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A holistic visualization for quality of Chinese materia medica: Structural and metabolic visualization by magnetic resonance imaging
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The causes of the neurodegenerative processes in Alzheimer's disease (AD) are not completely known. Recent studies have shown that white matter (WM) damage could be more severe and widespread than whole‐brain cortical atrophy and that such damage may appear even before the damage to the gray matter (GM). In AD, Amyloid‐beta (Aβ42) and tau proteins could directly affect WM, spreading across brain networks. Since hippocampal atrophy is common in the early phase of disease, it is reasonable to expect that hippocampal volume (HV) might be also related to WM integrity. Our study aimed to evaluate the integrity of the whole‐brain WM, through diffusion tensor imaging (DTI) parameters, in mild AD and amnestic mild cognitive impairment (aMCI) due to AD (with Aβ42 alteration in cerebrospinal fluid [CSF]) in relation to controls; and possible correlations between those measures and the CSF levels of Aβ42, phosphorylated tau protein (p‐Tau) and total tau (t‐Tau). We found a widespread WM alteration in the groups, and we also observed correlations between p‐Tau and t‐Tau with tracts directly linked to mesial temporal lobe (MTL) structures (fornix and hippocampal cingulum). However, linear regressions showed that the HV better explained the variation found in the DTI measures (with weak to moderate effect sizes, explaining from 9% to 31%) than did CSF proteins. In conclusion, we found widespread alterations in WM integrity, particularly in regions commonly affected by the disease in our group of early‐stage disease and patients with Alzheimer's disease. Nonetheless, in the statistical models, the HV better predicted the integrity of the MTL tracts than the biomarkers in CSF.
Traditional Chinese medicine (TCM) has significantly contributed to protecting human health and promoting the progress of world civilization. A total of 2,711 TCMs are included in the 2020 version of the Chinese Pharmacopoeia, which is an integral part of the world’s medical resources. Tu Youyou and her team discovered and purified artemisinin. And their contributions made the values and advantageous effects of TCM more and more recognized by the international community. There has been a lot of studies on TCM to treat diseases through antioxidant mechanisms, the reports on the new mechanisms beyond antioxidants of TCM has also increased year by year. Recently, many TCMs appear to have significant effects in regulating ferroptosis. Ferroptosis is an iron-dependent, non-apoptotic, regulated cell death characterized by intracellular lipid peroxide accumulation and oxidative membrane damage. Recently, accumulating studies have demonstrated that numerous organ injuries and pathophysiological process of many diseases are companied with ferroptosis, such as cancer, neurodegenerative disease, acute renal injury, arteriosclerosis, diabetes, and ischemia-reperfusion injury. This work mainly introduces dozens of TCMs that can regulate ferroptosis and their possible mechanisms and targets.
Morus alba Linn. is a plant with varieties of phytochemicals and commonly known as Mulberry tree or Nuni (in Assamese). Mulberry is
a perennial and woody plant, belongs to the family Moraceae consider as a native plant of China. In Assam, the leaves of mulberry tree are
out most important in horticulture department for feeding purpose. A literature survey was done out by using key words such as Morus
alba, or mulberry tree or phytoconstituents, or pharmacological activities, or ethnobotany on the search engine namely Scopus, Web of
Science, Google Scholars, PubMed. The purpose of this article is to forward a solid review about the plant profile, method of extraction,
phytochemicals constituents and pharmacological activities in reference to SARS-CoV-2. All the parts of Morus alba are phytochemically rich but highest number can be found in fruits and leaves. Fruits of Morus alba contains various class of phytochemicals such as alkaloids, anthocyanins, stillbenoids, flavones. Owing to presence of this compounds, it exhibits various pharmacological. Its leaves, fruits and root bark containing flavonoids, anthocyanins, alkaloids and stilbenoids, possess various pharmacological properties. Resveratrol is one of the most important compounds found in M. alba which possess SARS-CoV-2 inhibiting potential. Still there are some gaps remain in exploring whole plants parts and successive studies of toxicity.
Traditional Chinese medicine (TCM) provides therapeutic and health care effects through dietary intake. Owing to the susceptibility of plants to contaminations, a risk assessment system is urgently needed to ensure the safe use of TCMs. In this study, the contamination levels and risks associated with the dietary intake of short-chain chlorinated paraffins (SCCPs) and medium-chain chlorinated paraffins (MCCPs) were investigated in six kinds of frequently-used TCM herbs. The concentrations varied from 144.4 to 1527.8 ng·g⁻¹ dw for SCCPs and non-detect to 1214.1 ng·g⁻¹ dw for MCCPs, with mean values of 551.5 and 259.8 ng·g⁻¹ dw, respectively. A geographic distribution analysis indicated that the concentrations of CPs in TCMs were mainly associated with their levels of contamination in the ambient environment. Carbon atom-chlorine congener profiles of CPs were dominated by C10Cl7-8 and C14Cl7-8 congeners, accounting for 20.1% and 32.4% of the total SCCP and MCCP concentrations, respectively. Principal component analysis indicated that the TCM species might be the main factor influencing the accumulation of SCCPs congeners. Finally, a risk assessment reveals that the estimated daily intake and margin of exposure were far below levels that might pose a health risk, indicating an acceptable dietary intake of SCCPs and MCCPs in the studied TCMs. This is the first report of CPs in the TCM herbs and the obtained results are expected to aid in future evaluation of the quality of TCMs and ensuring diet and drug safety.
Graphical abstract
Fourier transform near-infrared (FT-NIR) spectroscopy is a nondestructive, rapid, real-time analysis of technical detection methods with an important reference value for producers and consumers. In this study, the feasibility of using FT-NIR spectroscopy for the rapid quantitative analysis and qualitative analysis of ‘Zaosu’ and ‘Dangshansuli’ pears is explored. The quantitative model was established by partial least squares (PLS) regression combined with cross-validation based on the spectral data of 340 pear fresh fruits and synchronized with the reference values determined by conventional assays. Furthermore, NIR spectroscopy combined with cluster analysis was used to identify varieties of ‘Zaosu’ and ‘Dangshansuli’. As a result, the model developed using FT-NIR spectroscopy gave the best results for the prediction models of soluble solid content (SSC) and titratable acidity (TA) of ‘Dangshansuli’ (residual prediction deviation, RPD: 3.272 and 2.239), which were better than those developed for ‘Zaosu’ SSC and TA modeling (RPD: 1.407 and 1.471). The results also showed that the variety identification of ‘Zaosu’ and ‘Dangshansuli’ could be carried out based on FT-NIR spectroscopy, and the discrimination accuracy was 100%. Overall, FT-NIR spectroscopy is a good tool for rapid and nondestructive analysis of the internal quality and variety identification of fresh pears.
Tissue clearing methods are increasingly essential for the microscopic observation of internal tissues of thick biological organs. We previously developed TOMEI, a clearing method for plant tissues; however, it could not entirely remove chlorophylls nor reduce the fluorescent signal of fluorescent proteins. Here, we developed an improved TOMEI method (iTOMEI) to overcome these limitations. First, a caprylyl sulfobetaine was determined to efficiently remove chlorophylls from Arabidopsis thaliana seedlings without GFP quenching. Next, a weak alkaline solution restored GFP fluorescence, which was mainly lost during fixation, and an iohexol solution with a high refractive index increased sample transparency. These procedures were integrated to form iTOMEI. iTOMEI enables the detection of much brighter fluorescence than previous methods in tissues of A. thaliana, Oryza sativa, and Marchantia polymorpha. Moreover, a mouse brain was also efficiently cleared by the iTOMEI-Brain method within 48 h, and strong fluorescent signals were detected in the cleared brain.
Hypercholesterolemia is a well-known etiological feature for cardiovascular diseases and a common indication of maximum categories of metabolic disorders. Para methoxy cinnamic acid is one of the cinnamic acid derivatives as a natural product obtained from the rice bran oil as an active constituent and has the antioxidant property. The present study was designed to evaluate the hypolipidemic activity of P-methoxy cinnamic acid against high fat diet induced hyperlipidemia in experimental rats. Male Wistar albino rats were divided into five groups (n = 6), and high fat diet was used to induce the hyperlipidemia for 28 days. P-methoxy cinnamic acid was used in two different doses (40 and 80 mg/kg body weight), and they were administered orally to the rats for 28 days during high fat diet. Atorvastatin (5 mg/kg) was used as reference standard. A significant elevated level of lipid abnormalities and tissue antioxidant parameters were reversed from normal level by the treatment of P-methoxy cinnamic acid in both the doses. Histopathological evidence further supported the protective action. Based on the initial findings, it was concluded that P-methoxy cinnamic acid was able to offer significant protection against high fat diet induced atherosclerosis. Future studies were recommended to identify the molecular mechanism of P-methoxy cinnamic acid against atherosclerosis protection.
The aim of the study was to challenge the common techniques utilized in the quality control of herbal products for the authentication of the two selected polyherbal products. Two polyherbal formulations, Voltarit® and Rheumax® commonly trade in the market to treat arthritis and related disorders, had been investigated regarding their quality control through selecting representative marker components from each drug. Different chromatographic procedures had been applied of which, a new validated HPLC method was developed for the simultaneous quantification of three active components (curcumin, demethoxycurcumin, bisdemethoxycurcumin, quercetin, and myristicin). Additionally, GC-FID was used for the relative quantification of fatty acids known for their anti-inflammatory activity in the hexane extract of the selected products. LC-MS-MS and GC-MS techniques were applied to tentatively identify some naturally occurring COX-2 inhibitors in methanol extract and fatty acid of the selected products, respectively. One of the products was adulterated by curcumin and surprisingly had no limonene or myristicin even it had celery seed as the pharmaceutical company said. The validation showed good linearity (R is between 0.9990 and 0.9998), sensitivity (LODs 0.1-0.4 μg), precision (RSD < 2%), and accuracy (99.82-101.4%) of the method. The developed HPLC method was proved to be a useful tool in the quality control of complex matrices of the selected polyherbal formulations. Many active components with anti-inflammatory effects were identified in the fixed oils, methanol, and hexane extracts of the HMs, so patients could take these herbal products with confidence about good quality and no chemical adulteration.
Background
Panax notoginseng is a highly valued medicinal herb used widely in China and many Asian countries. Its root and rhizome have long been used for the treatment of cardiovascular and hematological diseases. Imaging the spatial distributions and dynamics of metabolites in heterogeneous plant tissues is significant for characterizing the metabolic networks of Panax notoginseng, and this will also provide a highly informative approach to understand the complex molecular changes in the processing of Panax notoginseng.
Methods
Here, a high-sensitive MALDI-MS imaging method was developed and adopted to visualize the spatial distributions and spatiotemporal changes of metabolites in different botanical parts of Panax notoginseng.
Results
A wide spectrum of metabolites including notoginsenosides, ginsenosides, amino acids, dencichine, gluconic acid, and low-molecular-weight organic acids were imaged in Panax notoginseng rhizome and root tissues for the first time. Moreover, the spatiotemporal alterations of metabolites during the steaming of Panax notoginseng root were also characterized in this study. And, a series of metabolites such as dencichine, arginine and glutamine that changed with the steaming of Panax notoginseng were successfully screened out and imaged.
Conclusion
These spatially-resolved metabolite data not only enhance our understanding of the Panax notoginseng metabolic networks, but also provide direct evidence that a serious of metabolc alterations occurred during the steaming of Panax notoginseng.
Vanda roxburghii has been used in traditional medicine to treat nervous system disorders including Alzheimer’s disease (AD). We reported earlier a high acetylcholinesterase inhibitory and antioxidant activity in the chloroform fraction of this plant. Therefore, this study was designed to explore the compounds with acetylcholinesterase inhibitory and antioxidant activities from the chloroform fraction of Vanda roxburghii. Phytochemical investigation led to the isolation for the first time of a fatty acid ester: methyl linoleate (1), and three phenolics: syringaldehyde (2), vanillin (3), and dihydroconiferyl dihydro-p-coumarate (4) along with the previously reported compound gigantol (5). Among the isolates, vanillin (3) and dihydroconiferyl dihydro-p-coumarate (4) were found to significantly inhibit the activity of acetylcholinesterase, scavenge the free radicals, exhibit the reducing power and total antioxidant activity, and effectively reduce the peroxidation of lipid. Gigantol (5) and syringaldehyde (2), despite lacking the activity against acetylcholinesterase, exhibited antioxidant activity. Among the compounds, gigantol (5) appeared to be the most potent antioxidant. These findings revealed that V. roxburghii contained compounds with potential acetylcholinesterase inhibitory and antioxidant activity, which support its traditional use in the treatment of AD.
Searching for bioactive agents from medicinal plants, eleven constituents were isolated from Polyscias guilfoylei stem for the first time, including a nucleoside uracil (1), two sterols β-sitosterol (2) and daucosterol (3), a saponin androseptoside A (4), two lignans (+)-pinoresinol (5) and (+)-syringaresinol (6), four phenolic acids protocatechuic acid (7), methyl protocatechuate (8), caffeic acid (9), and 5-O-caffeoylquinic acid (10), and a flavonoid quercitrin (11). Metabolites 1, 4, and 6–11 have never been observed in genus Polyscias before. Phenolic compounds 7 and 9 possessed the respective IC50 values of 21.33 and 13.88 µg/mL in DPPH (2,2-diphenyl-1-picrylhydrazyl) antioxidative assay, as compared with that of the positive control resveratrol (IC50 = 13.21 µg/mL). From density functional theory (DFT) calculated approach, the DPPH free radical scavenging capacity of two compounds 7 and 9 can be explained by the role of OH groups at carbons C-3 and C-4. Antioxidative actions of these two potential agents are followed HAT (H atom transfer) mechanism by OH bond disruption in gas, but SPLET (sequential proton loss electron transfer) mechanism in solvents water and methanol. Compared to 4-OH group, 3-OH group showed better bond disruption enthalpies and better kinetic energies since it reacted with HOO• and DPPH radicals. Sterols 2–3 and flavonoid 11 induced the IC50 values of < 2.0 µg/mL better than the positive control acarbose (IC50 = 184.0 µg/mL) in α-glucosidase inhibitory assay. Their interactions with human intestinal C- and N-terminal domains of α-glucosidase were explored using molecular docking study. The obtained results proved that compounds 2, 3, and 11 bind relatively stronger with the C-terminal domain than to the N-terminal domain through pivotal residues in the binding site and could be hypothesized as mixed inhibitors.
Graphic abstract
Bletilla striata is a well-known traditional Chinese herb with anti-inflammatory properties that is widely used in the treatment of lung conditions such as silicosis, tuberculosis, and pneumogastric hemorrhage. However, little information on the anti-inflammatory ingredients and their activities is available. In this study, an effect fraction of Bletilla striata (EFBS) was enriched, and its anti-inflammatory activities and underlying mechanisms were investigated. EFBS was enriched by polyamide column chromatography and characterized by HPLC; an LPS-induced acute lung injury model was used to evaluate the anti-inflammatory activities of EFBS. Meanwhile, the main anti-inflammation-contributing ingredients and possible molecular mechanism of anti-inflammatory activity in EFBS were verified by component-knockout method combined with LPS-induced RAW264.7 cell model. The EFBS mainly consisted of coelonin (15.88%), batatasin III (32.49%), 3-O-methylbatatasin III (6.96%), and 3-hydroxy-5-methoxy bibenzyl (2.51%). Pretreatment with the EFBS (20 mg/kg and 60 mg/kg) for five days prior to the administration of LPS resulted in decreases in wet-to-dry lung weight ratio, neutrophil number, MPO activity, total protein concentration, NO level, and MDA level, as well as IL-1β, IL-6, MCP-1, and TNF-α concentrations in the bronchoalveolar lavage fluid. Western blot analysis demonstrated the increased expressions of iNOS, COX-2, and NF-κB p65 in the LPS treatment group, all of which were ameliorated by EFBS pretreatment. Histological examination confirmed the protective effect of the EFBS. Additionally, component-knockout assay confirmed that these four quantitative components contributed significantly to the anti-inflammatory effect of EFBS. Coelonin, batatasin III, 3-O-methylbatatasin III and 3-hydroxy-5-methoxy bibenzyl were the main anti-inflammatory components of EFBS and could regulate the expression of downstream inflammatory cytokines by inhibiting p65 nuclear translocation. These findings uncover, in part, the molecular basis underlying the anti-inflammatory activity of Bletilla striata.
1. Introduction
Bletilla striata (Thunb.) Reichb. f. is a perennial herbaceous orchid known as Baiji (白及) in China. Its dried pseudobulb has been used for millennia in traditional Chinese medicine as an astringent hemostatic agent for the treatment of hemoptysis, hematemesis, traumatic bleeding, and chapped skin [1].
Many conditions can lead to the occurrence of hemoptysis, such as lung cancer [2], tuberculosis [3], and silicosis [4]. Clinical reports have indicated that Bletilla striata is beneficial for treating tuberculosis and silicosis [5, 6]. Accordingly, four Bletilla striata-related products have been approved by the China Food and Drug Administration (CFDA) [7] for use in the treatment of gastric ulcers and lung-related conditions such as pertussis, tuberculosis, silicosis, chronic tracheitis, and emphysema. However, the active ingredients in these products are largely unknown or not well characterized.
Researchers have attempted to elucidate the pharmacologically active substances in Bletilla striata (and their mechanisms of action) from various perspectives. The polysaccharides in Bletilla striata are the most widely and deeply studied components, exhibiting wound-healing [8], antiulcer [9], hemostatic [10], and immune modulation activities [11]. However, a growing body of evidence indicates that the small-molecule components of Bletilla striata may play more important therapeutic roles. For example, the n-butyl alcohol fraction of a 70% ethanol Bletilla striata extract was demonstrated to exhibit excellent platelet-aggregation activity [12] while the terpenoid and stilbenoid compounds from Bletilla striata were shown to exhibit angiogenesis [13] and antitumor activities [14], respectively. Furthermore, the present authors’ previous work has demonstrated that the ethanol extract of Bletilla striata effectively prevents silica-induced lung fibrosis by regulating the antioxidation system, the immune system, and cytokine levels [15]. Importantly, this extract was found to be more effective than the polysaccharides in Bletilla striata [16]. Clearly, the small-molecule components of Bletilla striata are worthy of further research.
Silicosis, an incurable disease, characterized by chronic lung inflammation and progressive fibrosis, affects tens of millions of workers involved in dusty occupations in many countries [17]. Therefore, it is of great significance to explore the pathogenesis of silicosis, develop drugs to prevent, treat, or relieve the symptoms, and improve the quality of life of silicosis patients. Research has indicated that sustained inflammation plays a critical role in silica-induced pulmonary fibrosis [18], and downregulation of inflammation can ameliorate pulmonary fibrosis [19]. Macrophages are key regulators during this process and become the ideal target of anti-inflammatory drug development [20]. The lipopolysaccharide- (LPS-) induced RAW264.7 cell inflammation model in vitro and LPS-induced acute lung injury (ALI) model in vivo are two accepted methods for investigating the underlying mechanisms of inflammation and screening anti-inflammatory drugs. Based on these models, medicinal plants have been reported to possess numerous secondary metabolites with potent anti-inflammation effects and demonstrated therapeutic promises for inflammation-related diseases [21]. Therefore, medicinal plants are important resources of anti-inflammatory compounds [22]. And our previous studies confirmed that the ethanol extract of Bletilla striata has the preventive and therapeutic effect on silica-induced pulmonary fibrosis in rats [15]. We further isolated and identified a series of anti-inflammatory compounds from the ethanol extract of Bletilla striata [23, 24], guided by LPS-induced RAW264.7 cell inflammation model, of which coelonin has the strongest anti-inflammatory activity [24, 25]. Meanwhile, an effective fraction of Bletilla striata (EFBS) with significant anti-inflammatory activity was prepared [25]. Accordingly, the purpose of this study was to assess the therapeutic efficacy of EFBS in an ALI mouse model, so as to reveal the anti-inflammatory molecular mechanism and provide scientific basis for the establishment of the pharmacodynamic index components of the Chinese herb Bletilla striata.
2. Materials and Methods
2.1. EFBS Preparation
The pseudobulbs of Bletilla striata were collected from Meichuan, Wuxue, Hubei province, China, and authenticated by Dr. Zhishan Ding of Zhejiang Chinese Medical University. A voucher specimen (BS No. 20171028) was deposited in the College of Life Science at Zhejiang Chinese Medical University. EFBS were prepared according to our previous study [25]. Briefly, 100.0 g of the tuber powder was reflux extracted in 1 L of 80% ethanol, and this was repeated three times. The filtrate was concentrated under vacuum to 600 mL, and an equal volume of distilled water along with 30.0 g polyamide (100-200 mesh, Taizhou Luqiao Sijia Biochemical Plastics Factory, Zhejiang, China) was added. The suspension was then further concentrated under vacuum to 600 mL. Finally, the suspension was packed into a suitable column and successively eluted with water, 20% ethanol, and 40% ethanol, and the 40% ethanol elution was collected, concentrated, and dried under vacuum to obtain the EFBS under investigation. The EFBS was characterized, and the main component contents were obtained by HPLC. Briefly, each sample was injected (10 μL) and analyzed using a Dionex Ultimate™ 3000 HPLC system (Dionex, California, USA) with pulsed amperometric detection at 193 nm. An Acclaim® 120 C18 (, 5 μm) HPLC column protected with a Phenomenex security guard column (C18, ) operated at 30°C was used, and the flow rate was maintained at 1 mL/min. The elution solvents were acetonitrile (A) and 0.1% acetic acid (B). Samples were eluted according to the following gradient: 0-5 min 10% to 40% A, 5-12 min 40% A isocratic, 12-16 min 40% to 45% A, 16-22 min 45% A isocratic, 22-25 min 45% to 48% A, and 25-33 min 48% A isocratic, and finally washing and reconditioning of the column.
2.2. The Four Main Compounds Knockout-EFBS (KO-EFBS) Preparation
To better reveal the contribution of four main components to the anti-inflammatory activity of EFBS, the four compounds were removed by semipreparative liquid chromatography on a Dionex Ultimate™ 3000 semiprepared HPLC system (Dionex, California, USA) to obtain sample KO-EFBS. A Welch Ultimate® XB-C18 (, 10 μm) HPLC column operated at 30°C was used, and the flow rate was maintained at 5 mL/min, and detection at 193 nm. Samples were eluted with acetonitrile (A) and 0.1% acetic acid (B) as the following gradient: 0-26 min 32% A isocratic, 26-28 min 32%-55% A, 28-38 min 55%A isocratic, 38-39 min 55%-90% A, 39-43 min 90% A isocratic, 43-44 min 90%-32% A, and 44-54 min 32% A isocratic. And meanwhile, the four main component peaks were also collected and mixed to prepare mixed-compounds 1-4 fraction (MC1-4). Both KO-EFBS and MC1-4 were characterized by the HPLC method described above.
2.3. Animals and Cell Culture
Male Institute of Cancer Research (ICR) mice were obtained from the SIPPR/BK Laboratory Animal Company (Shanghai, China) and were used at 6–8 weeks of age (20-25 g). The mice were caged under pathogen-free conditions (, 12 h light/12 h darkness cycle, 45-55% relative humidity) and allowed free access to water and food during the study period. All experiments were performed in full compliance with standard laboratory animal care protocols approved by the Institutional Animal Care Committee of Zhejiang Chinese Medical University.
RAW264.7 cells (ATCC) were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were maintained in DMEM medium (Gibco, USA) containing 10% heat inactivated fetal bovine serum (Gibco, USA), 100 units/mL penicillin, and 100 μg/mL streptomycin. They were cultured at 37°C in a humidified atmosphere of 5% CO2 incubator.
2.4. Drug and LPS Administration
The mice were randomly divided into five groups (control group, model group, DEX (5 mg/kg, Sigma) group, EFBS (20 mg/kg) group, and EFBS (60 mg/kg) group). The control and model groups were administrated equal volumes of vehicle orally, and the DEX and EFBS groups were administered intraperitoneally and orally, respectively. All groups were administered continuously for five days, and 5 mg/kg LPS was infused 1 h after the last administration. Mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg, Sigma), and then LPS (5 mg/kg, Sigma) was intratracheally instillated. The control group was administered an equal volume of PBS. All the mice were sacrificed 6 h after LPS administration.
2.5. Lung Wet-to-Dry Weight Ratio
Lung edema was calculated by reference to the lung wet-to-dry weight ratio (W/D). The right lung was ligated and excised, and the wet weight of the upper lobes of right lung was determined. The lungs were then placed in an incubator at 60°C for three days to obtain the dry weight, and the W/D weight ratio was then calculated.
2.6. Bronchoalveolar Lavage Fluid Collection and Cell Count
Following the removal of the right lung, the bronchoalveolar lavage fluid (BALF) was collected by lavaging with of PBS containing 0.1 mM EDTA. Then the BALF samples were centrifuged at for 10 min at 4°C, and the supernatants were collected for cytokine determination. The cell pellets were resuspended in 1 mL of PBS to count the total cells and neutrophils using a hemocytometer and staining by the Wright-Giemsa method.
2.7. Assessment of Capillary Leakage
Inflammation factors dramatically increase the permeability of alveolar-capillary barriers and thus following inflammation cell and protein infiltration. Hence, the total protein concentration in the BALF supernatant was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, China).
2.8. Measurement of MPO Activity and Oxidant Stress
As an index of neutrophil infiltration, the BALF MPO activity was measured with assay kits (Multi Sciences Biotechnology, Shanghai, China) according to the manufacturer’s instructions.
The concentrations of NO and MDA in the BALF supernatant were determined using commercially available colorimetric assay kits (Beyotime Biotechnology, China) according to the manufacturer’s instructions.
2.9. Measurement of BALF Cytokine Levels
Levels of IL-1β, TNF-α, IL-6, and MCP-1 in the BALF were quantified using the CBA method according to the manufacturer’s protocols (BD, USA) using a BD Accuri™ C6 flow cytometer (BD, USA).
2.10. Lung Histopathology
The lower lobes of right lung were fixed with 10% neutral formalin overnight at room temperature, and specimens were dehydrated in graded alcohol and embedded in paraffin. Then, 4 μm-thick paraffin sections were obtained and stained with hematoxylin and eosin (H&E) according to the standard protocol. The degree of pathological injury was scored based on neutrophils infiltration, edema, hemorrhage, and disorganization of lung parenchyma, as the scoring system described by Kiyonari et al. [26]. The damage degree of each index was scored with values from 0 to 4, and higher scores suggest more severe damage presented. Finally, total histological scores were calculated [27].
2.11. In Vitro Cell Viability Assay
RAW264.7 cells were seeded into 96-well plates at a density of cells/well and were treated with different concentrations of EFBS for 48 h. Then, cell viability was determined using CCK8 assay according to the manufacturer’s instructions. Briefly, 10 μL CCK8 reagent (Dojindo, Tokyo, Japan) was added to each well and incubated for 3 hours at 37°C. Absorbance was measured at 450 nm on a microplate reader.
2.12. Cell Apoptosis Assay
RAW264.7 cells were seeded into 6-well plates at a density of cells/well and were treated with increasing concentrations of EFBS for 48 h. Cells were detected using the Annexin V-FITC apoptosis detection kit (556547, BD Pharmingen, USA) and analyzed on a Beckman CytoFLEX S flow cytometer (Beckman Coulter, CA.USA).
2.13. Measurement of Cell Culture Supernatant Cytokine Levels
RAW264.7 cells were pretreated with EFBS, KO-EFBS, MC1-4, or KO-EFBS combined with MC1-4 for 1 h and followed by stimulation with LPS (200 ng/mL) for 12 h. The culture supernatant was collected for IL-6 and TNF-α detection. The remaining cells were then treated by 1 mM ATP for additional 15 min at 37°C [28]; then, supernatants were collected for IL-1β detection. All cytokines were quantified using the Cytometric Beads Array (CBA) method according to the manufacturer’s protocols (BD, USA).
2.14. Western Blot Analysis
The middle lobes of right lung tissue were washed twice in ice-cold PBS, cut into small pieces, and homogenized in cold protein lysis buffer (Thermo Fisher Scientific, USA) with protease and phosphatase inhibitors (Roche, Germany). Total proteins were obtained by incubating the lysates at 4°C for 30 min with vortex mixing, followed by centrifugation at for 15 min at 4°C, and the supernatant was transferred to a new tube for analysis.
For cell culture samples, RAW264.7 cells were pretreated by EFBS, KO-EFBS, MC1-4, KO-EFBS combined with MC1-4, or single compounds for 1 h and then stimulated with LPS (200 ng/mL) for 30 min or 24 h. Cells were collected, and whole-cell proteins were extracted with M-PER Mammalian Protein Extraction Reagent (78503, Thermo Fisher Scientific, USA) at 4°C for 30 min. Then, the samples were centrifuged at for 15 min, and the supernatant was transferred to a new tube to analyze the expression of iNOS and COX2 (24 h treated samples) and the phosphorylation of JNK, ERK1/2, and p38 (30 min treated samples). And nuclear proteins were extracted in accordance with the instruction of the Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime Biotechnology, China) for the detection of the nuclear translocation of p65.
Before blotting, the protein was quantified using the BCA method. Simple western immunoblotting was performed on a Simple Wes system (ProteinSimple, California, USA) using a Size Separation Master Kit with Split Buffer (12-230 kDa) according to the manufacturer’s standard instruction and using anti-iNOS (EPR16635) (ab178945, Abcam, USA), anti-NF-κB p65 (E379) (ab32536, Abcam, USA), anti-COX2 (EPR12012) (ab179800, Abcam, USA), anti-p38 (ab170099, Abcam, USA), anti-p38(phospho T180+Y182) (ab195049, Abcam, USA), anti-JNK1+JNK2+JNK3 (ab208035, Abcam, USA), anti-JNK1+JNK2+JNK3(phospho T183+T183+T221) (ab124956, Abcam, USA), anti-ERK1+ERK2 (ab54230, Abcam, USA), anti-Erk1(pT202/pY204)+Erk2 (pT185/pY187) (ab50011, Abcam, USA), anti-Lamin A/C antibody (2032S, CST, USA), and anti-β-actin (4970S, CST, USA) antibodies. The Compass software (version 4.0.0, ProteinSimple) was used to program the Simple Wes and for presentation (and quantification) of the western immunoblots. Output data were displayed from the software as calculated averages of seven exposures (5-480 s).
2.15. Statistical Analysis
All data are presented as , and eight animals are included in each group. Statistical analysis was carried out using Graph Pad Prism 6.0 (La Jolla, CA) and performed using a one-way ANOVA followed by Dunnett’s test. is accepted as the level of significance.
3. Results
3.1. Characterization and Quantification of EFBS
The EFBS was prepared and characterized by HPLC. It is mainly composed of stilbenes [6, 29] and the compounds we previously identified [23]. Quantitative analysis of the four main components coelonin, batatasin III, 3-O-methylbatatasin III, and 3-hydroxy-5-methoxy bibenzyl [24] in EFBS was performed, and their contents were revealed to be 15.88%, 32.49%, 6.96%, and 2.51%, respectively (Figure 1(a)). A standardized EFBS sample (Figure 1(b)) was used for subsequent pharmacological studies.
(a)
Apple is a rich source of bioactive phytochemicals that help improve health by preventing and/or curing many disease processes, including cancer. One of the apple polyphenols is phloretin [2′,4′,6′-Trihydroxy-3-(4-hydroxyphenyl)-propiophenone], which has been widely investigated for its antioxidant, anti-inflammatory and anti-cancer activities in a wide array of preclinical studies. The efficacy of phloretin in suppressing xenograft tumor growth in athymic nude mice implanted with a variety of human cancer cells, and the ability of the compound to interfere with cancer cells signaling, have made it a promising candidate for anti-cancer drug development. Mechanistically, phloretin has been reported to arrest the growth of tumor cells by blocking cyclins and cyclin-dependent kinases and induce apoptosis by activating mitochondria-mediated cell death. The blockade of the glycolytic pathway via downregulation of GLUT2 mRNA and proteins, and the inhibition of tumor cells migration, also corroborates the anti-cancer effects of phloretin. This review sheds light on the molecular targets of phloretin as a potential anti-cancer and anti-inflammatory natural agent.
Five coumarins (5,7,8-trimethoxycoumarin (1), sabandin (2), cubreuva lactone (3), 5,7-dimethoxycoumarin (5) and braylin (6)), seven furoquinoline alkaloids (isopimpinelin (4), pteleine (7), maculine (8), skimianine (10), robustine (11), y-fagarine (12) and dictamine (13) and the furofuran type lignin syringaresinol (9)) have been identified for the first time in the roots of Zanthoxylum tingoassuiba A. St.-Hil., Rutaceae. Pure compounds 1, 6, 9, 12 were tested against Leishmania amazonensis parasites and epimastigotes forms of Trypanosoma cruzi. All the tested products displayed an antiparasitic activity similar to that of the positive controls (benznidazole and amphotericin B). Compound 9 was the most active against both parasites with IC50 values of 11.98 μM and 7.55 μM against L. amazonensis and T. cruzi, respectively.
Context: Seaweeds are rich in bioactive compounds in the form of vitamins, phycobilins, polyphenols, carotenoids, phycocyanins and polysaccharides; many of these are known to have advantageous applications in human health. 3-Hydroxy-4,7-megastigmadien-9-one (comp) was isolated from Ulva pertusa (U. pertusa) Kjellman (Ulvaceae), which is a familiar edible green seaweed.
Objective: This study evaluates the anti-inflammatory activity of comp in CpG DNA-stimulated bone marrow-derived dendritic cells (BMDCs).
Materials and methods: For evaluating the effect of comp on cytokines production, BMDCs were treated with doses of comp (0, 0.5, 1, 2, 5, 10, 25 and 50 μM) for 1 h before stimulation with CpG DNA (1 μM). Cytokine production was measured by ELISA. Western blotting was conducted for evaluating effect of comp (50 μM) on MAPKs and NF-κB pathways. Luciferase reporter gene assay was conducted for effect of comp (0, 5, 10 and 25 μM) on transcriptional activity of AP-1 and NF-κB.
Results: Comp exhibited strong inhibition of interleukin (IL)-12 p40, IL-6 and TNF-α cytokine production with IC50 values of 6.02 ± 0.35, 27.14 ± 0.73, and 7.56 ± 0.21 μM, respectively. It blocked MAPKs and NF-κB pathways by inhibiting the phosphorylation of ERK1/2, JNK1/2, p38 and IκBα. In addition, it strongly inhibited the transcriptional activity of AP-1 and NF-κB with IC50 values of 8.74 ± 0.31 and 12.08 ± 0.24 μM, respectively.
Discussion and conclusion: Taken together, these data suggest that comp has a significant anti-inflammatory property and warrants further studies concerning the potential of comp for medicinal use.
Optical projection tomography (OPT) is a well-established method for visualising gene activity in plants and animals. However, a limitation of conventional OPT is that the specimen upper size limit precludes its application to larger structures. To address this problem we constructed a macro version called Macro OPT (M-OPT). We apply M-OPT to 3D live imaging of gene activity in growing whole plants and to visualise structural morphology in large optically cleared plant and insect specimens up to 60 mm tall and 45 mm deep. We also show how M-OPT can be used to image gene expression domains in 3D within fixed tissue and to visualise gene activity in 3D in clones of growing young whole Arabidopsis plants. A further application of M-OPT is to visualise plant-insect interactions. Thus M-OPT provides an effective 3D imaging platform that allows the study of gene activity, internal plant structures and plant-insect interactions at a macroscopic scale.
Recently the number of studies investigating triterpenoid saponins has drastically increased due to their diverse and potentially attractive biological activities. Currently the literature contains chemical structures of few hundreds of triterpenoid saponins of plant and animal origin. Triterpenoid saponins consist of a triterpene aglycone with one or more sugar moieties attached to it. However, due to similar physico-chemical properties, isolation and identification of a large diversity of triterpenoid saponins remain challenging. This study demonstrates a methodology to screen saponins using hyphenated analytical platforms, GC-MS, LC-MS/MS, and LC-SPE-NMR/MS, in the example of two different phenotypes of the model plant Barbarea vulgaris (winter cress), glabrous (G) and pubescent (P) type that are known to differ by their insect resistance. The proposed methodology allows for detailed comparison of saponin profiles from intact plant extracts as well as saponin aglycone profiles from hydrolysed samples. Continuously measured 1D proton NMR data during LC separation along with mass spectrometry data revealed significant differences, including contents of saponins, types of aglycones and numbers of sugar moieties attached to the aglycone. A total of 49 peaks were tentatively identified as saponins from both plants; they are derived from eight types of aglycones and with 2–5 sugar moieties. Identification of two previously known insect-deterrent saponins, hederagenin cellobioside and oleanolic acid cellobioside, demonstrated the applicability of the methodology for relatively rapid screening of bioactive compounds.
Tree shrews are small mammals now commonly classified in the order of Scandentia, but have relatively closer affinity to primates than rodents. The species has a high brain-to-body mass ratio and relatively well-differentiated neocortex, and thus has been frequently used in neuroscience research, especially for studies on vision and neurological/psychiatric diseases. The available atlases on tree shrew brain provided only limited information on white matter (WM) anatomy. In this study, diffusion tensor imaging (DTI) was used to study the WM anatomy of tree shrew, with the goal to establish an image-based WM atlas. DTI and T2-weighted anatomical images were acquired in vivo and from fixed brain samples. Deterministic tractography was used for three-dimensional reconstruction and rendering of major WM tracts. Myelin and neurofilaments staining were used to study the microstructural properties of certain WM tracts. Taking into account prior knowledge on tree shrew neuroanatomy, tractography results, and comparisons to the homologous structures in rodents and primates, an image-based WM atlas of tree shrew brain was constructed, which is available to research community upon request.
Nuclear magnetic resonance imaging can provide high resolution images of internal structures of fruits and vegetables and can be used for nondestructive evaluation of various internal quality factors, such as bruises, dry regions, worm damage, stage of maturity, and presence of voids, seeds, and pits. Experimental parameters have profound effects on image enhancement of specific features of the specimen.
In this study, magnetic resonance imaging (MRI) and bulk nuclear magnetic resonance (NMR) were used to track the progressive development of intact oil palm fruit (variety Tenera). Fresh fruits were harvested at 4, 12, 16 and 21 weeks after anthesis (WAA) and all measurements of spin-spin relaxation times (T 2 -values) were performed at 2.35 Tesla and 20ºC. MR imaging data were fitted to mono-exponential decay curves, which depicted a progressive increase in the mean T 2 –values for mesocarp (18-32 ms) and kernel (13-74 ms) with WAA. The same data were also fitted to bi-exponential decay curves for both the mesocarp and kernel, which showed a decrease of the T 21 -values from 12-10 ms and 10-9 ms, respectively and an increase of the T 22 -values from 83-118 ms and 129-139 ms, respectively with WAA; those two components are assigned to the protons of water (T 21) and to those of the oil (T 22). The multi-exponential fitting of the bulk NMR T 2 relaxation data for the intact fruit demonstrated three distinct components; T 21 (intermediate component, 23-30 ms) and T 22 (long component; 64-144 ms), which are assigned to water protons and oil, respectively and T 23 (short component; 3-6 ms) related to the shell. The bulk NMR T 2 relaxation data for the kernel also demonstrated three components; T 21 (intermediate component, 12-22 ms) and T 22 (long component, 59-135 ms), which was assigned to water bound in the endosperm (kernel) and oil content respectively, while short component T 23 (3-2 ms) was associated to the shell.
Mature leaves of plants transferred from low to high light typically increase their photosynthetic capacity. In Arabidopsis thaliana, this dynamic acclimation requires expression of GPT2, a glucose 6-phosphate/phosphate translocator. Here, we examine the impact of GPT2 on leaf metabolism and photosynthesis. Plants of wild-type and of a GPT2 knockout (gpt2.2) grown under low light achieved the same photosynthetic rate, despite having different metabolic and transcriptomic strategies. Immediately upon transfer to high light, gpt2.2 plants showed a higher rate of photosynthesis than wild-type (35%) however, over subsequent days, wild-type plants acclimated photosynthetic capacity, increasing photosynthesis 100 after 7 days. Wild-type plants accumulated more starch than gpt2.2 plants throughout acclimation. We suggest that GPT2 activity results in the net import of glucose 6-phosphate from cytosol to chloroplast, increasing starch synthesis. There was clear acclimation of metabolism, with short terms changes typically being reversed as plants acclimated. Distinct responses to light were seen in wild-type and gpt2.2 leaves. Significantly higher levels of sugar-phosphates were seen in gpt2.2. We suggest that GPT2 alters the distribution of metabolites between compartments and that this plays an essential role in allowing the cell to interpret environmental signals.
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In this paper, we propose a novel method for parcellating the human brain into 193 anatomical structures based on diffusion tensor images (DTIs). This was accomplished in the setting of multi-contrast diffeomorphic likelihood fusion using multiple DTI atlases. DTI images are modeled as high dimensional fields, with each voxel exhibiting a vector valued feature comprising of mean diffusivity (MD), fractional anisotropy (FA), and fiber angle. For each structure, the probability distribution of each element in the feature vector is modeled as a mixture of Gaussians, the parameters of which are estimated from the labeled atlases. The structure-specific feature vector is then used to parcellate the test image. For each atlas, a likelihood is iteratively computed based on the structure-specific vector feature. The likelihoods from multiple atlases are then fused. The updating and fusing of the likelihoods is achieved based on the expectation-maximization (EM) algorithm for maximum a posteriori (MAP) estimation problems. We first demonstrate the performance of the algorithm by examining the parcellation accuracy of 18 structures from 25 subjects with a varying degree of structural abnormality. Dice values ranging 0.8-0.9 were obtained. In addition, strong correlation was found between the volume size of the automated and the manual parcellation. Then, we present scan-rescan reproducibility based on another dataset of 16 DTI images - an average of 3.73%, 1.91%, and 1.79% for volume, mean FA, and mean MD respectively. Finally, the range of anatomical variability in the normal population was quantified for each structure.
Scope
Gastro‐AD (GAD) is a soy flour derived product that undergoes an industrial fermentation with Lactobacillus delbrueckii R0187 and has demonstrated clinical effects in gastroesophageal reflux and peptic ulcer symptom resolution. The aim of this study is to describe and link GAD's metabolomic profile to plausible mechanisms that manifest and explain the documented clinical outcomes.
Methods and results
¹ H NMR spectroscopy with multivariate statistical analysis is used to characterize the prefermented soy flour and GAD products. The acquired spectra are screened using various resources and the molecular assignments are confirmed using total correlation spectroscopy (TOCSY). Peaks corresponding to different metabolites are integrated and compared between the two products for relative changes. HPLC and GC are used to quantify some specific molecules. NMR analyses demonstrate significant changes in the composition of various assigned bioactive moieties. HPLC and GC analysis demonstrate deglycation of isoflavones after fermentation, resulting in estrogenically active secondary metabolites that have been previously shown to help to reduce inflammation.
Conclusion
The identification of bioactive molecules, such as genistein and SCFAs, capable of modulating anti‐inflammatory signaling cascades in the stomach's gastric and neuroendocrine tissues can explain the reported biological effects in GAD and is supported by in vivo data.
Amino compounds are widely present in complex mixtures in chemistry, biology, medicine, food, and environmental sciences involving drug impurities and metabolisms of proteins, biogenic amines, neurotransmitters, and pyrimidine in biological systems. Nuclear magnetic resonance (NMR) spectroscopy is an excellent tool for simultaneously identifying and quantifying these in-mixture compounds but has a limit-of-detection (LOD) over several micromolarities (>5 μM). To break such a sensitivity barrier, we developed a sensitive and rapid method by combining the probe-induced sensitivity enhancement and nonuniform-sampling-based 1H–13C HSQC 2D-NMR (PRISE-NUS-HSQC). We introduced two 13CH3 tags for each analyte to respectively increase the 1H and 13C abundances for up to 6 and 200 fold. This enabled high-resolution detection of 0.4–0.8 μM analytes in mixtures in 5 mm tubes with a 5 min acquisition on 600 MHz spectrometers. The method is much more sensitive and faster than traditional 1H–13C HSQC methods (∼50 μM, >10 h). Using sulfanilic acid as a single reference, furthermore, we established a database covering chemical shifts and relative-response factors for >100 compounds, enabling reliable identification and quantification. The method showed good quantitation linearity, accuracy, precision, and applicability in multiple biological matrices, offering a rapid and sensitive approach for quantitative analysis of large cohorts of chemical, medicinal, metabolomic, food, and other mixtures.
Resveratrol is a dietary polyphenol that interacts with gut microbiota to possess various biological activities. To identify the microbial metabolites of resveratrol, fresh feces from 12 volunteers were cultured in vitro. Their urine samples were collected after taking a commercial capsule containing 600 mg of resveratrol. Metabolites were characterized and quantified by UPLC-Q-Exactive plus orbitrap MS/MS. The results showed that dihydroresveratrol, 3-(4-hydroxyphenyl)-propionic acid, and lunularin were the major microbial metabolites of RSV with interindividual differences. 3-(4-Hydroxyphenyl)-propionic acid significantly attenuated the inflammatory response of LPS-treated RAW264.7 cells and DSS-induced colitis in antibiotics-treated pseudo-germ-free mice by regulating MAPK and NF-κB pathways. In contrast, dihydroresveratrol did not exhibit significant anti-inflammatory effects, and lunularin exhibited pro-inflammatory effects in cells. This study may help to better understand the health effects of resveratrol and its microbial metabolites.
Positron emission tomography (PET) is an imaging technology that measures 3D spatial distribution and kinetics of radio-tagged biomolecules in a living subject quantitatively and nondestructively. Commonly used positron-emitting radionuclides include 11C, 13N, and 15O, which are essential elements for plant growth. Combining radiotracer techniques with PET, this in vivo molecular imaging capability offers plant biologists a powerful tool for molecular phenotyping research. While PET is widely used clinically for cancer diagnosis and pre-clinically for drug development, it is an unfamiliar imaging tool for plant biologists. This chapter introduces the basic principles of PET, factors that affect the quantitative accuracy of PET when imaging plants, and techniques for administering radiotracers to plants for a variety of molecular plant imaging applications.
The moistening process of Rehmanniae Radix was characterized quantitatively by moisture phase, texture properties, and component content based on water absorption kinetics and expansion kinetics. Non-linear fitting of water absorption kinetics and expansion kinetics in the moistening process of Rehmanniae Radix was carried out. Low-field nuclear magnetic resonance and imaging(LF-NMR/MRI) technology was used to investigate the phase state and distribution changes of water during the moistening process. The Texture Analyzer was used for the determination of texture properties. The correlations between water absorption rate, expansion rate, water phase state, hardness, and compression cycle work of Rehmanniae Radix at different moistening time were analyzed. The results showed that the water absorption kinetics and expansion kinetics of Rehmanniae Radix were in accordance with the first-order kinetics. Moreover, the water absorption rate and expansion rate increased with the increase in temperature but decreased with the increase in the size of the medicinal materials.In the moistening process, the moisture was transferred from the outside to the inside, and the proportion of the moisture phase changed significantly.Within 16 hours, free water increased from 0.825% to 97.7%,while bound water decreased from 99.2% to 2.33%.Within 28 hours, the texture properties, such as hardness and compression cycle work, decreased gradually with the prolongation in moistening time.At 32 hours, water was evenly distributed throughout the whole medicinal material, and the texture properties also tended to be stable.Pearson correlation bivariate analysis showed that moistening time, water absorption rate, expansion rate, the relative content of free water and bound water, hardness, and compression cycle work were significantly correlated, suggesting that water absorption kinetics and expansion kinetics, LF-NMR/MRI,and Texture Analyzer could directly and quantitatively characterize the moistening process.This study is expected to provide a scientific basis for clarifying the scientific connotation of the moistening process of Rehmanniae Radix.
Glycyrrhiza uralensis is a popular medicinal plant worldwide. Its roots and rhizomes are used as the traditional Chinese medicine Gan-Cao. However, little is known on medicinal potential and chemistry of the other parts of the plant. In this work, the biological activities and chemical components of the roots, stems, leaves, and seeds of G. uralensis were investigated comparatively. The four parts exhibited different but noticeable biological activities. The chemicals in the four parts were globally characterized by liquid chromatography mass spectrometry (LC/MS) on a Thermo Vanquish UHPLC system connected to a Q-Exactive quadrupole Orbitrap mass spectrometer. By integrating molecular networking, compound spectral matching, MS2LDA-based substructure recognition, and reference standards comparison, a total of 1301 compounds were rapidly characterized. Three flavonoid C-glycosides were purified and their structures were identified by NMR spectroscopic analysis. Orthogonal partial least squares-discriminate analysis (OPLS-DA) further revealed 196 differential chemicals for the four parts. This work will promote the medicinal resource utilization of G. uralensis.
The constituents of Ceratocarpus arenarius L., as a traditional anticancer medicine of Kazakh, were firstly profiled with UHPLC-QTOF-MS/MS. The potential compounds against EGFR-TK were virtually screened. As a result, forty-four compounds were analyzed, including 18 flavonoids, 8 steroids, 4 phenolic acids, 9 fatty acids, 1 coumarin and 4 other compounds. Among them, 9 flavonoids, N-trans-Feruloyltyramine (5), stigmasterol (11) and carthamone (38) were recognized as potential key anti-tumor constituents of C. arenarius through docking to active site of EGFR-TK. It indicated that the compounds formed moderate to strong interactions with EGFR-TK contributing to the antitumor activity through a synergetic actions. Besides, the anticancer effects of C. arenarius was verified with in-vitro anti-tumor activity investigation against A549. Our results firstly reveals the active constituents basis of C. arenarius against cancer and provides novel insights into the further application of effective constituents and mechanism of C. arenarius.
The study of the organ structure of plants and understanding their physiological complexity requires 3D imaging with subcellular resolution. Most plant organs are highly opaque to light, and their study under optical sectioning microscopes is therefore difficult. In animals, many protocols have been developed to make organs transparent to light using clearing protocols (CPs). By contrast, clearing plant tissues is challenging because of the presence of fibers and pigments. We describe progress in the development of plant CPs over the past 20 years through a modified taxonomy of CPs based on their physical and optical parameters that affect tissue properties. We also discuss successful approaches that combine CPs with new microscopy methods and their future applications in plant science research.
The improvement of the harvest period standards is critical in the quality control of Chinese medicinal materials. The present study statistically analyzed the harvest period standards of plant medicinal materials in the 2020 edition of Chinese Pharmacopoeia(Vol.Ⅰ) and put forward the existing problems and suggestions based on herbal records and modern research to provide references for the improvement of the standards. According to the statistical analysis, in 499 types of plant medicinal materials, harvest period standards are recorded under 486 types, accounting for 97.4%, and are lacking in the remaining. Only one medicinal material(Stellariae Radix) is recorded with the standard of the harvest year. The standards of the harvest season and phenological period are recorded under 233 types, accounting for 46.7%. For 237 types, only harvest season is specified, accounting for 47.5%, and for 15 types, only harvest phenological period is specified, accounting for 3.0%. Among 222 types mainly derived from cultivation and 51 types from wild resources and cultivation, only 11 types are recorded with harvest period of cultivated products. Only Stellariae Radix is recorded with the harvest period standards for the wild and cultivated products separately. The harvest period standards of plant medicinal materials with different medicinal parts have certain rules to follow. The main problems about the harvest period standards are discovered. Specifically, no harvest period standards are recorded under 13 types of plant medicinal materials. Almost all perennial cultivated medicinal materials are not recorded with harvest year standard. No phenological period standard is found under 250 types of plant medicinal materials. There is no clear distinction between the harvest period standards of cultivated and wild products. The evidence for harvest period standards of 26 types of plant medicinal materials that can be harvested all year round is insufficient. As a result, it is proposed to strengthen basic research in response to the above-mentioned problems and improve the harvest period standards as soon as possible to ensure the quality of Chinese medicinal materials.
When investigating the potential use of plants as a raw material for an all-natural cosmetic formulation, the main parameters are the chemical composition, antioxidant potential, antimicrobial action, and toxicity. Additionally, the production of natural cosmetics should also consider the availability of primary materials and the environmental and socioeconomic impact. Gliricidia sepium is a species that produces a large amount of plant material, being cultivated in the agroforestry system. However, studies of phytochemical composition and chemical spatial distribution are scarcely using the MALDI MS (matrix-assisted laser desorption ionization mass spectrometry) and MALDI MSI (mass spectrometry imaging) techniques. A methodology was developed to optimize ionization parameters and analysis conditions by evaluating the efficiency of three matrices: α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid (DHB), and 2-mercaptobenzothiazole in MALDI MS analysis. All results were compared to ESI MS (electrospray ionization mass spectrometry), and afterward, MALDI MSI analysis was performed on the leaf surface. This study showed through phytochemical analysis that G. sepium leaves are composed of polyphenols and tannins, concluding that the methanolic extract had a higher amount of flavonoid content. Four compounds were identified on the leaf surface, and their spatial distribution was analyzed by MALDI MS using DHB as a matrix. Kaempferol, isorhamnetin, and some fatty acids showed potential applicability for cosmetical use. All the extracts presented antioxidant activity or antimicrobial action and no cytotoxicity. Therefore, extracts of G. sepium could be used as raw materials in cosmetics.
The mortality of sepsis-induced cardiac dysfunction (SICD) is very high due to the complex pathophysiological mechanism. Syringaresinol (SYR) is a natural abstract which possesses anti-inflammatory property. The present study aims was to identify the protective impact of SYR on sepsis-induced cardiac dysfunction and investigate the specific mechanisms. We found that SYR improved the cardiac function and alleviated myocardial injury in mice that subjected to cecal ligation and puncture, in addition, SIRT1 expression was significantly elevated after SYR treatment compared to sepsis group both in vivo and in vitro, along with suppression of NLRP3 activation and proinflammatory cytokines release. However, SIRT1 inhibitor EX427 abolished the impact of SYR on LPS-induced pyroptosis in cardiomyocytes. Furthermore, molecular docking analysis predicted that there is high affinity between SYR and estrogen receptor (ER), ER inhibitor ICI182780, the specific ERβ inhibitor PHTP and the specific ERαinhibitor AZD9496 were used to examine the role of ER in the protective effect of SYR against SICD, and the results suggested that ER activation was essential for the cardioprotective function of SYR. In conclusion, SYR ameliorates SICD via the ER/SIRT1/NLRP3/GSDMD pathway.
Defining the spatial distributions of metabolites and their structures are the two key aspects for interpreting the complexities of biosynthesis pathways in plants. As a means of obtaining information on the spatial distribution of metabolites, a strategy is needed that has high sensitivity and allows visualization. Toward this goal, we carried an untargeted metabolomics to obtain detailed metabolic information on different plant parts of Salvia miltiorrhiza, the roots of which are widely used in traditional Chinese medicine. Systematic optimization of desorption electrospray ionization mass spectrometry imaging (DESI-MSI) including parameter selection and sample preparation were carried out to improve the sensitivity of the method for plant samples. Guided by the metabolomics data, the spatial distributions of diverse metabolites, including phenolic acids, flavonoids, tanshinones, carbohydrates, and lipids, were characterized and visualized for both the underground and aerial parts. To integrate the information pertaining to the spatial distribution of metabolites, the flavonoids and phenolic acids (phenylpropanoid metabolic pathway) were chosen as examples for in-depth study the biosynthesis pathways in S. miltiorrhiza. The complementary data obtained from the metabolomics study and mass spectrometry imaging enabled the identification of key reactions involved in flavonoid biosynthesis in flowers, which lead the changes in metabolite distribution. The analysis also identified the core precursor for phenolic acid biosynthesis in Salvia species. Therefore, the powerful combination of metabolomics and mass spectrometry imaging provides a basis for obtaining detailed information on spatial metabolome and constitutes a platform for deep understanding the biosynthesis of bioactive metabolites in plants.
Camellia sinensis (tea) is an evergreen plant having bioactive compounds associated with various pharmacological effects, including anti-cancerous activity. These phytochemicals are variedly distributed in plant tissues. A detailed study to understand chemical composition within the economically underutilized tea tissues is required to generate value. Therefore, a comprehensive chemical profiling of underutilized C. sinensis parts [coarse leaves, flowers, fruits (immature); n = 9] was performed by NMR techniques. NMR (1D and 2D) spectroscopy ambiguously identified and quantified fifty-seven metabolites (Coarse leaves: 35, flowers; 42, immature fruits; 45). The statistical analysis showed apparent tissue-specific similarities (26 metabolites) and variations. Further, HPLC-DAD revealed absolute quantification of catechins, caffeine and theanine among the different parts of C. sinensis. Moreover, cytotoxicity studies of tea tissues against colorectal cancer cell lines showed anticancer potentials. This chemical information and anticancer activity of underutilized C. sinensis parts will help to develop value added nutraceutical and cosmeceutical products.
Objective: To study the chemical constituents from the active ethyl acetate part of the stems of Dendrobium officinale. Methods: The compounds were isolated and purified by column chromatographies on macroporous resin, MCI, silica gel, Sephadex LH-20, ODS, preparative thin-layer chromatography and preparative RP-HPLC. Their structures were identified by the analysis of their spectra data of ¹H-NMR, ¹³C-NMR, MS and the physical and physiochemical properties. Results: A total of 34 compounds were isolated and identified from the ethyl acetate fraction of D. officinale as moscatin (1), loliolide (2), naringin (3), hesperetin (4), liquiritigenin (5), 2-(4-hydroxy-3-methoxyphenyl)-3-(2-hydroxy-5-methoxyphenyl)-3-oxo-l-propanol (6), isoliquiritigenin (7), (E)-4-hydroxycinnamic acid (8), (Z)-4-hydroxycinnamic acid (9), coniferyl p-coumarate (10), sinapyl p-coumarate (11), tinosporaic acid A (12), N-trans-coumaroyltyramine (13), N-trans-feruloyltyramine (14), syringaresinol (15), pinoresinol (16), medioresinol (17), lirioresionol (18), 2(5H)-furanone 5-hydroxy-3,4-dimethyl-5-pentyl (19), syringic acid (20), palmitic acid (21), 4-hydroxybenzoic acid (22), p-hydroxybenzaldehyde (23), ferulic acid (24), dihydroconiferyl alcohol (25), N-heptadecane (26), 4-hydroxy-3,5-dimethoxy trans cinnamaldehyde (27), dihydro-p-cinnamic acid (28), (+)-(4S)-(2E)-4-hydroxy-2-nonanoic acid (29), 1,3-benzenediol (30), hydroquinone (31), salicylic acid (32), p-hydroxyacetophenone (33), and vanillin (34). Conclusion: Compound 2 is isolated from Dendrobium for the first time, and compounds 3-7 are found from D. officinale for the first time. © 2021, Editorial Office of Chinese Traditional and Herbal Drugs. All right reserved.
Dendrobium huoshanense is one of the rare Chinese medicinal herbs with high quality and excellent efficacy. However, its effective chemical basis is still unclear. Of note, D. officinale is the most widely utilized among the Dendrobium species. Therefore, the current study systematically investigated the chemical constituents of methanolic extracts and different polar fractions of aqueous extracts from the two herbs by HPLC‐ESI‐MSn, and then compared in vitro antioxidant activities of their 5 different polar extracts. Consequently, 61 and 49 compounds were identified from D. huoshanense and D. officinale, respectively, of which, 43 compounds were common to both species. Besides, 17 out of 22 different compounds were identified only in D. huoshanense. Moreover, the peak areas of some shared identical compounds of D. huoshanense were significantly larger than that of D. officinale. In vitro antioxidant evaluation results showed that n‐BuOH‐soluble fraction of the two herbs exhibited remarkable antioxidant activity. Furthermore, the antioxidant activity of different fractions of D. huoshanense were separately superior to that of D. officinale, which may be attributed to its variable and high contents of flavonoids, bibenzyls and phenanthrenes. These results provide the evidence for the high quality and efficacy of D. huoshanense.
Aim
Brain microvascular endothelial cells (BMVECs), as the important structure of blood-brain barrier (BBB), play a vital role in ischemic stroke. Pyroptosis of different cells in the brain may aggravate cerebral ischemic injury, and PGC-1α plays a major role in pyroptosis. However, it is not known whether BMVECs undergo pyroptosis after ischemic stroke and whether PGC-1α activator Medioresinol (MDN) we discovered may be useful against pyroptosis of endothelial cells and ischemic brain injury.
Methods
For in vitro experiments, the bEnd.3 cells and BMVECs under oxygen and glucose-deprivation (OGD) were treated with or without MDN, and the LDH release, tight junction protein degradation, GSDMD-NT membrane location and pyroptosis-associated proteins were evaluated. For in vivo experiments, mice underwent transient middle cerebral artery occlusion (tMCAO) for ischemia model, and the neuroprotective effects of MDN were measured by infarct volume, the permeability of BBB and pyroptosis of BMVECs. For mechanistic study, effects of MDN on the accumulation of phenylalanine, mitochondrial reactive oxygen species (mtROS) were tested by untargeted metabolomics and MitoSOX Red probe, respectively.
Results
BMVECs underwent pyroptosis after ischemia. MDN dose-dependently activated PGC-1α, significantly reduced pyroptosis, mtROS and the expressions of pyroptosis-associated proteins (NLRP3, ASC, cleaved caspase-1, IL-1β, GSDMD-NT), and increased ZO-1 and Occludin protein expressions in BMVECs. In tMCAO mice, MDN remarkably reduced brain infarct volume and the permeability of BBB, inhibited pyroptosis of BMVECs, and promoted long-term neurobehavioral functional recovery. Mechanistically, MDN promoted the interaction of PGC-1α with PPARα to increase PPARα nuclear translocation and transcription activity, further increased the expression of GOT1 and PAH, resulting in enhanced phenylalanine metabolism to reduce the ischemia-caused phenylalanine accumulation and mtROS and further ameliorate pyroptosis of BMVECs.
Conclusion
In this study, we for the first time discovered that pyroptosis of BMVECs was involved in the pathogenesis of ischemic stroke and MDN as a novel PGC-1α activator could ameliorate the pyroptosis of endothelial cells and ischemic brain injury, which might attribute to reduction of mtROS through PPARα/GOT1 axis in BMVECs. Taken together, targeting endothelial pyroptosis by MDN may provide alternative therapeutics for brain ischemic stroke.
Six lignans (1-6) were isolated from salted Aconiti lateralis Radix Praeparata for the first time. These isolates were elucidated as hedyotisol-A (1), (7“R,8”R)-8“-syringaresinol-4”-hydroxy-3“,5”-dimethoxyphenyl-7“,9”-propanediol (2), lariciresinol-4-O-β-D-glucopyranoside (3), (7S,8S)-4-hydroxy-3-methoxy-7,8-(2′,1′-O-β-D-glucopyranosyl)phenyl-propanetriol (4), (+)-isolariciresinol (5), and (+)-lyoniresinol (6) by analyzing extensive and comprehensive spectral data and compared with the data described in the literature, respectively. Compounds (1-6) were evaluated for their neuroprotective activities against corticosterone-induced cell death in PC12 cells with desipramine as the positive control drug. Among them, compounds 1 and 2 showed moderate neuroprotective activities, which increased the survival rates of PC12 cells from 45.50 ± 2.23% to 65.98 ± 1.29%, 58.19 ± 2.94% at 10 μM, respectively.
Eventhough the development of vaccine against COVID-19 pandemic is progressing in different part of the world a well-defined treatment plan is not yet developed. Therefore, we investigate the inhibitory activity of a group of dietary bioactive flavonoids against SARS-CoV-2 main protease (Mpro), which are identified as one of the potential targets in the drug discovery process of COVID-19. After the initial virtual screening of a number of bioactive flavonoids, the binding affinity of three compounds - Naringin, Naringenin and Amentoflavone - at the active site of Mpro was investigated through MD Simulations, MM-PBSA and DFT Binding Energy calculations. From the MD trajectory analysis, Amentoflavone and Naringin showed consistent protein-ligand interactions with the aminoacid residues of the active site domains of Mpro. The excellent inhibitory activity of Amentoflavone and Naringin was established from its MM-PBSA binding energy values of −190.50 and −129.87 kJ/mol respectively. The MET165 residue of Mpro is identified as one of the key residue which contributed significantly to MM-PBSA binding energy through hydrophobic interactions. Furthermore, the DFT binding energy values of Amentoflavone (-182.92 kJ/mol) and Naringin (-160.67 kJ/mol) in active site molecular clusters with hydrogen bonds confirmed their potential inhibitory activity. These compounds are of high interest because of their wide availability, low cost, no side effects, and long history of use. We can prevent the severity of this disease for home care patients using these effective dietary supplements. We are hopeful that our results have implications for the development of prophylaxis of COVID-19.
Communicated by Ramaswamy H. Sarma
Two new dihydrophenanthrofurans (1 and 2) and two new bisbibenzyl derivatives (3 and 4) were isolated from the traditional Chinese medicinal plant Dendrobium nobile, along with four known compounds (5–8). The absolute configurations of compounds 1 and 4 were elucidated through extensive NMR and ECD spectroscopic analyses. New compounds showed no antimicrobial activity against four gram-positive bacterial strains and four gram-negative bacteria at the concentration of 1 mg/mL, but displayed significant cytotoxic activity against HepG2 human hepatic cell line with the IC50 values ranging from 1.25 μM to 19.47 μM.
Keywors: Dendrobium nobile, Orchidaceae, bisbibenzyl, dihydrophenanthrofuran.
Objective To study the moisture transfer laws of Chaenomeles sinensis in different drying processes. Methods Using the non-destructive and non-invasive technique of low field-nuclear magnetic resonance (LF-NMR), the transverse relaxation time (T 2 ) inversion spectrum of C. sinensis slice was monitored under different drying methods (hot air drying, drying after evaporation, segmental drying and drying in the shade) to analyze the changes of moisture migration. Results There were three different types water that were detected in C. sinensis (free water > bound water > immobilized water). The internal water distribution and water content changed during drying process. The moisture changes were similar in hot air drying, drying after steaming, and drying in shade, the total water gradually decreased, and the combining degree between moisture and non-water components enhanced. Steaming promoted the water loss rate of C. sinensis slice, the water loss rate was higher in drying after steaming than in hot air drying, and the difference was significant (P < 0.05). During the intermittent drying, the conversion of different states of water would occur in order to return to a relatively stable equilibrium. During the low temperature drying process, immobilized water content decreased and free water content increased. The low-temperature drying has less damage to the tissue, which is more conducive to the conversion of immobilized water into free water, and thus the water dissipated faster. During the early of drying, high temperature caused tissue structure damage, the bonding force between water and non-aqueous tissue would be strengthened because of the tissue shrinkage. Conclusion The three different types water content and peak area in T 2 was positively correlated. The LF-NMR technique would provide useful guides for the investigation of water distribution and variation of C. sinensis, which will provide a theoretical basis for C. sinensis processing. © 2018, Editorial Office of Chinese Traditional and Herbal Drugs. All right reserved.
Nitrogen is an essential, often limiting, factor in plant growth and development. To regulate growth under limited nitrogen supply, plants sense the internal and external nitrogen status, and coordinate various metabolic processes and developmental programs accordingly. This coordination requires the transmission of various signaling molecules that move across the entire plant. Cytokinins, phytohormones derived from adenine and synthesized in various parts of the plant, are considered major local and long-distance messengers. Cytokinin metabolism and signaling are closely associated with nitrogen availability. They are systemically transported via the vasculature from plant roots to shoots, and vice versa, thereby coordinating shoot and root development. Tight linkage exists between the nitrogen signaling network and cytokinins during diverse developmental and physiological processes. However, the cytokinin-nitrogen interactions and the communication systems involved in sensing rhizospheric nitrogen status and in regulating canopy development remain obscure. We review current knowledge on cytokinin biosynthesis, transport and signaling, nitrogen acquisition, metabolism and signaling, and their interactive roles in regulating root-shoot morphological and physiological characteristics. We also discuss the role of spatio-temporal regulation of cytokinins in enhancing beneficial crop traits of yield and nitrogen use efficiency.
The microscopic morphology of plant cells and their ergastic substances is an important standard for the identification of Chinese traditional medicine. We have developed a new method, X-ray phase-contrast imaging (XPCI) based on the microfocus X-ray tube, to explore microstructures of Chinese herbal medicine. The results indicate that XPCI is capable of distinguishing the structures commonly used in the identification. Non-destructive detection and high sensibility are counted among the major advantages of XPCI. The possibility of future applications of XPCI in the field of medicine identification is discussed.
Constituents of Ephemerantha fimbriata (BL.) P. F. HUNT et SUMMERH, which is used as a source plant of the Chinese crude drug 'Shi-Hu', were examined and two new phenanthrenes, fimbriol-A (2) and fimbriol-B (8), a new dihydrophenanthrene, ephemeranthol-C (6), and dihydroconiferyl dihydro-p- coumarate (5) were isolated together with denbinobin (1), (+)-pinoresinol (3), (+)-syringaresinol (4), 3,4,5-trimethoxybenzoic acid (7), lusianthridin (9), and dihydro-p-coumaric acid (10). Structures of the new compounds were elucidated by the use of spectroscopic methods including two-dimensional NMR techniques.
Background/aim:
Phenolic compounds isolated from Dendrobium ellipsophyllum Tang & Wang (Orchidaceae) have been shown to possess potential pharmacological activity; however, their anticancer as well as anti-metastasis activities are largely unknown. The aim of the present study was to isolate active compounds from D. ellipsophyllum and to explore the possible effects of phenolic compounds isolated from the plant for cytotoxic as well as anti-metastatic properties.
Materials and methods:
The compounds were isolated by using chromatographic techniques including silica gel and Sephadex LH20. Each of the isolates was evaluated for their cytotoxicity on H292 human lung cancer cell lines by 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay. The cytotoxic compounds were further evaluated for apoptosis-inducing and anoikis-sensitizing effects.
Results:
Ten phenolic compounds were isolated, 5,7-dihydroxy-chromen-4-one (1:); 4,5-dihydroxy-2,3-dimethoxy-9,10-dihydrophenanthrene (2:); moscatilin (3:), 4,4'-dihydroxy-3,5-dimethoxybibenzyl (4:); 4,5,4'-trihydroxy-3,3'-dimethoxybibenzyl (5:); (2S)-homoeriodictyol (6:); (2S)-eriodictyol (7:); chrysoeriol (8:); phloretic acid (9:); and luteolin (10:). Compounds 4:, 5:, 8: and 10: exhibited appreciable cytotoxic activity with 50% inhibitory concentration values less than 250 μM. These compounds also showed potential apoptosis induction and anoikis-sensitizing effect at non-toxic concentrations.
Conclusion:
Compounds 4:, 5:, 8: and 10: are responsible for cytotoxic and anti-metastatic activities of D. ellipsophyllum.
Regional heterogeneity in cortical cyto- and myeloarchitecture forms the structural basis of mapping of cortical areas in the human brain. In this study, we investigate the potential of diffusion MRI to probe the microstructure of cortical gray matter and its region-specific heterogeneity across cortical areas in the fixed human brain. High angular resolution diffusion imaging (HARDI) data at an isotropic resolution of 92-μm and 30 diffusion-encoding directions were acquired using a 3D diffusion-weighted gradient-and-spin-echo sequence, from prefrontal (Brodmann area 9), primary motor (area 4), primary somatosensory (area 3b), and primary visual (area 17) cortical specimens (n = 3 each) from three human subjects. Further, the diffusion MR findings in these cortical areas were compared with histological silver impregnation of the same specimens, in order to investigate the underlying architectonic features that constitute the microstructural basis of diffusion-driven contrasts in cortical gray matter. Our data reveal distinct and region-specific diffusion MR contrasts across the studied areas, allowing delineation of intracortical bands of tangential fibers in specific layers—layer I, layer VI, and the inner and outer bands of Baillarger. The findings of this work demonstrate unique sensitivity of diffusion MRI to differentiate region-specific cortical microstructure in the human brain, and will be useful for myeloarchitectonic mapping of cortical areas as well as to achieve an understanding of the basis of diffusion NMR contrasts in cortical gray matter.
Two new ionone derivatives, named rhododendrone and rhododendronside, were isolated from the alcoholic extract of the aerial parts of Rhododendron przwalskii Maxim. Their structures were elucidated on the basis of spectroscopic analysis Keywords: Rhododendron przewalskii Maxim., Ericaceae, rhododendrone, rhododendronside. Rhododendron przewalskii Maxim. has been used as a folk medicine in China for the treatment of hypertension and coronary heart disease 1 . As a part of our continuing program on the study of plant-derived bioactive compounds, the chemical constituents of R. przewalskii Maxim. growing in Gansu were investigated. In the previous paper 2 , we reported eight components from this genus. The present paper deals with the structure elucidation of two new ionone derivatives named rhododendrone 1 and rhododendron-side 2 isolated from the EtOH extract of the aerial parts of R. przewalskii.
Bruising of the mesocarp in avocado fruit is an important postharvest issue for the industry. Proton magnetic resonance imaging (1H-MRI) was used as a non-destructive tool to monitor bruise expression over time in avocado cv. Hass fruit. 1H-MRI clearly identified fruit morphological features and bruised mesocarp tissue. The pixel intensity value of T2 weighted spin echo 1H-MRI images of avocado fruit pericarp changed over time with fruit softening. Bruised mesocarp tissue in impacted fruit appeared relatively hyperintense (brighter) in T2 weighted 1H-MRI images. For firm ripe fruit impacted from 25 cm drop height (0.38 J ± 0.004) and for firm ripe fruit impacted from 50 cm drop height (0.81 J ± 0.011), hyperintensity in the mesocarp beneath the impact point was evident immediately after impact. However, visible symptoms of bruising in the form of flesh browning did not appear in parallel serial destructive assessments until after day 1 following impact on day 0. The brown, bruised mesocarp volume in ripe fruit increased progressively over the assessment period of 3 days. This trend was evident in destructive assessments as well as in 1H-MRI images. In hard green mature fruit impacted from 100 cm drop height (1.68 J ± 0.020), contrast between mesocarp tissue beneath the impact site and surrounding sound mesocarp was evident in T2 weighted 1H-MRI images from day 0. However, no bruise symptoms were evident as flesh browning upon serial destructive assessments of fruit over the 3 days assessment period. The average pixel intensity values at the impact site in T2 weighted 1H-MRI images for both firm ripe and hard green mature fruit decreased over the period of assessment. In contrast, the pixel intensities in the T2 weighted 1H-MRI images of diseased flesh increased over time.
