pseudobulbs and leaves, respectively. And in total, 80 Unigenes encoding eight key enzymes for GAS biosynthesis, were identified. Particularly, glycosyltransferase, the key enzyme of the last step in the GAS biosynthetic pathway had 39 Unigenes candidates, of which, transcript28360/f2p0/1592, was putatively identified as the most likely candidate based on analysis of co-expression, phylogenetic analysis, and homologous searching. The metabolomics and transcriptomics of pseudobulbs versus roots showed that 8,376 DEGs and 345 DEMs had a substantial association based on the Pearson's correlation. This study notably enriched the metabolomic and transcriptomic data of P. chinensis, and it provides valuable information for GAS biosynthesis in the plant.

The brain of the tree pangolin (Manis tricuspis). X. The spinal cord.

The spinal cord of the tree pangolin is known to be very short compared to the overall length of the body and tail. Here, we provide a description of the tree pangolin spinal cord to determine whether the short length contributes to specific structural, and potentially functional, differences. The short spinal cord of the adult tree pangolin, at around 13 cm, terminates at the midthoracic level. Within this shortened spinal cord, we could identify six regions, which from rostral to caudal include the prebrachial, brachial, interramal, crural, postcrural, and caudal regions, with both the brachial and crural regions showing distinct swellings. The chemoarchitecture of coronal sections through these regions confirmed regional assignation, being most readily delineated by the presence of cholinergic neurons forming the intermediolateral column in the interramal region and the sacral parasympathetic nucleus in the postcrural region. The 10 laminae of Rexed were observed throughout the spinal cord and presented with an anatomical organization similar to that observed in other mammals. Despite the shortened length of the tree pangolin spinal cord, the regional and laminar anatomical organization is very similar to that observed in other mammals. This indicates that the functional aspects of the short tree pangolin spinal cord can be inferred from what is known in other mammals.

The brain of the tree pangolin (Manis tricuspis). VIII. The subpallial telencephalon.

The current study provides a detailed architectural analysis of the subpallial telencephalon of the tree pangolin. In the tree pangolin, the subpallial telencephalon was divided into septal and striatopallidal regions. The septal region contained the septal nuclear complex, diagonal band of Broca, and the bed nuclei of the stria terminalis. The striatopallidal region comprised of the dorsal (caudate, putamen, internal and external globus pallidus) and ventral (nucleus accumbens, olfactory tubercle, ventral pallidum, nucleus basalis, basal part of the substantia innominata, lateral stripe of the striatum, navicular nucleus, and the major island of Calleja) striatopallidal complexes. In the tree pangolin, the organization and numbers of nuclei forming these regions and complexes, their topographical relationships to each other, and the cyto-, myelo-, and chemoarchitecture, were found to be very similar to that observed in commonly studied mammals. Minor variations, such as less nuclear parcellation in the bed nuclei of the stria terminalis, may represent species-specific variations, or may be the result of the limited range of stains used. Given the overall similarity across mammalian species, it appears that the subpallial telencephalon of the mammalian brain is highly conserved in terms of evolutionary changes detectable with the methods used. It is also likely that the functions associated with these nuclei in other mammals can be translated directly to the tree pangolin, albeit with the understanding that the stimuli that produce activity within these regions may be specific to the life history requirements of the tree pangolin.

The brain of the tree pangolin (Manis tricuspis). VII. The amygdaloid body.

Here, we describe the cytoarchitecture and chemoarchitecture of the amygdaloid body of the tree pangolin. Our definition of the amygdaloid body includes the pallial portions of the amygdala, and the centromedial group that is a derivative of the subpallium and part of the extended amygdala. The remainder of the extended amygdala is not described herein. Within the amygdaloid body of the tree pangolin, we identified the basolateral group (composed of the lateral, basal, and accessory basal amygdaloid nuclei), the superficial, or cortical nuclei (the anterior and posterior cortical nuclei, the periamygdaloid cortex, and nuclei of the olfactory tract), the centromedial group (the central amygdaloid nucleus and the medial nuclear cluster), and other amygdaloid nuclei (the anterior amygdaloid area, the amygdalohippocampal area, the intramedullary group, and intercalated islands). The location within and relative to each other within the amygdaloid body and the internal subdivisions of these groups were very similar to that reported in other mammalian species, with no clearly derived features specific to the tree pangolin. The only variation was the lack of an insular appearance of the intercalated islands, which in the tree pangolin were observed as a continuous band of neurons located dorsomedial to the basolateral group similar in appearance to and almost continuous with the intramedullary group. In carnivores, the closest relatives of the pangolins, and laboratory rats, a similar appearance of portions of the intercalated islands has been noted.

Three new phenanthrenes from Pholidota chinensis Lindl. and their antibacterial activity.

Three new phenanthrenes were isolated from Pholidota chinensis Lindl. Their structures were elucidated on the basis of spectroscopic techniques and comparison of their data to the values reported in the literature. From the 95% EtOH extract, three new compounds, namely 9, 10-dihydro-2, 4, 6-trihydroxy-7-methoxyphenanthrene (1), 11-methoxyflaccidin (2), and 2-methoxy-3,7-dihydroxy-5H phenanthro[4,5-bcd] pyran (3), were identified. Compound 3 showed an MIC50 of 68.39 µM against Staphylococcus aureus subsp. Aureus.

The gut microbiome of the Sunda pangolin (Manis javanica) reveals its adaptation to specialized myrmecophagy.

BACKGROUND: The gut microbiomes of mammals are closely related to the diets of their hosts. The Sunda pangolin (Manis javanica) is a specialized myrmecophage, but its gut microbiome has rarely been studied. METHODS: Using high-throughput Illumina barcoded 16S rRNA amplicons of nine fecal samples from nine captive Sunda pangolins, we investigated their gut microbiomes. RESULTS: The detected bacteria were classified into 14 phyla, 24 classes, 48 orders, 97 families, and 271 genera. The main bacterial phyla were Firmicutes (73.71%), Proteobacteria (18.42%), Actinobacteria (3.44%), and Bacteroidetes (0.51%). In the PCoA and neighbor-net network (PERMANOVA: pangolins vs. other diets, weighted UniFrac distance p < 0.01, unweighted UniFrac distance p < 0.001), the gut microbiomes of the Sunda pangolins were distinct from those of mammals with different diets, but were much closer to other myrmecophages, and to carnivores, while distant from herbivores. We identified some gut microbiomes related to the digestion of chitin, including Lactococcus, Bacteroides, Bacillus, and Staphylococcus species, which confirms that the gut microbiome of pangolins may help them to digest chitin. SIGNIFICANCE: The results will aid studies of extreme dietary adaption and the mechanisms of diet differentiation in mammals, as well as metagenomic studies, captive breeding, and ex situ conservation of pangolins.

Chemical Constituents and their Antioxidant, Anti-Inflammatory and Anti-Acetylcholinesterase Activities from Pholidota cantonensis.

Alzheimer's disease (AD) has the third highest health expenditures after heart disease and cancer. It has emerged as a serious global health issue. The discovery of new drugs to prevent and treat AD is of utmost importance. Pholidota cantonensis is an edible medicinal plant consumed in China. It is widely used in traditional Chinese medicine to treat various diseases. P. cantonensis has been reported to have antioxidant, anti-inflammatory, antitumor and antibacterial activities. Among these properties, its potent antioxidant activity has attracted our attention, since oxidative stress is one of the important pathological mechanisms involved in AD. This study aimed to isolate the compounds from the active extract and evaluate their bioactivities. Fifteen compounds, including one new compound, were obtained. The isolates were tested for 2,2'-diphenyl-1-picrylhydrazyl (DPPH)/2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities, anti-acetylcholinesterase (anti-AChE) activities and inhibitory effects on nitrogen monoxide (NO) release in the BV-2 cells. Compounds 1, 2, 4, 6, 8, and 13-15 exhibited two kinds of AD-associated bioactivities. More importantly, compound 13 showed more potent NO inhibitory activity (IC50 = 0.72 ± 0.08 µM) than the positive control quercetin (IC50 = 12.94 ± 0.08 µM). Compound 13 also had a higher inhibitory rate (99.59 ± 0.43%) on AChE than that of the positive control galantamine (78.32 ± 1.16%) at the concentrate of 50 µg/mL. Our studies provide new insights into this plant in terms of its potential in the development of new multi-target anti-Alzheimer's disease (anti-AD) drugs.

Genomic evidence for the parallel regression of melatonin synthesis and signaling pathways in placental mammals.

Background: The study of regressive evolution has yielded a wealth of examples where the underlying genes bear molecular signatures of trait degradation, such as pseudogenization or deletion. Typically, it appears that such disrupted genes are limited to the function of the regressed trait, whereas pleiotropic genes tend to be maintained by natural selection to support their myriad purposes. One such set of pleiotropic genes is involved in the synthesis ( AANAT, ASMT) and signaling ( MTNR1A, MTNR1B) of melatonin, a hormone secreted by the vertebrate pineal gland. Melatonin provides a signal of environmental darkness, thereby influencing the circadian and circannual rhythmicity of numerous physiological traits. Therefore, the complete loss of a pineal gland and the underlying melatonin pathway genes seems likely to be maladaptive, unless compensated by extrapineal sources of melatonin. Methods: We examined AANAT, ASMT, MTNR1A and MTNR1B in 123 vertebrate species, including pineal-less placental mammals and crocodylians. We searched for inactivating mutations and modelled selective pressures (dN/dS) to test whether the genes remain functionally intact. Results: We report that crocodylians retain intact melatonin genes and express AANAT and ASMT in their eyes, whereas all four genes have been repeatedly inactivated in the pineal-less xenarthrans, pangolins, sirenians, and whales. Furthermore, colugos have lost these genes, and several lineages of subterranean mammals have partial melatonin pathway dysfunction. These results are supported by the presence of shared inactivating mutations across clades and analyses of selection pressure based on the ratio of non-synonymous to synonymous substitutions (dN/dS), suggesting extended periods of relaxed selection on these genes. Conclusions: The losses of melatonin synthesis and signaling date to tens of millions of years ago in several lineages of placental mammals, raising questions about the evolutionary resilience of pleiotropic genes, and the causes and consequences of losing melatonin pathways in these species.

The scale of Nigeria's involvement in the trans-national illegal pangolin trade: Temporal and spatial patterns and the effectiveness of wildlife trade regulations.

The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) prohibits commercial trans-national trade in pangolin specimens. However, African pangolins are continually trafficked to Asia for traditional medicine, with Nigeria considered a key hub. Using reported Nigeria-linked pangolin seizure data and interviews with Nigerian law enforcement officials, we a) characterised Nigeria's involvement in global pangolin trafficking January 2010-September 2021, particularly observing trafficking trends after pangolin's CITES Appendix I listing; b) estimated the minimum number of pangolins whose scales are in Nigeria-linked seizures January 2010-September 2021, and; c) assessed ongoing efforts within Nigeria to curb pangolin trafficking. Nigeria-linked seizures involved 190,407 kg of pangolin derivatives (99.9% scales) from a minimum of 799,343 pangolins (95% confidence interval; 625,944-996,353) of four species (see caveats in Methods). All shipments confiscated in transit were destined for Asia, with a rapid increase in the mass of maritime shipments over time. Furthermore, stockpiling of pangolin derivatives for overseas shipment is perhaps a prominent trafficking model in Nigeria. Nigeria's law enforcement efforts improved from 2017, the same year Nigeria apparently began playing a hub role. The impact of pangolin's CITES Appendix I listing on pangolin trafficking was unclear, as the marked rise in seizures from 2017 when the listing became effective, coincided with improvements in Nigerian law enforcement efforts. COVID-19-induced travel restrictions likely reduced trafficking activities in 2020 but activities may have fully resumed in 2021. This study provides new information to inform effective enforcement and policy formulation efforts to protect African pangolins.

Vitamin A deficiency and its treatment in captive Sunda pangolins.

The high incidence of disease in captive pangolins is a major obstacle in pangolin-conservation breeding programs. Therefore, elucidating pangolins' susceptibility to disease is the key to conservation progress. At the Pangolin Research Base for Artificial Rescue and Conservation Breeding of South China Normal University (PRB-SCNU), vitamin A deficiency was diagnosed in 14 captive Sunda pangolins. Typical eye signs included lacrimal eyes, keratopathy and a blank, milky orb. The afflicted pangolins were treated with vitamins A and D for 15-30 days; all individuals recovered. We report the detection and treatment of vitamin A deficiency in captive Sunda pangolins at the PRB-SCNU. Our results could provide guidance for the future prevention and treatment of vitamin A deficiency and associated diseases in pangolin species, both to reduce the incidence of these diseases in captive pangolins and to aid conservation efforts.

Qualitative and quantitative analysis of phenolic compounds by UPLC-MS/MS and biological activities of Pholidota chinensis Lindl.

Pholidota chinensis Lindl. is commonly used as functional tea or folk medicine in China, but few studies have been conducted on its phenolic composition and bioactivities. This study was aimed to investigate the phenolic profiles of P. chinensis extracts by different solvents (petroleum ether, ethyl acetate, n-butanol and water) and their in vitro antioxidant, antidiabetic, and anti-inflammatory activities. The results showed that a total of 42 phenolic compounds were accurately identified, 30 of them were further quantified in P. chinensis extracts by UPLC-MS/MS. Among four extracts, ethyl acetate extract not only had the highest contents of total phenolics and 21 individual phenolic compounds, but also possessed the strongest antioxidant (DPPH, 1.43 mmol AAE/g; ABTS, 5.38 mmol TE/g; FRAP, 2.58 mmol FSHE/g), α-glucosidase inhibition (13.29 mmol ACAE/g) and anti-inflammation (IC50 = 18.71 µg/ml) activities. Also, the antioxidant, antidiabetic, and anti-inflammatory activities were significantly correlated with total phenolics and individual phenolic compounds. Orientin and vitexin can be considered as the potential quality markers of P. chinensis. This study will provide a comprehensive phenolic profile of P. chinensis and relates them to their antioxidant, antidiabetic, and anti-inflammatory activities.