Repeated mechanical damage enhanced Aquilaria sinensis resistance to Heortia vitessoides through jasmonic acid.

Introduction: The leaf-chewing pest Heortia vitessoides severely threatens the growth and development of Aquilaria sinensis. In our previous study, we found that mechanical damage (MD) to stem enhanced A. sinensis sapling resistance to H. vitessoides larvae. Methods: To reveal the defense mechanisms underlying this observation, we analyzed the types and contents of volatile organic compounds (VOCs), phytohormone contents, and expression of phytohormone-related genes in response to MD and herbivory wounding(HW). Results: Here, we identified several VOCs, such as the pesticides fenobucarb and 2,4-di-tert-butylphenol, in mature leaf (ML) of MD-treated plants. Compared with salicylic acid (SA) or the ethylene (ET) pathway, jasmonic acid (JA) content and JA-related genes were more strongly upregulated. Interestingly, we found a dramatic difference between JA-related upstream and downstream genes expression in YL and ML, which confirmed that JA-Ile accumulation in MD-ML and HW-ML could be derived from local damaged site. Discussion: Taken together, we provide evidence that the JA pathway plays a dominant role in the A. sinensis response to MD and HW.

Specific mating behavior of Malayan pangolin (Manis javanica) in captivity.

Pangolin is a mysterious animal in the Family Pholidota, Mammalia. Malayan pangolin (Manis javanica) is one of eight existing species and is listed in Manis. With the number of wild pangolins (Manis spp.) rapidly decreasing, captive breeding has become an important way to protect them from extinction. The research on mating behavior of pangolins is an important content to understand its reproductive characteristics and develop breeding management. From 2016 to 2022, a total of 360 mating events were observed in six males and 24 females through closed circuit television (CCTV) surveillance. The results show that males do not engage in complex courtship behavior before mating. In addition, we found that male pangolins adopted a ventrolateral mating position. Once males selected the side (left/right) of the female pangolin from which to approach to mate, they usually remained on the same side for subsequent mating, suggesting that male pangolins may have a preference in mating position. Finally, all mating events were observed at 1.72 ± 1.47 (n = 83, Mean ± SD) days after cohabitation and adjustment time before mating (from the male touching the female to intromission) took 4.98 ± 3.86 mins (n = 323). During mating, males hugged females and remained still for 47.37 ± 10.08 seconds (n = 323), which is the ejaculation and post-ejaculation quiescent time. Remarkably, we observed for the first time two peak mating times, 19:00 to 22:00 and 1:00 to 3:00, suggesting that they may have a preference for mating times. This study provides new insight into the mating behavior of M. javanica and contributes to the development of scientific conservation measures to improve the reproductive capacity of M. javanica.

Influence of steroidal glycosides from Cynanchum auriculatum on antioxidant indicators in H2O2-damaged PC12 cells.

The root of Cynanchum auriculatum Royle ex Wight is a traditional Chinese medicine, which is rich in C21 steroidal glycosides by phytochemistry research. In this study, the antioxidant effect of 27 C21 steroidal glycosides isolated from the root of C. auriculatum by our group was evaluated using the H2O2-treated PC12 cells. As the result, all tested compounds altered the activities of lactate dehydrogenase, superoxide dismutase, catalase and glutathione peroxidase at concentrations as low as 1 µM in H2O2-treated PC12 cells. They also decreased the levels of intracellular reactive oxygen species and Ca2+. Further, the correlation between their structural features described by molecular descriptors and the indicators of bioactivity was analyzed by partial least squares analysis, displaying those six bio-indicators were positive correlated with 13 molecular descriptors and providing some guidance for further study of relationships between steroid structure and antioxidant activity.

Weaning period and growth patterns of captive Sunda pangolin (Manis javanica) cubs.

This study tracked and recorded the weight changes of 13 captive Sunda pangolin cubs from lactation to maturity to explored the appropriate weaning time and reveal the rules of its weight growth. SPSS 25.0 was used to build a cubic equation model to fit the body weight change rules of 4 individuals who nonvoluntarily ingested artificial feed (NIAF) at 127 days after birth and 5 individuals who voluntarily ingested artificial feed (VIAF) at 86-108 days after birth. The body weight of NIAF cubs aged 0-120 days and VIAF cubs aged 0-150 days were estimated according to the fitting model. An independent sample T-test was performed on the mean body weight of the two groups during the late lactation period. The results showed that at 105 days after birth, the body weight of the VIAF group was significantly higher than that of the NIAF group (P = 0.049), and the body weight of the VIAF group was extremely significantly higher than that of the NIAF group at 114 days (P = 0.008); The peak cumulative body weight of the NIAF cubs during lactation appeared around 130 days of age (n = 3); The mortality rate was 66.7% (n = 3) after about 150 days if the feed was continuously consumed nonvoluntarily. It was concluded that the milk secretion period of the mother is about 0-5 months after giving birth; the weaning period of the cubs should be 4-5 months after birth. If the cubs don't follow the mother to eat artificial feed for 3 months after birth, it can start be induced with artificial diet which adds termites, and the time point cannot be later than 130 days, otherwise it is not conducive to the survival of the cubs; When sexually mature, the body length and body weight of female cubs account for about 84% and 60% of the adult, respectively; the body maturity and body weight of female cubs tend to be stable about 15.3 months and 16.4 months, respectively. Finally, a special needle-shaped nipples and nursing patterns of female Sunda pangolins were also recorded in this study. These findings play an important role in guiding the nursing of captive Sunda pangolin cubs and other pangolin cubs. It is expected to improve the survival rate of the cubs by exploring the appropriate weaning time and the rules of weight growth. By scientifically planning the reproductive cycle of the female Sunda pangolins, our goal is to expand the population size and eventually release to the wild, meanwhile improving knowledge of this critically endangered species.

Response of Properties and Chemicals to Preparation Parameters in the Oxidation Processing of Refined Montan Wax.

Refined montan wax (RMW) is a lignite-based chemical product with wide application and high added value. However, research on its processing and performance is very limited. Currently, four parameters in the key preparation procedure for the oxidation bleaching of RMW, including the concentration of two oxidants (H2SO4 (P1) and CrO3 (P2)), oxidation time (P3), and the mass ratio of CrO3 used in two oxidation steps (P4), were systematically evaluated in regard to their impact on the properties and chemistry of RMW. The results showed that the four tested parameters visibly affected RMW, and each parameter had a different impact on the properties of RMW by range analysis, of which P1 showed a greater influence on its acid value; P2 influenced its friability, specific surface area, and aperture; P3 affected its color, initial melting point, and saponification value; and P4 had a higher impact on its final melting point, melting range, and hardness. Gas chromatography with flame ionization detection-mass spectrometry analysis revealed that the compounds found in RMW samples (RMWs) under different oxidation conditions differed significantly, with major differences in the content and amount of these components. Among the compounds in RMWs, 16 different compounds (variable importance of projection > 1) were found by the orthogonal projections to latent structures discriminant analysis method, nine of which have a strong relationship to the different performances of RMWs. This work provided a basis for the development of performance-oriented preparation processing technology for RMW.

Successful captive breeding of a Malayan pangolin population to the third filial generation.

Pangolins are threatened placental mammals distributed in Africa and Asia. Many efforts have been undertaken in the last century to maintain pangolins in captivity, but only a few of them succeeded in maintaining and keeping this species in a controlled environment. This study reports the first systematic breeding of the Critically Endangered Malayan pangolin (Manis javanica) in captivity. Our captive breeding approach successfully improved the reproductive rate for both wild and captive-born female pangolins. From 2016 to 2020, we had 33 wild pangolins and produced 49 captive-born offspring spanning three filial generations. The female offspring further bred 18 offspring, of which 14 (78%) were conceived during the first time of cohabitation with males, and four offspring were conceived during the second cohabitation event, suggesting that they may practice copulation-induced ovulation. We observed that captive-born female pangolins could reach sexual maturity at 7-9 months (n = 4), and male pangolins could mate and successfully fertilise females at nine months age (n = 1). We also observed a female pangolin conceiving on the eighth day after parturition (the fifth day after the death of its pup). Our captive pangolins had a female-biased sex ratio of 1:0.5 at birth, unlike other known captive-born mammals. Also, captive-born pangolins were generally more viable after successful weaning and had a similar gestation length (~185 days) to wild pangolins. Most importantly, we report the first self-sustaining captive population of Malayan pangolins, and this species has an efficient reproduction strategy. These advances provide more comprehensive information for people to understand pangolins, and have implications for conserving endangered Malayan pangolins and providing scientific guidance to the management of other pangolin species.

The effect of microorganisms on the biomodification of montan resin from lignite.

Montan resin (MR) is an industrial by-product or solid waste generated during the production of refined montan wax and is not typically reused. In this paper, a bio-modification method using three strains of microorganisms, Acinetobacter venetianus (AV), Pseudomonas aeruginosa (PA), and Phanerochaete chrysosporium (PC), was studied to promote the performance and bio-function of MR so that MR could be recycled. MR can be degraded by these three microorganisms, and their weight loss rates were similar over the treatment period of 15 days. Compared with the original MR, the hydrophilicity of modified MRs was improved, which was related to the increase in apparent oil-water partition coefficients (Kows) and oxygen-containing and hydrophilic groups in modified MRs based on IR and GC-MS analysis. The bio-function of modified MRs by the three strains in terms of promoting maize seed germination and seedling growth was greater compared with untreated MR. Overall, these findings indicate that biomodified MRs might have useful agriculture applications.Implications: An environmentally-friendly method using microorganisms to achieve recycle of solid waste, montan resin (MR) was established in this study. Through this bio-treatment, the performance and bio-function of MR were both improved, that is the appearance and hydrophilicity of modified MRs were better than thoes in before, and the modified MRs treated by three strains showed the better promoting effects on maize seed germination and seedling growth than untreated MR, indicating the modified MRs have the certain potential of agricultural utilization in the future.

Angiogenic effects of low molecular weight organic acids present in fulvic acids of different sources.

Fulvic acid (FA) is a natural mineral medicine with a long medical history in folk. However, the active chemicals of FA remain unknown due to its diversity of sources and the complexity of compositions, which have become a bottleneck in quality control and medicinal development. Based on the traditional effect on angiogenesis, FAs from eight different coal sources were prepared and their active fractions were investigated by the CAM model, resulting that most of acetonitrile dissolved parts of these FAs (DFAs) produced angiogenesis effects. Through chemical analysis on DFAs by GC-FID/MS, six shared organic acids with low molecular weights were identified and quantified, which showed the promoting effects on capillary areas, VEGF, b-FGF, and Ang-1 at different degrees. The PCA analysis showed that the five shared organic acids with high recognition are the active chemicals in different sources of FAs which may be responsible for the angiogenesis effects.

Effects of Chronic Stress on the Fecal Microbiome of Malayan Pangolins (Manis javanica) Rescued from the Illegal Wildlife Trade.

Pangolins (scaly anteaters, Pholidota) are among those mammals that are most affected by the international, illegal wildlife trade. Recently, wildlife rescue centers in China became dedicated to rehabilitate confiscated pangolins and prepare them for reintroduction to the wild. Chronic stress is thought to be the main reason for a disturbed microbiota community and a higher mortality rate of pangolin in captivity. In this study, we compared the cortisol levels and the fecal microbiome of Malayan pangolin (Manis javanica) born and reared in captivity (PCB; n = 7) with those rescued from the wildlife trade (PCT; n = 16). Results show that the level of cortisol in PCT was significantly lower than that observed in PCB. There were also significant differences in the composition of the fecal microflora between the two groups, and the diversity of intestinal microbiota was higher in PCB than in PCT. At the phylum level, the bacteria with significant difference between the two groups included Firmicutes and Bacteroides. At the genus level, bacteria such as Bacteroides, Parabacterides, and Clostridium showed significant differences between the two groups. This study proves that chronic stress has a considerable effect on the diversity and composition of fecal microbiota in Malayan pangolin.

Neuroprotection of two C21 steroidal glycosides from Cynanchum auriculatum against H2O2-induced damage on PC12 cells.

Neuroprotection under conditions of oxygen stress of two C21 steroidal glycosides, cynsaccatol Q (1) and saccatol K (2), isolated from Cynanchum auriculatum Royle ex Wight, and their potential mechanisms were studied using the model of H2O2-induced damage on PC12 cells in this paper. Experiments showed that compounds 1 and 2 can both regulate the activities of antioxidant enzymes, including GSH-Px, LDH, CAT, SOD, decrease the levels of intracellular ROS and Ca2+, reduce cell apoptosis, and regulate the expression of HO-1 and NQO1 through the Nrf2-ARE pathway, thus play the role of neuroprotection against oxidative damage.

Analysis of chemical characteristics of lignite upgrading wastewater and its agricultural utilization.

The lignite upgrading wastewater (LUW) produced in the drying and upgrading process of lignite cannot be discharged directly. Conventional wastewater treatment methods are usually costly and unable to achieve efficient utilization of water resources which are rich in activity components. In this study, the water quality analysis showed that LUW belonged to seriously polluted waters with low pH and very high total nitrogen content. Fifty-five compounds, mainly phenols and organic acids, were identified by gas chromatography-mass spectrometry (GC-MS) analysis. The study confirmed that the LUW, after being diluted to an appropriate concentration, could significantly promote the growth of wheat seedlings. The phenols and organic acids were the activity material basis of LUW, which promoted seed germination possibly through playing a role similar to plant hormones and simultaneously enhancing the utilization of nutrient elements. LUW had the natural advantages of directly developing high-end liquid fertilizers in terms of its physical form, chemical composition, biological activity, safety and economy. This study confirmed the feasibility of applying LUW to agricultural field as liquid fertilizer only through simple dilution without other treatments. Applying LUW as liquid fertilizer can not only supply a fertilizer product with low production cost and outstanding efficacy, but also provide an efficient and green way for the treatment of upgrading wastewater, which utilize the LUW as natural resources instead of purifying and discharging.

Chemical modification of montan resin by peracetic acid and agricultural application of its modified products.

Montan resin (MR) is a by-product produced during the refinement process of montan wax extracted from lignite and has no usage yet. Chemical modification is an effective method to change the material property for expanding or converting the application area of the material itself. Our previous study found that the high hydrophobicity of MR is the primary limiting factor for its utilization in agriculture. Based on this point, this study attempted to chemically modify MR using the oxidation of peracetic acid, resulting that the MR hydrophilicity was significantly improved, and a water-soluble product (WSP) was obtained. The optimized oxidation conditions of MR, including the reaction temperature (X1), reaction time (X2), weight ratio of oxidant and montan resin (X3), and oxidant concentration (X4), were determined using single-factor experiments and response surface analysis. The modification degree was evaluated using elemental and oil-water partition coefficient analyses, infrared (IR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS), revealing that the oil-water partition coefficient of the modified product decreased and that the number of chemical constituents with oxygen-containing functional groups clearly increased after modification. Furthermore, the WSP was tested its effects on germination and seedling growth of the wheat seed. Compared with the control group, the WSP showed a promoting effect on the growth and germination of wheat. The WSP concentrations of 600 mg·L-1 and 300 mg·L-1 had the most substantial effect on the root and seedling growth of wheat, respectively. Implications: Montan resin, a useless by-product produced from crude montan wax, was chemically modified via oxidation of peracetic acid. Its hydrophilicity was significantly improved, and a water-soluble product was obtained after the chemical modification. The optimized oxidation conditions of montan resin were determined using single-factor experiments and response surface analysis. The amount of chemical constituents with oxygen-containing functional groups increased in the modified products after modification, as determined by IR and GC-MS analysis, among other methods. The water-soluble modified product showed an obvious effect in promoting growth and germination of wheat at 600 mg·L-1 and 300 mg·L-1, respectively.

Analysis and identification of the parent coal sources of fulvic acid according to convention, spectroscopy and chemometrics.

Fulvic acid (FA) is a kind of organic and complex water-soluble components mainly extracted from low rank coals with small molecular weight, active physical properties (such as cation exchange capacity, pH-buffering alkalinity) and positive biological functions. However, the performance of FA varies greatly, mainly induced by its different sources of raw coals. Thus, classifying the fulvic acid obtained from different coal samples is required. According to their chemical differences, two methods are developed in this paper to distinguish the origin of coal in China in combination with chemometric tools. First, the ash content, elemental composition, ultraviolet-visible (UV-Vis) and fluorescence spectra of sixteen fulvic acid samples from peat, lignite and weathered coal are measured and fifteen parameters are obtained from each sample. In the first Linear Discriminant Analysis (LDA) strategy, Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) and stepwise LDA are employed to reduce variables. A discriminant function (DF) constructed only by EEt/Bz and FI is obtained, with its accuracy verified by clustering and leave-one-out cross validation (LOOCV) with an accuracy of 87.5%. In another machine learning tactics, Pearson correlation and principal component analysis (PCA) reduce the dimensions of all variables. In the end, all sixteen samples are divided into three groups by support vector machine (SVM), with an accuracy of 100%. In conclusion, based on the differences in the chemical composition of FA from different sources, the method for combining UV-Vis and fluorescence with LDA or SVM can effectively classify the coal sources of FA.

REPORT- Anti-inflammatory effect of austroyunnane B on RAW264.7 cells.

Austroyunnane B, a highly oxidized guaianolide-type sesquiterpenoid isolated from the whole plants of Artemisia austro-yunnanensis by our group, revealed significant inhibition against NO production on the model of lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In this study, its anti-inflammatory effect on some inflammatory cytokines and proteins was further evaluated using the same model. As results, co-treatment with different concentrations (0.39-100µmol /L) of austroyunnane B on LPS-induced RAW264.7 cells displayed reduction of the nitric oxide (NO) generation in a dose dependent manner. It also showed a concentration-dependent inhibition against TNF-α, IL-1ß, IL-6 and IL-10, and down-regulation of protein expressions of iNOS and COX-2 on that model at the range of 0.625-10µmol/L, suggesting that austroyunnane B can be used as a lead compound for the development of inflammatory drug.

Sesquiterpene lactones from Artemisia austroyunnanensis suppresses ROS production and reduces cytokines, iNOS and COX-2 levels via NF-KB pathway in vitro.

The inhibitory effects of six sesquiterpene lactones (1-6) isolated from Artemisia austroyunnanensis Ling & Y. R. Ling were studied on the LPS-induced inflammatory mediator production in RAW264.7 cells. The results showed they can decrease the level of ROS in a concentration-dependent response. The compounds down-regulate the expression of iNOS and COX-2 and a series of classical inflammatory factors, including TNF-α, IL-1ß, IL-6 and IL-10. These effects can be partially explained by the supression of the phosphorylation of NF-κB. Additionally, the relationship between their structures and inflammatory factors was analyzed and discussed.

Rapid determination of antioxidant molecules in volatiles of rose tea by gas chromatography-mass spectrometry combined with DPPH reaction.

Volatiles have been regarded as active substances in many foods, whose chemicals can be analyzed by GC-MS qualitatively and quantitatively. However, the activities of volatiles are often studied as a whole, and it has no an effective method to determine that which molecule is active in volatiles by far. In order to identify the antioxidant molecules in volatiles, a rapid determination method was developed by GC-FID/MS combined with DPPH radical reaction in this study. Three antioxidant molecules were identified and validated among 20 components in rose tea infusion. Their activity validation and the methodological evaluation indicated this method could be used for distinguishing antioxidant molecules in volatiles rapidly and effectively.

Antidiarrhoeal mechanism study of fulvic acids based on molecular weight fractionation.

As the important component of humus, fulvic acids (FA) have a good antidiarrhoeal effect on animals and humans, and have been worldwide used in animal husbandry and even clinical practice for a long time. Due to the extremely complex chemical composition and structure of FA, the material basis and mechanism of its antidiarrhoeal activity have not been fully elucidated. In this study, we used ultrafiltration technique to fractionate this heterogeneous mixture into a series of relatively uniform fractions. The main structural features of FA and its fractions were characterized, and at the same time their antidiarrhoeal activities on drug-induced diarrhoea model mice were evaluated and the collagen content in the intestine of mice were determined. Through contrastive study of the relative variations between structure characteristics and antidiarrhoeal activities with the change of molecular weight, we found that the oxygen-containing functional groups especially phenolic hydroxyl groups, molecular weight distribution, colloidal properties and astringency were the material basis of the antidiarrhoeal activity. Fulvic acid substances had a dual antidiarrhoeal mechanism acting on the intestinal mucosa. The components with low molecular weight (< 5 K) mainly acted on the inside of intestinal mucosa and the components with high molecular weight (> 5 K) acted on the surface, and they could simultaneously exert the antidiarrhoeal effects.

Chemical components of the volatile oil from leaves of Cananga odorata and its anti-oxidant activity.

Cananga odorata (Lamk.) Hook. f. et Thoms., belonging to Annonaceae, is an evergreen tree. The oils extracted from its flower are a famous perfume and used in daily chemical and food industry. Although this plant has been widely cultivated in tropical regions of the world, the yield of oils from its flower is very limited. In order to develop the other parts of this plant, the chemical constituents of the volatile oils from the leaves of C. odorata was analyzed by gas chromatography/flame ionization detector (GC-FID) and GC/mass spectrometry (GC-MS). And the volatiles showed nitric oxide (NO) inhibitory activity with an IC50 value of 37.61µg/mL and anti-oxidant activity with an IC50 value of 3.84mg/mL, respectively.

Biological Modification of Montan Resin from Lignite by Bacillus benzoevorans.

The montan resin (MR) is a solid waste produced during the industrial process of refined montan wax from lignite, and usually disposed by landfill and incineration, which easily cause environmental pollution and resource waste. Based its physicochemical properties, our study attempted to modify MR by Bacillus benzoevorans to achieve ecological utilization of MR. As results, the weight loss rate of MR, expressed as modification degree, was found to increase with the increase of B. benzoevorans-incubated time. The apparent oil-water partition coefficient (Kow), used to evaluate the improvement on hydrophilicity of MR, significantly increased (P < 0.01) after modification. IR analysis showed the functional groups of -OH and C=O in modified MR were more than those in MR. Meanwhile, comparison of the chemical changes between MR and modified MR by relatively quantitative analysis of gas chromatography-mass spectrometry (GC-MS) revealed that the content of some chemical components in the latter decreased, and the newly appeared chemical components all had more oxygen-containing functional groups. The bioactivity of the modified MR in agricultural application was evaluated regarding germination and seedling growth of maize seed preliminarly. Compared with the original MR-treated group, the modified MR showed an obvious effect on promoting the growth and germination of maize.

Neuroprotective Effects of Extracts from the Radix Curcuma aromatica on H2O2-induced Damage in PC12 Cells.

Aim & Objectives: Curcuminoids are characteristic constituents in Curcuma, displaying obviously neuroprotective activities against oxidative stress. As one of the Traditional Chinese Medicines from Curcuma, the radix of Curcuma aromatica is also rich in those chemicals, but its neuroprotective activity and mechanism remain unknown. The aim of the current study is to evaluate the neuroprotective effects of extracts from the radix of C. aromatica (ECAs) on H2O2-damaged PC12 cells. MATERIAL AND METHODS: The model of oxidative stress damage was established by treatment of 400 µM H2O2 on PC12 to induce cell damage. After the treatment of ECWs for 24 h, the cell viability, LDH, SOD, CAT and GSH were measured to evaluate the neuroprotection of ECAs on that model. The potential action mechanism was studied by measurement of level of ROS, cell apoptosis rate, mitochondrial membrane potential (MMP), morphologic change, the intracellular Ca2+ content (F340/F380) and the expressions of Bcl-2, Bax and Caspase-3. Additionally, the constituents from tested extracts were analyzed by HPLC-DAD-Q-TOF-MS method. RESULTS: Compared with a positive control, Vitamin E, 10 µg/ml of 95% EtOH extract (HCECA) and 75% EtOH extract (MCECA) can markedly increase the rate of cell survival and enhance the antioxidant enzyme activities of SOD, CAT, increase the levels of GSH, decrease LDH release and the level of ROS, attenuate the intracellular Ca2+ overloading, reduce the cell apoptotic rate and stabilize MMP, down-regulate Bcl-2 expression, up-regulate Bax and caspase-3 expression, and improve the change of cell morphology. The chemical analysis showed that diarylheptanoids and sesquiterpenoids are the major chemicals in tested extracts and the former were richer in HCECA and MCECA than others. CONCLUSIONS: These findings indicated that the effects of HCECA and MCECA on inhibiting the cells damage induced by H2O2 in PC12 are better than other extracts from the radix of C. aromatica, and the active constituents with neuroprotective effects consisting in those two active extracts are diarylheptanoids.